Proc. Natl. Acad. Sci. USAVol. 76, No. 12, pp. 6632-6636, December 1979Medical Sciences

a- and f3-adrenergic stimulation of arachidonic acid metabolism incells in culture

(phospholipase A2/prostaglandins/adrenergic antagonists/norepinephrine/adrenergic receptors)

LAWRENCE LEVINE* AND MICHAEL A. MOSKOWITZtt*Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154; tLaboratory of Neuroendocrine Regulation, Department of Nutrition andFood Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and *Section of Neurology, Peter Bent Brigham Hospital, HarvardMedical School, Boston, Massachusetts 02115

Communicated by H. N. Munro, September 12,1979

ABSTRACT Madin-Darby canine kidney cells (MDCK)synthesize prostaglandin (PG) F2, PGI2 (measured as 6-keto-PGEia), PGE2, PGD2, and thromboxane A2 (measured asthromboxane B2). When incubated in the presence of norepi-nephrine (6 p&M), the syntheses of these arachidonic acid me-tabolites are stimulated -fold. Norepinephrine's effect can beantagonized by the addition of a-adrenergic receptor blockingagents (phenoxybenzamine>phentolamine>yohimbine>di-benamine>tolazoline) but not by the 0-adrenergic blocking drugpropranolol. Norepinephrine's stimulation is also inhibited bylow concentrations of dihydroergotamine, bromocryptine, er-gocryptine, and ergotamine. The stimulation of PG synthesisby norepinephrine is reversible, continues during the 24 hr ofincubation, and requires the presence of norepinephrine at thereceptor site but it is not blocked by the addition of colchicine,cytochalasin B, or cycloheximide. Neither phenoxybenzaminenor ergotamine at concentrations that block norepinephrine'sstimulation of PG biosynthesis suppresses the increase in PGsynthesis induced by exogenous arachidonic acid, suggestingthat the a-adrenergic regulation is not occurring primarily atthe cyclooxygenase step in the metabolism of arachidonic acid.In mouse lymphoma cells (WEHI-5), low concentrations ofisoproterenol or norepinephrine stimulate the synthesis ofthromboxane, an effect that can be blocked by the addition ofpropranolol but not by relatively high concentrations ofphenoxybenzamine or ergotamine. Taken together, these resultssuggest that a-adrenergic receptor stimulation promotes thedeacylation of phospholipids by MDCK cells whereas 0-adrenergic mechanisms lead to activation of similar pathwaysin WEHI-5 cells.

The mechanism by which catecholamines stimulate the bio-synthesis of prostaglandin-like substances in adipose tissue (1,2), spleen (3-6), lungs (7), phrenic diaphragm (8,9), brain (10),kidney (11), and skin (2) is poorly understood. It is possible thatthese compounds stimulate via receptor-mediated mechanisms;for example, treatment with the a-adrenergic receptor blockingagent phenoxybenzamine inhibits the appearance of prosta-glandin-like material from dog spleen (3, 4, 6) and rabbit kidney(11) after the administration of norepinephrine (NE). It is alsopossible that prostaglandin-like substances are released as aresult of tissue contraction (e.g., splenic capsule) induced by thecatecholamines (6). On the other hand, stimulation of prosta-glandin (PG) synthesis by the catecholami-nes may simply re-flect their properties as cofactors for the cyclooxygenation ofarachidonic acid, as shown by studies using microsomes pre-pared from seminal vesicles (12).

In the present study, we examined the relationship betweencatecholamine receptors and PG synthesis by cells in culture.

We now report that the regulation of PG biosynthesis is con-trolled, in part, by a- or f3-adrenergic receptors which, whenstimulated, promote the deacylation of phospholipids andsubsequent metabolism of arachidonic acid.

MATERIALS AND METHODSCell Cultures. Exponentially growing dog kidney (MDCK)

cells were treated with 0.25% trypsin and seeded at 2 X 105 cellsper 60-mm Falcon tissue culture dish in 4 ml of Eagle's minimalessential medium containing 2 mM i-glutamate and supple-mented with 10% (vol/vol) fetal bovine serum, 250 units ofpenicillin per ml, and 250 jg of streptomycin per ml; they wereincubated for 24 hr. In the experiments described below, thecells were washed twice with 2 ml of the medium lacking fetalbovine serum and incubated with 4 ml of the medium lackingthe fetal bovine serum but containing the experimental re-agents.

Assay of Arachidonic Acid Metabolites. Thromboxane A2(TBXA2) (measured as TBXB2), PGE2, PGF2., and PGD2 weremeasured in culture fluids by radioimmunoassay using antiserawhose serologic properties have been described (13, 14). Pros-tacyclin, measured as 6-keto-PGF1a, was also measured byradioimmunoassay. In this system, 10 pg of 6-keto-PGFia in-hibited the binding of 6-keto-[3H]PGFia to anti-6-keto-PGFiaby 50%; PGE2, PGF2a, and PGA2 crossreacted less than 1%. Insome experiments, separation and quantitation of arachidonicacid metabolites were performed as reported (15). Briefly,conditioned medium from MDCK cells was first acidified topH 3.5 and the arachidonic acid metabolites were adsorbed ontoXAD-2 resin (ISOLAB, Akron, OH). The resin was washed with20 ml of H20, and arachidonic acid metabolites were elutedwith 100% ethanol. The eluates were dried under nitrogen atroom temperature and resuspended in ethanol. Samples werethen clarified by centrifugation, concentrated by drying undernitrogen, and subjected to high-pressure liquid chromatographyusing a reversed-phase system (15). The eluted fractions wereassayed by radioimmunoassay. In other experiments, the con-ditioned media were analyzed directly by radioimmuno-assay.

Chemicals. Drugs used in this study were purchased fromSigma except as noted. The tritiated arachidonic acid metab-olites were purchased from New England Nuclear.

Stock solutions of a-adrenergic antagonists in dimethylsulfoxide were stored in the dark at -20°C. Dilutions weremade in medium lacking fetal bovine serum just prior to theexperiment, and the appropriate amount was added to the

Abbreviations: NE, norepinephrine; PG, prostaglandin; TBX,thromboxane.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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culture dish. The highest level of dimethyl sulfoxide used (0.1%)had no effect on MDCK cells or production of PGs. Solutionsof the a- and f3-adrenergic agonists as well as propranolol werefreshly made for each experiment in the medium lacking fetalbovine serum. None of the reagents at the concentrations usedinterfered with the radioimmunoassays.

RESULTSThe MDCK cells synthesized PGF2a, PGI2 (measured as 6-keto-PGFIa), PGE2, PGD2, and' TBXA2 (measured as TBXB2)(Fig. 1). In order to maximize arachidonic acid metabolism sothat the metabolic profile could be clearly demonstrated, in theexperiment shown in Fig. 1 the cells were stimulated to me-tabolize polyenoic acids by incubation with a tumor-promotingphorbol diester [the phorbol diester stimulates deacylation ofcellular lipids (16, 17) and provides the polyenoic substratesrequired for the synthesizing enzymes of PG, TBX, and pros-tacyclin]. Such treatment stimulates PG production 5- to 10-fold(16,'17). Radioimmunoassay of arachidonic acid metabolitesbefore and after separation by high-pressure liquid chroma-tography gave similar values. [Radioimmunoassay of condi-tioned media from cells established from mouse lymphoma,bovine aorta, rabbit aorta smooth muscle, normal humanforeskin, normal human embryonic lung, and a rat adult typeII alveolar cell before and after separation also gave comparable

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values (unpublished data).] Hence, all subsequent analyses wereperformed by radioimmunoassay of the conditioned mediawithout separation, unless otherwise indicated.NE in doses as low as 2-10 ,tM stimulated the production of

PGE2, PGF2., PGI2, TBXA2, and PGD2 without affecting thenumber or viability of cells. When measured after 24 hr of in-cubation, 6 AM norepinephrine stimulated synthesis of all ofthese products 3-fold (Fig. 2). The NE effect continued for atleast 24 hr but was not inhibited by addition of cycloheximide(0.2 ,ug/ml), colchicine (1 Mug/ml), or cytochalasin B (0.1 ug/ml)and therefore does not depend upon the synthesis of protein orintegrity of microtubules or microfilaments. The possibility thatNE was inhibiting catabolism of PGE2, PGF2a, TBXA2, PGI2,and PGD2 by 15-hydroxydehydrogenase and A13-reductase wasunlikely because the metabolites of PGE2 and PGF2a, the15-keto- and 13,14-dihydro-15-keto derivatives, are not foundin MDCK cells or their culture fluids (15). Epinephrine anddopamine also stimulate PG production. At 10 ,uM, epinephrinewas the most potent of the agonists tested; isoproterenol did notstimulate PG production at any of the concentrations tested(2-20 MM).The effects of NE (6 ,M) could be blocked by addition of low

concentrations of the a-adrenergic receptor blocking agentsphenoxybenzamine or ergotamine (Fig. 3). The blockade (likethe stimulation by NE) seems to occur at either the cyclooxy-genase or phospholipase reaction because all of the arachidonicacid metabolites were inhibited to a similar extent. To distin-guish between these two possible loci of activity, PG synthesiswas stimulated by incubating the cells in the presence of ara-chidonic acid. The arachidonic acid-induced stimulation wasnot blocked by high concentrations of phenoxybenzamine (3MM) or ergotamine (0.7 MM) (Table 1). Because it has beenshown (17) that little, if any, free arachidonic acid exists inMDCK cells, it seems most likely that NE promotes the deac-

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FIG. 1. High-pressure liquid chromatogram of cyclooxygenaseproducts of arachidonic acid metabolism by MDCK cells. MDCK cells(2 X 105 cells per 60-mm tissue culture dish) were incubated in min-imal essential medium (4.0 ml) containing 12-O-tetradecanoylphorbol13-acetate (1 ng/ml) for 24 hr. The media from 20 dishes were pooledand the arachidonic acid metabolites were adsorbed on and elutedfrom XAD-2 resin. The concentrated eluate was subjected to high-pressure liquid chromatography and the fractions were assayed byradioimmunoassay. After similar analysis, essentially the samemetabolic profile was obtained in a second experiment. Also, in at least50 experiments with MDCK cells, similar metabolic profiles have beenobtained after radioimmunoassay of the culture fluids before chro-matographic separation.

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FIG. 2. Effect of NE on PGE2, PGF2a, PGI2 (6-keto-PGFia"), andTBXB2 synthesis by MDCK cells. Multiple dishes of MDCK cells (2X 105 cells per 60-mm tissue culture dish) were incubated in minimalessential medium lacking 10% fetal bovine serum (-) or in minimalessential medium lacking 10% fetal bovine serum but containing 6 AMNE (0). At various periods of time the medium from three dishes wasremoved and assayed with antisera of the appropriate serologicspecificity. Each point gives the mean for the three dishes (±SD). Thisexperiment was done three times. The times of incubation variedamong the experiments, but in each experiment the degree of stim-ulation of each arachidonic acid metabolite was the same.

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FIG. 3. Effect of phenoxybenzamine (A) and ergotamine (B) onNE stimulation of PG synthesis by MDCK cells. Multiple dishes ofMDCK cells (2 X 105 cells per dish) were incubated for 24 hr withminimal essential medium alone, with 6,uM NE, or with 6,MM NE plusincrements of phenoxybenzamine or ergotamine. The medium was

removed and assayed for PGF2q (0), 6-keto-PGFIa (0), PGE2 (A),and TBXB2 (a). The data are expressed as the percentage of inhi-bition ofNE stimulation. Duplicate dishes were used and the valuesobtained were within 20% of the mean value. This experiment withincrements of each drug was done twice. Concentration-dependentinhibition was found. Experiments with the two drugs at a singleconcentration have been done at least five times.

ylation of phospholipids (and the availability of arachidonicacid) by perhaps stimulating a phospholipase pathway ratherthan by affecting the cyclooxygenase enzyme. Additionalstudies are needed, however, to confirm experimentally the siteof NE action.

Other a-adrenergic antagonists suppressed the ability of NEto stimulate the entire spectrum of arachidonate metaboliteswith the following order of potency: phenoxybenzamine->phentolamine>yohimbine>dibenamine>tolazoline (Table2). Among the ergot alkaloids, bromocryptine, ergocryptine,dihydroergotamine, and ergotamine were effective inhibitors.L-Ergothioneine and ergonovine were not effective blockers

Table 1. Effect of phenoxybenzamine and ergotamine on

stimulation ofPG production by exogenous

arachidonic acidMean + SD,

ng/ml culture fluidDishes, 6-Keto-

Treatment no. PGF2a PGFia

Minimal essential medium 18 0.87 0.11 0.31 i 0.06Arachidonic acid (2 M^g/ml) 6 3.86 0.32 1.56 + 0.14Phenoxybenzamine (3.2 IiM) 3 0.92 : 0.13 0.33 + 0.09Phenoxybenzamine (0.64,gM) 3 0.91 ± 0.08 0.30 + 0.04Phenoxybenzamine (3.2,uM)+ arachidonic acid (2 Mg/ml) 3 3.47 ± 0.33 1.44 + 0.08

Phenoxybenzamine (0.64 MM)+ arachidonic acid (2,ug/ml) 3 4.33 0.47 1.27 : 0.17

Ergotamine (0.76 MM) 3 0.83 0.08 0.34 + 0.09Ergotamine (0.76 MM)+ arachidonic acid (2 Mg/mi) 3 3.72 i 0.22 1.59 + 0.11

MDCK cells (2 X 105 cells per 60-mm dish) were incubated for 24hr in minimal essential medium lacking 10%/ fetal bovine serum butcontaining the indicated reagents. The medium was collected andsubjected to radioimmunoassay. This experiment was done twice. Thenormalized mean values obtained agreed within 20% of the mean

values shown.

Table 2. Inhibition of NE stimulation of arachidonic acidmetabolism by a-adrenergic receptor antagonists

Antagonist Ic50, MDihydroergotamine 1.8 X 10-8Bromocryptine 2.1 X 10-8Ergotamine 3.0 X 10-8Ergocryptine 3.8 X 10-8Phenoxybenzamine 7.9 X 10-8Phentolamine 1.2 X 10-7Yohimbine 2.7 X 10-7Dibenamine 1.4 X 10-6Tolazoline 7.6 X 10`6Ergothioneine >1 X 106Ergonovine >1 X 1o-6

The inhibition of stimulation of PGF2,, PGE2, 6-keto-PGFia, andTBXB2 synthesis by 6 MM NE was determined as described in Fig.3. The concentrations inhibiting stimulation 50%6 were determinedby interpolation of such inhibition curves. Experiments were doneat least twice with each drug and more than five times with somedrugs. The Ic5o values agreed within 20% of the mean values shown.

even at concentrations of 4.4 and 2.3 AM, respectively. Theability of NE to enhance PG synthesis appears to depend uponits occupation of the receptor site. When MDCK cells wereincubated for 3 hr with 6 iM NE and then washed and rein-cubated for 21 hr in the absence of the agonist, the levels ofsynthesized products did not increase much beyond those ob-served in the unstimulated state (Fig. 4). Addition of 1 ,LM di-hydroergotamine to the culture after incubation for 3 hr with6 IAM NE suppressed the ability of NE to stimulate PG synthesisover the next 21 hr. By contrast, the addition of 1 MiM pro-pranolol, a f3-adrenergic receptor blocker, did not affect thesubsequent stimulation by NE.Dopamine appears to stimulate PG synthesis by a different

mechanism of action, because the addition of phenoxybenza-mine, ergotamine, or propranolol (5 MM) did not alter do-pamine's ability to increase PG synthesis by these cells. Additionof an equivalent amount of other amines (serotonin, histamine)was without effect.To determine the ability of another cell line to respond to the

addition of adrenergic compounds, cells established from amouse lymphoma (WEHI-5) were incubated with NE, dopa-mine, or isoproterenol. In the presence of 6 ,MM NE or isopro-terenol but not dopamine, the synthesis of TBX was stimulated3- to 4-fold. Propranolol at 1 MiM suppressed this stimulationwhereas 3 ,M phenoxybenzamine or 0.7 ,uM ergotamine didnot inhibit this effect (Table 3). Thus, in contrast to the MDCKcells, PG biosynthesis in WEHI-5 cells may result from a directaction of NE on a f3-adrenergic receptor that controls PG syn-thesis, most likely by stimulating the deacylation of cellularlipids.

DISCUSSIONPGs, prostacyclin, and TBX are synthesized in most mammaliancells after the deacylation of membrane phospholipids by theenzyme phospholipase A2. This first step provides the cell witharachidonic acid, the PG precursor that normally does not existfree in cells but is bound to the glycerol moiety of phospho-glycerides. Once cleaved, arachidonic acid serves as substratefor the cyclooxygenase to form cyclic endoperoxides and li-poxygenase to form hydroxy fatty acids. The short-lived en-doperoxides then become rapidly converted to TBX, PGs, andprostacyclin by specific enzymes and nonenzymatic degrada-tion.A number of hormones and other metabolically active

compounds have been shown to release PGs from organs and

Proc. Natl. Acad. Sci. USA 76 (1979)

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FIG. 4. Requirement for the presence of NE to stimulate thesynthesis of PGs during incubation with MDCK cells, and the effectof 1 MM dihydroergotamine or 1 uM propranolol on NE-inducedstimulation when added at 3 hr. Multiple dishes ofMDCK cells (2 X105 cells per dish) were incubated in minimal essential medium alone(0) or containing 6 ,M NE (@). At 3 hr of incubation, the mediacontaining NE were removed and replaced with 4.0 ml of minimalessential medium. The cells in these dishes were incubated for 21 morehr (N). Also at 3 hr, propranolol (o) or dihydroergotamine (A) wasadded to the cells incubating with NE (final concentrations of thepropranolol and dihydroergotamine, 1 MM). These cells were incu-bated for 21 additional hr. Propranolol and dihydroergotamine alsowere added at 3 hr to dishes containing only minimal essential me-dium and these dishes were incubated another 21 hr; in addition, at3 hr, the cells incubating in minimal essential media were given an-other 4.0 ml of fresh medium to measure the effects of these variablesin the absence of NE. There was no significant change in PGF2aproduction. Each point gives the mean (±SD) for three culture dishes.This experiment was done twice. Propranolol had no effect; the ef-fective inhibition of stimulation by dihydroergotamine or removalofNE was found. In addition, in two experiments in which dihydro-ergotamine was added or NE was removed after 1 hr, propranolol hadno effect but in the presence of dihydroergotamine or after removalof NE, PG production was not stimulated.

tissues (18). Most "releasing compounds" probably act bystimulating PG biosynthesis because PGs, TBXs, and prosta-cyclins are not stored in cells or tissues to any extent. One such"releasing" group is the catecholamines (1-11). For example,the addition of NE to synaptosomes stimulates the productionof the PG precursor arachidonic acid and other fatty acids (19).Sympathetic nerve stimulation releases large amounts of PGsin the effluent of isolated perfused organs such as the spleen (3,4) and kidney (20). NE, epinephrine, and methoxamine stim-ulate the release of PGE or PGF from contracted splenic cap-

sular tissue; pretreatment with phentolamine blocks these ef-fects (6). NE and epinephrine also stimulate the release of PGsfrom isolated perfused rabbit kidney; phenoxybenzamine, butnot propranolol, blocks this release (11). Although suggestiveof an a-adrenergic receptor-mediated response, these latterexperiments do not exclude the possibility that PG release isevoked by the drug-induced mechanical stimulation and notprimarily by the action of the drug itself. Other studies alsoprovide suggestive evidence of a cause-and-effect relationshipbetween the catecholamines and the biosynthesis of PGs(21).Our data establish a clear relationship between NE and the

stimulation of PG synthesis by cells in culture. Whereas theMDCK cells are stimulated by a-adrenergic receptor mecha-nisms, the WEHI-5 cells appear to become activated by occu-

Table 3. Stimulation by NE and isoproterenol ofTBX synthesisby WEHI-5 cells and the effects of a- and f-adrenergic

antagonists on NE stimulationTBX,

ng/ml cultureTreatment fluid

Minimal essential medium 0.028 + 0.004

Norepinephrine (6 tM) 0.088 h 0.009Isoproterenol (6,MM) 0.114 I 0.013Dopamine (6 MM) 0.031 I 0.002

Phenoxybenzamine (3MM) 0.031 + 0.001Phenoxybenzamine (0.6 MM) 0.036 + 0.002Phenoxybenzamine (3MM) + NE (6 MM) 0.087 + 0.012Phenoxybenzamine (0.6 MM) + NE (6 ,uM) 0.081 + 0.011

Ergotamine (0.8 MM) 0.030 + 0.003Ergotamine (0.15 MM) 0.036 + 0.006Ergotamine (0.8 ,M) + NE (6MuM) 0.087 + 0.005Ergotamine (0.15,MM) + NE (6AM) 0.081 + 0.005

Propranolol (3.9 AM) 0.031 + 0.006Propranolol (3.9 MAM) + NE (6 AM) 0.044 I 0.002

WEHI-5 cells (5 X 105 cells per 60-mm dish) were incubated for 24hr in 4 ml of minimal essential medium lacking 10% fetal bovine serumbut containing the indicated reagents. The media were collected andassayed for TBX with an antiserum to TBXB2. The results given arethe mean (4SD) of three dishes. This experiment has been done threetimes. In each experiment, NE and isoproterenol stimulated PGproduction and dopamine did not; propranolol inhibited NE stimu-lation and phenoxybenzamine and ergotamine did not.

pation of the f3-adrenergic receptor by NE. NE stimulation ofPG production by MDCK cells is dose dependent, reversible,and not dependent upon protein synthesis or microtubular ormicrofilament integrity, but it requires the presence of NE atthe receptor site. The ability of a-adrenergic receptor blockingagents to inhibit NE-induced stimulation correlates with thepotency of these drugs in vvo and their ability to inhibit ra-dioligand binding in vitro (22). MDCK cells respond to NE byincreasing, to the same extent, the synthesis of all arachidonatemetabolites made by the cell. This suggests that receptor-mediated stimulation occurs at the cyclooxygenation of ara-chidonic acid or deacylation of phospholipids; Bradykinin,thrombin, tumor-promoting phorbol diesters, adriamycin, ar-omatic polycyclic hydrocarbons, retinoids, epidermal growthfactor, and polypeptide component of bee venom (melittin)have been found to stimulate PG production in vitro by in-creasing the deacylation of cellular lipids (23). This was notsurprising because it had already been found that virtually allavailable arachidonic acid is incorporated by MDCK cells intophospholipids (23).Thus it seems likely, but not certain, that NE, like the com-

pounds listed above, activates phospholipase A2 to liberatearachidonic acid. Further indirect evidence for such a mech-anism is provided by experiments which showed that the a-adrenergic receptor blocking agents were unable to block thestimulation of PG synthesis induced by arachidonic acid, al-though they were effective inhibitors of NE activity.The ability of NE to stimulate the deacylation of phospho-

lipids and biosynthesis of PGs may or may not be related todrug-induced changes in membrane permeability reported forMDCK cells. These cells retain in culture many features char-acteristic of distal tubular epithelial cells such as tight junctions,transport of ions, permeability to water, and sensitivity to sev-eral hormones (24). The addition of pharmacological doses ofPGE1 or PGE2 causes a marked increase in the activity ofadenylate cyclase and formation of cyclic AMP by MDCK cells,

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a property shared with other hormones such as vasopressin,oxytocin, and glucagon and with cholera toxin. PGs have pre-

viously been thought to modulate cellular permeability to waterand ionic fluxes induced by hormones such as vasopressin (25).In cortical collecting tubules, NE has been shown to affectadenylate cyclase activity and to modify water and electrolytetransport in various epithelia (26). It is possible that the PGsthemselves alter membrane properties and account for NE-induced changes in permeability or ion transport; alternatively,receptor activation and PG biosynthesis, or the resulting gen-

eration of lysophosphatides within the membrane, may becoupled to methylation of membrane phospholipids to modifymembrane structure and function (27). Recently, 3-adrenergicreceptor-mediated mechanisms have been reported to increasephospholipid methylation of erythrocyte membranes, therebyaltering membrane viscosity and enhancing membrane fluidity(28). The extent that a-adrenergic receptor-mediated PGsynthesis modifies membrane function in transport epitheliumremains to be determined.

L.L. is a Research Professor of Biochemistry of the American CancerSociety (Award PRP-21). M.A.M. is a recipient of Teacher-InvestigatorAward 11081 from the National Institute of Neurologic and Com-municative Diseases and Stroke. This is publication no. 1279 from theDepartment of Biochemistry, Brandeis University, Waltham, MA02254.

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