hydrolysis in chromaffin cells

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1 Br. J. Pharmaol. (199), 11, Influene of phorbol esters, and diaylglyerol kinase and lipase inhibitors on noradrenaline release and phosphoinositide hydrolysis in hromaffin ells J. Alison Jones, P. Jane Owen & 'Mihael R. Boarder \It&/,'-. Mamillan Press Ltd, 199 Department of Pharmaology and Therapeutis, University of Leiester, P.O. Box 138, Medial Sienes Building, University Road, Leiester LEI 9HN 1 We have investigated the modifiation of ateholamine efflux and inositol phosphate formation in ultured adrenal hromaffin ells by tetradeanoyl phorbol aetate (TPA) and inhibitors of diaylglyerol kinase (R 59 22) and diaylglyerol lipase (RG 8267), the two prinipal pathways of diaylglyerol metabolism. 2 TPA (1 nm to 1 pm) eliited a slow, alium-dependent, sustained release of noradrenaline, whih was partially bloked by the dihydropyridine alium hannel bloker (-) and potentiated by the hannel enhaner (+) R enhaned noradrenaline efflux at 3 and 5pM, while the lipase inhibitor RG 8267 failed to eliit release. 4 Neither R nor RG 8267 affeted bradykinin- or histamine-stimulated release, but both drugs substantially attenuated niotine- and high K+-stimulated release. 5 Pretreatment for 1min with TPA (but not the relatively inative 4-methoxyTPA) or the non-phorbol protein kinase C stimulator mezerein potently inhibited bradykinin- and histamine-stimulated aumulation of total [3H]-inositol phosphate; inhibition of [3H]-inositol phosphate formation was also seen with 24 h TPA treatment. 6 Neither R nor RG 8267, separately or together, affeted bradykinin-stimulated [3H]-inositol phosphate formation. 7 Thus while the mehanism exists for inhibition of formation of inositol phosphates by stimulation of protein kinase C, these studies failed to show that this mehanism is ativated by agonists ating on phospholipase C linked reeptors. Introdution Chromaffin ells maintained in primary ulture release ateholamines in response to ativation of a variety of ell surfae reeptors. This inludes reeptors whih, in addition to stimulating release of noradrenaline, stimulate hydrolysis of polyphosphoinositides, generating inositol phosphates and diaylglyerol, suh as reeptors to bradykinin, histamine, angiotensin II and prostaglandin (Livett & Marley, 1986; Zimlihman et al., 1987; Noble et al., 1988; Plevin & Boarder, 1988; Koyama et al., 1988; Owen et al., 1989a; Plevin et al., 199). The generation of diaylglyerols whih may ativate protein kinase C (PKC) is likely to be of signifiane in stimulus-seretion oupling. The learest indiation of this is the demonstration that PKC ativating phorbol esters suh as tetradeanoyl phorbol aetate (TPA) an themselves stimulate release, in a alium-dependent manner, both in intat (Broklehurst et al., 1985; Pootte et al., 1985) and permeabilised ells (Knight & Baker, 1983; Pootte et al., 1985; Broklehurst & Pollard, 1985). The studies with permeabilised ells demonstrate that PKC ativation may play a role in exoytosis at the level of fusion of granules with the ell membrane, while Bittner & Holz (199) have shown that more than one mehanism may be responsible for enhanement of release from permeabilized ells. A seond possible role for PKC in stimulus-seretion oupling may be at the level of the reeptor. Ativation of phospholipase C (PLC) may ause a diaylglyerol-mediated stimulation of PKC whih feeds bak to inhibit the agonistindued ativation of PLC. Evidene for suh a mehanism falls into two ategories. Firstly, the inhibition of agonistindued stimulation of PLC by exogenous PKC ativators suh as phorbol esters. Seondly, evidene that inhibitory feedbak loop is ativated by an agonist, suh as the enhane- ' Author for orrespondene. ment of agonist-stimulated PLC by prior down-regulation of PKC, or attenuation of agonist-stimulated PLC by bloking diaylglyerol breakdown. Studies in a variety of preparations have shown that phorbol esters an attenuate agoniststimulated formation of inositol phosphates; in hromaffin ell preparations it has been shown that histamine-indued inositol phosphate aumulation an be attenuated by phorbol ester (Wan et al., 1989). Several reent studies have provided evidene that suh a feedbak loop is ativated by agonists ating on PLC-linked reeptors in platelets, smooth musle and epithelial ells (Helper et al., 1988; King & Rittenhouse, 1989; Pfeilshifter et al., 1989; Crouh & Lapetina, 1989). In the present study, we used bovine ultured adrenal hromaffin ells to haraterize the effet of phorbol esters and inhibitors of diaylglyerol metabolism on release of ateholamines. We demonstrated the inhibition of agonist-stimulated PLC by phorbol esters and, also, sought to provide evidene for the regulation of inositol phosphate responses by agoniststimulated diaylglyerol prodution. Methods Freshly obtained bovine adrenal medullae were digested with ollagenase/protease as desribed by Marriott et al. (1988). Chromaffin ells were purified to about 9% by entrifugation and differential plating and ultured on 24-well 'Primaria' plates in omplete medium as desribed earlier (Owen et al., Cells for release studies were washed twie with HEPES-buffered balaned salt solution (BSS) ontaining (mm): NaCI 125, KCI 5.4, NaHCO3 16.2, N-2-hydroxyethylpiperazine-N'-2-ethanesulphoni aid 3, NaH2PO4 1, MgSO4.8, CaCl2 1.8 and gluose 5.5, gassed with 95% 2: 5% CO2 and buffered to ph 7.4. Release was measured by inubating the ells at 37 C in BSS in the presene or absene of drugs. A preinubation period of 1min, in the presene or absene of drugs, was introdued where appropriate. At the

2 522 J.A. JONES et al. end of the inubation period the medium was removed, entrifuged and the supernatant was aidified with.1 M HCI. The ell ontents were extrated into.1 M HCl and the noradrenaline and adrenaline ontents of both supernatant and ell extrat were estimated by high pressure liquid hromatography with eletrohemial detetion. Similar patterns of release were seen for both noradrenaline and adrenaline, but only the release of noradrenaline is shown, expressed as either pmol per well or as % of ell ontent. Eah result is the mean of quadrupliate determinations from a single ell preparation whih has been repeated in similar or idential form three or more times on different ell preparations. To estimate stimulation of total inositol phosphate formation, ells were inubated with 1 uci myo-[2-3h]-inositol (15Cimmol-1) at 37C for 32-4h in.5ml of modified BSS ontaining (mm): NaCl 125, KCl 5.4, NaHCO3 16.2, NaH2PO4 1, MgSO4.8, CaCl2 1.8, gluose 5.5, GIBCO nonessential and essential amino aids at 1%, glutamine 27mg looml-1, streptomyin 5pg looml-1, peniillin 5iu 1mP-1, ytosine arabinoside 5pM, in 5% C2 95% air. Cells were then washed, and preinubated with 1mm lithium hloride in BSS in the presene or absene of drugs for 1 min. Drugs were then added for the duration of the inubation period as appropriate in BSS plus lithium. The reation was stopped with old methanol. Chloroform extration was followed by isolation of [3H]-inositol phosphates on Dowex-l (Cl-') essentially as desribed by Rooney & Nahorski (1986). Dihydropyridine, phorbol esters and diaylglyerol kinase and lipase inhibitors were diluted with BSS from stok solutions in dimethylsulphoxide (DMSO). Materials Cell ulture supplies were from GIBCO, Paisley, Sotland exept for Primaria plates (Falon) whih were from Beton- Dikinson, Oxford. myo-[2-3h]-inositol was from New England Nulear. The diaylglyerol kinase inhibitor R (6 - [2 - [4 - [(4 - fluorophenyl)phenylmethylene] piperidinyl] - ethyl]-7-methyl-sh-thiazolo[3,2-a]pyrimidin-5-one) was purhased from Janssen, Olen, Belgium. The diaylglyerol lipase inhibitor RG 8267 (1,6-bis(ylohexyloximinoarbonylamino) hexane) (Revlon) was a kind gift of Rorer Central Researh, Washington, Pasadena, USA, while the isomers of (4- (benzoxadiazolyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinarbonate-isopropylester) were kindly donated by Sandoz Ltd, Basle, Switzerland. Other hemials and drugs were from Sigma Chemial Co. pl, Poole, Dorset, U.K. or Fisons pl, Loughborough, U.K. Results Effet of TPA, diaylglyerol kinase inhibitor and diaylglyerol lipase inhibitor on noradrenaline release The time ourse, dose-response urve and alium-dependene for the release of noradrenaline in response to TPA is shown in Figure la,b and, respetively. The rate of release was harateristially slow and onsequently subsequent experiments used a 3min inubation. The EC5 for TPA was between 8 and 35 nm (3 determinations). Consistent with previous observations, phorbol ester-stimulated release was dependent on added extraellular alium (Figure i), being maximal at.3 mm. The dependene on extraellular alium suggests that alium entry may play a role in TPA-stimulated release, thus the effets of two dihydropyridine alium hannel drugs were studied (Table 1). The alium hannel bloker (-) redued noradrenaline release in response to TPA while its stereoisomer, (+) , a alium hannel enhaner, aused a small release alone but had a greater than additive effet when ombined with TPA. The endogenous PKC ativator diaylglyerol is metabo- 4+) 1 C. J --a 2',If 1 Uo )- o k o 3-2 'a m 1- o- U) a, 12 " ql -~~~~~~~ Time (min) ~~~~ TPA on. (nm) Calium on. (mm) Figure 1 Charateristis of tetradeanoyl phorbol aetate (TPA)- stimulated release of noradrenaline. ( Release in response to inubation for inreasing time with 1,UM TPA () or dimethylsulphoxide ontrol (). (b) Release after 3min inubation with inreasing onentrations of TPA. () Release in response to inubation for 3min with 1 nm TPA in inreasing onentrations of extraellular alium. Basal release in the absene of added alium was %. Release is expressed as % of total noradrenaline ell ontent. Values are means and vertial bars show s.e.mean of quadrupliates from a single experiment. lised through either phosphorylation to phosphatidi aid, by diaylglyerol kinase (DG kinase), or deaylation at the 2 position to monoaylglyerol, by diaylglyerol lipase (DG lipase). It was therefore of interest to see whether omparable effets to TPA ould be produed by inhibition of one or both of these pathways. Hene, we investigated the effet of a DG kinase inhibitor (R 59 22) and a DG lipase inhibitor (RG 8267) on noradrenaline release. Neither the vehile alone (dimethylsulphoxide.3%) nor the DG lipase inhibitor RG 8267 (up to SOpUM) had any effet on release over a period of 3min. The DG kinase inhibitor R59 22 did indue enhaned effiux at 3pM and above (Figure 2, after inubation periods of over 8 min (Figure 2b). The DG lipase inhibitor failed to stimulate release at any time up to 6 min (Figure 2b). Sine bradykinin and histamine stimulate inositol phosphate prodution in hromaffin ells as well as release (Plevin & Boarder, 1988), then they must also stimulate formation of diaylglyerol. It was therefore possible that inhibition of dia- Table 1 Effet of dihydropyridine on tetradeanoyl phorbol aetate (TPA)-stimulated release TPA (1 nm) (-) (+) Data shown are pmol noradrenaline released per well over a 3min inubation period, with dihydropyridine present during a 1min preinubation as well as during the inubation period. Dimethyl sulphoxide was present at.1% throughout. Values are means + s.e.mean of quadrupliates from a single experiment.

3 PROTEIN KINASE C RELATED COMPOUNDS AND CHROMAFFIN CELLS 523 a 4-3- Basalm KI 3 F~M // LI 3MBL KI + L Ni 3 FiM / / / / / // KI + Ni Sg/ Z 2- C 5) o 1 a O (D 6- m U) ṅ 5- -a 4- so z Conentration R (>M) 1 KI + LI + Ni S//v K 6 mmy////////////// LI + K KI + LI + K 4/,// Mean release of noradrenaline (% of ell ontent) Figure 3 Noradrenaline release in response to niotine (Ni) and high K+ (K): effet of diaylglyerol (DG) kinase (KI) and DG lipase (LI) inhibitors. The inhibitors were present where appropriate during a 12min preinubation period as well as during the 6min inubation period, in the presene or absene of 3pM niotine or 6mM K+, during whih release was measured. Both inhibitors were present at 3AM, and dimethylsulphoxide at.3% was inluded where inhibitors were not added. Values are means and horizontal bars show s.e.mean of quadrupliates from a single experiment. Time (min) Figure 2 Release of noradrenaline in the presene of inhibitors for diaylglyerol (DG) kinase and diaylglyerol lipase. ( Noradrenaline release (% of ell ontent) in response to inreasing onentrations of the DG kinase inhibitor R59 22 (). The dimethylsulphoxide vehile up to.3% had no effet on release. (b) Release (% of ell ontent) after various times of inubation with 3pFM RG 8267 (O), 3uM R () or.3% dimethylsulphoxide (. Values are means and vertial lines show s.e.mean of quadrupliates from a single experiment. ylglyerol breakdown would modify release by these agents. It was found that bradykinin-stimulated release was unaffeted by either the kinase inhibitor or the lipase inhibitor, added separately or together at 3AM (data not shown). The histamine-stimulated release was redued by 3pM kinase inhibitor (data not shown); this was, however, the expeted onsequene of the antihistamine nature of the drug and was unrelated to its metaboli effets. Noradrenaline release stimulated by either niotine or high K+ (depolarization) was inhibited by preinubation with either inhibitor (3pM). This is illustrated by results from one suh experiment shown in Figure 3; with a stimulation period of 6 min it was found that the niotine-evoked stimulation ould be almost eliminated by either R or RG The high K+-stimulated release was similarly affeted by the kinase inhibitor R 59 22, but was redued to a lesser extent by the lipase inhibitor RG Thus, it appears that neither inhibitor of diaylglyerol metabolism inhibited bradykinin-stimulated release, but they both substantially attenuated release stimulated by niotine and high K+ depolarization. Effet of TPA, diaylglyerol kinase inhibitor and diaylglyerol lipase inhibitor on agonist-stimulated inositol phospholipid breakdown In the experiments to investigate the effets of TPA on agonist-stimulated formation of total inositol phosphates (in the presene of lithium), the pretreatment time with the phorbol ester was either 1Omin (to stimulate PKC) or 24 h (to downregulate PKC). In eah ase the phorbol ester was also present during the 3min inubation period in the presene of the agonist. We have previously shown that histamine and bradykinin stimulation of total inositol phosphate formation is linear for 3min (Plevin & Boarder, 1988). The influene of 1iUM TPA pretreatment for 1min and 24h is illustrated by the results in Tables 2 and 3 respetively. Surprisingly, both pretreatment periods produed a substantial loss in agoniststimulated formation of inositol phosphates. The effet of 1min TPA pretreatment was harateristially greater with histamine stimulation than with bradykinin stimulation, while 24h TPA pretreatment almost eliminated (histamine) or substantially redued (bradykinin) the inositol phosphate response to agonists (Table 3). We investigated the effets of different TPA onentrations, of the relatively inative 4-methoxy TPA and of the struturally disparate PKC ativator mezerein on bradykinin- and Table 2 Effet of tetradeanoyl phorbol aetate (TPA) pretreatment for 1min on bradykinin and histamine stimulation of total inositol phosphate formation Bradykinin Histamine DMSO ontrol TPA (1FM) Data shown are d.p.m. of [3H]-inositol phosphate aumulated in the presene of 1mM lithium during a 3min inubation period. TPA was present where appropriate, with dimethyl sulphoxide (DMSO) vehile at.1%, during a 1min preinubation as well as the inubation period. Values are means + s.e.mean of quadrupliates from a single experiment. Table 3 Effet of 24h pretreatment with tetradeanoyl phorbol aetate (TPA) on bradykinin- and histaminestimulated total inositol phosphate formation Bradykinin Histamine DMSO TPA (1pFM) Data shown are d.p.m. of [3H]-inositol phosphate aumulated in the presene of 1mM lithium during a 3min inubation period. TPA was present, where appropriate, with dimethylsulphoxide (DMSO) vehile at.1% during a 24h preinubation as well as the inubation period. Values are means + s.e.mean of quadrupliates from a single experiment.

4 524 J.A. JONES et al. Basal His 3 fm -i TPA 1 nm + His TPA 1 nm + His TPA 1 nm + His TPA 1 AXM + His TPA 1 FtM MeOTPA 1 ILm MeOTPA 1 ALM + His Mez 1 AM + His I h Basal BK 1 nm TPA InM + BK TPA1nM + BK TPA 1 nm + BK TPA 1 p.m + BK TPA 1 p.m MeOTPA 1 p.m MeOTPA 1 p.m + BK Mez 1 p.m + BK IP aumulation (d.p.m. per well) Figure 4 Total [3H]-inositol phosphate (IP) formation in response to stimulation for 3min with ( 3M histamine (His) or (b) 1nM bradykinin (BK). Tetradeanoyl phorbol aetate (TPA) was present at the onentrations shown, 4-methoxy TPA (MeOTPA) 1 jim or mezerein (Mez) 1 fm were present during a 1min preinubation as well as the 3 min inubation period. Values are means and horizontal bars show s.e.mean of quadrupliates from a single experiment. histamine-stimulated formation of inositol phosphates. These results are illustrated in Figure 4. When stimulation was with histamine substantial effets of TPA ould be seen at 1 nm, with essentially maximal effets at 1nm. 4-MethoxyTPA at 1 fim was no more effetive than TPA at 1 nm. Mezerein (1 pm), while having no effet of its own, effetively inhibited histamine-stimulated formation of inositol phosphates. A very similar pattern of results was obtained when stimulation was with bradykinin (Figure 4b). These results are onsistent with the regulation by PKC of histamine- and bradykininstimulation of inositol phosphates. The indiation that phorbol ester stimulation of PKC might regulate the hromaffin ell response to histamine and bradykinin led us to investigate the role of endogenously ativated PKC, using the inhibitors of diaylglyerol kinase (R 59 22) and lipase (RG 8267). When inluded before and during inubations with bradykinin, R was without effet (Table 4). Results of experiments in whih the effet of R on histamine stimulation was investigated are not presented sine it is known that R is an H1-reeptor antagonist (de Chaffoy de Courelles et al., 1985). RG 8267 had no effet on either bradykinin- or histamine-stimulated inositol phosphate formation. A ombination of the two inhibitors also failed to influene the aumulation of inositol phosphate in response to bradykinin. Disussion The initial observation of Knight & Baker (1983), who used permeabilized ells to show that TPA indued an inreased sensitivity of the exoytoti proess for alium, presumably by ativating PKC, was influential in direting attention to the role of stimulation of this enzyme in exoytosis. However, here we are onerned with the role of PKC in the initiation of stimulus seretion oupling at the ell membrane. The time ourse of TPA-stimulated release desribed was found to be very slow and sustained and dependent on extraoliular alium, in onfirmation of previous observations (Broklehurst et al., 1985). There are perhaps two explanations for this alium-dependeny. Firstly, inubation in nominally alium-free medium results in a redution in free intraellular alium levels, to whih TPA-stimulated release is sensitive (Broklehurst et al., 1985; Pootte et al., 1985). Seondly, TPA leads to an enhaned alium influx, without whih TPAstimulated release annot our. We found that in hromaffin ells the release in response to TPA was partly bloked by the dihydropyridine antagonist (-) Chromaffin ells have learly been shown to possess L-type dihydropyridine sensitive voltage gated alium hannels whih may mediate stimulus-seretion oupling (e.g. Garia et al., 1984; Boarder et al., 1988; Owen et al., 1989b) and so one explanation for this observation is that TPA-stimulated release is, in part dependent upon an enhaned opening of these hannels aused, diretly or indiretly, by stimulated PKC ativity. The data we present here ontain one additional indiation that TPA is affeting alium entry through L-type hannels: the alium hannel agonist (+) enhaned the release stimulated by TPA. This is onsistent with the demonstration of dihydropyridine-sensitive phorbol ester-stimulated alium influx in loned rat pituitary ells (Albert et al., 1987). Effets of TPA on alium fluxes in other ell types inlude an enhanement of alium hannel opening in Aplysia (De Riemer et al., 1985), failitation of depolarization enhaned alium influx in intat adrenal medulla of the rat (Wakade et al., 1986), inhibition of depolarization-enhaned alium flux in PC12 ells (Harris et al., 1986; Di Virgilio et al., 1986) and attenuation of alium influx in neutrophils (MCarthy et al., 1989). An interesting alternative to TPA as an ativator of PKC is the inhibition of DG kinase and/or DG lipase, in an attempt to elevate the levels of diaylglyerol, an endogenous ativator (alongside alium) of PKC. The DG kinase inhibitor R has been shown to potentiate thrombin-indued diaylglyerol prodution in platelets and inhibit phosphatidi aid prodution in neutrophils (de Chaffoy de Courelles et al., 1985; Mege et al., 1988), while RG 8267 has been shown to inrease basal diaylglyerol levels and potentiate hormonal stimulated diaylglyerol prodution (Sutherland & Amin, Table 4 Effet of inhibitors of diaylglyerol lipase and kinase on bradykinin- and histamine-stimulated inositol phosphate prodution Bradykinin (1 nm) Histamine (3puM) RG 8267 and RG 8267 R R ± * * The diaylglyerol lipase inhibitor RG 8267 was 1puM and the diaylglyerol kinase inhibitor R was 5jM. Data shown are d.p.m. of [3H]-inositol phosphates produed during a 3min inubation period with the inhibitors present during both this inubation period and a preeding 1min pre-inubation. Values are means + s.e.mean of quadrupliates from a single experiment. * Effet of R on histamine stimulated [3H]-inositol phosphate prodution was not valid due to antihistamine nature of the inhibitor.

5 PROTEIN KINASE C RELATED COMPOUNDS AND CHROMAFFIN CELLS ; Chang et al., 1988). In experiments run in parallel with those desribed here, we have shown that both these metaboli inhibitors inrease the aumulation of diaylglyerol in hromaffin ells at 3M and 5uM, with the effet of R being greater than that of RG 8627 (Owen & Boarder, unpublished observations). Here we showed that the DG kinase inhibitor was able to enhane release at these onentrations. By ontrast, the lipase inhibitor was not able to eliit inreased noradrenaline efflux. Combined with the effet of these ompounds on diaylglyerol aumulation in hromaffin ells (Owen & Boarder, unpublished observations), these results may indiate a relationship between enhanement of diaylglyerol aumulation and the stimulation of release. Bradykinin-stimulated noradrenaline release was unaffeted by the DG kinase and lipase inhibitors suggesting that aumulation of diaylglyerol plays little role in bradykininstimulated release. This is onsistent with the differing sensitivity of bradykinin- and TPA-stimulated release to dihydropyridines: the TPA-stimulated release is partially sensitive (this paper) while bradykinin-stimulated release is insensitive (Owen et al., 1989) to dihydropyridine alium hannel blokers. The ontrasting observation that niotineand high K+-stimulated release is inhibited by both kinase and lipase inhibitors is most likely due to an effet at the level of alium entry, sine the lak of effet on responses to bradkykinin shows that the exoytoti proess is not impaired. The inhibition of niotine- and high K+-stimulated release may be through PKC ativation and subsequent phosphorylation of a omponent of alium entry, or a 'non-speifi' membrane effet perturbing hannels and/or niotini reeptors. A possible role for bradykinin-stimulated PKC in hromaffin ells is modulation of reeptors and assoiated effetor mehanisms. For example, we have previously provided evidene that angiotensin II enhanes prostaglandin stimulation of adenylate ylase in ultured adrenal medulla ells, by a mehanism involving diaylglyerol prodution and the ativation of PKC (Boarder et al., 1988). In addition, a number of examples exist in a variety of ell types of inositol phospholipid-linked reeptor responses whih are downregulated by phorbol ester-stimulated PKC (e.g. Rittenhouse & Sasson, 1985; Drummond, 1985; Aiyar et al., 1986), with indiations that endogenously produed PKC ativation may be involved (Helper et al., 1988; King & Rittenhouse, 1989; Crouh & Lapetina, 1989; Pfeilshifter et al., 1989). We showed that the inositol phosphate response to bradykinin and histamine in hromaffin ells was attenuated by 1min prior ativation of PKC. These results indiate that stimulation of PKC an result in a redued response to these agonists, and raise the possibility that enhaned prodution of diaylglyerol may play a feedbak role in the response. Pretreatment for 24h with phorbol esters, a protool whih auses loss of PKC ativity in a variety of ell types, inluding PC12 ells and adrenal hromaffin ells (Matthies et al., 1987; Bittner & Holz, 199), might therefore be expeted to enhane the inositol response, as shown reently in different ell types (Helper et al., 1988; Pfeilshifter et al., 1989). However, our observation of redued responses for both bradykinin and histamine stimulation is diffiult to interpret: it may be due to a redued response to the agonist following a long period of prior PKC stimulation by TPA, or a possible dependeny of the response on intat PKC ativity in the ells. A further investigation is needed to monitor the hanges in PKC ourring in these ells on lengthy treatment with phorbol esters. As previously disussed, diaylglyerol is metabolised by kinase or lipase pathways, so a further strategy is to potentiate the onsequenes of inreased synthesis of diaylglyerol by inhibiting one or both of these pathways. Agonist-enhaned diaylglyerol formation may only be able to ativate a feedbak loop when diaylglyerol breakdown is impaired. This would result in a redued inositol phosphate formation in response to agonists in the presene of the diaylglyerol kinase and/or lipase inhibitors. However, we found no effet of either or both of these inhibitors on bradykinin-stimulated aumulation of inositol phosphates. Furthermore, when histamine was used to stimulate these ells, there was no effet on the DG lipase inhibitor. These results demonstrate that protein kinase C ativation by phorbol esters an inhibit the reeptor-stimulated synthesis of inositol phosphates in hromaffin ells. However, the results provide no support for the suggestion that diaylglyerol produed as a result of reeptor stimulation in hromaffin ells ats to down-regulate the inositol phospholipid reeptor effetor system. This may help to explain why in hromaffin ells the agonist stimulation of inositol phosphate prodution is linear for 45 min (Plevin & Boarder, 1988), while other systems whih have this feedbak loop desensitize rapidly (e.g. Helper et al., 1988). Referenes AIYAR, N., NAMBI, P., WHITMAN, M., STASSEN, F.L. & CROOKE, S.T. (1986). Phorbol ester mediated inhibition of vasopressin and /1- adrenergi responses in a vasular smooth musle ell line. Mol. Pharmaol., 13, ALBERT, P.R., WOLFSON, G. & TASHJIAN, A.H. (1987). Diaylglyerol inreases ytosoli free alium onentrations in rat pituitary ells. J. Biol. Chem., 262, BOARDER, M.R., PLEVIN, R. & MARRIOTT, D.B. (1988). 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