r0LS Of CALCIUM AND PHOJPHOLiPIDS IN TiANSEPITHELIAL SODIUM ION AND

WATERR TRAft>PORT IN1 AIMPHIBIAN EPITHELIA

THESIS

Pres-nted to tne Graduate Council of the North Texas State University

in Partial Fullfiliiment of the Requirements

For the Degreo of

MATER OF SCIENCE

3y

Nlimman Tarapoom, B.A.

Donton, Texas

August, 1963

larapoom, Nimman., Role of Calcium and Phospholipids in

Transepithejial Sodium Ion and Water Transport in Amphibian

Epithelia. Master of Basic Health Science (Pnarmacology), August,

19o3, 123 pp., 10 tables, 31 figures, bibliography, 139 titles.

The present investigation is concerned with determining the role

of calcium, phospholipids, and phospnoiipid metabolites on

transapithelial sodium and water transport in response to

antidiuretic hormone (ADH). These studies utilize the frog skin for

determining sodium transport and amphibian urinary bladder for water

fUow measurements and scanning electron microscopy of cell surface

morpnology.

ine results demonstrate tnat phospholipids and phospholipid

metabolites containing arachidonic acia stimulate transepithelial

sodium transport through amiloride sensitive channels and the action

of rhese lipids involves tne synthesis of prostaglandins. These

lipids also inhibited the increase in water flow induced by ADH, and

this effect was prevented with prostaglandin synthesis inhibitors.

Prostaglandins alter intracellular calcium concentrations and agents

effecting calcium metabolism alter cell surface morphology and the

changes in surface substructure induced by ADH. These observations

support the hypothesis that alterations in membrane permeability to

water and ions :ay involve metabolism of membrane phospnohipids and

prostaglandin biosynthesis.

TABLE OF CONTENTS

PageLIST OF TABLES.................. .............. v

LILT OF ILLUSTRATIONS ...... .. .......... vi

INTRODUCTION........ .... . ........ ... .1

Effects of ADH on the Human Kidney. . . .1Role of Calcium Ions in ADH Action.......... ... 4Pnospholipid Metabolism. ..a...............11Role of Microtubules and Microfilaments in ADH Action. .21

hE..D................. . .. ......... 27

Animals................ . .............. 27Solutions,............ . . ...... ... 27Drugs. ... .......... ....... .26Osmotic Water Flow, ...... ... . ..... .... 29Heasurment of Electrical Properties Across Frog SKin . .30Seperation of Toad Urinary Bladder Epithelial Cells. . -36Oxygen Consumption. ................ ... . .......... 37Scanning Electron Microscopy, .-..................... 39Protein Determination... . ........... . .4oStatistical Analysis.0. ... . .......... . 42

RESULTS.................. . ........... 43

Effects of Phospholipids on Sodium Ion Transport. . . . 43Effects of Phospholipids on Water Transport... .... 56Effects of ADH, Verapamil, Calcium Ionophore onSurface Pvorphology of Frog Urinary Bladder.. .........68

DISCUSSIO0N..0.0...-- --- .. . ....... .......95

SUMMARY...................* .. ..0. .0....... .......108

BIBLIOGriAPHY...................... .... .. .. .. .. ... 111

iv

LISP OF TABLES

Ta bl Page

I. Effects of DAG on tne Electrical Properties Across FrogSkin . . . . . . . .0 .0 .0 .0 .0 .0 .0 .0 .0 .a .0 .a .0 .0 .0 .0 . 48

II. Effects of DAG on Unidirectional Sodiumi Flux Across FrogSkin................... ......... ... 50

III. Effects of Phospholipids and PhospholipidMetabolites on the Electrical Properties of Frog skin

. .. .. ................ .... . ... .. ...... 52

IV. Effects of DAG on ADH Hydro-Osmotic Response . ..... 58

V. Effects of PhosphotidyIcncline on Water FlowAcross Toad Bladder.-.-.-.-.-.-..............59

VI. Effects of Phosphopipids and PhospholipidMetabolites on Water Flow Across Toad Bladder . . .60

VII. Effects of Prostaglandin E2 and Arachidonic Acid onWater Flow Across Toad Bladder. . .......... .03

VIII. Effects of Indomethacin on Arachidonic Acidinhibited ADH-Induced Water Flow.. ........ 65

IX. Effects of Arachidonic Acid and PGE2 on CyclicAMP-Induced Iater Flow.-.-.-.-.-............66

X. Effects of Arachidonic Acid on ADH-Stimulated WaterFlow in tne Presence of Low Calcium.b.7..... ...67

V

LIST OF ILLUSTRATIONS

Figure Page

1. Biosyntnesis Pathway of Phospholipids . . . . . . . . 12

2. Synthesis Patfnway of Phospholipids fromPhosphaidic Acid . . . . . . . .. . . . . . . . . 14

J. Synthesis Pathway for PhosphatidyicholineBiosyntnesis. . . . . . . . . . . . . . . . . . . .14

4. Sites of Pnospholipase Attack on Phospholipids

5. Pathways of Arachidonic Acid Metabolism . . . . . . . 19

-. Toad Bladaer Preparation to Measure Water Flow . . . .31

7. Using Chamber Preparation of Short-CircuitTechnique.............-.-.-.-.-.. ....33

b. Oxygen Electrode and Sample Chamber to MeasureOxygen Consumption .-.-.........-.-.... 36

9. Standard Curve for Protein Determination. . . . . . . 41

10. Diagram of Open and Snort-Circuited Epithelium . . . .44

11. Effect of DAG on Electrical Properties on Frog SKin* ..... . . . . . . .. .......... . . . ..47

12. Effect of DAG and Indomethacin on Frog Skin . . . . . 54

13. Effect of SAG and Indomethacin on Frog Sdin. . . . . ...55

14. SEM of Frog Bladder, Control. . . . . . . . . . . . . 70

15. SE of Frog Bladder, Control. . . . . . . . . . . . . 71

1b. SEN of Frog Bladder, Control, Hypotonic. . . . . . . .72

17. SEP of Frog Bladder, ADH-...-.............. 75

10. SEM of Frog Bladder, ADH.-.....-.-... .. ......76

vi

19. SEl of Frog Bladder, ADH, hypotonic. . . . . .. 0 .77

SEd of Frog Bladder,

SEM of Frog Bladder,

SEm of Frog Bladder,

SEM of Frog Bladder,

SE- of Frog Bladder,

SEm of Frog Bladder,

SEP of Frog Bladder,

SEM of Frog Bladder,

SEM of Frog Bladder,

SE of Frog Bladder,

SEM of Frog Bladder,

SEll of Frog Bladder,

Ionophore.

lonophore.

Ionophore.

lonophore,

lonophore,

Ionophore,

lonophore,

Verapail.

Verapamil,

Ionophore,

Verapaiiil,

Verapamil,

79

.. . ...80

~61

Hypotonic....... .. 3

ADH.... .. . .......84

ADH............

ADH, Hypotonic . 86

.. . ....89

ADHO.a............92

ADH, Hypotonic. . . . 8

Hypotonic........91

ADH, hypotonic. . . . 93

vii

2AJ

21.

22.

23.

214.

2' .25.0

2o.

21.

2b.

29.

30.

31.

I PRODUCTION

ie mecnanism by whicn Antidiuretic hormone (ADH) alters the

permeability of membrane to ions and water is not completely

understood. The present study was designed to determine the role of

phospholipias and calcium in the membrane permeability changes

occuring due to stimulate by ADH. The principle hypothesis is to

demonstrate that the coupling between receptor activation by ADH and

tne cascaae of cellular events invoked may involve alteration in

cellular pnosphoiipid metabolism, prostaglandin synthesis and changes

in intracellular calcium concentrations. There cellular effects of

ADd may then lead to cnanges in membrane permeability.

ThE Effectof'ADH on the Human Kid

Vasopressin or ADH is secreted by the posterior lobe of the

pituitary gland and regulates water retention by the kidney.

Osmoreceptors in the supraoptic nuclei of the hypothalamus of the

brain respond to alterations in osmotic pressure and regulate ADH

release from the pituitary gland. AD which is released by the

pituitary will affect the kidney by stimulating the reabsorption of

water in the late distal tubule and tne collecting ducts of the

1

2

nepnrons in the kidney.

formally, in tne absence of' ADH, the distal tubule and the

collecting duct are not permeable to water. In the presence of ADd,

tne permeability of the distal tubule and collecting duct to water

increases. The mechanism by which ADH causes an increase in water

flow nas been the object of increased investigation. Handler and

Orloff (61) suggested that ADH binds to the basal membrane of the

collecting duct and stimulates the activity of adenyl cyclase and the

formation of cyclic adenosine 3', 5'-monophosphate (cAhP). The cAMP

activates a protein kinase which may be involved in the

pnospoorylation of a protein component of the collecting duct apical

membrane which may result in an increased permeability to water.

ADH nas also been shown to have tne property of increasing the

permeability of isolated membranes including frog skin, toad skin and

frog or toad urinary bladder to water and sodium (Na+). These

tissues have been used extensively as a model for studying the

mechanism of action of ADh.

ie process by wfich ADH stimulates Na and water transport are

through entirely different mechanisms. This evidence is derived from

studies using various inhibitors of these two processes. Agents such

as cytochalasin B (26, 115), colchicine (115), and vinblastine (115)

innibit the hydro-osmotic response of toad urinary bladder to ADH

witnout affecting the stimulatory effect of hormone on Na+ transport.

Similar results have been reported with the anesthetics, halothane

and methoxyflurane (65). The diuretic amiloride (13) blocks the

-y

stimulation of Na transport by ADH, but not the increase in water

transport produced in toad bladder by either ADH or cyclic AMP.

Thus, the ADH increases in water flow and on sodium transport appear

to involve separate mechanisms.

The mechanism of action of ADH on Na transport is not well

understood. There are some studies involving toad urinary bladder

and frog skin which show that ADH decreases the resistance for

passive Na transport at the luminal (mucosal) membrane of epithelial

cells (34, 32, 112). The increase in intracellular Na+ concentration

stimulates the sodium-potassium activated adenosine triphosphatase

(Na+-K +ATPase) localized at the basolateral membrane which moves

sodium from inside the cell to the extracellular fluid. ADH binds to

receptors on the basolateral membrane, which results in an activation

of adenyl cyclase. The activation of adenyl cyclase results in an

increase of cAMP synthesis from ATP. This increase in intracellular

cAMP appears to be involved in the subsequent increase in luminal

membrane permeability to Na+ ions and water (58). Dousa,et al.,(35)

snowed that vasopressin through cAMP activates a protein kinase in

the intact renal bovine medulla and other studies have demonstrated

this action in the mammalian renal medulla (5, 110). The mechanism

by which cAMP increases the permeability of the apical membrane may

involve the phosphorylation of membrane proteins (37).

Exogenous cAMP can mimic the effect of vasopressin by increasing

the permeability of the membrane to Na and water. When vasopressin,

exogenous cAMP or theophylline is applied to the serosal side of the

toad urinary bladder, an increase in transepithelial Na+ and water

results (96). Both vasopressin and theophylline, an inhibitor of

phosphodiesterase, increase the concentration of cAMP in the bladder

(60).

Evidence from past studies supports the hypothesis that the

vasopressin-induced increase in Na+ and water permeability is

mediated through an increase in cAMP and an activation of a protein

kinase with a resultant increase in membrane permeability to Na+ and

water.

The Role of Calcium Ions in ADH Action

Activation by ADH of its membrane receptor and the increase in

membrane permeability that results is dependent on the concentration

of calcium ions (Ca+2) inside the cell (102). Calcium ions play a

universal role in cellular function, including roles in the blood

clotting system, cellular adhesion and integrity, membrane stability,

Oone and teeth formation and the regulation of enzyme and second

messenger production. Calcium ions are necessary for the maintenance

of membrane integrity, membrane structure, and ion and water

transport functions in frog skin and toad urinary bladders. Bentley

(12), in toad urinary bladder, showed that active sodium transport,

as measured by a short-circuit current technique, was depressed when

calcium was witndrawn from the bathing media. Under Ca+2 free

conditions, this membrane failed to demonstrate its usual increase in

snort-circuit current in response to vasopressin (12). Curran et

al., (33) reported a decrease in net sodium transport across frog

skin treated with EDTA, while Hays and his co-workers (63) observed

marked structural changes in bladder epithelial cells when incubated

in calcium-free solutions. The epithelial cells become detached from

eacn other due to a loosening of tight junctions. This observation

corresponds to a decrease in short-circuit current, electrical

potential and resistance, but an increase in the permeability of the

membrane to water, chloride and urea. Futhermore, these epithelial

cells do not respond to ADH. Oxygen consumption in these cells was

not impaired when calcium was removed from the media (63). Zerahn et

al., (82, 136) have demonstrated that a significant portion of the

total oxygen consumption of the toad bladder accounts for the active

transport of sodium by the Na -K+ ATPase. Thus it was suggested that

calcium-free solutions do not effect the sodium pump but possibly

interact at some other step in transmural sodium transport.

Calcium thus plays an important role in the actions of ADH.

There is increasing evidence that ADH may in fact alter calcium

metabolism. Recently, ADH has been shown to increase calcium efflux

from toad urinary bladder prelabeled with4 5 Ca+2 (35, 109). Pietras

et al., (99) have suggested that ADH increases total cellular calcium

in isolated frog bladder epithelial cells, whereas Cuthbert and Wiong

(35) have shown an increased calcium exchange in response to ADH or

cAMP. Burch and Halushka (25) observed that ADH, theophylline and

6

exogenous cAMP alter the efflux of 45Ca+ 2 from isolated toad bladder

45 +2epithelial cells preloaded with Ca+. ADH and theophylline

45 +2increase the efflux of Ca , whereas cAMP inhibits efflux. Using

compartmental analysis, they found that vasopressin, theophylline and

cAMP, all of which enchance toad bladder permeability to water and

sodium, decrease the size of the slowly exchanging pool of calcium in

the cells. PGE2 , which inhibits the ADH-induced increase in water

flow, caused an increase in Ca+2 efflux in toad bladder cells. This

effect may nowever, be due to an increase in calcium uptake by the

cells and PGE2 also increased the size of the slowly exchanging Ca+2

pool. Tney suggested that the effect of ADH, theophylline, exogenous

cAMP and PGE2 on water transport is correlated to changes in the size

of tne slowly exchanging calcium poo..

Alterations in the cellular calcium concentration effects sodium

and water transport differently. Bentley (11) observed that

increases in the calcium concentration of the bathing media inhibits

tne response to ADH on water flow in toad urinary bladder, whereas

raising calcium failed to inhibit the response to ADH on sodium

transport (12). Petersen and Edelman (89) postulated two active

sites of ADH action, one for water transport which is sensitive to

calcium and the other for sodium transport which is unaffected by

changes in calcium concentration.

Further results elaborating the role of calcium on water

transport have been obtained using the calcium ionophore A23187 which

is a carboxylic acid antibiotic. A23187 has the specific property of

7

complexing divalent cations in a two to one ratio, thus forming a

neutral lipid-soluble complex (71, 103). A23187 has the capability

of increasing the cellular calcium concentration (from 10-7"to 10-6

M) by transporting calcium from the extracellular space (10-3M).

A23167 may also release calcium from intracellular stores, such as

from mitochondria (103). The concentration of calcium inside the

cells is important with respect to the actions of ADH. William et

al., (129) observed that bathing the serosal surface of toad urinary

bladder with 10 m calcium, inhibits the ADH hydro-osmotic response.

Humes et al., (69) showed that verapamil, which inhibits calcium

uptake in toad bladder cells, attenuated the hydro-osmotic response

to AD and theophylline but increased the hydro-osmotic response to

exogenous cAMP. This effect may be due to a direct inhibitory effect

of verapabLil on phosphodiesterase activity. The inhibition of

verapamil on water flow was calcium dependent and it was suggested

that calcium ions act as an important coupling factor for ADH action.

lonophore A23187 has no effect on water transport induced by

exogeneous cAIP (135). Several studies have shown that vasopressin

stimulates the release of prostaglandins from toad bladder at the

same concentrations whicn stimulate the hydro-osmotic response (23,

137). Yorio et al., (134) suggest that the increase in calcium

inside the cell stimulates tne production of prostaglandins.

Prostaglandins, like A23187, inhibit the hydro-osmotic response

mediated by ADH and theophylline, but not that induced by exogenous

cAMIP (56, 97, 135). Yorio et al., (135) observed that indomethacin,

a prostaglandin synthesis inhibitor, blocks the effect of calcium

ionopnore A23107 in decreasing ADH and methylxanthine-stimulated

water flow. They also found that ionophore-like prostaglandins

decrease cAMP concentrations in the toad urinary bladder cells when

both ADH stimulated or unstimulated conditions. The role of

prostaglandins in the inhibition of the hydro-osmotic response may be

the result of their inhibition of adenylate cyclase which in turn

lowers the cAMP content (135). This may explain why ionophore or

prostaglandins can inhibit the hydro-osmotic response mediated by ADH

and theopnylline but not that of exogeneous cAMP.

Studies concerning the role of calcium in ADH action also

demonstrate the importance of cAMP in the mechanism of ADH-induced

sodium and water transport. It is widely accepted that ADH activates

a protein kinase which is involved in the phosphorylation of plasma

membrane proteins. A cAMP-dependent protein kinase has been

identified in the renal medulla (5, 37). Schlondorff et al., (106)

reported an ADH dose-dependent activation of a cAMP dependent

protein kinase in the toad bladder and otners demonstrated similar

effects on the mammalian collecting tubule (38, 42).

Studies of platelets have shown that the first step of thrombin

formation is the stimulation of the enzyme phospholipase C which

cleaves phosphatidylinositol giving 1,2 diacylglycerol which is

converted into phosphatidic acid by the enzyme diacylglycerol kinase.

Ionopnore A23187 can bypass this step and act directly on

phospholipase A which is the enzyme that causes the release of2

9

arachidonic acid. Arachidonic acid is then acted on by the

cyclo-oxygenase system giving rise to the prostaglandin series (77,

78). Phospnolipase A, which cleaves arachidonic acid from

phospnatidylcholine, phosphatioylethanolamine or phosphatidylinositol

requires a higher calcium ion concentration for activity than

phospnolipase C or phosphatidic acid specific phospholipase A (17).

These phospholipases are the enzymes involved in the metabolism

of membrane phospholipids. There is increasing evidence which

suggests tnat the metabolism of phospholipids by phospholipases

occurs in response to several hormones and neurotransmitters (16).

Hokin and Hokin (66) have suggested that phospholipids are involved

in the transport of ions across the lipid membrane. Yorio and

Bentley (131) have shown that phospholipase C can increase sodium

transport in frog skin. This increase in sodium transport by

phospholipase C was associated with a unidirectional increase in

sodium influx which was amiloride sensitive (131). Several

phospholipids, particularly phosphatidic acid and cardiolipin, have

been shown to have ionophoretic capability, especially phosphatidic

acid, which can act as a calcium ionophore and regulate intracellular

calcium concentration (119). Arachidonic acid, a metabolite of

phospholipase action, stimulates the short-circuit current in the

isolated toad urinary bladder (59). This study also showed that an

increase in prostaglandin synthesis paralleled the increase in

short-circuit current. Several other studies have also shown that

exogenous prostaglandins increase the short-circuit current in frog

10

skin (6, 47) and toad urinary bladder (87). Vasopressin increases

the synthesis of prostaglandins in the kidney (124) and the toad

urinary bladder (137). Zusman and Keiser (138) have shown that

angiotensin II, bradykinin, and vasopressin stimulate arachidonic

acid release and prostaglandin synthesis in the kidney.

Indomethacin, a prostaglandin synthesis inhibitor, inhibited the

synthesis of prostaglandins produced in response to these hormones,

without effecting the release of arachidonic acid. Mepacrine, a

phospholipase inhibitor, prevented the arachidonic acid from being

released and reduced prostaglandin synthesis (138). Prostaglandin

synthesis inhibitors, such as acetylsalicylic acid, mefenamic acid,

paracetamol and phenylbutazone have been shown to decrease the

short-circuit current in frog skin (57) and toad urinary bladder

(133). Tnese observations suggest that prostaglandins may play an

important role in transepithelial sodium transport and may regulate

hormonal activity.

Another aspect of prostaglandin hormone interaction is on

hormonal-induced increase in water transport. It has been suggested

that prostaglandins may act as regulators of the hormonal response by

a feedback mechanism due to inhibition of adenylate cyclase and

reduction of cAMP levels (135). The following observations are

consistent with this hypothesis. Indomethacin and meclophenamate

enhance the ADH-induced increase in water flow (54). Berl et al.,

(15) showed that indomethacin and aspirin potentiated the action of

ADH both in man and rat. Prostaglandins may play a major role in ADH

11

action in sodium and water transport. Precursors for the synthesis

of prostaglandins are fatty acids released from membrane

phospnolipids. The actions of ADH therefore may also involve the

metabolism of phospholipids in the membrane. The next section

concerns the relationships of phospholipids, the composition of

epitnelial membranes and the synthesis of prostaglandins.

Tne Stimulation of Phospholipase, Phospholipid Metabolism, and the

Composition of Epithelial Cell Membranes

load urinary bladder and frog skin, like other tissues, have as

the major lipid component of their plasma membrane, pnospholipids.

The major groups of phospholipids are phosphatidylcholine,

phosphatidylethanolamine, phosphatidylserine and

phosphatidylinositol. The other components of the lipid bilayer are

sphingolipids and sterols. Different epitnelial membranes are

composed of unique percentages of phospholipids, sphingolipids and

sterols.

In eucaryotic cells, most of the cellular phospholipids are

synthesized in the rough endoplasmic reticulum. Some studies have

reported that phospholipids, such as phosphatidic acid,

pnosphatidylglycerol and diphosphatidylglyceride, are synthesized in

the mitochondria (2).

Tne synthetic pathways are interrelated. A summary of the

12

2 Fatty-Acids2 CoASH Glycerol 3-Phosphate

Fatty AcylCoA /Glycerol-3-Phosphate Acyltransferase + Acyl-CoAt 1-Acyl-Glycerol3-Phosphate Acyl Transferase

Phosphatidic Acid

ATP CTP

Phosphatidate Phosphohydrotase Cytidine 5'-Tri-Phosphate

Diglyceride Kinase Phosphatidate Cytidylyl'

Diglyce ide CDP-Diglyceride Transferase

PhosphatidylserineSynthetase

Triglyce ide Serine Phosp atidyl

CDI Choline DF Ethanolamine GlycerolInositol DiphoJ hatidyl

Fhosphatidylcholine Phosphatidylethanolamine Phosphat ylserine Glycerol

2 Phosphatidylinositol

Phosphatidylethanolamine Phosphatidylserine Phosphatidylinositol-S-Adenosylmethionine Decarboxylase Kinase iADP

Methyltransferase DiphosphatidylinositolATP

S-Adenosylmethionine ADP

Triphosphati ylinositol

FIJURL 1. A summary enart showing interrelated pathways of

pnaospnolipid biosyntnesis. From Thompson, .A., 1980.

13

pathways is illustrated in Figure 1.

The major source for phospholipid synthesis is the phosphatidic

acid pool in the cell membrane. Cellular phosphatidic acid is

produced by the acylation of glycerol-3-phosphate by fatty acyl-coA.

Cnain lengths and degree of unsaturation of the fatty acid may

determine its association with specific phospholipids.

There are two steps involved in phosphatidic acid synthesis.

The first step is the formation of the ester linkage at position one

by giycerol-3-phosphate acyltransferase, while the second step

involves another acylation at position two by the enzyme acyl-coA or

1-acyl-glycerol-3-phosphate acyl transferase. These enzymes are

located primarily in the microsomal membrane and on the outer

mitochondrial membrane.

Phosphatidic acid is metabolized by two principle reactions.

The first, is dephosphorylation of phosphatidic acid to diglyceride

by the enzyme phosphatidate phosphohydrolase which is present in the

endoplasmic reticulum, mitochondria, lysosomes (22, 70) and also in

plasma membranes (29, 30). Phosphatidic acid can be resynthesized by

the enzyme, diglyceride kinase, which is distributed in both the

soluble fraction and in the plasma membrane. There is some

suggestion that the enzyme is found predominantly in the plasma

membrane (48, 67, 79).

Diglycerides can be utilized in the synthesis of

pnosphatidylcholine, phosphatidylethanolamine and triglyceride. A

summary pathway of these phospholipids is shown in Figure 2.

14

Ehosphatidic Acid

Di glyceride

CDFP-CholineCDP-Choline:F-Diacylglycerbl CD-Cholinephospho~

Ethanolaminetransferase

Ehosphatidylcholine Triglyceride

CDP-EthanolamineDiacylglycerolEthanolamine-phosphotransferase

F'hosphatidylethanolamine

FIGURE 2. A diagram snowing synthesis pathways of phospholipids from

pnospnatidic acid. From Thompson, G.A., 1960

.+ 'CDP-Cholineu.~ ir rC

CTP-PhosphocholineCytidyltransferase

FiGUE ,. A pathways for producing a cosubstrate for

phospnatidylcholine synthesis. From Thompson, G.A., 1980.

P* sphocholine + Prir rp T)

15

From the review by Thompson (118), the rate limiting step for

pnospholipid synthesis is the availability of CDP-choline (cytidine

diphosphate) or CDP-ethanolamine, which are produced by the

CTP-phosphocholine cytidyl-transferase. Tne reaction is shown in

Figure 3. Tne rate of phospholipid synthesis is not dependent upon

diglyceride availability. Infact, Iritani et al., (72) have shown

tnat excess diglyceride increases the rate of triglyceride formation

rather than phospholipid synthesis.

Phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine

and pnosphatidyinositol are synthesized primarily with palmitic acid,

a saturated fatty acid at the number one position of the phospholipid

backbone,and with oleic acid or linoleic acid, unsaturated fatty

acids, at the two position. However, at the end of the synthetic

process, phosphatidycholine, pnosphatidylethanolamine,

phosphatidylserine and phosphatidylinositol may also have the

following composition; a stearoyl residue at position one and an

arachidonyl residue at position two (68). The latter phospholipids

are involved in the synthesis of prostaglandins with the release of

aracnidonic acid by the action of the phospholipases.

There is another pathway for phosphatidylethanolamine and

phospnatidylcnoline synthesis from phosphatidylserine and this

pathway involves a methylation reaction. Phosphatidylserine is

decarboxylated to produce phosphatidyethanolamine by the enzyme

pnosphatidylserine decarboxylase. This enzyme is found to be

associated with tne inner mitochondrial membrane.

1t

Phosphatidyletnanolamine is tnen methylated into phosphatidylcholine

by enzymes found in the microsomes.

Pnospnatidic acid is also utilized in the formation of

CDP-Diglycerides. CDP-Digiycerides are the precursors for the

synthesis of phosphatidylinositol and phosphatidylglycerol in many

animal tissues. The formation of phospnatidylglycerol appears to

occur in the inner mitochondrial membrane. Phosphatidyiglycerol can

also be converted to dipnosphatidylglycerol by enzymes located on the

inner mitochondrial membrane. The formation of phosphatidylinositol

is catalyzed by enzymes in the endoplasmic reticulum.

Phosphatidylinositol can be phosphorylated to form diphosphoinositide

and further phosphorylated to triphosphoinositide. The synthesis of

diphospnoinositol is catalyzed by the enzyme phospnatidylinositol

kinase which is usually found in plasma membranes (48, 90, 128). The

further phosphorylaticn of diphosphoinositide to

tripnosphoinositidecan be accomplished by a similar kinase and this

has been demonstrated in brain (72, 74) and also in the plasma

membranes in the kidney (31) and in erythrocytes (4).

Degradation of phospholipids occurs through the action of

phospnolipases, which are lipolytic enzymes that hydrolyze the ester

bonds of fatty acids and diester bonds of phosphates. There are

several types of phospholipases which are particular to the organism,

organ or membrane.

There are four major types of phospholipases. Each

phospholipase hydrolyzes the bond in a specific region of the

17

pnospnolipid as shown in Figure 4.

Phospholipase A is the lypolytic enzyme which hydrolyzes the

ester bonds of fatty acids. There are two types of phospholipase A

enzymes. Pnospholipase A1 which is the enzyme that hydrolyzes the

acyl group at position number one of the phosphoglyceride backbone cf

tne phospnolipids. This enzyme is found mainly in microsomal

particles (105, 123) and has been identified in fractions from

several tissues such as rat brain (125) and heart (127).

Phospholipase A2 , on tne otherhand, will cleave off the free fatty

acid at the number two position of the phospholipid backbone. As

stated previously, the major, free fatty acid at this number 2

position is the unsaturated fatty acid, arachidonic acid, which

represents the precursor for prostaglandin biosynthesis.

Arachidonic acid is a twenty carbon length unsaturated fatty

acid and must be in the nonesterified form for prostaglandin

biosynthesis (80, 122). The cascade for the metabolism of

arachidonic acid is shown in Figure 5.

Tne level of the nonesterified form of arachidonic acid is quite

low in cellular tissues. Tne major source of free fatty acids is the

intracellular lipid pools, especially the phospholipids. Free fatty

acids are released by the action of the hydrolytic enzyme

pnospholipase A (76). Exogenously administered phospholipase

enzymes can cause the release of arachidonic acid with the subsequent

synthesis of prostaglandins (7, 97).

The synthesis of prostaglandins occurs in several tissues and

16

C

H3)C 0 C

Phospholipase A

J 'C 0 CHPhospholipase

A2

H2C 0 0

Phospholipase C hospholipase D

FIGUR.E 4. Tne principle sites of hydrolytic attack on pnospholipids

by .nzymes. The letters A , A , C, and D, represent phospholipiases

A , A , C, and D, respectively.

COOH

Arachidonic acid -

S-Lipoxygenase 02

OOH OHOH-COOH

5-HPETE 5-HETE

C+ c0COOH -)

Leukotriene A 4

Glutathione Le i

OHCOOH

0 H OHN^-- N IIH H 0

Leukotriene C4

OHCOOH

sN "

0

Leukotriene D4

)H, COOH

H

ukotriene B4

Arachidonic COOHacid-0

Cyclooxygenase 02

PGG2I C CjO H-COOO

OOH

Peroxidlase ProstacyclinPeroxree esynthase

radical

0.... OH OH

PGH2 - COON ProsiacyclinThromboxane

IH . ~ sy nthasea- COOH

Endoperoxide-E 0Endoperoxide -D loomerase Endoperoxide OH

Isomerase reductase Thromboxane A2

OH 0 OH-~COOH %COON -< ' COOH

O OH OH OH OH OHPGD 2 PGE 2 PGF2 0

P1GUAE 5. Cyclo-oxygenase an '5-Lipo-Oxygenase pathway of

Arachidonic Acid Metabolism. From Oates, J.A., Science,218 (1982).

20

tne type produced is dependent upon the stimulus, which may be

physical or pnarmacological. The release of prostaglandins doesn't

occur via a storage pool, but rather is the result of synthesis in

response to the stimulus. This process has been shown to occur in

spleen (55), lung, adrenal, intestine (101) and kidney (3).

In tne kidney, the major site of prostaglandin synthesis is in

the renal medulla which shows about a ten fold higher rate of

production than the cortex (53). In the renal cortex predominant

synthesis involves prostaglandin E 2, prostaglandin I2, and

prostaglandin F2 (81, 100, 130).

The synthesis of prostaglandins results when arachidonic acid is

released from lipid stores. Two enzymes can act on arachidonic acid.

One is cyclo-oxygenase whicn yields cyclized, oxygenated products,

the endoperoxides, prostaglandin G 2 and prostaglandin H 2 The other

enzyme is lipoxygenase which yields noncyclized oxygenated products,

such as HETE (12L-hydroxy-5,8,11,1 4-eicosatetraenoic acid).

Prostaglandin H 2 is unstable and is rapidly converted to thromboxane

A 2 or prostacyclin. Prostaglandin H 2 can also be converted to

prostaglandin E prostaglandin F 2 and prostaglandin D Z The amount

of trese prostaglandins may vary depending upon which chemical or

stimulus is present (27, 53).

In transporting epithelia, prostaglandins have been shown to

play a role in regulating the actions of ADH on osmotic water flow

and also stimulate transepithelial sodium transport. The precursor

to prostaglandin synthesis, arachidonic acid, has been shown to

21

increase Na transport in toad bladder (59) and this effect is

blocked by eicosatetracynoic acid, a prostaglandin synthesis

inhibitor. The present study was designed to examine the changes in

pnospholipid metabolism and alterations in membrane permeability by

determining the effects of various phospholipids and phospholipid

metabolites (including prostaglandins) on ion and fluid transfer

across isolated epithelia.

Role of icrotubule and Microfilament in ADH Action

As stated earlier, the ADH induced increase in water flow

involves a series of complex steps, including the role of

microtuoules and microfilaments in the expressions of hormone induced

alterations in membrane permeability. Recently, Burch and Halushka

(24) have presented evidence which suggests that colchicine-induced

redc tion of the ADH response may be due to an increase in

prostaglandin synthesis.

This same laboratory has also shown that prostaglandins

increased the concentration of intracellular calcium in the toad

bladder epithelium (25). Calcium plays an important role in many

cellular processes and it has been shown that calcium and calcium

regulatory protein is involved in the control of the

microtubule-microfilament assembly-disassembly process (88).

Using freeze fracture and scanning electron microscopy (SEM),

22

Levine et al., (66) have shown that trifluroperazine (TFP), a calcium

binding protein antagonist, decreased the frequency of

intramembranous particle aggregates which normally appear in response

to ADH and that TFP may produce this by blocking an effect of calcium

on tne microfilament function.

Recent advances in the technology of electron microscopy have

improved the study of the cell surface morphology of toad urinary

bladders, frog bladders and frog skin. This technique can be

utilized to study the interaction between membrane probes and

hormones, on permeability changes and morphological alterations.

The epithelium of the toad and frog bladder consists of three

layers:

(1) A mucosal epithelium which faces the urine, and has been

reported to be involved in transport activity;

(2) Tne sub-mucosal layer which is composed of connective

tissue, smooth muscle and blood vessels;

(3) A serosal epithelium which faces the peritonial cavity.

Using transmission electron microscopy, one can identify four

types of epithelial cells in the urinary bladder epithelium.

Granular cells (GR) constitute approximately eighty-five percent of

the total number of cells. Mitochondria-rich cells (MR) comprise ten

percent of the total epithelia and goblet cells (GO) or mucous

producing cells make up only five percent of the epithelium. These

three cell types include the cells which face the lumen of the

bladder. The fourth cell type is the basal cell which represents the

cell that faces the peritonial cavity (28, 44, 75). Danon et al.,

(30) described the granular cells as large, flat, polygonal shaped

cells, covered with finger-like microvilli, whose densities vary from

cell to cell. Mitochondria-rich cells are characterized by their

oval smallness, and their densely packed microvilli. These cells

appear to be responsible for the increase in the level of cAMP by ADH

(110). Goblet cells are characterized by long microvilli, with an

opening in the center. Each mitochondria-rich cell is found to be

contacted with three to five granular cells forming a floral, roset

shape. The granular cells join to form ridges which seem to be

sequences of microvilli adhering to each other, forming elevated

borders. The ratio of granular cells to mitochondria-rich cells is

four to one (36).

Miills and Malick (93) studied the morphology of the toad urinary

bladder in response to vasopressin. They concluded that the mucosal

epithelium surface changes due to the hydro-osmotic response but

these changes were not associated with active sodium transport. They

found that in response to stimulation by ADH, the granular cells

transform the ridge like appearance into individual microvilli.

These microvilli stick out from the epithelial surface. The granular

cell is the only cell type that undergoes this morphological change.

Dibona et al., (44) suggested that the granular cell is the major

site of action of the hydro-osmotic response to vasopressin.

Spinelli et al., (114) showed that in response to vasopressin,

exogenous cAMP and theophylline, the morphological changes of the

24

mucosal surface appear similar in tne presence of an osmotic

gradient. The most apparent changes in the surface of the granular

cells is the reduction of the ridge-like network to the microvilli.

They suggested that this change may be due to the swelling of the

cells, causing a reduction of the microvilli during hydro-osmotic

flow. In contrast, Davis et al., (40) observed changes in the

surface structure of toad urinary bladder following exposure to ADH

both in the presence and absence of an osmotic gradient, similarly to

that observed by Dratwa et al., (45). Le Furgey and Tisher (63)

demonstrated that tne appearance of microvilli coincided with the

increase in the water permeability of the luminal membrane in

response to ADH.

Tne alteration in microvilli formation may relate to the state

of microtubular function. Taylor et al., (117) showed that

colchicine, an inhibitor of microtubule polymerization, inhibited the

action of vasopressin-induced water flow. Cytochalasin B, an

inhioitor of microfilament formation, also inhibited the action of

vasopressin on the hydro-osmotic response. Taylor et al., (117)

suggested that microfilaments and microtubules may play an important

role in the water flow response of ADH. Similar results on the

effect of cytochalasin B have been reported by other investigators

(43, 115). Taylor et al., (117) also found that the inhibition by

cytocnalasin B is only on water flow, as it has no effect on basal or

ADh-induced sodium transport. Colchicine, podophyllotoxin and

vinblastine also reduce the response of bladders to cAMP (116). The

25

preSence of microfilaments was first noticed by Choi in the

epitnelial cells of the amphibian bladder, Bufo marinus (28).

Carasso and his co-workers (26) nave distinguished two classes of

microfilaments in the granular cells of Rana esculata. Taylor (115)

also found the distribution of fine and coarse filaments, especially

the fine filaments which are 50-70 A, dispersed beneath the apical

membrane of granular cells of Bufo marinus. From the study of Burch

and Halushka (24), colchicine and vinblastine can stimulate

prostaglandin synthesis in toad urinary bladders. When the bladders

are incubated with the prostaglandin synthesis inhibitor

indomethacin, meclofenamate or naproxen, the inhibition by colchicine

of ;he nydro-osmotic response to vasopressin is prevented. They

suggested that the inhibitory effect of colchicine is due to the

synthesis of prostaglandins inside the toad bladder cell which can

tnen act by negative feedback inhibit the water response to ADH (24).

Le Furgey et al., (84) revealed that colchicine and cytochalasin

B have no effect on the projection of microvilli on granular cells in

response to ADH or exogenous cAUP. They also found that colchicine

ana cytochalasin B have no effect on the structure of the microridges

found on tne mucosal surface in unstimulated toad urinary bladders.

They suggested that the cytoskeleton, microfilament and microtubule

systems are not essential for maintenance of normal surface structure

of toad urinary bladders nor are they involved in the mechanism in

response to ADH or exogenous cAMP. In contrast, Davis et al., (41)

reported that cytochalasin B causes a more pronounced effect on the

26

granular cells of toac bladder in the presence of ADH, with the

granular cells demonstrating more swelling and some times rupture.

Davis et al., (41) suggested that cytochalasin B may cause the

trapping of water inside the cells.

The role of microfilaments and microtubules in the mechanism of

ADH is still controversial and more studies are needed to clarify

their involvement in the response to vasopressin. In the present

investigation, the relationship between calcium, prostaglandins and

hormone actions on cellular morphology will be examined using the

calcium antagonist, verapamil, and the calcium ionphore A23187.

METHODS

Animals.

in these experiments the abdominal skin of the frog and the

urinary bladder of the toad were used. These epithelia represent

models for the late distal tubule and collecting duct of the

mammalian kidney. Botn animals were kept without feeding and were

used within one month of arrival and both sexes were used.

Frogs, Rana pipiens, (Northern grass frogs) were obtained from a

commercial supplier (Lake Champlain Frog Farm, Vermont) and kept in a

container with tap water at room temperature (25 C).

Toads, Bufo marinus were obtained from NASCO (Ft. Atkinsin,

Wisconsin) and Kept in terraria at room temperature (25 C).

Solutions

The composition of Ringer's solution was as follows: 111 mM

NaCI, 3.35 mM KCI, 2.70 u CaCl, 4.0 rM NaHCO3, 5.0 mM Glucose. The

solution was aerated and the pH was 7.8-8.0. The solution had an

osmolality of 225 MOsmol (131).

27

2d

Medium A consisted of 90 mM NaCi, 3.5 mM KCl, 25 mM NaHCO,, 0.5

mM MgSO4 , 0.5 mM KH2PO4 , 1.0 mM CaCI2 , 6.0 mM Glucose. The solution

was bubbled under 95% 02 and 5% CO2 and had a pH of 7.4-7.5, and an

osmolality of 220 mOsmol (104).

Medium B consisted of 90 mM NaCl, 3.75 mM KCl 25 mM NaHCO3 , 0.5

mM KH2PC), 14 mM Glucose, 10 mM HEPES. The solution was bubbled with

95 02 and 5 CO2 to a PH of 7.4-7.5, and had an osmolality of 230

mOsmol (104).

Medium C consisted of medium B plus 0.1 mM Cacd2 , 1.2 mM MgCl2

and collagenase 100 mU/ml (104).

Medium D consisted of medium B plus 0.5 rM EGrA (104).

Medium E consisted of medium D plus 4% BSA (Bovine Serum

Albumin) (104).

Low Calcium Ringer's solution; consisted of 111 mM NaCl, 3.35 mM

KCl, 0.027 mM CaCl2 , 4 mM NaHCO3 , 5 rmM Glucose.

Cnoline Ringer's solution; consisted of 115 mM Choline Cloride,

3. 35 mMI KHCO 3 , 2.7 mM CaCl 2' 5 mM Glucose.

Drugs

Drugs used were purchased from the chemical suppliers:

Arginine vasopressin (ADH), PGE2 , Pnosphatidic Acid,

Cardiolipin, Diparuitoyl glycerol, Dioleoly Phosphatidylcholine,

Stearoyl-OC-lysophosphatidylcholine, Collagenase (Sigma Chemical

29

Company, St. Louis, Mo.); Stearoylarachidonyl glycerol (SAG) and

Diarachidonyl Phosphatidyl glycerol (DAG) (Avanti Polar Lipids Inc.

Birmingham, Al.); Diarachidonyl Phosphatidylcholine and

Lysopnospnatidylcholine arachidonyl (Serdary Research Laboratory,

London, Ontario Canada); 1,2 Diolein (P-- Biochemicals Inc.

Milwaukee, Wisconsin); Arachidonic Acid and Calcium Ionophore A23187

(Calbiochem-Behring Corp., La Jolla Ca.); Verapamil (A gift from

Knoll Pharmaceutical Company Whippany N.J.)

All of the phospholipids suspended in chloroform, were dried

with nitrogen gas and sonicated in Normal Ringer's solution prior to

incubation with the toad urinary bladders and frog skins.

Mecasurementof Osmotic Water Flow

In these experiments, toad urinary bladders were used. The

animals were double pithed, dissected and the bilobed bladder

removed. The bladders were then cut in half, tied with silk thread

and attached onto the end of a glass tube with the mucosal (urinary)

side facing inward. Since each toad provided two lobes, one lobe was

used as a control, while the other was used for the experimental

procedure (some drug or hormone added to it). The size of the

bladder sacs were kept constant to minimize variation of surface

area. Each sac was filled with four milliliters of Normal Ringer's

solution and placed in test tubes which contained 30 ml of Normal

30

Ringer's solution. The bladders were equilibrated for at least 30

minutes prior to the start of the experiment. This preparation is

shown in Figure 6.

Measurement of Water Movement

Water movement was measured gravimetrically according to Bentley

(10). Water transfer is measured in tne presence of an osmotic

gradient (1/10 diluted Normal Ringer's solution inside the sac).

Following an incubation period, the bladder sacs were weighed to the

nearest 0.1 milligram on the Mettler balance. The bladder was then

immersed in 30 ml of Normal Ringer with drug (experiment side) or

without drug (control side). After 30 minutes of incubation, the

bladder was removed from the bath and reweighed. The amount of water

lost was calculated by subtracting the final weight from the initial

weight. Water is lost as fluid moves from inside the sac to the

outside solution along the osmotic gradient.

Measurement of Electrical Properties Across Frog Skin

Frogs were double pithed and the skin dissected away from the

underlying connective tissue as one continuous sheath and placed in a

petri dish containing Normal Ringer's solution. Tne frog skin was

31

FiGuhE b. Photograph of the toad bladder preparation used to measure

water flow.

32

mounted on Ussing-type divided chambers. To protect the skin from

damage, a silicone and parafilm ring were put on each side of the

chamber. The chamber was filled with 10 ml of Normal Ringer's

solution and each side was aerated which promoted oxygenation and

stirring. This apparatus is shown in Figure 7.

The amount of sodium ion transported across frog skin was

determined by using a short-circuit current system. The electrical

potential difference (P.D.) across the skin was measured using

Ringer-agar bridges connected through calomel cells to a

potentiometric recorder (Heath model SR-205). The short-circuit

current (lsc) was obtained by passing a current, from an external

battery connected through a pair of silver-silver choloride cells and

Ringer-agar bridges across the tissue to bring the spontaneous

potential difference to zero. An automatic voltage clamp allowed the

short-circuit current to be continuously recorded on a single pen

onart recorder (Heath model SR-205). Experiments were begun only

after a steady state was reached, i.e. constant current flow, which

usually occured within 1-2 hours. The P.D. was obtained directly

from tne recording. The resistance was calculated using Ohm's Law.

V=IR or R=P.D./Isc

The Isc is represented as the amount of current measured,

divided by the area of the chamber (Tr/Cm2).

In order to determine the amount of sodium ions that move

33

FIGURE 7. Photograph of Us&Lng chamber preparation of tne frog skin

used in the snort-circuiting technique.

transepithelially, the radioisotope Na was used. ( Na was

obtained from New England Nuclear, Boston, Ma.) This measurment was

a unidirectional flux (from mucosal to serosal surfaces or from

serosal to mucosal surfaces). After steady state was achieved, the

isotope (luci/ml) was added to either side of the chamber (mucosal

or serosal surface) and represented the hot side. A sample was

collected every 30 minutes, for 4 periods, from the cold side or

opposite compartment from where the Na was added before adding the

drug to obtain a baseline reading. This procedure was repeated for 4

periods after adding the drug. The sample was collected by taking 2

ml of fluid from the cold side and the same volume of Ringer's

solution was added to replace that lost through sampling. A hot

sample was obtained by taking 50 ul from the hot side and this was

mixed with 1.95 ml of cold Normal Ringer's solution to obtain a total

volume of 2 ml. Tne hot sample was usually taken after the first

sample in order to allow 30 minutes for radioisotope equilibration.

The second not sample was taken after adaing the drug or after the

fourth sample. The samples were placed into plastic, liquid

scintillation vials and counted in a Beckman gamma counter. The hot

sample was used to determine the isotope specific activity and for

determining ion movements or flux. Flux is the rate of ion movement

across a membrane and the flux was represented as uMole/cm 2-hr or

uequivalent/cm 2-hr.

Net flux (Jnet) is the difference between the forward flux from

mucosal to serosal (Jm-s) and the backflux from serosal to mucosal

34

(Js-m).

J (net) = J m-s - J s4m

The movement of ions may be represented as specific ion current

(I) rather tnan flux. Current and flux are related in the following

equation.

I =(Z)(F)(J)

where:

I = Electric Current (Amp/Cm2 or Coulomb-Cm2/ sec or uAmp/Cm2

or uCoulomb-Cm 2/sec )

Z = Valence (equivalent/mole or uequivalent/umole )

F = Faraday's constant (96,500 Amp-sec/equivalent or

Coulomb/equivalent or uAmp-sec/ uequivalent or Coulomb/ uequivalent)

J = Cnemical flux (mole/Cn'?-hr or equivalent/Cm2 - hr or umole/Cm2

-hy or uequivalent/Cm2-hr)

Chemical flux can be converted to electrical units by the

following equation:

I = Z(J)(96,500/36,000)

I = Z(J)(26.8)

(The time unit in Faraday's constant was converted from seconds

to hours)

3o

Method for Separating Epithelial Cells of Toad Urinary Bladder

Tnis method utilizes both chemical and mechanical forces. Toads

were double pithed and Oladders were collected as described above.

Each lobe of the bladder was tied onto the end of a glass rod and the

interior was filled to capacity with medium A. The sac was immersed

in a small beaker which contained 30 ml of medium A and was aerated

for an hour. Following this period the fluid both inside and outside

was changed to medium C which contained collagenase (100 units/ml)

Tne bladders were incubated in medium C for an additional 45 minutes

and then the fluid on both sides was changed to medium D which

contained EGTA (0.5 mM). The sacs were further incubated in this

medium for 15 minutes. At the end of this incubation period, the

bladder sacs were rubbed between two fingers to release the

epithelial cells from tne underlying connective tissue. The cells

were collected in centrifuge tubes, centrifuged at 3000 rpm. for 10

minutes and the cell pellet was collected. Tne cells were layered

over 4. BSA gradient and centrifuged for 15 minutes at 1000 rpm. The

pellet was collected and resuspended in medium A and incubated for 15

minutes under 95 0 2+ 5% CO2 . These cells were ready to use for

scanning and transmission electron microscopy (SEM) and for C)

consumption measurements.

37

Measurement of Oxygen Consumption

In this experiment a biological oxygen monitor (YSI, model 53,

Yellow Spring) was utilized. This apparatus contains an oxygen

electrode to measure the change of oxygen saturation of the bathing

solution. A suspension of toad bladd- epithelial cells was placed

in a chamber in which the oxygen probe was emersed. A picture of

this apparatus is shown in Figure 8. The chamber contains 5 ml of

Normal Ringer's solution saturated with air at 25 C. At the bottom

of this chamber lies a magnetic stirring bar which helps mix the

bathing solution. This apparatus is connected to a recorder (Heath,

Single pen chart recorder, model SR-205) which monitors the amount of

the oxygen in the solution. The cells were allowed to equilibrate

with Normal Ringer's at least 30 minutes. Oxygen consumption gas

recorded with and without the drug. Oxygen consumption was

linearally stable and was measured over 5 minute periods. The amount

of oxygen consumed was determined from the amount initially present.

Normal Ringer's solution at 25 C contains 5.78 ul of oxygen at STP

per rmilliter of solution when saturated with air. Therefore if the

cells were incubated with 5 ml of Ringer's solution, this solution

contained 5 x 5.78 = 2b.9 ul of oxygen. The amount of oxygen

consumed in 5 minutes times the amount of oxygen in that solution

equals the amount of oxygen that the cells consumed. The data was

normalized by measuring protein content of the cell suspension and 0

PLUNGER

RUBBER ' RINGS (2)

TEST CHAMBER

PROBE TIP

PROBE CL A MP

HOLD DOWYN RING

SHOULDER.

BATH COV

OVERFLOW GROOVE

ACCESS SLOT

FlGURE3 . Diagram of the oxygen electrode and sample chamber used to

measure oxygen consumption of thfe epithelial cells of toad urinary

bladoer.

38

SCREW

ER

MAGNETIC STIRRER

14

39

consumption represented as ul of 0 2 consumed/mg protein.

The Preparation of Frog Urinary Bladder for Scanning Electron

Microscopy (SEM)

Frog and toad urinary bladders have similar mucosal epithelial

morphology. The small size of frog bladders makes them suitable for

SEM, and therefore frog urinary bladders were chosen for these

experiments. In each experiment, six bilobed bladders were used.

Frogs were double pithed and the bladders collected. The bladders

were put into small beakers which contained Normal Ringer's solution

which was continuously aerated. One hemibladder was incubated

separately in Normal Ringer's containing 10 mU/ml ADH or 10 6 M Ca+2

Ionophore or 1i6 M Verapamil or 1C-6 M Ca +2 Ionophore plus 10 mU/ml

ADH or 10-6 M verapamil plus 10 mU/ml ADH while the other hemibladder

served as a control. All of these bladders were maintained under

isotonic conditions with Normal Ringer's solution.

In another set of experiments, hemi-bladders were exposed to

hydro-osmotic conditions. The bladders were tied into a sac as

described previously for toad bladder preparations and the sac was

filled with 1/10 Normal Ringer's solution. The outside solution

bathing tne bladder contained isotonic Ringer's solution. These

bladders were exposed to the same agents, at the same concentrations,

as in the other, set isotonic series. At the end of the incubation

40

periods, the bladders were collected and fixed in cold, 2%

glutaraldehyde and S-Collidine buffer. This buffer has no ions and

is best suited for SEM studies. The bladders were fixed for 24 hours

by leaving them in a refrigerator overnight. The bladders were then

washed twice in S-Collidine buffer and dehydrated in a graded alcohol

and amyl acetate solution (50, 75, 95%, 100%). The bladders were

dried by a critical point drying method using liquid C02 in a

Polatron critical point drying apparatus. Each bladder was coated

witn gold palladium to allow for increased conductivity. This tissue

was then ready for SEM.

Protein Determination Method

Assay of protein concentrations of toad urinary bladder cells

utilized the Bradford Assay method (21). After testing for 02

consumption, the epithelial cells were centrifuged and resuspended in

2 ml of' 0.1 N NaOH. The suspension then was sonicated and

centrifuge again. Aliquots of the supernatant were collected for

protein determinations.

A standard curve, was established using bovine serum albumin. A

protein solution containing 0-100 ug of protein was pipeted into test

tubes, adjusted to 1 ml with 0.1 N NaOH and 5 ml of Coomassie Blue

Reagent, vortexed and absorbance measured at 595 nM with a

spectrophotometer (Hitachi model 100-40). The concentration of

Ao2StoV'

O.4w

- a

-u e. ,. ...

4. .h .. .. .....

40t - 4 - - +-. 4,--e 4 -~~~~7 . . -- . . . - . . - - - -. - -- - . - -- - - - - -- - '- - - - - --. . ..... ..

t ....... -... .... -.-

;4 0 -!* ::- ........ .. .. -r 3 -. *- -- + + -- -- - 4 - -. - -.-- - - - - - .+.. - - . .. . - . * -. - --. --..

-...- ++.- ..--- ---. -.- --- -- . .+.-.-..- .. --.. --

- - - +- -+- +~~~~~7.=77 7 1..-... .. ,--.-. ?e. . .- - . -

---- - - - - --. +-. * **-... -.. . -.-.-.-.. .

+. .--. . -.. .. --...- .-.- +-- :A . * *-- * - -' - '

-.... .. ... - .t 7.:-:. e ... . . . ... .. . . . . e.. . .. . . .. -.-....---. ....-------.- 4- --.4 ..4- : : ::: 1 :: : . ...- --. , - - - . - - - - -. .- - - .-- .- - -- --- --- --4 - -. ... -. . -.. , . ..--. .-- . . - - + -- - - - - - -- . e - - - -. - + - -. -... -.. -.. -.-...

- -e-4 ....,- ... .... .---- ..... . . - ..- . --. ... . + 4 - - . . . . -- - - -

- - . 4- ...-.~ ~. . .. .... . ..-.. .,- .. . . ... . .. -- ----. .. -- . ---- -. + 4 --- ----e+ . + .-.-. - -t-.++ ----.. -. ++-. - l.. ... .. +. .. .. .. . . . . -,-. . -.-. -. . . . . .. ----.... -. -- -*- ' -. -... *-- -

T T ' i "1 - - - + - - -+ .. . -I + t I, . -- . -..- .. . - -- - --....-. - -- -- - -- . - -- - -- -4.., ... ...,.. .... . .. +g. -......... .+ . - e . . . --- --. I- I --- -I - -T . T -- . ,- - - -- .-

0 ~~ ...... ..... .. ... ... , . . . -.....-...... ....... ....... ..................... - -.-

- - - + . - -- -- - - - - - - - - - - - - . - - - - . - - - - - - -

. .. .. . . . .. . -- - 4 - - - - -~ ~~ ~~~~~. . . ... . --... . -+ - . - - . - - - - - - - - - e - - - -

- 4 .. . -. . . .. . .. ...- ... . .......--.- - . -. - - --. ..-- .. - -- + . ... - -. -.

- - -- -

LOO

C ..

( 0 .. .... .... .....Cot-r - f . . - - ---

1.1 . S R 5

. . .r.... ........... .

-- ------------t- -- ---

-- uq dpt

FIGUh- 9. Graph snows a standard curve for protein determination,

eorrilation coeffecient is .997613.

41

&.1t4I....

--

-- :

-. -.-

-1

r r , _U: -- -- -- 41 -- ; : -1: -I ;g;.-; -. I -.; ; -- I : - --I :: -.- -- -. P-1; ;4 i -; 1- 4.-: -,_-I -- :: 1; -. -. -. V. -.- .- I .- -- . -1 -. . . -. I .- - -. .- i -- -

- ....

r. ACIIIL -I * I t'' ... . 1. - ! 1 1

(- I I ....... .......

. .........4 1-tid

7.717: 77 77

......-.-

t , e 4.. --- -...

42

protein was plotted versus absorbance and a standard curve generated.

The concentration of protein in the samples from toad bladder cells

was determined from its absorbance and the standard curve. A typical

standard curve is shown in Figure 9.

Statistical Analysis

The statistical analysis was performed using a Radio Shack

TRS-60 microcomputer using statistical program package. A student's

t-test for paired observations between two sample means was applied

to the data.

RESULTS

Effects of Phospholipids on the Sodium Ion Transport Across the Frog

Skin

The main function of frog skin epithelium is to conserve salt

and water for osmoregulation. This epithelium, when mounted as a

flat sheet in a lucite Ussing chamber and bathed on both sides with

Ringer's solution, generates an electrical potential difference

(P.D.) ranging between 10 to 100 mV between the pond and blood

surfaces. The blood side is positive relative to the pond surface.

This electrical potential difference across frog skin is due to a net

positive sodium ion transport against an electrochemical gradient

(121). The frog skin can be represented as a circuit (Figure 10),

where the active transport pathway is depicted as ENathe

electromotive force of the active sodium transport mechanism. The ENa

is represented by a battery with positive side inward. The internal

resistance of the sodium pathway ( a ) is analogous to the high

resistance created by the tight junctions. A current, consisting of

positively-charged sodium ions, moves from left to right through the

upper branch of the circuit. In parallel, the passive branch is

represented by Rna (the low resistance opposing the passive movement

43

ENo

EXTERNALN INTENALB

SURFACE Ron sURFACE

Rex Rex

VD

FIGURE 10. Diagram of an equivalent electrical circuit analogous to

the frog skin epitnhlium (open circuit) and te snort-circuited

hi small diagram (in square) represents the open circuit, the

aiectromnotive force responsible for the active inward flux of sodium

Na is represented by a battery. RNais the high resistance created

by the Light junctions. R represents the resistance of the passive

transport pathway. PD is measured between points A and B. The

short-circuit system consisted of VD, represents an automatic voltage

clamp ana R represents tne external resistance due to theexRinger-agar bridges and the electrodes. Tne current registered in

the amrineter (A) is the short-circuit current (Isc). From Herrera,

1911.

45

of ions). Positive current is represented mainly by a left to right

movement of anions, mainly the movement of chloride ions through the

bottom branch of the circuit. Transepithelial potential difference

is measured between points A and B. Thus, this part of the circuit

represents the open circuit system. In the short-circuit current

system, the voltage clamp (VD) is connected to the circuit. The P.D.

across the epithelium is automatically reduced to zero by the VD (the

potential difference between A and B to zero). When the potential

difference equals zero, no current will flow through the passive

pathway, assuming no chemical gradient exists (the same volume of

identical solutions bathing both sides of the membrane) and therefore

the passive movement of ions is in equilibrium. The current, which

is generated by the active transport pathway, will flow through the

ammeter (Am) and external resistance (Rex). The amount of current

that is read from the ammeter is exactly equivalent to that generated

by active sodium transport. The short-circuit current and the

amount of external current applied to bring the P.D. to zero is

equivalent to the net active sodium transport from the outside

surface to the inside surface across the epithelium. The measurement

of P.D. and Isc across the frog skin provides information on the

movement of ions across this epithelium. The electrical properties

of the membrane can be used as an index of the action of drugs or

hormones.

These experiments were designed to test the effect of several

phospholipids or phospholipid metabolites on transepithelial sodium

transport using the short-circuited frog skin epithelium. When

diarachadonyl glycerol (DAG) and 1-steroyl-2-arachidonyl glycerol

(SAG) at 100 ug/ml were applied to the serosal bathing medium or the

blood side of the skin, a sustained increase in short-circuit current

(Isc) and potential difference (P.D.) was produced, with no apparent

change in transepithelial resistance (Figure 11, Table I). The

effect was prolonged and the Isc returned to normal base line when

tne phospholipid was removed from the bathing solution. When SAG and

DAG were placed in the bathing solution on the external pond or

mucosal sides, they had no effect on transmural sodium transport.

The Isc in these epithelia normally represents active sodium

transport across these membranes. The diuretic, amiloride, applied

to tne mucosal bathing solution blocked the increase in Isc

stimulated by DAG (Figure 11). A similar response was also observed

with SAG. These observations suggest that DAG and SAG increase

sodium ion transepithelial transport through amiloride sensitive

channels. This effect is similar to the effect of phospholipase C

that was reported by Yorio and Bentley (131).

Transepithelial sodium transport is stimulated by antidiuretic

hormone and aldosterone. This stimulation is due to an increase in

sodium ion influx with no change in the efflux. To determine if the

phospnolipid metabolites have a similar response, unidirectional

sodium fluxes were measured using 22Na as the radioisotope. 22Na was

applied to the mucosal or serosal bathing solution and the samples

were taken from the opposite side of the radioisotope administration.

47

70PD (mV)

or 60- AmilorideIsc (PA/Trcm2

50-

40-

30-

20 - 4DAG

10 -

io io ~time (min) 9 2

FIGURE 11. The effects of diarachidonylglyceroi (DAG) (100 ug/ml on

the corium side) on the PD ( mV) and Isc (uA/TCI 2) across the frog

skin In vitro. The skins were mounted as described in the text.

Ailorde (10 4) was added to the epidermal side of the frog skin.

Tne isc is represented as the continuous line and PD as the filled

circles.

TABLE I

EFFECTS OF DEGLYCERIDES ON POTENTIAL DIFFERENCE (PD),SHORT-CIRCUIT CURRENT (Isc) AND ELECTRICALRESISTANCE (R) ACROSS ISOLATED FROG SKIN

LIPID 20 MIN 20 MINBEFORE AFTERLIPID LIPIDTREAT. TREAT.

(7) Stearoyl-arachidonyl glycerol (SAG) ++PD (mV) 2 51 !.5 77 7Isc (uA/C ) 21 2 30+ 3bR (k . 'CM ) 2.5 t 0.3 2.2 tO.2

(7) Diarachidonyl glycerol (DAG)+bPD (mV) 28 5 43 7bIsc (uA/C2 ) 12 1 2 22 t 4R (kQ. CM ) 2.2 10.2 1.9 10.6

(7) Diarachidonyl phosphatidyl choline + bPD (mV) 2 2 6 411Isc(uA/C ) 10O2 21 3R (k . CH ) 2.1 +O.4 1.9 IQ.4

p<.05 for difference from initial period, using at-test.

paired student

Diglycerides were added to the serosal (blood side ) solution atconcentrations of 100 uG/ml. The values represent the mean I S.E. of20 min readings before and after administration of the drug. Thenumber of experimints are indicated within the parenthisis. Thecomposition of the bathing solutions was: 111 mM NaCl, 4 mM NaHCO3 ,2.54 mM CaCl2 , 3,35 mM KCl and 5 mM glucose, final pH, 8.0.

Samples were taken for four, 30 minute, consecutive periods before

adding the agents and then for four periods following the addition of

tne drugs. The data presented are the means of 30 minutes before and

after adding the agents plus or minus the standard error. A stable

flow of isotope was used as an index of isotope equilibration. The

increase in Isc produced by DAG may be accounted for by an increase

in the unidirectional influx of sodium from pond to blood side with

no change in the backflux of sodium from blood to pond side, (Table

II). The 22 Na influx before DAG was administered was 1.2 + 0.5

ueq/cm 2/hr and 30 minutes, and after DAG, the Na+ flux increased to

2.1 t 0.09 ueq/cm2/ hr (mean + S.E. of nine experiments; P < 0.05

using a paired student t-test).

ueq/cm2/hr before and 0.69 + 0.

addition of DAG (mean + S.E. of

significant, P > 0.05 ; using a

From these results DAG and

influx of Na which accounts for

effect on sodium influx through

that produced by the endogenous

Phosphotidylinositol appears in

The sodium backflux was 0.68-+0.05

10 ueq/cm2/hr 30 minutes after the

nine experiments, not statistically

paired student t-test).

SAG apparently stimulates the

the increase in Isc observed. This

amiloride sensitive pathways mimicks

hormones and phospholipase C.

the membrane of many tissues as

1-steroyl-2-aracohidonyl-phosphatidylinositol(8). DAG and SAG are

phospholipid metabolites which alter the membrane permeability and

consist of the same fatty acid (arachidonic acid) at number two

position as that normally found in the membrane.

In the following experiments other phospholipid compounds were

TABLE II

EFFECTS OF DIACYLGLYCEROL ON UNIDIRECTIONAL SODIUM FLUXES ACROSSISOLATED FROG SKIN

30 min before 30 min afterDAG DAG

ueq/CM 2/hr ueq/CM 2/hr

(9) Sodium influx 1.2 .05 2.1 t .09

(pond to blood) p<.05

(9) Sodium outflux 0.68 .05 0.89 1 .10

(blood to pond) N.S.

I ________ I

51

investigated to determine whether or not they have the ability to

alter sodium transport across frog skin. Phosphatidic acid and

cardiolipin had no effect on transepithelial transport of sodium

across frog skin as measured by the Isc. Both of these agents showed

no effect on short-circuit current and potential difference (Table

III). 1,2, Diolein as well as dipalmitoylglycerol also caused no

change in Isc and P.D. Dipalmitoyl phosphatidylcholine, dioleyl

phosphatidylcholine and stearoyl-lysophosphatidylcholine also had no

effect on the electrical properties of frog skin (Table III) whereas

phosphatidylcholine containing arachidonic acid, and diarachidonyl

phosphatidylcholine caused an increase in Isc and potential

difference across the frog skin (Table I). The Isc in the presence

of this drug was 21 + 3 uA/cm compared with the beginning period

without drug, in which the Isc was 10 12 uA/cm 2(mean + S.E. of seven

experiments; P < 0.05 using a paired student t-test). These

observations suggested that phospholipid-stimulated sodium transport

required the presence of arachidonic acid at the two position of the

phospholipid or diacylglycerol.

Arachidonic acid is the precusor to the synthesis of

prostaglandins. Previous studies have shown that prostaglandins

alter tne permeability of epithelial membranes (9, 57, 59). Hall et

al., (57) and Halushka et al., (59) observed an increase in Isc

across the frog skin and isolated toad urinary bladder when

arachidonic acid or prostaglandins were applied to the serosal

bathing solution. It has been suggested that phospholipase

52

TABLE III

EFFECTS OF PHOSPHOLIPIDS AND PHOSPHOLIPID METABOLITES ONTHE ELECTRICAL PROPERTIES OF THE ISOLATED FROG SKIN

LIPIDS

A. Pnosphatidic Acid (6)PD (mV)Iso (uA/CM 2 )

B. Cardiolipin (8)PD (mV)Iso (uA/CM 2 )

C. 1,2, Diolein (8)PD (mV)Isc (uA/CM 2)

D. Dipalmitoyl glycerol (6)PD (mV)Iso (uA/Ct12 )

E. Dipalmitoyl pnosphatidylcholine (8)

PD (mV)Isc (uA/C42)

F. Dioleoyl phosphatidylcholine (8)

PD (mV)Isc (uA/CM 2 )

G. Stearoyl- lysophosphatidylcholine (8)

PD (,",)Ise (uA/Ct42)

20 MINSBEFORELIPIDTREATMENT

63 + 1928 1 6

29 + 316 2 2

43 1 827 + 4

36 + 323 + 3

22 + 822 1 2

23 + 212 1 3

46 1 933 t 7

20 MINSAFTERLIPIDTREATMENT

46 11320 1 8

29 + 314 1 2

40 + 824 + 3

35 + 324 ; 2

2322

+

+

81

22 1 214 +

39 11329 1 b

The value presented represent the mean I S.E. of 20 minutes beforeand after administration of drug. Drugs were added to the serosal(blood side ) solution at concentration 100 ug/ml. The number ofexperiments are indicated within the parenthesis.

activation and the release of arachidonic acid is the rate limiting

step for the synthesis of prostaglandins (49). These studies imply

that phospholipase activation and phospholipid metabolism could

generate the arachidonic acid which serves as a precursor for

prostaglandin synthesis which subsequently increases transepithelial

sodium transport. In platelets, it has been suggested that the

arachidonic acid is released from diacyiglycerol by diacylglyceride

lipase following a phosphatidylinositol specific phospholipase C

effect (19). However Billah et al., (17) suggested that arachidonic

acid release by phospholipase A occurs after the actions of

phospholipase C on phosphatidylinositol. The generated

diacylglycerol is quickly phosphorylated by diacyglycerol kinase to

phosphatidic acid which is then acted on by a specific phospholipase

(A2 ) to release arachidonic acid (17).

The following study examines whether prostaglandin formation

contributes to the increase in sodium transport previously observed.

Preteatment of frog skin with indomethacin, a cyclo-oxygenase

inhibitor, prevented the increase in Isc produced by DAG.

Indomethacin (10 5 M) was then applied to the serosal bathing

solution and the Ise was 10 +2 uA/cm. Twenty minutes after the

addition of DAG, the Isc was 11 -3 uA/cm , (meant+ S.E. of six

experiments). Indomethacin also prevented the increase in

transepithelial sodium transport produced by SAG (Figures 12 and 13).

These results suggest that membrane permeability can be

regulated by phospholipids, phospholipid metabolites and

54

PDI(mV) 40.44 Y

I (A/ 7r C )30

20~

Isc (uA/cm 2 )

10~indo Ind. DAG

PD (mV) DAG

0-T

30 60 90Time (min)

FIGUtE 12. The effects of diarachidonylgiycero1 (DAG) (100 ug/ l on

the corium side) on the PD (GV) and Isc (uA/TrCM 2) across tne frog

skin in vitro in the presence of indomethacin (10 5M). The Isc is

represented as tne continuous line and PD as the filled circles.

80

PD (mV) 60-or

Isc(uA/;, CM2)

40-

*dNDO Q SA Go

20-

20 40. 60 80 100 120time

FiGUrxE 13. Tns effects of stearoyl araohidonylglycerol (SAG) (100

ug/zl on ne corium side ) on tne PD (mV) an Isc (uA/rCM 2 )across

;he frog sKin In vitro in the presence of indoretnacin (10 5 AM).

Indometnacin was fresiily prepared using 0. 1 M sodium phosphate buffer

and was added to tne corium side of the frog skin. The Isc is

represented as the continuous line and PD as the filled circles.

56

prostaglandins. Thus pnospholipids and phospholipid metabolism may

play an important role in ion transport activity

Effect of Phospholipids and Prostaglandins on Transepithelial Water

Transport Across Toad Urinary Bladder.

The toad's (Bufo marinus) urinary bladder responds to

vasopressin (ADH) or vasotocin by increasing its reabsorption of

water. To understand more about the mechanisms involved in the

increase in water flow induced by ADH, toad urinary bladders were

used as a model for the mammalian collecting tubule. The gravimetric

method of Bentley (10) was used in determining the quantity of water

transfered across the bladder in response to hormone. Previous

studies have shown a relationship between hormone receptor activation

and an increase in phospholipid metabolism in liver (18).

Alterations in phospholipid metabolism may play a role in the

translation of the initial hormonal receptor interaction to the

subsequent permeability changes. The present series of experiments

were designed to test the effects of different phospholipids and

their metabolites on transepithelial water transport.

Diarachidonyl phosphatidylglycerol (DAG) was applied to the

serosal side of the toad urinary bladder at 100 ug/ml. DAG alone did

not have any effect on osmotic water movement in the absence of

hormone. In the presence of DAG water flow was 51+- 7 mg/30 minutes

57

whereas in the control tissue water flow was 66+ 27 mg/30 minutes

(mean - S.E. of 12 experiments p>.05 using a paired student t-test)

(see Table IV). When ADH was applied to the serosal side of the

tissue, ADH induced a significant increase in transepithelial water

transport (Table IV). Preincubation of the bladders with DAG

decreased the hydro-osmotic response to ADH (Table IV).

Diacylglycerol is a product of phospholipase C action. Phospholipase

C has been shown to attenuate ADH-induced increase in transepithelial

water flow (35). The inhibition of water flow by DAG is similar to

of the effects of prostaglandins on water flow (62). The same effect

is found with phospholipase C on transepithelial water transport

(131). The rate limiting step in the synthesis of prostaglandins is

the activation of phospholipase causing the release of arachidonic

acid (49). Exogenous PGE 2 inhibits the hydro-osmotic effect of ADH

(139). DAG contains two molecules of arachidonic acid which may be

released and serve as the precusors for prostaglandin synthesis. To

determine at which position arachidonic acid is being released by

endogenous phospholipases, 1-steoryl-2-arachidonyl

phosphatidylglycerol (SAG) was applied to the bathing medium on the

serosal side. SAG, like DAG, didn't have any effect on basal osmotic

water flow even at a concentration of 200 ug/ml (Table VI). SAG at

100 ug/ml caused a significant inhibition of ADH-stimulated water

flow. This inhibition was lower than that observed with DAG. SAG at

higher concentrations (150 ug/ml and 200 ug/ml) did not have any

effect on the ADH response (Table VI). These results suggest the

TABLE IV

EFFECTS OF DIARACHIDONYLGLYCEROL ON THE ADH HYDRO-OSMOTICRESPONSE. WATER FLOW MG! 30 MIN.

LIPID

(12) DAG(100 uG/ml)

(12) CONTROL

(12) DAG +INDO%ETHACIN(10 )

(12) DAG

PERIOD I PERIOD II+ ADH(10 mU/mi)

SIGNIFICANCE

I -~ 4.

51 + 7

66 % 21

54 + 7

44 + 7

482 57

1106 95

698 + 50

329 : 54

p<.O1

p<.O1

L I

The values presented represent the meanand after administration ADH. The numberindicated within the parenthesis.

S.E. of 30 minutes beforeof experiments are

59

TABLE V

EFFECTS OF PHOSPHATIDYLCHOLINE ON TRANSEPITHELIAL WATERACROSS TOAD URINARY BLADDER ( MG/30 MINUTES)

LIPID

(6) Phosphatidyl-choline Diarachidonyl(50 uG/ml)

(6) Phosphatidyl-choline Diarachidonyl(100 uG/mi)

(6) Lysophosphatidylcholine Arachidonyl(100 uG/ml)

(6) L- - Lyso-Phosphatidylcholine(100 uG/mi)

(6) Phosphatidyl-choline Dioleoyl(100 uG/mi)

bp<.05 from TREAT.cp>.05 from TREAT.

TREAT. 1NO LIPID

TREAT. 2+ LIPID

~42 ! 6.3 58 ! 15C

36 ! 5.6

40 !7.1

3 ! 2.7

44 ! 118

'

47! 7.8c

73 ! 13..

60 ! 9 -3b

15 105ci

TREAT. 3ADH(10MU/ML)NO LIPID

1290!61

1366!-73

1563!-103

1514!65

1300!85

1I TEIT 3.

1 or TREAT. 3.1 or TREAT. 3

The values presented represent the tean I.S.E. of 30 minutes beforeand after administration of drug and 30 minutes after ADHadministration. The number of experiments are indicated within theparenthesis.

TRANSPORT

TREAT. 4ADH+ LIPID

1170!61c

1109!54 b

1065!118.4b

877+118

1228,130c

60

TABLE VI

EFFECTS OF PHOSPHOLIPIDS AND PHOSPHOLIPID METABOLITES ONTRANSEPITHELIAL WATER TRANSPORT ACROSS TOAD

URINARY BLADDER (MG/30 MIN)

LIPID

(11) Cardiolipin(50 uG/ml)

(11) Stearoyl-arachidonylglycerol(SAG) (50 uG/rul)

(18) Stearoyl-arachidonylglycerol(SAG)(100 uG/ml)

(12) Stearoyl-arachidonylglycerol(SAG) (150 uG/ml)

(5) stearoyl-arachidonylglycerol(SAG) (200 uG/ml)

(6) Phosphatidicacid (50 uG/ml)

I I T

TREAT. 1NO LIPID

45 1-8.5

33 121

41 13.3

36 1 7

111177

b- _ _ _

p<.05 from TREAT.Cp>.05 from TREAT.

TREAT. 2+ LIPID

47 16.3 c

35 7.5c

45 + 3.5C

44 +8.3

149184 c

1 or TREAT. 31 or TREAT. 3

TREAT. 3ADH(1imU/ml)NO LIPID

1805188

10061t120

148963

16291-51

13231117

898+ T55

The values presented represent the mean 1S.E. of 30 minutes beforean after administration of drug and 30 minutes after ADHadminintration. The number of experiments are indicated within theparenthesis.

24TREAT.ADH +LIPID

+

2004198 c

1016195C

1309157 b

16061,53 C

14081.87 c

7121123 c

61

presence of arachidonic acid at the number two position was

sufficient to produce an inhibition, although it is highly likely

that for DAG, both arachidonic acid molecules probably play a role in

the response.

In the following studies, the effects of several kinds of

phospholipids were tested on their ability to alter water transport.

Diarachidonyl phosphatidylcholine at 50 ug/ml had no effect on basal

osmotic water flow or that induced by ADH (Table V), whereas at 100

ug/ml diarachidonyl phosphatidylcholine inhibitred the ADH-induced

water flow (Table V). Lysophosphatidylcholine arachidonyl, which has

arachidonic acid attached at the number one position of the

phospholipid backbone, inhibited the permeability of toad urinary

bladder induced by ADH. This inhibitory effect was also seen with

lysopnosphatidylcholine from egg, in which the fatty acid composition

at position one was unknown. Phosphatidylcholine dioleoyl, which

contains oleic acid at the one and two positions, showed no effect on

water flow in both the presence and absence of ADH. Cardiolipin (50

ug/mI) and phosphatidic acid (50 ug/ml) had no effect on basal water

flow or the hydro-osmotic response to ADH.

Phospholipids containing arachidonic acid inhibit the

hydro-osmotic response to ADH. Zusman and Keiser (136) observed that

ADH stimulated prostaglandin E biosynthesis in the renal medulla and

it nas been shown that exogenous PGE and PGE inhibited the water

permeability response to ADH but not that induced by cAMP (97). The

inhibitory effect of these phospholipids could be due to the release

62

of arachidonic acid for subsequent prostaglandin synthesis. To test

this possibility, indomethacin (10-5), a prostaglandin synthesis

inhibitor, was added to the serosal side of the toad bladders prior

to adding diarachidonyl glycerol. Indomethacin has no effect on

basal water flow or that induced by ADH (135). Indomethacin in

combination with diarachidonylglycerol had no effect on basal water

flow (Table IV) but indomethacin prevented the inhibition normally

observed with DAG on ADH-stimulated water flow (Table IV).

To further probe the relationship between phospholipids,

prostaglandins and water flow, the effect of prostaglandin E2 and

arachidonic acid on ADH-stimulated water flow was studied. PGE2

inhibited ADH-induced water flow at 10-8 M concentration but at higher

concentrations of PGE2 at 10-5M, this was not observed. Similar dose

effects of prostaglandins have been observed previously (135). It

has been suggested that PGE2 inhibits the ADH response by acting on

2adenyl cylcase as PGE2 failed to inhibit exogenous cAMP-stimulated

water flow (9). Yorio et al., (135) also observed that PGE2 at low

concentrations (10-8M) decreased the cAMP concentrations in the

presence and absence of ADH. However, at higher concentrations (10-5

M) PGE2 caused an increase in the cellular cAMP concentrations even

in tne presence of ADH. Arachidonic acid produced the same effect as

observed witn PGE 2 . At 25 ug/ml, arachidonic acid produced no

significant inhibition of the ADH response, but at higher

concentrations, such as 50 ug/ml, a fifty percent inhibition of the

ADd-stimulated water flow was produced (Table VII). Higher doses,

63

TABLE VII

EFFECTS OF PROSTAGLANDINS E AND PROSSTAGLANDIN PRECUSOR ONTRANSEPITHELIAL WATER TRANSPORT ACROSS TOAD

URINARY BLADDER (MG/30 MIN)

PERIOD 1 PERIOD 2 PERIOD 3 PERIOD 4NOPGE2 + PGE ADH ADH +OR AA 2 OR AA2 (10mU/ml) PGE

NO PGE OR iAOR AA 2

( 10) PGE 2 (10-$)

(6) PGE 2 (10~4)

(6) Arachidonic acid(25 uG/ml)(AA)

(6) Arachidonic acid(50 uG/nml) (AA)

57 t 24

43 + 14

(6) Arachidonic acid 42 + 12(100 uG/ml) (AA)

t<(. 05 from period 1 or periodCp>.05 from period 1 or period

74 t 30

52 + 10

38 ! 5.5c

1239 +120

1568+ 41

136924

820+139

1218+-134

3-.

33

1445 U125C

1356- 47b

964+106b

3 5 0 !53b

743+138b

Tne values presented represent the mean tS.E. of 30 minutes beforeand after administration of drug and 30 minutes after ADHadministration. The number of experiments are indicated within theparenthesis.

64

suctl as 100 ug/ml produced similar inhibitions. Arachidonic acid

inhibition of the ADH-induced water flow may be due to the generation

of prostaglandins. Indomethacin was used in the following experiment

to show that this inhibitory effect of arachidonic acid was, in fact,

due to prostaglandin synthesis. Several doses of indomethacin were

added to determine at what concentration indomethacin prevented the

inhibitory effect of arachidonic acid on the ADH-stimulated and

unstimulated water transport. Indomethacin at 5x 1T5 M and 1l7 4 M

completely prevented the arachidonic acid inhibition of the

ADH-stimulated water flow (Table VIII) and at this concentration

indomethacin did not alter osmotic water flow in the absence of ADH.

These results reinforce the idea that arachidonic acid or

phospholipids containing arachidonic acid inhibited ADH-stimulated

water transport by stimulating prostaglandin biosynthesis. Past

studies nave shown that prostaglandin E 2 inhibited the ADH response

at the level of adenyl cyclase by preventing cAMIP production (135).

Arachidonic acid at 50 ug/ml showed a significant inhibition on the

effect of cAMP in increasing transepithelial water flow (Table IX).

This effect of arachidonic acid on cAMP was not observed with

prostaglandin F . PGE 2 (1 rn8 M) when applied to the serosal side of

toad urinary bladder failed to inhibit cAMP-stimulated water flow

(Table IX). Schlondorff et al ., (107) have shown that at higher

prostaglandin concentrations, the hydro-osmotic response to cAMP was

inhibited. They suggested that this inhibition may occur at a site

different from the cAMP system, such as at the level of microtubule

TABLE VIII

EFFECTS OF INDOMETHACIN ON ARACHIDONIC ACID INHIBITEDADH-INDUCED WATER FLOW MG/ 30 MIN.

TREATMENT

(6) Indomethacin (10-5M)+ Arachidonic acid

50 uG/mi)

(6) Control

(6) Indomethacin (5x105M)+ Arachidonic acid( 50 uG/ml)

(b) Control

(5) Indomethacin (10 4 M)+ Arachidonic acid( 50 uG/ml)

(5) Control

PERIOD INO TREAT,

i-

PERIOD II* ADH(10 MU/ML). TREAT.

SIGNIFICANCE

1 4 1.

25 t 5.4

9.3 t 2.2

15 + 8.7

20 2 4.7

9.6 + 2.4

9.5 1 2.2

715 t14 9

1036 +104

857 + 208

1196 t195

1024 +7.5

1164 2155

p<.05

p>.05

p>.05

Tne values presented represented the mean S.E. of 30 minutes beforeand after administration of ADH. The bladders were incubated inindomethacin for 30 minutes before adding the arachidonic acid. thenumber of experiments are indicated within the parenthesis.

I I

66

TABLE IX

EFFECTS OF ARACHIDONIC ACID AND PGE2 ONINDUCED TRANSEPITHELIAL WATER TRANSPORT

CYCLIC AMP-(MG/60 MIN)

TREATMENT

(9) Arachidonic acid(50 uG/ml)

(9) Control

(6) PGE 2 (10~I)

(6) Control

PERIOD I PERIOD II+ cAMP

4

45 t 3.5

33 5.7

28 + 5.7

26 7 8.5

249 t 40

730 1192

574 +155

801 12 2 8

SIGNIFICANCE

p<.05

P.05

The values presented represent the mean + S.E. of 30 minutes beforeand after administration of drugs and 60 minutes after cyclic AMPadministration. Tne number of experiments are indicated within theparenthesis.

I

I I

67

TABLE X

EFFECTS OF ARACHIDONIC ACID ON ADH-STIMULATED WATER FLOWIN THE PRESENCE OF LO4 CALCIUM BATHING SOLUTION

(0.027mM) (MG/30 MIN)

TREATMENT

(6) Low calcium (O.027m1i)t I(6) Low calcium (0. 027riMv),in serosal medium.

(6) Control

(6) Aracnidonic acid(50 uG/ml) + Low calcium(0.027mi') in serosalmedium-.

(L) Arachidonic acid(50 uG/ml) + N. Ringer(2.7 )raU)

PERIOD I PERIOD II+ ADH(10MU/AL)

SIGNIFICANCE

30.3t 4.5 1388t 114

30.14 3.51 1440 + 48

30 290 30

36. 4 t 3.2 660 t69

P>.05

p<.05

The values presented represent the mean ' S.E. of 30 minutes beforeand after administration of drugs and 30 minutes after ADHadministration. The number of experiments are indicated within theparenthesis.

66

function. Burch and Haluhka (25) have shown that PGE increased

calcium uptake in toad bladder cells. Experiments were therefore

designed to test whether the arachidonic acid inhibition required the

presence of calcium ion for activity. Water flow was measured in the

presence of a low extracellular calcium concentration (0.027mM).

This concentration was chosen so as to maintain cellular integrity.

Tne lower calcium concentration of the bathing media did not have any

effect on the ADH nydro-osmotic response (Table X). In the presence

of this low calcium media, arachidonic acid produced a significant

increase in osmotic water movement in the absence of hormone (Table

X), however arachidonic acid even in the presence of low calcium

still produced an inhibition of the ADH-stimulated water flow (Table

X). Perhaps, the amount of calcium in the external media was still

substantial to allow for an increase in calcium uptake by these cells

by prostaglandins. In the complete absence of calcium the integrity

of the epithelial cells becomes questionable with tight junctions

becoming loose, and thus each experiments are not possible.

Effects of Antidiuretic Hormone, Verapamil and Calcium Ionophore on

Ilucosal Epithelial Surface of Frog Urinary Bladder

Since calcium plays an important role in ADH action and in

microtubular function, experiments were designed to probe this

relationship further.

09

The mucosal surface of the urinary bladder of the frog, Rana

pipiens, is covered by epithelial cells with structural features

similar to those of the toad, Bufo marinus. These epithelial cells

occur in clusters, with each cluster containing numerous granular

cells, a few mitochondria-rich cells, and goblet cells. Figure 14 is

a low, power scanning electron micrograph of several clusters of

mucosal epithelium of frog urinary bladder revealing the surface

substructures of many, large polygonal, granular cells and a few

rounded, mitochondria-rich cells (MR). In control tissues subjected

to no experimental procedure other than rinsing in normal isotonic

Ringer's solution, the substructural morphology of the mucosal

epitnelium reveals a smooth surface with a continuous distribution of

granular, mitochondria-rich, and goblet cells. The surface of the

predominent granular cells displays very, uniformly distributed

microridges over the entire surface of the cell (Figure 15).

Mitocnondria-rich cells also display microridges but they

occasionally appear slightly coarser than the ones present on

granular cells. Although the presence of microvilli is detected

occasionally in the granular cells, their general absence on the

epithelial surface may be regarded as a consistent feature. The cell

borders also lack microvilli and appear with thin demarcation,

regardless of their presence in granular cells or MR cells. Urinary

bladder tissue, when subjected to an osmotic gradient, undergoes very

little alteration with no apparent morphological changes other than

the loosening of the microridges (Figure 16). The microridges may

70

FIGURE 14. Control specimen. Scanning electron microgapn

of the Naucosal surface epithelium of frog (Rana pipiens)

urinary bladder showing cell clusters, and the smooth

surface of the control cells with a distribution of fine

microridges and a lack of microvilui. (X 3000)

71

FIGURE . Control specimen. Enlarged surface view of the

mucosal epithelial cells of urinary bladder of frog showing

extensive wel-defined distribution of microridges over the

granular cells and lack of microvilli. (X 9000)

72

FIGURE 16. Control specimen treated in hypotonic Ringer's

solution on the mucosal surface and isotonic solution on

serosal surface reveals loose microridges over the granular

cells and the microvilli in the upper mitochondria-rich

cell. (X 9750)

73

occasionally appear continuous between two adjacent granular cells

(Figure 16, see arrows). A hypotonic osmotic gradient on the mucosal

surface of the lumen occasionally causes formation of microvilli on

MR cells. Occasionally a few granular cells center a MR cell forming

a floral rosette pattern (Figure 16).

Effect of Antidiuretic Hormone (ADH) With and Without an Osmotic

Gradient

ADH increases transepithelial water flow and in the presence of

an osmotic gradient, the water flow reaches peak capacity in 15 min

which may include an increase of ten to twenty fold. Water flow in

the absence of hormone is 30 mg/30 min whereas in the presence of

hormone water flow increases to 1.5 to 2 g/30 min. Figure 17 is a

SEM picture taken of the mucosal epithelial surface of frog urinary

bladder exposed to 10 mU/ral of ADH for 15 mins prior to fixation in

the absence of a mucosal to serosal osmotic gradient. In the

presence of ADH, prominent cell borders are induced with an extensive

network of microridges in the granular cells. Such microridges, when

viewed under higher magnification, appear to consist of an

anastomosing network, resembling the venation of a board leaf (Figure

18). Microvilli become distinctly visible at this higher

magnification and are strictly localized at the junctions of the

microridges (Figure 18). Tne presence is also noticeable over the

74

celi borders. The microvilli of the small, rounded,

mitochondria-rich cells become very prominent in the hemibladdep

tissue because of ADH action. here tne microvilli appear compact and

slightly more elongated than those on the granular cells. Figure 16

shows a cell at the lower left (arrow), similar in size to a granular

cell, containing tightly-packed microvilli. Such cells were also

occasionally found in control tissues. These cells may represent

either transitional granular cells or perhaps a new type of cell

occuring infrequently in the mucosal epitheliu of the frog urinary

bladder.

In tne presence of an osmotic gradient and simultaneous exposure

to ADH, the mucosal surface undergoes marked alterations. The

granular cells swell due to ADH stimulation. This has also been

report-d in the urinary bladcder of the toad (93, 83). This swelling

results in a considerable expansion of the microridges, which may

possibly act like cisternae to accumulate water, ions, and

macromolecules (Figure 19). In the presence of an osmotic gradient,

ADH inauces an apparent loosening of microridges and a decrease of

microvilli in the granular cells. Some of these cells may show a

complete loss of their microriages and microvilli with a resulting

blistering effect. Similar effects have been reported by others

(114).

75

FIGURE 17. Scanning electron micrograph of mucosal

epitnelial cells treated with ADH (10 mU/ml) for 15 mins in

isotonic Ringer's solution before fixing the tissue in 21

giutaraldenyde to show cell swelling, prominent cell

borders, extensive network of microridges in the granular

cells and microvilli in the mitochondria-rich cell (arrow).

(X 3300)

FIGURE 18. Scanning electron micrograph of mucosal surface

of thie urinary bladder of frog exposed to ADH for 15 mins

in isotonic Ringer's solution. The cell in lower left

(arrow), contains tightly-packed microvilli and may

represent a transitional granular cell. (X9900)

T7

FIGURE 19. Scanning electron micrograph of a mucosal

surface of frog urinary bladder epithelium showing

blistering effect associated with ADH exposure with an

hypotonic osmotic gradient on the mucosal lumen. (X 9600)

7b

Effect of Calcium Ionophore A23187

In the absence of a transmural osmotic gradient, the effect of

calcium ionophore A23167 is more marked on urinary bladder surface

morphology than that induced by ADH stimulation. Figure 20

represents a surface view of the epithelium of the mucosal lumen of

tne frog bladder exposed to 10-l6ionophore for 15 minutes. The

ionopnore causes a significant swelling of the granular cells such

that these cells assume the shape of balloons. The granular cells

show a markedd increase in the appearance of microvilli which appear

as finger-like projections into the bladder lumen. Te ballooning

effect as observed on the granular cells, due to ionophore action, is

not as noticeable in the MR cells. Possibly, this is why the MR

cells appear dwarfed and sunken in the midst of granular cells

(Figure 21, see arrow). The ionophore also causes a considerable

degree of anastomosing of the microridges in the granular cells,

thereby revealing a rosette pattern and network on the cell surface

(Figure 22). However, the MR cells retain their usual microvilli

with visible cell borders.

Wnen the urinary bladder is subjected to combined experimental

treatment, with calcium ionophore A23187 and a transmural osmotic

gradient across the mucosal membrane, a significant change in the

surface substructure is revealed in the scanning electron

micrographs. T e most distinct effect of the ionopnore under an

osmotic gradient is the induction of numerous blisters, possibly

79

FIGURE 20. Surface contour revealed by scanning electron

microscope of the mucosal epithelium of urinary bladder of

frog exposed to 1(g 6 H Ca+ 2 ionophore for 15 mins in

isotonic Ringer's solution. (X9600)

FiGURE 21. Scanning electron micrograph of the surface

morphology of the ucosal epithelial cells exposed to 1(J6

Ca +2 ionophore in isotonic Ringer's solution for 15 mins

snowing pronounced induction of the microvilli from the

junctions of the microridges of the cells and the

submersion of the small mitochondria-rich cells (arrow) by

enlarged granular cells. (X9900)

80

1

FIGURE 22. Surface sub-structures obtained by SEM from the

mucosal epithelial surface exposed to 10-6M Ca+2 ionophore

for 15 rins in isotinic Ringer's solution. (X9900)

causea by the loss of a large proportion of microridges and

microvilli from the surface of the epithelial cells (Figure 23).

These cells also show microridges and microvilli loosely distributed

over the cell surface. Although the swelling of the cells is

accompanied by ionophore and osmotic gradient, tne cell border

occasionally shows some degree of excavation.

Effect of Calcium Ionophore A23187 and Antidiuretic Hormone

The ballooning effect, caused by calcium ionophore alone, is

observed to a lesser extent with the combined stimulation of

ionophore plus ADH. Urinary mucosal cells still retain their

swelling even in the presence of ADH, but tne counteraction of ADH on

ionophore is considerable, showing an overall reduction in swelling

of the mucosal epithelial cells (Figure 24). Upon closer scrutiny of

tne individual cell surface, under higher magnification using SEM,

one can observe . large number of microvilli emerging at points where

the microridges merge to form a continuous network over the entire

cell surface (Figure 25). However, the microvilli are not

promineriiy projected into the urinary lumen as finger-like

projections as in the case of calcium ionophore treatment alone.

Sucn cells also show a certain degree of blistering (Figure 25, see

arrow) as witn ADH or ionophore alone. When a transmural osmotic

gradient is introduced, the cell swelling is largely retained. A low

j-2

63~

FIGURE 23. Surface view of the mucosal epitheliun showing

cells with swelling and blistering due to Ca+2 ionophore

treatment in the presence of a hypotonic osmotic gradient.

(X9900)

FIGURE 24. SEl of the urinary bladder mucosal epithelium

depicting the retention of the vast number of microvilli in

cells exposed to Ca+2 ionophore plus ADH in isotonic

Ringer's solution. Microvilli in general are not

prominently projected into the bladder lumen as in the case

of Ca+2 ionophore exposure alone. (X3300)

FIGURE 25. A higher magnification of the mucosal

epithelial cells of frog urinary bladder showing the

retention of a large population of microvilli resulting

+2.from Ca ionophore plus ADH treatment under isotonic

conditions. The microvilli are typically localized at

corners of microridges and on cell borders. (X9600)

86

FIGURE 26. A low power SEM depicting a large area of the

mucosal surface of the epithelium of frog urinary bladder

after exposure to 1(a 6 M Ca +2 ionophore plus 1OmU/ml ADH in

hypotonic Ringer's solution (1/10) bathing the mucosal

surface. (X 320)

87

power SEW picture (Figure 26) depicts such cellular expression,

snowing a vast majority of cells with inhibited swelling imposed by

the combined effects of ionophore plus ADH. In an enlarged surface

view (Figure 29), taken from the same area as in Figure 26, there is

some degree of swelling in granular and MR cells and the retention of

some microvilli and microridges. Tne swelling and the treatments

with ionophore and ADH under a hypo-osmotic gradient may have caused

the appearance of considerable blistering of the cell surface and

excavation of cell margins adjacent to the cell borders (Figure 29).

Effect of Calcium Antogonist, Verapamil

Verapamil, a calcium antagonist, prevents entry of calcium into

cells, and has been shown to inhibit the ADH-induced increase in

osmotic water flow (69). Since the effect of verapamil on mucosal

epithelium of urinary frog bladder is largely unknown, a SEM picture

of the mucosal surface of this tissue is presented in Figure 27.

This micrograph represents the surface morphology of frog urinary

bladder exposed to 10-6 verapamil on the serosal side for 15 mins in

isotonic Ringer's solution. The cell surface reveals the occurrence

of a few microvilli occurring typically at the contact points of the

reticular icroridges. These slightly elevated microvilli occuring

within the network of microridges give the impression of a smooth

cell. An apparent effect of verapamil on the mucosal epithelium is

FIGURE 29. Enlarged SEA of the same tissue as in Figure 26

obtained to show the swelling of granular and

mitochondria-rich cells and induction of microvilli

stimulated witn Ca+2 ionophore plus ADH. Notice the

blistering of cell surface and the excavation of cell

proper as well as cell borders due to hypotonic conditions.

(X 9900)

FIGURE 27. Pronounced projection of the mitochondria-rich

cells is shown in this SEM view taken from the mucosal

epithelial cells subsequent to exposure to 10-6M verapamil

for 15 mins under isotonic conditions. (X9900)

90

that te microridges of MR cells tend to separate from the

neighboring granular cells and proliferate above the level of

granular cells (Figure 27). The tendency for microridge elongation

of MR cells appears to be induced by verapamil and not observed with

treatments of ADH or ionophore, and may be regarded as a unique

feature associated with verapamil stimulation. Pronounced microridge

projections are restricted only between MR cells and granular cells

as the latter still maintain their normal cell to cell contacts.

The effect of verapamil, in tne presence of a transmural hypotonic

osmotic gradient, induces very few alterations in the surface

substructures of the epithelial cells (Figure 30). This is expected

as verapamil has little effect on basal transepithelial water flow.

The ceils are slightly swollen but are unlike that seen with ADH or

ionopnore. Sucn treatment with verapamil,under an osmotic gradient,

induced formation of fewer microvilli, prominent cell borders and a

slight excavation of cells adjacent to the cell borders.

Effect of Verapamil Plus ADH

Figure 26 is a surface view of the epithelial cells of the frog

urinary bladder exposed to 10-6 M verapamil plus 10 mU/mI of ADH on

the serosal side. There is an extensive, reticulate network of

microridges interspersed by microvilli. The general smooth surface

of the granular and MR cells appears practically identical after

91

FIGURE 30. SEM view of mucosal epithelial cells of the

frog bladder exposed to 10 i verapamil for 15 mins under

hypotonic conditions. Notice the organized structure of

the microridges with some microvilli interspaced at

junctions of the microridges. (X9300)

92

FiGURE 2o. $urface view of the mucosal epithelial membrane

exposed to 1o0Ai verapamil plus 10 mU/mi of ADH under

isotonic conditions showing extensive reticulate structure

of microridges mingled with microvilli. The cell borders

tend to separate from one another. (X9900)

93

FIGURE 31. Scanning electron micrograph taken from the

surface of the mucosal epithelium exposed to verapamil plus

ADH in the presence of a hypotonic osmotic gradient (1/10

Ringer's solution) on the mucosal lumen. Frequent

excavation both in the cell proper and cell borders, occurs

with a loss of microvilli and microridges and appearance of

blisters apparent. (X 9900)

94

exposure to verapamil alone, but the appearance of microvilli are

more prominent in MR cells with ADH present. The number of

microvilli occuring in these cells appears to be greatly reduced as

compared to ADH alone. The combined effect of veraparail and ADH

introduces a tendency of microridge elongation in MR cells similar to

that produced by verapamil treatment alone, but the apparent

separation among the neighboring granular cells is greatly

accentuated in this treatment (Figure 28). In the presence of an

osmotic gradient, tne epithelial cells still undergo some degree of

cell separation and cell swelling because of the stimulation of

verapamil plus ADH (Figure 31). This enlarged view of tne SEM

picture further reveals that only the cell swelling accompanies the

treatment with verapamil plus ADH under osmotic gradient, but this

treatment also frequently induces excavation between the MR cells and

granular cells. There is a general loss of microvilli and

microridges from the cell surface, and as a result, the granular

cells display pronounced blisters over the their surface.

DISCUSSION

The present results demonstrate that phospholipids and

phospholipid metabolites containing arachidonic acid stimulate

transepithelial sodium transport in frog skin epithelium and inhibit

the hydro-osmotic response to ADH in toad urinary bladder.

'When the phospholipid metabolites, diacylglycerol, and either

diarachidonyl (DAG) or steroylarachidonyl (SAG), were placed in the

solution bathing the coriu or blood side of the frog skin, it

produced a sustained increase in short-circuit current (Isc) and

potential difference (P.D.) with no apparent change in resistance

(R). This effect was prolonged and the Isc returned to initial

baseline values when the lipid was removed from the medium. When DAG

and SAG were added to the epidermal surface, a minimal effect was

observed. The Isc in these epithelia is normally an indication of

active sodium transport. The increase in Isc produced by SAG or DAG

added to the inner side was prevented by the diuretic amiloride.

These observations suggest that diacylglycerides sitmulated

transmural sodium transport through amiloride sensitive pathways,

similar to that observed previously with the enzyme, phospholipase C

(131). Endogenous hormones, vasopressin (ADH), and aldosterone can

also stimulate sodium transport by increasing sodium entry across the

apical membrane of amphibian epithelia. This is reflected in an

95

increase in the unidirectional sodium influx with no change in efflux

of sodium. In the present study, the increase in Is produced by DAG

was accounted for by an increase in sodium transport. DAG increased

the unidirectional influx (from pond to blood side ) of 22Na with no

apparent effect on the backflux (from blood to pond side). These

observations suggest that DAG specifically stimulates the active

transmural transfer of sodium through amiloride-sensitive channels,

mimicking that produced by endogenous hormones and phospholipase C.

The fatty acid composition of PI predominantly appears in some

tissues as steoryl-arachidonyl-phosphatidylinositol (8). DAG and SAG

have arachidonic acid at the number two position of the glycerol

molecule. Wihen this fatty acid was substituted with other fatty acid

constituents, the effect on transepithelial sodium transport was

lost. It was observed that dioleoylglycerol and dipalmitoylglycerol

had no effect on the electrical properties across frog skin.

Phospholipids containing arachidonic acid also were found to

stimulate the Isc of isolated frog skin epithelium, whereas

substitution of the arachidonic acid with oleic or palmitic acid

removed this stimulatory property of the phospholipid. These results

again support the idea that stimulation of transepithelial ion

transport by phopholipids may involve the release of arachidonic

acid.

Pnospholipid turnover is under the influence of the action of

phospholipase enzymes, and it has been suggested that phospholipid

activation and the release of arachidonic acid is essential for

96

97

prostaglandin synthesis (57). The administration of arachidonic acid

or prostaglandins (PGE) increases sodium transport in these epithelia

(59). The increase in Isc by DAG or SAG may be mediated by

prostaglandins. Pretreatment of frog skins with indomethacin, a

cyclo-oxygenase inhibitor, prevented the increase in short-circuit

current produced by DAG or SAG.

These results support the hypothesis that the change in membrane

permeability produced by diglycerides may be mediated through the

production of prostaglandins. Yorio and Bentley (133) reported the

presence of phospholipase enzymes in frog skin epithelium and that

this enzyme activity was enchanced by the mineralocorticoid hormone,

aldosterone. Preteatment of the frog skin with mepacrine, a

phospholipase inhibitor, prevented the increase in sodium transport

produced by SAG (Yorio, personal communication). This data

reinforced the idea that the release of arachidonic acid from these

lipids is essential for the increase in sodium transport observed.

The release of arachidonic acid from membrane phospholipids is

controversial. In platelets, it has been suggested that the

arachidonic acid is released from diacylglycerol by diglyceride

lipase following a phosphatidylinositol specific phospholipase C

action (19). However, Billah et al., (17) have suggested that

arachidonic acid release occurs sequentially following the action of

pnospnolipase C on PI. The diacylglycerol product is quickly

phospnorlyated by diacylglycerol kinase to phosphatidic acid with the

subsequent release of arachidonic acid occurring through a

98

phospholipase A2 enzyme specific for phosphatidic acid. Further

investigation is necessary to determine what sequence of enzyme

action occurs in amphibian epithelia. The observations that

diacyiglycerol compounds stimulated ion transport suggests that the

actions of phospholipase C followed by phospholipase A or a

diglyceride lipase may contribute to the release of arachidonic acid

in these epithelia.

The control of electrolyte transport by endogenous hormones and

second messengers is complex and incompletely understood. The

present observations suggest that phospholipid metabolites can alter

membrane permeability and the increase in sodium transport observed

is mediated through the release of arachidonic acid and the synthesis

of prostaglandins. This may represent one of the steps involved in

the mechanism of alteration in membrane permeability induced by

endogenous normones. The breakdown of phospholipids have been

implicated in a variety of cellular responses that are mediated

through hormones and neurotransmitters. It is possible that this

step may be involved in transepithelial ion transport.

Tne role of prostaglandins in the actions of ADH has been the

object of increased attention. This is particularly true concerning

ADH effects on transepithelial water movement (62). The actions of

ADH on water flow and sodium transport are mediated through different

mechanisms (62). This can be shown with the use of various

innibitors of the two processes (see Introduction). The effects of

prostaglandins on these two actions of ADH is also different and

99

controversial. Several studies have reported that prostaglandins

inhibit the ADH stimulated water flow by inhibiting cAMP formation

(95). Zusman et al., (137) reported that vasopressin stimulated

prostaglandin biosynthesis in toad bladder epithelium and suggested

that prostaglandins may act as a negative feedback in the ADH

response on water flow. In the present study additional evidence is

presented in support of this contention.

VWhen DAG was applied to the bathing solution on the corium side,

an inhibition of the ADH induced-water flow was observed. A similar

response was observed with SAG but it was not as effective. The

effects of DAG and SAG mimics the response reported for phospholipase

C on transepithelial water transport (131). Several other fatty

acid, substituted, phospholipid metabolites were investigated. The

results on water flow are similar to what was observed with these

agents on sodium transport. Cardiolipin and phosphatidic acid had no

effect on osmotic water movement in the absence or presence of ADH.

Pnosphatidylcholine groups, such as phosphatidylcholine

diarachidonyl, lysophosphatidylcholine arachidonyl and also

lysophosphatidylcholine inhibited the effect of ADH on water

transport, whereas phosphatidylcholine dioleyl had no effect on water

flow. Therefore, the phospholipids containing arachidonic acid were

active in preventing the increase in water movement induced by ADH.

The exception to this generalization was the action of

lysopnosphatidylcholine from egg lecithin. The fatty acid

constituent in this compound is unknown, it may contain arachidonic

100

acid. Alternatively, lysophosphatidylcholine has detergent like

effects and it also has been reported to inhibit adenylcyclase in

bovine renal medulla (4) and in the rat liver (105). This may be

contributing to the inhibitory response. Similarly, the actions of

diarachidonyl phosphatidylcholine may be due to the lyso metabolite

following the action of phospholipase A2 . Dioleoyl

phosphatidylcholine had no effect on water flow, again demonstrating

the importance of having arachidonic acid as the fatty acid. The

effects of DAG and SAG on the ADH response were prevented by

indomethacin. These results suggest that diglycerides containing

arachidonic acid attenuate the ADH-stimulted water transport across

toad urinary blaader by inducing prostaglandin synthesis.

Prostaglandins have been shown by several investigators to block the

ADH response by inhibiting adenyl cyclase (97, 135). Yorio et al.,

(135) measured cAMP concentrations in toad bladder epithelial cells

and found that PGE2 (10-8 M) decreased cyclic AMP formation, whereas

at higher concentrations of PGE2 (10N), cyclic AMP was increased.

PGE2 at low concentrations (1OA4) produced an inhibition of the

ADH-stimulated water flow, whereas PGE2 atlO-M had no effect on the

ADH response. These results agree with past studies on the

relationship between prostaglandins and ADH (62). Prostaglandin

synthesis has been reported to depend upon phospholipase and

cyclo-oxygenase activity (49). Additional information regarding

ADH-prostaglandin interactions was obtained by examining the effect

of arachidonic acid on water flow. Arachidonic acid produced a

101

dose-dependent inhibition of the hydro-osmotic response to ADH with a

maximum effect observed at 50 ug/ml (10-4M). Incubation of the toad

bladder with indomethacin prevented the effect of arachidonic acid on

ADH-induced water flow. This result also supports the hypothesis

that the inhibitory effects of arachidonic acid may be due to an

increase in the synthesis of prostaglandins. POE2 has been reported

to inhibit ADH-stiumlated water flow by inhibiting adenyl cyclase

(56, 56). The present observations concerning the action of PGE2 and

arachidonic acid on the cAMP response are somewhat confusing. PGE 2

10- 8 M nad no effect on the cAMP stimulated water flow which is

similar to what others have reported (56, 58, 139), whereas

arachidonic acid at 50 ug/ml attenuated the hydro-osmotic response to

cAMP. The reasons for this difference are not clear. Arachidonic

acid may be converted to prostaglandins other than PGE2 and exert its

action through this mechanism. It has previously been shown that

exogenously administered PGE1, PGE2 , PGF2 and PGA2 can inhibit the

ADH induced osmotic water flow (120). Recently Schlondorff et al.,

(107) suggested that exogenous PGE2 at high concentrations interferes

with vasopressin's response on water flow at a step distal from

cyclic nucleotide formation. Arachidonic acid at 10 4 M may give rise

to figh concentrations of prostaglandins which may interact at a site

beyond the adenyl cyclase-cAiAP system.

The effects of prostaglandins and some phospholipid metabolites

have been suggested recently as playing a role in regulating

intracellular calcium concentrations. Calcium ion has been shown to

102

be an important coupling factor in the ADH response on water flow.

Verapamil, which is a calcium antagonist, prevents the increase in

water movement normally accompanying the addition of ADH in toad

urinary bladder (69). Burch and Halushka (25) reported that the

regulation of the cellular calcium pool may, in part, mediate the

effects of vasopressin. Recently Levine et al., (86) observed the

effect of trifluoperazine, an inhibitor of the calcium-dependent

activation protein, calmodulin, also inhibited vasopressin's

hydro-osmotic effect on the toad bladder at a site distal to the

generation of cAMP. Schlondorff et al., (107) suggested that the

hydro-osmotic effect of vasopressin involves the activation of

several second messengers including cyclic AMP and calcium. They

also reported the activation of cAMP dependent protein Kinase by PGE

(107). Tnis activation did not lead to an increase in water flow.

The substrate for this kinase in unknown. Taylor et al., (117) have

strong evidence to suggest that microfilaments and microtubles play a

major role in ADH action, and Burch et al., (24) observed that the

colchicine induced inhibition of the ADH-stimulated water flow is

prevented by prostaglandin synthesis inhibitors. This suggested that

the function of the cytoskeletal system may be dependent on

prostaglandin formation. Marcum et al., (88) reported that calcium

and calcium dependent regulator protein play an important role in the

control of microtubule assembly and disassembly. Alterations in

cellular calcium may influence the function of the

microtubule-microfilament system. This system participates in the

103

morphological changes that usually accompany the increase in water

flow produced by ADH. As prostaglandins have been shown to alter the

concentration of calcium within the cell, the present study probed

this interaction further by examining the effects of agents that

alter cellular calcium on the morphological changes accompanying

hormone action in bladder epithelial cells.

Scanning electron microscopy (SEM) was used as a method to

examine bladder surface substructure following various treatment

protocols. The general morphology of the toad bladder has been

described previously using SEM (40, 41, 114). The observations

reported here on frog urinary bladder are in agreement with previous

reports on general cell morphology and extend beyond those reports by

including a role of calcium in the morphological changes accompanying

hormonal activation and drug action.

The mucosal epithelium of frog uinary bladder is composed

principally of polygonal granular cells interspersed with smaller

ovoid mitochondrial rich cells (MR). One MR cell is usually

surrounded by four to five granular cells as has been previously

reported for toad uinary bladder (39). Recently, Davis et al., (40)

examined the mucosal cell surface of toad urinary bladders using SEM

in the presence and absence of the neurohypophysial hormone

vasopressin. They reported that the most pronounced morphological

changes were localized to the granular cells, particularly when the

cells were exposed to an osmotic gradient plus the hormone. Specific

reference was made to the appearance and transformation of

104

microridges to microvilli when hormone was present and this was seen

only in the granular cells. The present observations are consistent

with Davis' work, however, it was also observed that transformations

and the appearance of microridges in hormone treated cells, in the

absence of an osmotic gradient are similar to that reported in the

toad bladder by Dratwa et al., (45).

The ability of ADH to augment water permeability in these

epithelia is influenced by a number of physiological and

pharmacological manipulations. Among these is the alteration in

cellular calcium concentrations. For instance, calcium ionophore

A231b7, inhibits the ADH and theophylline mediated hydro-osmotic

response but not that due to cAMP (135). This effect of the

ionophore has been attributed to an increase in prostaglandin

synthesis. The calcium antagonist, verapamil, also inhibits the

actions of ADH on water flow and the hydro-osmotic response to

exogenous cAMP (69). The administration of phenothiazines or W7,

inhibitors of calcium regulatory protein mediated processes, can

prevent the increase in water flow that usually accompanies ADH or

cAMP administration (Yorio, et al., personal communication),

indicatng an action of these agents subsequent to the formation of

tnis nucleotide. Collectively these observations support the

contention that calcium is an important intracellular messenger

involved in both the activation and regulation of the antidiuretic

hormone response. This study nas examined the mucosal surface of the

urinary bladder of the frog, Rana pipiens, using scanning electron

105

microscopy. Specific changes in the structure of the surface of the

epithelium were observed following exposure of the tissue to ADH,

calcium ionophore and the calcium antagonist verapamil.

As previously reported by others, the most marked morphological

change induced by ADH is the appearance of microvilli at the junction

of the microridges, predominantly in the granular cells. These

changes were observed even in the absence of an osmotic gradient,

differing from the results reported by Mills and Malick (93). The

reason for this discrepancy is unknown. Perhaps, it is the

differences in the responsiveness of tie frog versus the toad urinary

bladder. In the absence of an osmotic gradient, ADH increases the

permeability to water, although net water flow remains unchanged

(132). Therefore, these changes observed in the epithelial cell

components, due to the action of ADH may be attributed to the

biochemical events initiated by ADH regardless of the presence or

absence of a transmural osmotic gradient.

The calcium ionophore produced swelling of the mucosal granular

cell with the appearance of numerous microvilli that projected out

into the lumen, resembling a pin cushion. This response was observed

even in tne presence of an added hormone or when an osmotic gradient

is present. The production of such pronounced microvilli is similar

to the effects of high concentrations of calcium on wall microtubules

in pollen granules as reported by Flynn and Rowley (52). These

authors suggested that increased concentrations of calcium cause the

microtubules to contract and elongate, thus increasing cell surface

106

projections. Perhaps the ionophore increases cellular calcium by in

inducing microtubule elongation in the granular cell. Calcium and

calcium regulatory protein have been implicated in the control of the

microtubule-microfilament system and are essential for the expression

of the antidiuretic hormone response (117). It has been shown that

increases in intracellular calcium cause the disassembly of the

microtubular system (126). The effects of the ionophore or excess

calcium may be the result of their action on the microtubule system.

The disassembly of microtubules may cause the normal, rigid,

cytoskeletal system to be dissociated, thus allowing the microridge

network to project out into the lumen forming microvilli.

Thne calcium antagonist, verapamil, produced significant

organization of the structure of the microridges in the absence of

normone or osmotic gradient. The MR cells were also noticeably

affected by the presence of this calcium antagonist as the cell

contacts with the surrounding granular cells were markedly altered.

The MR cells appear to be separated from other cells and balloon out

into the lumen. The microvilli of the MR cells were not as prominent

and in most cases absent. This was particularly noticeable in the

presence of an osmotic gradient, where normally an osmotic gradient

induces MR cell microvilli formation. The lack of granular cell

microvilli formation was also observed when ADH was added in the

presence of verapamil. Thus the most distinguishing effect verapamil

has on the system is to prevent microvilli formation. This action of

verapamil may contribute to its inhibitory effects in the ADH induced

107

increase in water flow, and may also explain why the hydro-osmotic

effect of exogenous cAMP is also blocked by verapamil.

Collectively, these observations suggest that ionophore and

verapamil are inhibiting the actions of ADH on water flow through

different mechanisms. This may be the result of the actions of these

agents on different steps in the pathway of the regulation of

intracellular calcium and its relationship to the hormone response.

The role calcium plays in mediating the actions of ADH has received

considerable attention. ADH has been shown to enhance Calcium efflux

from prelabeled, intact, toad urinary bladders (25, 35, 110) and most

recently prostaglandins have been reported to increase intracellular

calcium concentrations (25). The present results, which demonstrate

that compounds which alter calcium metabolism produce profound

morphological changes in the mucosal epithelium, are consistent with

the previous findings on the importance of intracellular calcium and

the actions of antidiuretic hormone. Calcium localization, using

energy dispersive X-ray analysis, is planned to help differentiate

the locus of action of these agents with respect to the hormonal

effects and the actions of prostaglandin, phospholipids and

arachidonic acid on cell surface morphology. These future studies

may help explain the interactions between these cellular messengers

and the actions of antidiuretic hormone.

SUMMARYY

It was observed that phospholipid metabolites and pnospholipids

containing arachidonic acid (AA) stimulate transepithelial sodium

transport. The increase in transport was specific and occurred

through amiloride sensitive channels. Indomethacin and mepacrine

inhibited the increase in transport mediated by DAG or SAG,

suggesting that the response required the biosynthesis of

prostaglandins. The phospholipid metabolites also inhibited the

ADH-induced increase in water flow and prostaglandin biosynthesis.

Exogenous arachidonic acid and prostaglandins inhibit the

hydro-osmnotic response to added cyclic Ai4P (cAMP). Prostaglandins at

high concentrations failed to inhibit cAMP-induced water flow. The

reason for the difference in effect of PGE2 and arachidonic acid on

cAMP-induced water flow is not known. It was suggested that the

difference may be due to the high AA concentration used (10 M) which

may nave been converted to other AA metabolites other than PGE2 and

these metabolites effect the hydro-osmotic response to ADH or cAMP at

some step distal to cAMP production, perhaps with the

microtubule-microfilament system. Tne cytoskeletal system has been

suggested as playing an important role in the ADH- induced increase

in water flow. Changes in the mucosal epithelial surface of the

108

109

amphibian urinary bladders occur as a consequence of osmotic water

movement and from the actions of ADH. The cytoskeletal system may

function to maintain the surface structure morphology of epithelial

cells. The assembly and disassembly of microtubules and

microfilaments are affected by changes in intracellular calcium

concentrations. It was observed that changing intracellular calcium

concentrations in frog urinary bladders can alter the surface

morphology of the epithelial cell.

ADH in the absence of an osmotic gradient produced an

anastomosing network of microridges and the projection of microvilli

at the junction of the microridges on the granular cell surface. In

the presence of an osmotic gradient, ADH produced a more pronounced

affect on surface morphology, with expansion of the microridges and a

"blistering" effect on tne granular cell surface. The expansion of

tne microridges may function to accumulate water and ions in response

to ADd.

Calcium ionophore, A23187, in the absence of an osmotic gradient

produced numerous projections of microvilli on the granular cell

surface with no noticeable effect on mitochondria rich (MR) cell

morphology. The inducement of microvilli formation by A23187 may be

a consequence of the increase in intracellular calcium concentrations

causing microtubule elongation. Calcium ionophore inhibited the

hydro-osmotic response of ADH. In the presence of an osmotic

gradient and ADH, calcium ionophore inhibited the projections of

microvilli on MR cells which usually occurs as a result of the

110

nyarro-osmotic gradient.

Verapamil, a calcium antagonist, also inhibited the ADH

nydro-osmotic response, however, this effect is mediated through a

different mechanism than calcium ionophore. The most pronounced

effect of verapamil was on the elongation of the microridges in MR

cells suCn that the MR ceils separated from neighboring granular

cells. In the absence of an osmotic gradient, verapamil inhibited

the appearance of microvilli on granular cells and MR cells that are

normally induced by ADH. In the presence of ADH and an osmotic

gradient, verapamil inhibited both the projection of microvilli on MR

cells and granular cells and also produced an expansion of

microridges on granular cells resulting in a significant blistering

effect. Tnis effect of verapamil may be due to the trapping of water

inside the cells causing the swelling of microridges.

The present results demonstrate that compounds which alter

calcium metabolism produce profound morphological changes in the

mucosal epithelium, are consistent witn the previous findings on the

importance of intracellular calcium and the actions of antidiuretic

hormone. Calcium localization, using energy dispersive X-ray

analysis, is planned to help differentiate the locus of action of

these agents with respect to the hormonal effects and the actions of

prostaglandins, phospholipids and arachidonic acid on cell surface

morphology. These future studies may help explain the interactions

between these cellular messengers and the actions of antidiuretic

hormone.

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