I959

Ion Transport and Metabolism in Slices ofGuinea-Pig Seminal-Vesicle Mucosa

BY R. WHITTAM AND H. J. BREUERMedical Research Council Cell Metabolism Research Unit, Department of Biochemistry, University of Oxford,

and Chemische Abteilung, Chirurgische Universit0ts-Klinik, Bonn, Germany

(Received 5 January 1959)

Lardy & Phillips (1943), and Blackshaw (1953)found that bull and ram spermatozoa showoptimum motility in vitro only when at least5-10 mM-K' ions are present in the rpedium.Potassium also stimulates the metabolism ofspermatozoa, and Dott & Walton (1956) haveshown that both glycolysis and respiration areneeded for maximum motility. Potassium istherefore essential for the motility and metabolismof spermatozoa. It is present in the natural milieufor spermatozoa on ejaculation, namely seminalplasma, which is elaborated mainly by the seminalvesicles in the rat, guinea pig and bull (see Mann,1954). As a preliminary to the study of thesecretion of ions in seminal plasma, Breuer &Whittam (1957) tried to find out how the highpotassium concentration was maintained in slicesof seminal-vesicle mucosa of the guinea pig. Theyshowed that anaerobiosis had little effect on thetissue sodium and potassium concentrationsduring incubation at 370, but that inhibitors ofglycolysis anaerobically or of respiration aerobic-ally caused a loss of potassium and a gain ofsodium. Metabolic energy from either respirationor anaerobic glycolysis was therefore needed tosupport the concentration gradients of sodium andpotassium, which they suggested may depend upona supply of adenosine triphosphate, the formationof which is common to both metabolic pathways.The electrical potential difference across the cellmembranes is not known and it is not possibletherefore to decide whether both potassium andsodium are actively transported. Thus the intra-cellular potassium concentration could be main-tained either by a primnary active transport ofpotassium or by a primary active outward move-ment of sodium, which would generate a potentialdifference and cause a passive inward movementof potassium. The present work has shown that,whether sodium or potassium or both ions areactively transported, there is a correlation betweenthe retention of potassium and the exclusion ofsodium, and the concentration of easily hydrolys-able phosphate. Other experiments are describedwhich show that a reduction in the sodium con-centration of the medium caused a roughly pro-

portional fall in the tissue sodium concentration,without the tissue potassium concentration beingaffected. Under these conditions the intracellularpotassium is probably retained passively by non-diffusible anions.

METHODS

Materials and procedure. Guinea pigs weighing 400-600 g. were stunned and killed by bleeding from the carotidartery, and the seminal vesicles removed immediately.Slices of seminal-vesicle mucosa were made with a dryrazor blade as described by Breuer & Whittam (1957). Theslices of mucosa (30-50 mg.) were then placed in 3 ml. ofmedium (described below) in 15 ml. stoppered conical cups,which were shaken at 10, 170, 270 or 370 in a water bath.In anaerobic experiments, a stick of yellow phosphorus wasplaced in the centre well. After various periods, the sliceswere taken from the cups, blotted on Whatman no. 541filter paper and weighed.

Media. The phosphate medium of Krebs & Henseleit(1932) was used as the standard medium. Low-sodium andsodium-free media were prepared by partially or completelysubstituting 0 154M-choline chloride or 0 308M-sucrose forthe NaCl, and 10 mM-2-amino-2-hydroxymethylpropane-1:3-diol (tris) chloride buffer, pH 7 4, for the phosphatebuffer. Unless otherwise stated, glucose (20 mM) andsodium L-glutamate (10 mM) were added to the media assubstrates. Glutamate was added because Breuer &Whittam (1957) found that a higher potassium and a lowersodium concentration were maintained in the tissue in itspresence.

Determination of sodium and potassium. The weighedtissue slices were placed in 5 ml. of N-HNO3 and allowed tostand overnight (to leach the soluble salts from the tissue).Samples (1 ml.) of the acid extract of the tissue werediluted to 5 ml. for the flame-photometric estimation of Naand K with the flame-photometer attachment of theBeckman DU spectrophotometer.

Determination of chloride. Chloride was determined, on a1 ml. sample of the HNO, extract of the tissue, by thepotentiometric-titration method of Sanderson (1952).

Determination of easily hydrolysable phosphate. Mucosalslices (30-50 mg.) were placed in 1 ml. of 5% (w/v) tri-chloroacetic acid, broken up with a glass rod and allowedto stand at 40 during the night. The samples were thenshaken and centrifuged. Samples (0.4 ml.) of the super-natant acid extract were taken for the estimation of in-organic phosphate by the method of Berenblum & Chain(1936). For the estimation of easily hydrolysable phos-phate, 1 ml. of N-HCI was added to 0-2 ml. of the extract in

638

IONS AND METABOLISM IN SEMINAL-VESICLE MUCOSAtrichloroacetic acid, and the mixture heated for 7 min. ina boiling-water bath and the inorganic phosphate thenestimated. The value for easily hydrolysable phosphatewas obtained by subtraction of the inorganic phosphateconcentration before acid hydrolysis from that found afterheating for 7 min. at 1000 in N-HCa. Easily hydrolysablephosphate is taken to originate mainly from adenosinetriphosphate (ATP) and creatine phosphate.

Measurement of re8piration and anaerobic glycoly8is. Theconsumption of 0, and liberation of CO, by the mucosa wasmeasured in Warburg manometers at 10, 17°, 270 and 370in the usual way. The results are expressed as Q0, and Qao,(d./mg. dry wt./hr.).

Water content of ti8&ue. The tissue water was taken to beequal to the loss of weight on drying the tissue overnightat 105°.

RESULTS

Concentration8 of potasium and ea8ilyhydrolyeable pho8fphate in ti&8ue 81ce8

Previous experiments showed that anaerobiosis didnot prevent the retention of potassium and theexclusion of sodium by slices of seminal-vesiclemucosa, although cooling and inhibitors of respira-tion and glycolysis did prevent this (Breuer &Whittam, 1957). A maintenance of concentrationgradients in the same tissue, under both aerobicand anaerobic conditions, eliminates any mech-anism of cation transport depending on con-sumption of 0, and electron transport in the cyto-chrome system. An alternative possibility is thateasily hydrolysable phosphate (e.g. ATP or creatinephosphate) might support active cation transport,both aerobically and anaerobically, by energizinga carrier mechanism. In this case, low and highpotassium concentrations would be expected withlow and high concentrations of easily hydrolysable

phosphate respectively, irrespective of whetherpota#sium is actively transported inwards directlyor follows as a consequence of active sodium trans-port outwards. This expectation has been tested bymeasuring the concentrations of easily hydrolys-able phosphate in slices containing either low orhigh concentrations of potassium. These wereobtained by incubation at 10 or 370, and in thepresence or absence of metabolic inhibitors.To obtain enough tissue for incubation under the

six conditions shown in Table 1 the tissue from twoanimals was required. Slices were immersed in thestandard medium at room temperature until theyhad all been made (about 10 min.), and thentransferred to the conical cups for gassing andincubation. Analysis of the tissue immediatelyafter slicing showed that the potassium concentra-tion fell by 21 umoles/g. of whole tissue, and thatthe sodium concentration rose by a similar amount,during the immersion in the medium (Table 1).The reason for these changes is not clear, but theymay be partly due to a loss of potassium and a gainof sodium by damaged cells on the outside of theslices. This explanation requires that about 18% ofthe cells would be damaged. It is also suggested bythe similar loss of potassium that occurred afterimmersion in 0-308M-sucrose solution (see Table 4).This loss was then about the same after 2 min.(22 pnoles/g.) as after 45 min. (27 ptmoles/g.), aswould be expected from an equilibration ofdamaged cells with the bathing solution. For thisreason, the concentrations found after incubationhave been compared with the values for the tissueafter immersion in the medium at room tempera-ture and not with those for freshly preparedslices.

Table 1. Concentrations of ea8ily hydrolyaable pho&phate and of potassium and sodiumin s8oCes of seminal-vesie2e mucosa after incubation in vitro for 45 min.

20 mm-Glucose and 10 mm-sodium glutamate were added to the medium, pH 7-4. Mean values of at least two resultsare given. When added, the concentration of 2:4-dinitrophenol (DNP) was 0-2 mm and that of sodium iodoacetate (IAA)was 1 mm.

Concentrations(jumoles/g. of whole tissue)

Change during incubation(i,noles/g. of whole tissue)

Tissue after dry-slicingTissue before incubation

Tissue after incubation

Inhibitorsadded

NoneNoneDNP+IAAIAANoneNone

Easilyhydrolysablephosphate

8-9

K10988

Na Na+K35 14467 155

8-1 84 75 159 -0-88-8 75 85 160 -0-15-4 48 108 156 -3-54-7 50 104 154 -4-25-9 39 113 152 -3-05-7 35 115 150 -3-2

Easilyhydrolysablephosphate

Temp.37037373711

Gasphase0,N,03N,0,N,

K Na

-4-13-40-38-49-53

+8+18+41+37+46+48

Vol. 7:2 639

R. WHITTAM AND H. J. BREUER

The results of incubation for 45 min. at 370 showthat the concentration of easily hydrolysablephosphate (8-9 pmoles/g.) remained approximatelyconstant under both aerobic (8-1 umoles/g.) andanaerobic (8.8 ,umoles/g.) conditions (Table 1).After aerobic incubation, the tissue potassium con-centration was practically unchanged (84 tmoles/g.), wherea anaerobically it fell 15%, from 88 to75 ,umoles/g. The addition of 0-2 mM-2:4-dinitro-phenol plus mm-sodium iodoacetate aerobicallyand of iodoacetate anaerobically caused respectivelosses of 39 and 47 % in the easily hydrolysablephosphate concentration, and of 45 and 42% in thepotassium concentration. Under these conditions,the sodium concentration increased by an amountapproximately equivalent to the loss of potassium.After either aerobic or anaerobic incubation at 10,the loss of easily hydrolysable phosphate was 35%and that of potassium about 60%, and, again, theloss of potassium was balanced by an equivalentgain of sodium. The percentage losses of easilyhydrolysable phosphate and of potassium at 10 aretherefore only roughly correlated, possibly becausecooling retarded the rate of hydrolysis ofphosphateesters without markedly changing the rate of lossof potassium. The absence of major changes in theconcentrations of easily hydrolysable phosphateand of sodium and potamsium under both aerobicand anaerobic conditions at 370, and the com-parable losses of easily hydrolysable phosphate andpotassium, and the gains of sodium, produced bymetabolic inhibitors, suggest that the retention ofpotassium and the exclusion ofsodium are probablyfacilitated by labile phosphate esters.

Effect of temperature on the ti8sue ionic concentrationTo find whether a reduction of the temperature

of incubation would cause movements of tissuesodium, potassium and chloride to levels whichmight be correlated with the metabolic energysupplies, as shown by the Q0 and Qc, values,slices were incubated at 37°, 270, 170 and 10 underboth aerobic and anaerobic conditions. The resultsare shown in Table 2. The sum of the concentra-

tions of sodium plus potassium was approximatelyconstant at about 160 umoles/g., irrespective of thetemperature of incubation, which shows that anylosses of potassium were balanced by equivalentamounts of sodium. The Qo, and QNOS values wereeach about 7 after incubation at 37° and fell by52 and 34% respectively when the temperaturewas reduced from 370 to 27°. The ionic concentra-tions changed by only 7-14% on reducing thetemperature from 370 to 270, and are not thereforedirectly related to the metabolic quotients. Theenergy available from metabolism at 270 wasprobably sufficient to meet the demands of theactive transport processes. At 170, the Q09 andQao, values were approximately only 20 and 30%of those at 37°. The reduced energy supply thenprobably limited the active movements of ions, andaccounts for the lower concentration of potassium(58.5-61-0 ,moles/g.), and the higher concentra-tions of sodium (103-106 amoles/g.) and chloride(68-0-74-5 pmoles/g.) found aerobically and anaero-bically. At 10, the loss of tissue potassium and thegains of sodium and chloride were even greaterthan at 170, and the 02 consumption and C02output were immeasurably small with the Warburgapparatus.The intracellular concentrations of sodium and

potassium have also been calculated, a value of13 ml./100 g. of tissue being used to correct for theions in the extracellular space. This was the inulinspace previously found under both aerobic andanaerobic conditions at 370 (Breuer & Whittam,1957), and it has been assumed also to represent theextracellular space at the lower temperatures. Theintracellular potassium concentrations are likelyto be accurate since the correction for extracellularpotassium was small. The sodium concentration, onthe other hand, can be considered as only approxi-mate in view of the large correction for extra-cellular sodium and the likelihood of small varia-tions in the inulin space. Fig. 1 shows the results ofaerobic incubation and Fig. 2 those of anaerobicincubation. Both graphs show that whereas theconcentrations depended on the temperature of

Table 2. Effect of the temperature of incubation on the pota88ium, 8odium and chloride concentration8,and the, metabolic quotients of 8eminal-vesicle muco8a

Slices of mucosa (about 30-60 mg.) were shaken for 1 hr. in 3 ml. of phosphate or bicarbonate medium containing20 mM-glucose and 10 mM-glutamate, pH 7*4.Gas phase ... ... ... ... ...

Temp. .. . ... ... ... ...

QO0 or Qao ... ... ... ... ...

Ionic concentrations K+(Jumoles/g. of whole tissue) Na+

CN-Na+ +K+

02

370 270 1706-7

87-069*061-5158*0

3-279.579-066*0

158-5

1-458-5106-074.5

164*5

N2 + C02 (95:5)

1° 370 270 170 10

<0-240-0113*5153-5

7.374.585-044.5

159-5

4-869-092-052-0

161-0

2-461-0103-068-0

164-0

<0-235-0

115-0150.0

640 I959

IONS AND METABOLISM IN SEMINAL-VESICLE MUCOSAincubation they were not simply related to therates of respiration or of anaerobic glycolysis.A lower potassium concentration, at temperaturesbelow 370, was accompanied by a higher sodiumconcentration, and the sum of the two concentra-tions was approximately constant (148-165 jumoles/g. of cells). A similar dependence of chloride con-centrations on temperature was shown in slices ofguinea-pig-kidney cortex by Whittam (1956),without a simple relationship between concentra-tion and metabolic rate.

?11(-u04)

-WE

zz 61

+-0

0

L-i

Temperature

Fig. 1. Effect of the temperature of incubation on theintracellular sodium and potassium concentrations andthe Q02 of seminal-vesicle mucosa. Slices were incubatedfor 45 min. in phosphate standard medium.

-110 N

4)-90 66

E

+~~~K

41QN2~ ~ ~ ~ ~~~N

302

0 10 20 30 380Temperature

Fig. 2. Effect of the temperature of incubation on theintracellular sodium and potassium concentrations andthe Q"2 ofseminal-vesicle mucosa. Slices were incubatedfor 45 min. in bicarbonate standard medium.

41

Effect8 of lowering the 8odiqumconcentration of the medium

Ti8sue sodium concentration. The concentrationgradient of sodium between cells and medium in asodium-free solution, made isotonic with either anon-penetrating electrolyte or non-electrolyte, willfavour a movement of sodium from cells to mediuminstead of from medium into cells as in the mediumof Krebs & Henseleit (1932). The potassium con-centration of the tissue in the absence of externalpenetrating cations may be due to a Donnanequilibrium or to potassium movements eitherdirectly or indirectly dependent upon metabolism.To find the effect of the sodium concentration ofthe medium on the tissue sodium and potassiumconcentrations, slices of seminal-vesicle mucosahave been incubated aerobically at 370 in media inwhich sodium chloride had been partially or com-pletely replaced with choline chloride or sucrose.Choline choloride and sucrose were chosen asreplacements for sodium chloride as examples ofa relatively non-penetrating electrolyte and non-electrolyte. Although the extent of their penetra-tion into slices of seminal-vesicle mucosa was notdetermined, it seemed unlikely from results withother cells that this would be appreciable duringincubation times of 45 min.The results showed that the tissue sodium con-

centration was proportional to that of the mediumover the range 20-120 mm, and that it was constant(about 65 pimoles/g. of whole tissue) when themedium contained 120 and 155 mM-Na+ ions(Fig. 3). In sodium-free solution, the tissue sodiumconcentration was not significantly different fromzero. Similar results were found after incubation in

vs

:3on

.4)

-cv

-

z

0

o

v

ZLCc

0U~

75 -

50 0

250

oi x 80I 160 40 .80 120 1660

Concn. of Na+ in medium (mM)Fig. 3. Effect of sodium concentration in the medium on

the sodium concentration of slices of seminal-vesiclemucosa after aerobic incubation for 45 min. at 37°.0, Medium containing sucrose; 0, medium containingcholine chloride.

Bioch. 1959, 72

V01. 72 641

R. WHITTAM AND H. J. BREUER

the sucrose or choline chloride media. The resultsin Fig. 3 show that the cell membranes werepermeable to Na+ ions, which migrated from cellsto medium in the low-sodium and sodium-freesolutions. The lower tissue concentrations found athigher external sodium concentrations (120 and155 mM) must have been due to the active ex-trusion of sodium from the cells.To find the rate at which tissue sodium leaked

into sodium-free media, the tissue was analysed forsodium after incubation for various times. Fig. 4shows that the tissue sodium concentration fell tothe same extent in both the choline chloride andsucrose media. This fall represents a loss of sodiumfrom extra- and intra-cellular fluid, although aclear resolution into two components is not possiblefrom the curve. The graph shows, however, a rapidinitial fall gradually merging into a slower fall, aswould be expected from a more rapid loss fromextracellular fluid than from within the cells.

v. 804n

0-a 60

3o0bO

40

E

20z

%-Sucrose0

Standard medium

e or choline chloride mpedia15 30 45

0 is1 30 45U Time (min.)

Fig. 4. Effect on the sodium concentration in slices ofseminal-vesicle mucosa of aerobic incubation at 370 instandard medium (0) or in sodium-free sucrose (0) orcholine chloride (A) media.

'959Fig. 4 also shows that after about 30-45 min., thetissue sodium had become experimentally in-distinguishable from zero. In contrast with the lossof tissue sodium in sodium-free media, duringincubation in the medium containing 160 m -Na'ion, the tissue sodium concentration increasedalmost twofold in the first 2 min. and then stayedapproximately constant (70 pmoles/g. of wholetissue) for the remainder of the 45 min. incubation(Fig. 4).

Ti88ue potassium concentration. Results inTable 3 show that a partial or complete replace-ment of sodium with sucrose in the medium had nosignificant effect on the potassium concentration ofincubated slices, which was between 81 and86-punoles/g. of whole tissue irrespective of thesodium concentration of the medium. After incu-bation in the sodium-free choline chloride medium,the concentration was 72-5 tmoles/g. of wholetissue, which is lower than that found in tissuefrom the standard medium. The difference of131omoles/g. (85-5-72.5), with s.E. 2-8, is derivedfrom mean values from a number of experiments inwhich there was some scatter, and is significant(P < 0.01). It is probably due to an entry ofcholine into the cells in exchange for potassium, forMaizels & Remington (1958) have recently shownthat choline partially displaces potassium in renal-cortex slices, and it cannot be regarded as a com-pletely non-penetrating cation. No attempt hasbeen made to identify the anion which must leavethe cells with sodium during incubation in sodium-free sucrose medium although chloride seems themost likely. In choline chloride medium, a loss ofintracellular chloride is most unlikely, however, inview of the high external concentration, andsufficient choline may penetrate to balance the lossof sodium and the small loss of potassium.

Table 3. Effect of the replacement of 8odium chloride in the medium with either sucrose or choline chlorideon the potassium concentration, the water content and Q0, of 8lices of seminal-veswle mucosa

About 30-60 mg. of tissue was shaken for 45 min. at 370 in 3 ml. of medium, pH 7*4, containing 20 mm-glucose, 10 mm-glutamate and 5 mM-K+ ion. Gas phase was 03. Mean values and s.m. are given, and the figures in parentheses are thenumber of observations.

Compound with whichNaCl was replaced

Choline chloride

Sucrose

Control in standard medium

Concn. ofsodium inmedium(mM)

05

204080120

Q0,4-0±0-4 (5)

5-9±0-4 (6)

0 3-7±0-2 (10)2080 5-2±0-2 (9)120155 6-4±0-1 (36)

Concn. of potaasiumin tissue

(,umoles/g. ofwhole tissue)72-5±2-1 (20)71-5±1-1 (6)76-0±1-8 (8)75-0±1-8 (8)81-5+1-2 (8)78-5±0-9 (14)81-0±1-4 (8)85-0±0-8 (6)83-5±1-6 (8)83-0±0-9 (8)85-5+1-9 (16)

Water content(%, w/w)

79-1±0-7 (12)79-0±0-1 (10)80-2±0-3 (12)79-8±0-1 (12)80-4±0-3 (12)80-7±0-4 (10)79-3±1-0 (6)79-4±0-2 (8)79-5±0-5 (8)81-2±0-1 (8)80-5±0-4 (10)

642

IONS AND METABOLISM IN SEMINAL-VESICLE MUCOSA

Table 4. Sodium and potassium concentrations in slice of seminal-ve8icle muco8a after incubationat 370 in standard medium or in sodium-free, media

Slioes of tissue (about 50 mg.) were incubated in media, pH 7-4, containing 10 mm-tris buffer, 10 mM-glutamate, 20 mm-glucose and 5 mm-K+ ion. Gas phase was O. Mean values are given±s.E. and the figures in parentheses are the number ofobservations. Abnormal media are named after the principal solute.

Concn. of potassium (j.moles/g. of whole tissue)

Standard

108-7i1-5 (15)81-9±1-8 (6)77-2±2-1 (6)81-7±4-0 (6)86-7±3-4 (8)87-8±4-0 (12)83-9±1-6 (28)

Choline chloride

108-7±1-5 (15)85-6±3-7 (8)85-4±1-7 (8)79-2±2-9 (8)75-5±1-0 (8)72-4±1-5 (8)72-5±2-1 (20)

Sucrose

108-7±1-5 (15)86-7±2-0 (10)81-7±2-5 (10)83-5±2-7 (10)81-4±3-0 (6)81-3±3-2 (6)81-2±1-4 (8)

After incubation in either the standard mediumor in sodium-free media, the tissue lost potassium(about 20-30 umoles/g. of whole tissue) in the first2-4 min. (Table 4). This loss occurred in bothphosphate and bicarbonate media, and is probablydue to a loss of potassium from damaged cells on

the outside of the slice. Table 4 shows that thetissue potassium concentration was not significantlydifferent after incubation in the sodium-freesucrose medium and in the standard medium(81-2 and 83-9 pmoles/g. of whole tissue), althoughit was significantly lower in choline chloridemedium (72.5 mmoles/g. of whole tissue) (see alsoTable 3).

Rate of respiration. The rate of respiration of thetissue in standard medium at 370 of 6-4 pm./mg. drywt./hr. was reduced by 19% by a lowering ofsodium concentration of the medium to 80 mM(Q0 5.2-5-9), and by 42% in the sodium-free media(Qo2 3-7-4-0). Significant differences were notfound between the choline chloride and sucrose

media. [The tissue-water content (79-81 %) was

not changed by incubation in the low-sodium media(Table 3).]

Effect of metabolic inhibitors. The addition of 2:4-dinitrophenol to slices of seminal-vesicle mucosa

respiring in standard medium caused a loss ofpotassium, which was balanced by an uptake ofsodium, and this was part of the evidence indicat-ing a dependence of ionic concentrations on meta-bolism (Breuer & Whittam, 1957). The questionnow arises whether the potassium concentrationfound after incubation in sodium-free media is alsodependent on metabolism, whether directly or

indirectly, or whether it is due to a Donnan effectarising from the presence of non-diffusible anions.Fig. 4 shows that, after incubation in sodium-free,sucrose or choline chloride media, the tissue sodiumconcentration approached zero, and Table 4 that

the potassium concentration was approximatelyequal to that after incubation in standard medium.If retention of potassium in the cells in sodium-freemedia is a result of a Donnan equilibrium, inter-ference with metabolism should not affect it, but ifit is due to active transport, then metabolic in-hibitors would be expected to lower the concentra-tion.

Slices of mucosa have therefore been incubatedin the standard medium, and in the sodium-freesucrose and choline chloride media, in the presenceof 2:4-dinitrophenol and iodoacetate, which were

added to inhibit both respiration and glycolysis.The inhibitors added to the standard mediumcaused a lowering of the potassium concentrationby 24 mmoles/g. of whole tissue anaerobically, andby 28 jtmoles/g. of whole tissue aerobically, com-

pared with the control slices, which containedabout 75 1tmoles/g. of whole tissue (Table 5). Incontrast, the inhibitors caused only a small loss oftissue potassium (4-13 ,umoles/g. of whole tissue) inthe sucrose or choline choloride media, both aerobic-ally and anaerobically. The media contained10 mM-glucose and 10 mM-L-glutamate, and itseemed possible that this small loss might be due toa movement of glutamate accompanied bypotassium.

Effect of glucose and glutamate. To test thispossibility, slices were incubated aerobically ineach medium without the addition of glucose andglutamate. The potassium concentration was then70 umoles/g. of whole tissue after incubation in thesucrose medium, and 57 ,amoles/g. of whole tissueafter incubation in the choline chloride medium(Table 5). The values are 13 umoles/g. lower thanthose found after incubation with glucose andglutamate. These effects are almost identical withthose found after the addition of 2:4-dinitrophenolplus iodoacetate in the presence of glucose and

41-2

Medium ...

Time ofincubation

(min.)0248163245

643Vol. 72-

R. WIHITTAM AND H. J. BREUER

glutamate, and show that about 10-133,moles ofpotassium/g. of whole tissue, of the concentrationfound after incubation in sodium-free media con-taining glucose and glutamate, was due to thepresence of these compounds and to the avail-ability of metabolic energy. The results are com-patible with an active uptake of 10-13 ,umoles/g. ofglutamate accompanied by potassium, which wasabolished by 2:4-dinitrophenol plus iodoacetate.In contrast with the small effects of glucose and

glutamate on the tissue potassium concentrationsin sodium-free media, a large difference of 31 jumolesof potassium/g. of whole tissue was found in tissueincubated in the standard medium, with andwithout glucose and glutamate (Table 5). Again,this difference is similar to that found by adding2:4-dinitrophenol plus iodoacetate (28 pumoles/g.).If an active movement of glutamate accompaniedby potassium occurs to the same extent as in thetissue incubated in the sodium-free media, itwould explain only about 40% of the effects in thestandard medium. The greater effects in tissueincubated in the standard medium are undoubtedlydue to the presence of external sodium, which wasable to replace internal potassium in metabolicallydeficient tissues.

Effect of cooling. To test further whether a hightissue potassium concentration could be main-tained in the absence of metabolism, slices wereincubated at 10, since cooling should cause a lossof potassium maintained by a coupling to meta-bolism, but not when the potassium concentrationis due to a Donnan equilibrium. Table 5 shows thatthe tissue potassium concentration was 40 ,moles/g. of whole tissue after incubation at 10 in standard

medium, whereas that from sucrose medium was76 tmoles/g. of whole tissue, and that from cholinechloride medium 56 ytnoles/g. of whole tissue.The results in Table 5 therefore show that a high

tissue potassium concentration could be main-tained during incubation in sucrose or cholinechloride media in the virtual absence of metabolicenergy. In standard medium, only a low tissuepotassium concentration was then found since theintracellular potassium could be readily replaced byexternal Na+ ions (see Table 1). In the absence ofexternal Na+ ions, owing to a replacement withsucrose or choline, internal potassium was thereforeretained passively, probably by a Donnan equi-librium.

DISCUSSION

Easily hydroly8able pho8phate andpotas88ium and 8odium concentrations

The main result of the incubation of slices ofseminal-vesicle mucosa of the guinea pig inmedium at 370 is that, under both aerobic andanaerobic conditions, the concentrations of easilyhydrolysable phosphate (taken to be mainly ATP)and of potassium underwent only small changes.The easily hydrolysable phosphate concentrationwould not be expected to be as high anaerobicallyas aerobically, because the rates of oxygen uptakeand carbon dioxide output were the same, withQ and Q values about 7. Now, by assuming aphosphorylation quotient (P/O ratio) of about 3, itcan be shown that for the production of ATP a

QO, of 1 is approximately equivalent to a Qco, of 6(see Warburg, 1956). It follows therefore that theQao, was only about one-sixth of that required to

Table 5. Effect of cooling, the addition of gluco8e and glutamate and the addition of 2:4-dinitrophenol plusiodoacetate on the potassium concentration of 8eminal-vesicle muco8a after incubation in 8tandardmedium or in 8odium-free sucrose or choline chloride media

About 30-40 mg. of tissue was shaken for 45 min. in 4 ml. of solution in 15 ml. conical flasks containing 10 mm-trisbuffer, pH 7-4. Concentrations were glucose, 20 mM; L-glutamic acid, 10 mM; 2:4-dinitrophenol (DNP), 0-2 mm andiodoacetate (IAA), 1 mr. r

MediumStandard

Sucrose

Choline chloride

Concn. of potassium(/umoles/g. of whole tissue)

With glucose +glutamateNoADNPWith No glucose

No DNP With DNP and+IAA +IAA glutamate(a) (b) (c)76 48 4540 -74 50 4183 72 707676 7270 60 575673 60

Loss of potassium(jumoles/g. of whole tissue)

Glucose +glutamate

DNP +IAA omitted[(a) - (b)] [(a) - (c)] Cooling

28 31} 36

24 33

1131 7

4 _10 13} 14

13

Gaisphase0202N20202N,0202N,

Temp.3701

37371

37371

37

AAA I959

IONS AND METABOLISM IN SEMINAL-VESICLE MUCJOSAproduce ATP at the same rate anaerobically asaerobically. The similar concentrations found underthese conditions suggest that, for the synthesis tokeep pace with the loss of ATP, the rate of hydro-lysis anaerobically must be but one-sixth of therate aerobically.An increase in the efflux of sodium from squid

giant axons poisoned with cyanide, on the injectionof ATP (Caldwell & Keynes, 1957; Keynes, 1958;Caldwell, 1958), is the most direct evidence fora linkage of ATP with the active transport of acation (sodium), and a little less so are the parallelfalls in ATP concentration and potassium influx inglucose-free, human erythrocytes (Whittam, 1958).These are examples of cells which are respectivelypredominantly aerobic and exclusively anaerobicin their metabolism. The seminal-vesicle mucosa,on the other hand, maintains its potassium byenergy from either metabolic pathway and, in thisrespect, resembles frog skin, which can activelytransport sodium under anaerobic conditions atabout 60-80% of the rate under aerobic conditions(Leaf & Renshaw, 1957). It seems likely that ATPprovided by either respiration or glycolysis isutilized by the ion-transport mechanism in thesedifferent cells and tissues.Whatever unknown factors control the rate of

hydrolysis of ATP, the energy available fromanaerobic glycolysis was sufficient to support apotassium concentration almost as high anaerobic-ally as aerobically. Losses of easily hydrolysablephosphate and of potassium, and a gain of sodium,occurred when metabolic inhibitors (2:4-dinitro-phenol plus iodoacetate) were added at 370, and oncooling to 1°. The maintenance of the concentra-tions of easily hydrolysable phosphate and ofsodium and potassium both aerobically andanaerobically at 370, and the rough parallelismbetween the net losses of potassium and labilephosphate, suggest that the active transport ofsodium, and possibly of potassium, probablydepends on labile phosphate esters. These resultsdo not distinguish between whether both sodiumand potassium are subject to primary active-transport processes or whether potassium isdistributed passively as a consequence of activesodium transport. In either case, however, labilephosphate esters seem to be associated with theprimary active process.Under the conditions of incubation when the

concentration of labile phosphate fell, the possi-bility has to be considered that the orthophosphateproduced might leave the cells accompanied bypotassium and thus account for the fall in potas-sium concentration. This possibility can be dis-counted because the loss of potassium was fromthree to ten times greater than could be accountedfor by a loss of monobasic orthophosphate accom-

panied by potassium. (The discrepancy would beeven greater if account is taken of the mixture ofmono- and di-basic orthophosphate more likely tobe present.) The loss of potassium and gain ofsodium therefore seem to be due to a failure ofactive transport, which is correlated with hydrolysisof labile phosphate.

Tissues seem to fall into three classes from theway they depend on metabolic energy for the activetransport of ions. First, there are those tissues andcells which are predominantly aerobic in theirmetabolism, and which require respiratory energyfor the active transport of ions. Examples arechicken erythrocytes (Maizels, 1954), rat diaphragm(Creese, 1954) and skeletal muscle (McLennan,1955), guinea-pig brain and kidney cortex, retina(Krebs, Eggleston & Terner, 1951; Mudge, 1951;Whittam & Davies, 1953) and squid giant axons(Hodgkin & Keynes, 1955). Secondly, cells whichcan transport ions anaerobically and do not possessa respiratory pathway are human erythrocytes(Harris, 1941; Maizels, 1949). Thirdly, frog skin(Leaf & Renshaw, 1957), seminal-vesicle mucosa(Breuer & Whittam, 1957), duck erythrocytes(Tosteson & Johnson, 1957) and ascites tumour cells(Maizels, Remington & Truscoe, 1958) possess theability to transport cations actively both aerobic-ally and anaerobically, thus combining in the samecells the features of the first two types.

Pota8sium retention in the absence ofmedium in the 8odium

The finding that the replacement of sodiumchloride in the medium with either sucrose orcholine chloride caused almost a complete loss oftissue sodium shows that the cells in slices ofseminal-vesiclemucosaarepermeabletosodium. Theretentionoftissuepotassiumduringincubationinthesodium-free mediawas largely unaffected by cooling,the addition ofmetabolic inhibitors and the depriva-tion of substrates (glucose and glutamate), althoughthese factors caused a large loss of potassium fromtissue incubated in standard medium. This resultsuggests that non-diffusible, intracellular anions setup a Donnan equilibrium, which probably accountsfor the high tissue potassium found after incubationin the sodium-free sucrose and choline chloridemedia.

SUMMfARY

1. Conditions of incubation are described underwhich slices of seminal-vesicle mucosa of the guineapig retained most of their potassium in the absenceof oxygen and of sodium in the medium. A com-parison has been made between the ionic concentra-tions, the concentrations of easily hydrolysablephosphate and the rates ofrespiration and anaerobicglycolysis.

VoI. 72 645

646 R. WHITTAM AND H. J. BREUER I9592. The tissue maintained its initial concentra-

tion of easily hydrolysable phosphate, and retainedmost of its potassium during both aerobic andanaerobic incubation for 45 min. at 370 in standardmedium containing sodium chloride. The additionof 2:4-dinitrophenol and iodoacetate or a reductionof the temperature caused a loss of easily hydro-lysable phosphate and of potassium, and a gain ofsodium.

3. The rough correlation between the main-tenance of the concentrations of sodium andpotassium, and of easily hydrolysable phosphate, iscompatible with a dependence of active cationtransport on labile phosphate esters such asadenosine triphosphate.

4. Lowering the temperature of incubation from370 to 270 did not affect the concentrations ofsodium, potassium and chloride found in the tissueafter both aerobic and anaerobic incubation,although Q02 and Qo, were reduced by about50 %. Incubation at 170 and 10, however, causeda loss of potassium, gains of sodium and chlorideand a further fall in the metabolic quotients. Itappears that the energy supply was sufficient forthe active transport of ions at 270 but not at 170and 0°.

5. Tissue slices incubated in media in whichsodium chloride had been partially or completelyreplaced by either choline chloride or sucrose con-tained sodium in a concentration roughly pro-portional to that of the medium. Their potassiumconcentration, however, remained unchanged insucrose medium and fell only about 10 amoles/g. ofwhole tissue in choline chloride medium. Incuba-tion in sodium-free media caused a fall in Q0 from6 4 in standard medium to about 4 in the sucrose orcholine chloride media.

6. The tissue potassium concentration afterincubation in sodium-free media was lowered onlybetween 4 and 14,moles/g. of whole tissue by areduction of temperature from 370 to 10, or anaddition of 2:4-dinitrophenol plus iodoacetate or anomission of glucose and glutamate (which wereusually added to the medium). In contrast, thesefactors caused a large loss of potassium (24-36 pmoles/g. of whole tissue), which was balancedby a gain of sodium, after incubation in standardmedium.

7. The results suggest that potassium can beretained passively, by a Donnan equilibriumcreated by non-diffusible internal anions, in slicesof seminal-vesicle mucosa in the absence of meta-bolic energy, provided that sodium in the medium isreplaced by sucrose or choline chloride. Potasiumretention by the tissue in standard medium, how-.ever, depended on metabolic energy, because in itsabsence potassium was replaced by sodium.We wish to thank Professor Sir Hans Krebs, F.R.S., for

his helpful comments on the manuscript. The work wassupported by grants from the Rockefeller Foundation (inOxford) and the Deutsche Forschungsgemeinschaft (inBonn).

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