Angiotensin I1 Potentiates Prostaglandin Stimulation of Cyclic AMP ...

THEJOURNAL OF

Vol. 263, No.30, Issue of October 25, pp. 15319-15324,1988 Printed in U.S.A.

BlOLOCiCAL CHEMISTRY

0 1988 by The American Society for Biochemistryand Molecular Biology, Inc.

Angiotensin I1 Potentiates Prostaglandin Stimulation of Cyclic AMP Levels in IntactBovine AdrenalMedulla Cells but Not Adenylate Cyclase in Permeabilized Cells* (Received for publication, January 6, 1988)

Michael R. Boarder$, Robin Plevin, andDeborah B. Marriott From the Department of Pharmacology and Therapeutics, University of Leicester, P. 0. Box 138, Medical Sciences Building, University Road, Leicester LE1 9HN United Kingdom

The level of cyclic AMP in primary culturesof bovine synthesis (e.g. 2, 4). Bovine adrenal medulla tissue contains adrenal medulla cells is elevated by prostaglandin El. large amounts of prostaglandin receptors (5, 6). Cyclic AMP Angiotensin I1 is commonly reported to act on receptors levels in primary cultures of cells originating from this tissue linked tophosphoinositide metabolism or toinhibition are increased in the presence of PGEl (3). We have found of adenylate cyclase. We have investigated the effect that of several agents testedwhich act at cell surface receptors, of angiotensin I1 on prostaglandin El-stimulatedcyclic PGEl gave the largest stimulation of cyclic AMP levels in AMP levels in these primary cultures. Rather than these primary cultures. To further investigate cyclic AMP reducing cyclic AMP levels, we have found that angio- regulation by cell surface receptors, we looked for agonists tensin I1 powerfully potentiates prostaglandin El-stimwhich might reduce PGEl-stimulated cyclic AMP levels. Howulated cyclic AMP accumulation in intactcells, both in ever, some of those candidates for inhibitory influence over the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that adenylate cyclase were found instead to increase PGE1-stimfor stimulation of phosphoinositide breakdown by an- ulated cyclic AMP levels in these cultures. One of these is giotensin I1 in these cultures. The potentiation of stim- angiotensin 11, whose receptors have been shown in the past to be negatively linked to adenylate cyclase and, sometimes ulated cyclic AMP levels wasseen, although toa C (Ca”’/ apparently in the same cells (7), to phosphoinositide breaksmaller maximum, with the protein kinase phospholipid-dependent enzyme) activating phorbol down. We have recently shown (8) that angiotensin I1 stimester tetradecanoyl phorbolacetate and with the syn- ulates phosphoinositide breakdown in both adrenal chromafthetic diacylglycerol 1-oleoyl-2-acetyIglycero1;pre- fin cells and thenonchromaffin cells found in primary cultures phorbol ester, which from bovine adrenal medulla. treatment (24 h)withactive would beexpected to diminish protein kinase C levels, There are now a number of examples where the two major attenuated the angiotensin I1 potentiation of cyclic classes of second messenger receptor-effector mechanism, the AMP. Using digitonin-permeabilized cells we showed synthesis of cyclic AMP, and thegeneration of inositol phosthat adenylatecyclase activity was stimulated by pros- phates and diacylglycerols followingphosphoinositide breaktaglandin E1 with the same dose-response relationship down have been shown to regulate each other. Activation of as was cyclic AMP accumulation in intact cells, but the protein kinase C by phorbol esters or diacylglycerol has been permeabilized cells showed no response to angiotensin seen to reduce (9, 10) or enhance (11-15) the stimulation of 11. The results are discussed with respect to the hy- adenylate cyclase by a variety of agents. Several receptors pothesis that the angiotensin I1 influence on cyclic have been shown to be linked to stimulation of adenylate AMP levels is mediated, in part, by diacylglycerol stim- cyclase by an indirect mechanism. For example, studies on ulation of protein kinase C. several tissues have shown that histamine, acting on Hl receptors, is able to enhance cyclic AMP accumulation by an indirect influence on adenylate cyclase (16, 17). Histamine The adrenal medulla comprises approximately equal acting at H1 receptors has been shown to stimulate phosphoiamounts of secretory chromaffin cells and vascular endothe- nositide breakdown (18),so the influence on cyclic AMP could lial cells (1).Prostaglandins are known to influence various be a result of subsequent protein kinase C activation. In this report we show that angiotensin I1 has a powerful aspects of endothelial cell function (e.g. Ref. 2) and to influsynergistic action on PGEl-stimulated cyclic AMP accumuence stimulus secretion coupling in cultured chromaffin cells (3). Prostaglandin receptors, including that activated by lation in cells cultured from bovine adrenal medulla. We PGEI,’ are believed to be involved in the regulation of a consider evidence relating to the hypothesis that this synervariety of cell functions as a result of altering cyclic AMP gism derives from diacylglycerol-mediated activation of protein kinase C by angiotensin 11, enhancing the stimulatory * The costs of publication of this article were defrayed in part by effect PGEl has on adenylate cyclase. the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. ’ The abbreviations used are: PGE1, prostaglandin E,; TPA, 12tetradecanoyl phorbol 13-acetate; 4-OMeTPA, 4-O-methyl 12-tetradecanoyl phorbol 13-acetate; Me2S0,dimethyl sulfoxide; HEPES, 4(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitri1o)jtetraacetic acid; PIPES, l,.l-piperazinediethanesulfonic acid.

EXPERIMENTAL PROCEDURES

Primary Culture of Adrenal Medulla Cells Bovine adrenals were retrogradely perfused with collagenase (1 mg/ml, Sigma Type 111, protease (0.25 mg/ml, Sigma Type IV), and DNase (24 Gg/ml, Sigma Type I) in a balanced salt solution for 30 min. The medullae were then dissected free of cortical tissue, finely chopped, and stirred with the enzyme mixture for two consecutive

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Angiotensin 11and Cyclic AMP Synthesis

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30-min periods. Cells liberated during this procedure were purified on a Percoll gradient essentially as described by Kilpatrick et al. (19) and plated at 0.3 X lo6 cells/well in 24-well multiwell plates (Flow Laboratories, Irvine, Scotland). The culture medium was Dulbecco's modified medium supplemented with 10% fetal calf serum, 27 mg/ 100 ml glutamine, 1 m1/100 ml of 100 X nonessential amino acids, 5 pg/100 ml gentamycin, 250 pg/ml fungizone, 5 p~ fluorodeoxyuridine, and 5 p M cytosine arabinoside, all from GIBCO, Paisley, Scotland. Cells were maintained at 37 "C in 5% CO, and were used at 4-7 days in culture. Where cells were to be split into chromaffin-rich and chromaffinpoor fractions, the differential plating procedure of Waymire et al. (20) was used. Cells were placed in a 175-cm2tissue culture flask at lo6 cells/ml, 50 ml/flask, for 3 h at 37 "C. Unattached cells were decanted off, forming the chromaffin-rich fraction. Attached cells were removed by striking the side of the flask and were decanted off as the chromaffin-poor fraction. Catecholamines were estimated by high performance liquid chromatography with electrochemical detection (21) and protein by the method of Lowry et al. (22). Experiments Measuring Cyclic AMP in Intact Cells-Monolayers of cells were washed twice with 1 ml of a balanced salt solution (BSS: NaCl, 125 mM; KCl, 5.4 mM; NaHC03, 16.2mM; HEPES, 30 mM; NaH2P04, 1 mM; MgS04, 0.8 mM;CaC12, 1.8 mM; glucose, 5.5mM; buffered to pH 7.4, gassed with 5% CO2). Where phosphodiesterase inhibitors were used in experiments they were included in the final wash and in the subsequent incubation period. Following washing the cells were incubated with the drugs as indicated for 3 min in the above BSS; forskolin, TPA, andPGE1 were first dissolved in Me2S0 and diluted in BSS to give a final MeZSO concentration of no more than 0.1%. Numerous controls with 0.1% Me2S0 showed no effect on cyclic AMP accumulations. Angiotensin I1 and histamine were dissolved directly in BSS. All drugs were purchased from Sigma (Poole, Dorset, United Kingdom). The incubation was stopped by removal of the supernatant and extraction of cells into cold 0.1 M HC1. Cyclic AMP was measured using a protein binding assay (23). Estimation of Adenylate Cyclase in Permeabilited Cells-The permeabilization procedure was an adaptation from Terbush and Holz (24). The medium was removed from the cells and replaced with 20 p M digitonin in 139 mM potassium glutamate, 5 mM EGTA, 20 mM PIPES, 5 mg/ml bovine serum albumin, pH 6.4. After 10 min the cells were gently washed twice with 20 mM HEPES, 2 mM EGTA, pH 7.4, (HEPES-EGTA). Theadenylate cyclase assay was begun 20 mi, after theinitiation of permeabilization. The adenylate cyclase assay was an adaptation from Ref. 25. The HEPES-EGTA was replaced with 120 plof incubation mixture made up of Tris-HCl, pH 7.6, 50 mM; creatine phosphate, 5 mM; creatine kinase, 50 units/ml; MgCl,, 5 mM; cyclic AMP, 400 JLM;dithiothreitol, 1 mM; GTP, 30 p ~ isobutylmethylxanthine, ; 1 mM; EGTA, 1 mM; f3H]cyclicAMP, about 10,000 cpm in each incubation; ATP 0.5 mM to a specific activity of about 5 pCi/pmol [w3'P]ATP. To this was immediately added 30 p1 of 0.1% MezSO in water with or without various compounds as appropriate.Incubation for 10 min unless otherwise indicated at 37 "C was stopped by addition of200 pl of ATP (45 mM), cyclic AMP (1.3 mM), and sodium dodecyl sulfate ( 2 g/100 ml) neutralized with Tris base. After leaving at room temperature for at least 30 min, 1 ml of Hz0 was added and the mixture applied to small Dowex AG 50W-X4 (200-400) columns. The cyclic AMP was eluted with H 2 0 directly onto small alumina columns, which wereeluted with 0.1 M imidazole HCl, pH 7.3, into scintillation vials. Dual label counting for 3H and 32Penabled the calculation to accommodate recovery ofcyclic AMP, estimated from recovery of 3H-cyclic AMP, for each individual assay. Recovery of cyclic AMP through the procedure showed little variation within experiments, although it varied from 50 to 80% recovery between days. Chemicals were purchased from Sigma, and radiochemicals from Amersham Corp. RESULTS

Effect of Angiotensin 11 and Histamine on PGE1-stimulated Cyclic A M P Leuels in IntactCells-Consistent with our earlier observation (3) in the present series of experiments we have found that PGE, will raise cyclic AMP levels in adrenal medulla primary cell cultures in a dose-dependent manner with half-maximal stimulation at about 2 pM. In Fig. 1 the submaximal stimulation by 0.5 p~ PGEl gave cyclic AMP levels of about five times basal. Angiotensin I1 (at 100 nM)

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FIG. 1. Influence of angiotensin I1 and histamine on basal andPGEl-stimulated levels. PGE, was at 0.5 p ~ histamine , (HZST)at 10 ~ L M mepyramine , (MEP) at10 PM, angiotensin I1 (AZZ) at 100 nM, and (Sar', Thr') angiotensin I1 (SAR) at 10 pM. Values are means _+ S.E., n = 4.

alone also stimulated cyclic AMP levels by the same order of magnitude as 0.5 pM PGE1, but when 100 nM angiotensin 11 was combined with 0.5 pM PGEl therewas a clear potentiation of effects on cyclic AMPaccumulation (Fig. 1and subsequent figures). Half-maximal potentiating effects of angiotensin I1 were closeto 10 nM in the presence of 0.5 p~ PGEl (data not shown). The influence of phosphodiesterase inhibitors on these responses was studied with isobutylmethylxanthine and Ro 20-17-24. The increases in cyclic AMP and potentiation persist with isobutylmethylxanthine, shown here in the context of parallel experiments with permeabilized cells in Fig. 5A. Ontwo occasions concentration response curves have been constructed in the presence of phosphodiesterase inhibitors, for example with Ro 20-17-24 (50 JLM) present 3 min before and during the incubation with 0.5 p~ PGEland varying concentrations of angiotensin 11. This gave, in two separate experiments, an ECso of 6.4 and 9.0 nM (no inhibitor) or 8.5 and 6.5 nM (in the presence of the phosphodiesterase inhibitor). The failure to see a change in the concentrationresponse relationship occurred despite an increase in levels of cyclic AMP seen in the experiments with Ro 20-17-24. The potentiation in the presence of angiotensin I1 was not seen (Fig. 1) when 10 p M of the specific antagonist, (Sar', Thr') angiotensin 11, was present. Also shown in Fig. 1 is the ability of histamine alone to stimulate cyclic AMP accumulation. When included with PGE1, however,the increase with histamine was additive, not synergistic, in most experiments. The majority of the resultant stimulationof cyclic AMP levels

Angiotensin II and Cyclic AMP Synthesis I

FIG. 2. Influence of various compounds on cyclic AMP levels. TPA was at 100 nM, angiotensin I1 (AZZ) a t 100 nM, nicotine ( N I C ) a t 30 p ~ and , forskolin (FORSK) at 1 PM. Values are means & S.E., n = 4. The inset compares the TPA response in the presence of 1 p~ PGE, with that of 1-oleoyl-2-acetylglycerol(OAG) (25 p ~ ) phorbol 13-acetate (PHA) (100 nM), and 4-OMeTPA (100 nM), the results being expressed as stimulated cyclic AMP levels (ie. minus control levels).

could be antagonized with mepyramine, implicating H1 receptors in at least part of this response. Involvement of Protein Kinase C in Regulation of Cyclic AMP-To evaluate the possibility that protein kinase C is implicated in the regulation of cyclic AMP levels in these cells we have used the phorbol esters intwo different types of protocol. First, with acute usage during a3-min incubation to stimulate protein kinase C, we have directly shown that this treatment does cause an activation and translocationto membrane bound form in these cells' consistent with Terbush and Holz (24). Second, on prolonged exposure it is used to downregulate protein kinase C as reported by others (26, 27). This pretreatment should attenuate or eliminate protein kinase Cmediated responses. The effects of TPA were compared with that of the inactive or veryweakly active phorbol esters, phorbol 13-acetate or4-0-methyl-PMA (4-OMePMA) (28, 29). Fig. 2 shows results from an experiment investigating the influence of TPA and angiotensin I1 on cyclic AMP alone or together with various stimulants of cyclic AMP levels. The phorbol ester alone has no effect but it does clearly enhance the submaximal PGE, stimulation of cyclic AMP levels, but to a smaller degree than does angiotensin 11. Nicotine at 30 p M and forskolin at 1p M produce a similar elevation of cyclic AMP as PGEl at 0.5 p ~ We . have compared the effect of TPA and angiotensin I1 on these (Fig. 2). No substantial effect of either TPA or angiotensin I1 was seen on the nicotine-elevated cyclic AMP levels. Both these enhanced the submaximal stimulation of cyclic AMP by forskolin, again producing a larger response in the presence of angiotensin I1 than TPA, neither however was as larger as when combined with 0.5 p M PGEI. An example of an experiment comparing the effectiveness of TPA with the protein kinase C-activating diacylglycerol l-oleoyl-2-acetylglycerol,and the two inactive M. R. Boarder, R. Plevin, and D. B. Marriott, unpublished data.

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phorbolesters, phorbol 13-acetate, and 4-OMePTA, is shown in the inset to Fig. 2. While both TPA and 1-oleoyl-2-acetylglycerol are effective in enhancing the PGEl stimulation of cyclic AMP levels, phorbol 13-acetate, and 4-OMeTPA are seen to have no effect. We investigated the dose-response relationship for TPA enhancement of PGEl stimulation of cyclic AMP levels in two separate cell preparations and found an ECso from 8 to 20 nM, with maximal response at 50-100 nM. TPA then can, in part, mimic angiotensin 11, albeit with a quantitatively different response. After 24 h of treatment with 100 nM TPA, there was no change in protein content but the increase in cyclic AMP levels with angiotensin I1 alone and with angiotensin I1 and PGEl is considerably diminished, an effect not seen when 4-OMeTPA was used in place of TPA (Fig. 3). These pretreatment experiments have shown that stimulation by PGEl alone is diminished following 24 h of TPA pretreatment (Fig. 3). By contrast stimulation of cyclic AMP levels by forskolin is unaffected by pretreatment with TPA. WhichCells Are Responding-The cells in thestarting tissue (l), and therefore in the cell preparation used, are mainly chromaffin cells and vascular endothelial cells. In order to indicate which of these are contributing tothe responses seen here, we have differentially plated cells (20) to provide chromaffin cell-enriched and impoverished cultures, , as estimatedby catecholamine content/mg of protein. One of two such experiments, giving closelysimilar results, is shown in Table I. Here the protein content/well was in the same range for the two preparations (36 mg/well for chromaffinpoor and 49 mg/well for chromaffin-rich), andthe difference in noradrenaline content, expressed/mg protein, was 3.3-fold. The data are consistent with the major part of the PGEl response, both alone and enhanced by angiotensin I1 and histamine, being on the nonchromaffin cells. Assay of Adenylate Cyclase in Permeabilized Celk-In order to provide a more directestimation of adenylate cyclase activities, we have utilized a procedure of permeabilization of cells with digitonin, permitting access to the cell of [CX-~'P] ATP. The adenylate cyclase activity can then be directly r

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FIG. 3. Effect of 24-h pretreatment with phorbol esters. Cells in culture 24 h with 0.01% Me,SO (DMSO) (control) with either 4-OMeTPA or TPA at 100 nM as indicated, before measuring basal or stimulated cyclic AMP content (n = 4, f S.E.). PGE, was at 0.5 p M , angiotensin 11 ( A H ) was at 100 nM.

Angiotensin I I and Cyclic AMP Synthesis

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TABLE1 Cyclic AMP accumulation in Chromaffin cell-rich and chromaffin cell-poor cultures The chromaffin-rich and chromaffin-poor fractions are separated from a single starting culture. The figures are fmol cyclic AMP/mg protein, n = 4 _+ S.E. PGE, was a t 1 p M , angiotensin I1 a t 100 nM, and histamine a t 10 FM. Basal PGE, PGE, PGE,

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same day and with the same cell preparation, this time for responses to angiotensin I1 and histamine alone and in the presence of PGE,. The stimulation to PGEl andresponses to histamine and angiotensin I1follow the same pattern as shown previously with the cyclic AMP accumulation in intact cells (Fig. 5 A ) .On this occasion the experiment is done in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine to provide more similar conditions to those pertaining in the permeabilized cell experiments. However, while PGE, showed the expected stimulation of labeled cyclic AMP synthesis in permeabilized cells, the responses to histamine andangiotensin 11, alone or with PGE1, were lost (Fig. 5 B ) . Three essentially identical experiments were done on different cell preparations, giving the same results. DISCUSSION

Inthis report we have shown that angiotensin 11 can dramatically potentiate the increase in cyclic AMP levels caused by PGEI. Apart from reflecting a change in rate of synthesis this could be a result of altered phosphodiesterase activity or altered efflux of cyclic AMP from cells, both of which have been shown to be subject to regulation by cell surface receptors (30-32). The increase in cyclic AMP levels caused by angiotensin I1 and PGE1, as well as the potentiation 3 s 3 when added together, persists in the presence of phosphodiesterase inhibitors. Furthermore, the concentration-response m relationship remains unchanged by phosphodiesterase inhi2 = bition, despite the elevation in overall cyclic AMP levels which theseagents caused. The use of phosphodiesterase inhibitors is not simple in that they probably do not cause full inhibition of all enzymes potentially involved (33) and may be having other effects (e.g. Ref. 34). However, with regard to the elevation of cyclic AMP levels by PGE, alone 200 0 I I there is direct confirmation that increased rate of synthesis 0 0.3 1.0 310 10.0 3d.O is responsible from the adenylate cyclase assay with permeaIPGE 1 I uM bilized cells. The closely similar dependence on PGEl concenFIG. 4. Concentration response curve to PGEl for cyclic trations for the two procedures suggests that stimulation of AMP accumulation in intact ceIIs (open circles) and adenylate cyclase assay inpermeabilized cells ( fil2ed circles). Results are synthesis is quite adequate to explain the increased levels of cyclic AMP in intact cells. mean 4 SE, n = 4. An increase in cyclic AMP isnot normally considered within the repertoire of cellular responses to angiotensin I1 estimated by the rate of formation of labeled cyclic AMP. (although there is a previous report (35) of potentiation of Using a 10-min pretreatment with digitonin, cell interiors became accessible to trypan blue but remained attached to stimulation of adenylate cyclase in muscle cells). That the the multiwell. Longer treatment lead to detachment of cells. response is a powerful potentiation suggests not an influence In preliminary experiments we compared 2- and 10-min dig- on adenylate cyclase via stimulatory guanine nucleotide-binditonin treatments.In each case PGE,(at 1 and10 WM) ing protein but aninfluence downstream of another receptorstimulated the formation of cyclic AMP to the same degree effector mechanism. One of several possibilities is activation regardless of the duration of digitonin treatment. However, of protein kinase C following stimulated inositol phospholipid metabolism and diacylglycerol production. Stimulation of for10 PM forskolin, which gave a larger response than did 10 pM PGEI, showed astrongerstimulation of adenylate cyclase mation of inositol phosphates has been a widely described after 10 min than after 2 min of digitonin (data not shown). response to angiotensin 11,and using differentially plated cells In all other experiments reported here, digitonin treatment we have recently shown that angiotensin elicits this response was for 10 min. The time course of accumulation of labeled in both chromaffin cell and nonchromaffin cell components cyclic AMP was linear through 15 min, both basal and with of the adrenal cell culture used here (8).The concentration of 10 W M PGE,. There is a difference between the basal/stimu- angiotensin I1 giving 50% of the maximal inositol phosphate lated ratio using the adenylate cyclase assay in permeabilized response was in thelow nanomolar range, consistent withthe cells and the cyclic AMP accumulation in intact cells which hypothesis that the cyclic AMP potentiating effect seen here can be clearly seen in Fig. 4 and by comparing Fig. 5A with is a consequence of phospholipid breakdown. If potentiation is a result of protein kinase C activation B. PGE, at 1WM gives a stimulation several times above basal in the intactcell cyclic AMP procedure, but only twice basal then it should be possible to mimic the effect with the activating phorbol esters such as TPA. We see here that TPA, in the adenylate cyclase assay in permeabilized cells. A comparison of the response to different PGEl concentra- causing no increase in cyclic AMP alone, can indeed potentions using the two procedures on the same cell preparation tiate the effect of PGEl stimulation, as reported recently for is shown in Fig. 4,where it can be seen that thedose-response Swiss 3T3 cells (36).This effect is shown here to be mimicked relationship to PGEl is the same whether with permeabilized by the synthetic diacylglycerol 1-oleoyl-2-acetylglyceroland or intact cells. Fig. 5 again compares the two assays, on the is also seen with the protein kinase C mezerein (1 wM; data

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