Staurosporine inhibits inositol phosphate formation in bovine adrenal medullary cells

European Journal of Pharmacology

ELSEVIER

MolecularPharmacologySection290(1995)227-236

molecular p~har~m~

Staurosporine inhibits inositol phosphate formation in bovine adren

medullary cells Stephen J. Bunn *, Heather I. Saunders The Neuroscience Group, Facul(v of Medicine, The Unil'ersity of Newcastle, Callaghan, NSW 2308, Australia

Received 311November 1994; revised 14 March 1995;accepted 2 May 1995

Abstract

The effect of protein kinase C activators and inhibitors on histamine-stimulated phospholipase C in bovine adrenal medullary c been investigated. The protein kinasc C activators, phorbol 12,13-dibutyrate (PDB) or sn-l,2-dioctanoylglycerol (DOG), it histamine-stimulation of phospholipase C. This inhibition was prevented by the protein kinasc C-selective inhibitor Ro (3-{l-[3-(2-isothioureido) propyl]indol-3-yl}-4-(l-methylindol-3-yl)-3-pyrrolin-2,5-dione) but not the broad spectrum protein inhibitor staurosporine. Indeed staurosporine on its own inhibited both the histamine-stimulated resp~mse and, in permeabilize phospholipase C activated by Ca 2+. Staurosporine inhibition of phospholipase C is unlikely to be mediated via protein kina,, Ca 2 ~/calmodulin-dependent protein kinase because it was not reproduced by selective inhibition of these kinascs. Staurc treatment, however, reduced inositol phospholipid levels in stimulated cells. Thus staurosporine and Ro 31-8,220, two widely used kinase C inhibitors, have quite different effects on phospholipase C activation. Furthermore, staurosporine may cause this in through a reduction in the level of phospholipase C substrate. Kevwords: Histamine: Phospholipase C; Protein kinase C: Phorbol ester: Staurosporinc; Ro 31-8220; Adrenal chromaffin cell

1. I n t r o d u c t i o n Agonist-stimulated phospholipase C activity is well established as playing a key role in cellular signal-transduction. Following activation, phospholipase C mediates the hydrolysis of phosphatidylinositol 4,5-bisphosphate yielding inositol (l,4,5)trisphosphate (Ins(1,4,5)P3) and 1,2-diacylglycerol. These two intracellular second messenger molecules lead, in turn, to the mobilization of Ca 2+ from internal stores and the activation of protein kinase C, respectively (Berridge, 1993). While it is well established that this signalling pathway is present in a wide variety of cell-types and is activated by numerous receptor agonists, its precise role in any particular cell-type is often much less clear. For example, while it is evident that a range of agonists stimulate phospholipase C activity in the bovine adrenal medullary chromaffin cell the role of the resultant mobilization of intracellular Ca 2 ~ or enhanced protein kinase C activity is generally unclear. Thus the secretagogue histamine activates histamine H a receptors on these

• Corresponding author. Tel.: 61-49-21561; Fax: 61-49-216903.

cells to stimulate a profound increase in phospholi activity, with a rapid increase in lns(1,4,5)P3 levels elevation of intracellular Ca 2+ (Stauderman and 1990; Challiss et al., 1991; Boarder and Challiss, Bunn et al., 1994). Nevertheless, histamine-inducec tion of catecholamines was only transiently indepen extracellular Ca 2" (Bunn and Boyd, 1992). Simi] number of other studies have demonstrated that mq tion of intracellular Ca 2+ in the adrenal chromaf appears to be poorly coupled to the e x o c y t o t i c (Kim and Westhead, 1989; see, however, Augusti Neher, 1992). Protein kinase C (the other branch of the phosph C-generated signal), exists as a number of distinct isq which can be grouped into three families according mechanisms of activation. (Tanaka and Nishizuka, The so-called 'conventional' ( a , /3 n, /3 H, and 3,) 'novel' (6, e, 7/and 0) isozymes are Ca" '-depend Ca2+-independent forms of protein kinase C respe that require phospholipid and can be activated b,. and phorbol esters. In contrast, the mechanism of tion for the 'atypical' ( ~', A) protein kinase C isozyl currently unknown (Tanaka and Nishizuka, 1994.)

228

SJ. Bunn, lt.l. Saunder.s / European Journal of Pharmacology - Molecular Pharrnacoh~gq., Section 290 (1995) 227-236

adrenal medullary chromaffin cells express a representa rive of each of the protein kinase C families with protein kinase C o~, E and sr being detected by Western blot analysis (Pavlovic-Surjancev et al., 1993). There is some evidence that protein kinase C activation may have a role in facilitating secretion from the chromaffin cell although the isozymes involved have not yet been identified (Burgoyne et al., 1988; Knight et al., 1988). The data also indicate, however, that protein kinase C may also mediate a feedback inhibition of agonist-stimulated phospholipase (7 activity. Thus we and others have reported that protein kinase C-activating phorbol esters markedly inhibit the generation of [3H]inositol phosphates by histamine and agonists to other phospholipase C-coupled receptors in adrenal medullary cells prelabelled with [3H]inositol (Wan el al., 1989; Jones et al., 199[)). Such studies employing phorbol esters have an obvious limitation in that they provide a pharmacological and essentially chronic stimulation of protein kinase C which is quantitatively and perhaps qualitatively different from that provided by agonist generated diacylglycerol, In this current study therefore we have employed Ro 31-822(I, a cell-permeant protein kinase C-selective inhibitor, (Nixon et al., 1992) to determine firstly, whether this phorbol ester attenuation of phospholipid metabolism is indeed mediated by protein kinase C and secondly, whether this feedback pathway operates in response to endogenous (diacylglycerol-mediated) activation of protein kinase (7. In addition to activating protein kinase C the intracellular signalling cascade initiated by phospholipase C has the potential to stimulate other protein kinases including Ca-"/calmodulin-dependent protein kinase and cAMP-dependent protein kinasc. The possible modulatory influences that these kinases may exert on phospholipase C activity have been investigated using the broad-spectrum protein kinase inhibitor staurosporine, the protein kinase A-selective inhibitor, H89 ({N-[2-((3-(4-bromophenyl)-2propenyl)amino)ethyl]-5-isoquinoline suifonamide) (Chijiwa et al., 1990); the calmodulin antagonist W7 (IN(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide) (Hidaka et ai., 19811; and in permeabilized cells the Ca2'/calmodulin-dependent protein kinase II pseudosubstrate-inhibitor otCaZ+/caimodulin-dependent protein kinase 11273_302 (Malinow et al., 19891.

2. Materials and methods

2.1. Isolation and culture of bol'ine adrenal medullary cells Bovine adrenal medullary cells were isolated from bovine adrenal glands by collagenase/DNase digestion and the chromaffin cells purified by Percoll TM densitygradient centrifugation as described previously (Bunn and Boyd, 1992). Cells were plated onto rat-tail collagen coated

24-well tissue culture plates at a density of 0.5 × 10 per well (chromaffin cell number determined by i red staining; Livett, 1984) in Dulbecco's modified [ medium (DMEM) supplemented with 15 mM He mM L-glutamine, 5 mM glucose, 10% foetal calf 25 p.g/ml each of fluorodeoxyuridine and cytosine noside, 5 p.g/ml mycostatin, 50 /.tg/ml gentamyc I(X) p.g/ml each of penicillin G and streptomycil cultures were maintained at 37 ° in an incubator con' 5% ('O~ in humidified air.

2.2. Stimulation of phospholipase C actit'ity in intac After 3 to 8 days in culture the cells were loade [3H]inositol by removing the culture medium and ing it with 500 ~1 of medium M199 containing I% calf serum and 5 / x C i / m i [ 3 H ] i n o s i t o l ( 8 0 - 1 2 0 C i / i The cells were returned to the incubator for 48 h loading medium was then removed and each well two 5 min washes with ! ml of a buffer of the fol composition: (mM) NaCI 154, KCI 2.6, KzHPO ~ KH2PO 4 0.85 CaCI~ 2.2, MgSO 4 1.18, glucose 1~ raining 0.1% bovine serum albumin pt! 7.4 at 3~ experiments investigating the effect of various kinase inhibitors (staurosporine, W7 and H89) these were now introduced, at an appropriate concentrat 500 /xl of the same buffer for a 20 rain preincl period. In some experiments this initial preincubati~ followed by a second 10 min preincubation period in 500 /.tl of buffer, but now containing phorbol 12 butyrate (PDB), 4u-phorbol 12,13-didecanoate (40 or sn-i,2-dioctanoylglycerol (DOG) in the continue ence or absence of kinase inhibitor. Finally, the cell stimulated for 15 min with histamine (10 p,M) continued presence of the preincubation drugs in 501 the same buffer. The incubation was terminated moval of the stimulating buffer and the addition of. ice-cold 10% perchloric acid. After 1 h on ice t extract was collected into a plastic tube where the p ric acid was removed with i.5 ml of a 1:1 mix of tl tylamine and freon (Downes et al., 1986).

2.3. Stimulation of phospholipase C actici~, in pet lized cells Chromaffin cells, preloaded with [3H]inositoi, w~ meabilized using staphylococcus aureus o~-toxin scribed by Bader et al. (1986). Briefly, each well was washed twice for 5 min with 1 ml of Locke's followed by a 15 min preincubation with 1 ml of abilization buffer at 37 ° of the following com[ (mM) K~-glutamate 140, piperazine-N,N'-bis[2sulfonic acid 20, Mg2+-acetate 6, EGTA 0.5 an~ bovine serum albumin adjusted to pH 7.(I with 1 1~ The cells were then permeabilized by a 30 min inc with 200 p.I of the same buffer containing 5 mM A

S.J. Bunn, H.I. Saunders / European Journal of Pharmacology - Molecular Pharmacology. Section 290 (1995) 227-236

24 hemolytic units per ml a-toxin (hemolytic activity determined against 2.5% rabbits erythrocytes in 25 mM phosphate buffered saline). The permeabilized cells were then preincubated in 500 /xl of the same buffer containing the required concentration of a protein kinase inhibitor (staurosporine, W7, H89 or aCa2*/calmodulin-dependent protein kinase 11273_3o2) phospholipase C activity was then stimulated by replacing the a-toxin with 500 /zl of this preincubation buffer but now containing 5 mM ATP and a free Ca 2+ concentratiom routinely buffered to 470 nM with EGTA (5 mM EGTA and 2.0 mM Ca -~+)for 15min. This Ca 2+ concentration was calculated with the aid of a computer program (Equal, Biosoft) employing the equilibrium constants of Sillen and Martell (1971). The Ca 2+stimulation was terminated by the addition of 500 p,l 20% perchloric acid and the [~H]inositol phosphates extracted and analysed as described above. In some experiments chromaffin cells were rendered permeable with digitonin. The procedure used was essentially identical to that described above except the 30 min incubation with a-toxin was replaced with a 6 min incubation with 20 ,u,M digitonin,

2.4. Analysis of inositol phosphates The neutral aqueous phase of the above extract was diluted to 10 ml with water and applied to a 1 ml anion exchange column (Bio-Rad AG1-X8 resin 100-200 mesh formate form mixed 1:1 with water). After elution of the free [3H]inositol and [3H]glycerophosphoinositol with 10 ml of water and 60 mM sodium formate respectively the total [3H]inositol phosphates (mono, bis and trisphosphates) were eluted with 10 ml of 0.8 M ammonium formate in 0.1 M formic acid. The radioactive content of a sample of this fraction was determined by liquid scintillation counting (ASCII scintillant, Amersham). This method is based on that of Berridge et al. (1983) modified as described previously (Bunn et al., 1988).

chloroform/methanol (9:1)and 2 ml of water. Fol vigorous mixing and phase separation 2.7 ml of the phase was collected, neutralized, diluted to 10 m water and applied to an anion exchange column scribed above for inositol phosphate analysis. Free i was eluted from the columns with 10 ml of water ar the water soluble deacylation products glyceroph inositol, glycerophosphoinositol phosphate and g] phosphoinositol bisphosphate were eluted with 26 x mM ammonium formate containing 5 mM sodium t, rate, and 14 ml each of 300 mM and 1 M amrr formate in 0.1 M formic acid, respectively. The radi~ content of each fraction was determined by liquid sc tion counting of 5 ml aliquotes.

2.6. [SH]noradrenaline effiux studies [3H]noradrenaline effiux was assessed using a m( tion of the procedure described (Bunn and Boyd, Cultures were removed from the incubator and p bated twice for 10 min with 1 ml per well of a bt the following composition (mM) NaCI 144, K( MgSO 4 1.2, CaCI 2 2.2, Hepes 10 and glucose 10. serum albumin (0.1%) was added and the pH adju 7.4 at 37 °. The cultures were loaded with [3H]nor~ line (13-19 C i / m m o l ) by incubating with 500 /zl same buffer containing 0.3 mM ascorbic acid and 1/a [SH]noradrenaline. After a 60 min loading peri buffer was removed and the cultures given four washes with 1.0 mi of buffer. Cultures were then p bated for 20 min with staurosporine (100 nM) prior min stimulation with PDB (100 nM) in the co: presence of staurosporine. [3H]efflux was determi collecting the stimulation buffer and measuring rad: ity by liquid scintillation spectrometry (ASCII sci~ Amersham). Efflux was expressed in terms of initial cell [3H]content (Bunn and Boyd, 1992).

2. Z Analysis of data 2.5. Analysis of [~H] inositol phospholipids Inositol phospholipids were analysed by first scraping each well of cells up into 200 /zl of 1 M HCI followed by 200 /zl 1M HCI wash. The samples from two wells were pooled and the lipids extracted by vigorous mixing with 3.6 ml of chloroform: methanol (1:2). Phase separation was achieved by the addition of 1.2 ml each of chloroform and water. The chlorofrom extract was collected and the aqueous phase re-extracted with a further 1.2 ml of chloroform. The combined chloroform extracts were dried down under nitrogen before being re-dissolved in 1 ml of chloroform: methanol (1:4) and deacylated by mild alkaline hydrolysis. This was achieved by a 15 min incubation at 37 ° following the addition of 50 /.tl of 1.2 M NaOH (made up in a 1:1 mix of methanol and water). The deacylation was terminated by the addition of 2 ml of

Data is expressed as mean + S.E.M. for n detq tions on the specified number of different adrenal me cell preparations. Concentration-response data wel ysed by a one-way analysis of variance; where the gave a P < 0.05, comparisons between individual were made by the Bonferroni method. All other st: comparisons were made by unpaired Student's t-te

2.8. Drugs and materials (±)[7-3H]Noradrenaline (specific activity, 13 C i / m m o l ) and myo[3H]inositol (specific a c t i v i t y C i / m m o l ) were obtained from Amersham lnten (UK). Staphylococcus aureus a-toxin was from R Biochemicals International (MA, USA). Colla bovine serum albumin, antibiotics and antimitoti

S.J. Bunn, H.I. Saunders / European Journal of Pharntacologv - Molecular Pharmacology Section 290 ( 19951 22 7-236

2311

tamine, digitonin, PDB, 4a-PDD, DOG and W7 were obtained from Sigma Chemical (St Louis, MO. USA). Ro 31-8220 (3-{1-[3-(2-isothioureido) propyl]indol-3-yl}-4-(lmethylindol-3-yl)-3-pyrrolin-2,5-dione) was a generous gift from Dr. G. Lawton, Roche Products (Welwyn Garden City UK), the full structure of Ro 31-8220 is given as compound 3 in Davis et al. (1989). Okadaic acid was provided by Dr A. Sim (University of Newcastle, Australia) and the Ca2t/calmodulin-dependent protein kinase pseudosubstrate-inhibitor ( a C a 2+/calmodulin-dependent protein k i n a s e 11273_3(2) was kindly provided by Dr John Rostas (also at this University). Staurosporine and H89 f r o m Calbiochem (Sydney, Australia). All other tissue culture reagents were from Cytosystems (NSW Australia) and Percoll TM was from Pharmacia (NSW, Australia). All reagents were of analytical grade. Incubation buffers containing PDB, a-PDD, DOG, staurosporine, H89 or Ro 31-822(I were prepared from stocks of the drugs in 10t)°~ dimethylsulphoxide; care being taken to ensure rapid mixing upon dilution into an aqueous buffer.

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3.1. Effect of protein kinase C actit'ators on phospholipase C acticity

Fig. 1. The effect of PDB and DOG on histamine-stimulated a~. tion of [3H]inositol phosphates. Adrenal medullary cells. Ioac [3H]inositol, were preincubatcd for 10 min with the indicated c~

Incubation of bovine adrenal medullary cells with his-

tion of phorbol 12,13-dibutyratc (1) or ~n-l,2-dioctanoylglycc and then stimulated for 15 min with 10 p_M histamine. Total [3lq

tamine (10 ~ M for 15 min) stimulated a significant increase in accumulation of [3H]inositol phosphates (245 + 15% of that generated following incubation with buffer alone). Prior exposure to the protein kinase C activator PDB for 10 min caused a concentration-dependent inhibition of this response (Fig. 1) with an ICs0 of approximately 5 nM. In contrast, the biologically inactive phorbol ester 4a-PDD was without effect at concentrations up to 100 nM (100 +_ 4% of the response to histamine alone). The synthetic diacylglyceride DOG also fully inhibited histamine-stimulated phospholipase C-activity, although with a lower potency than PDB (1Cs0 approximately 200 /xM; Fig. 1).

3.2. Effect of staurosporine and the specific protein kinase C inhibitor Ro 31-8220 on phospholipase C activity The inhibition produced by a maximally effective concentration of PDB (100 nM) was prevented by preincubating the cells with the protein kinase C-selective inhibitor Ro 31-8220 (10 /zM for 20 min; Fig. 2). In addition to preventing PDB-inhibition of the histamine response, preincubation with Ro 31-8220 (10 /xM) caused a small, but significant, enhancement of the response to histamine alone (Fig. 2). In contrast to Ro 31-8220 the broad-spectrum protein kinase inhibitor staurosporine (100 nM for 20 min) not only failed to prevent the PDB inhibition of histamine-stimulated phospholipase C-activity but also

phosphates were analysed as described in Materials and methods experiment the mean basal response was subtracted from all data the residual net stimulated response expressed as a percentag( generated in the presence of histamine alone (% maximum). Dat mean ±S.E.M. of n = 6-t,~ determinations from 3 separate cell tions.

produced a significant partial inhibition of its own ( 100 nM staurosporine reduced the response to 57 _+ that generated in the presence of histamine alont 0.001). The protein phosphatase i and 2A inhibitor < acid also markedly inhibited the histamine-stir phospholipase C activity. A 20 min preincubation /.tM okadaic acid reduced the response to 18 + 8% found in the presence of histamine alone ( P < 0.00 2). The failure of staurosporine to prevent PDB-in of histamine-stimulated phospholipase C was not d~ excessive concentration of PDB. As can be seen i~ the presence of staurosporine did not inhibit, and potentiated, PDB-inhibition at all sub-maximal col tions of PDB tested producing a downwards shif concentration-response curve. Similarly, these d~ firm that staurosporine, in the absence of PDB, pro concentration-dependent inhibition of histamine-sti phospholipase C activity. In a separate series of ments it was demonstrated that this inhibition was cally significant at concentrations as low as 1.(I n

SJ. Bunn, H.I. Saunders / European Journal of Pharmacology - Molecular Pharmacolo~' Section 290 (1995) 227-236

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nM for 20 min) did not significantly affect basal [ flux (1.43 + 0.2%) but significantly inhibited that e by PDB to 2.6 + 0.2% ( P < 0.1)01 compared to [3H] evoked by PDB in the absence of staurosporine). lady, it has been previously demonstrated that PDB : lates protein phosphorylation in bovine adrenal met: chromaffin cells preloaded with - P i (Pocotte et al., Pretreatment with staurosporinc inhibited this PI duced protein phosphorylation; most noticeably tha 63 kDa protein band previously identified as ty hydroxylase (Bunn et al., 1992 and unpublished ob tions).

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In addition to inhibiting histamine-stimulated ph~ lipase C-activity in intact cells staurosporine also inl Ca2+-stimulated phospholipase C activity in a-toxi meabilized cells. Incubating permeabilized cells w creasing concentrations of free Ca 2÷ stimulated a c~ tration-dependent increase in the accumulation of [ ositol phosphates (data not shown). A 15 min incu with 470 nM C a 2 . (an approximately ECso concent

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Fig. 2. The effect of protein kinasc and protein phosphatase inhibitors on histamine-stimulated accumulation of [3H]inositol phosplaates. Adrenal medullary cells, loaded with [3tl]inositol, were preincubated for 20 min with 10 ,uM Ro 31-8220 ( + Ro), 100 nM staurosporine ( + Stauro) or 5 /.tM okadaic acid ( + O A ) prior to a further 10 min incubation with (shaded bars) or without (open bars) 100 nM phorba)l 12,13-dibutyrate. (_'ells were then stimulated for 15 rain with 10 /.tM histamine and the

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total [3H]inositol phosphates were analysed as described in Materials and methods. In each experiment the mean basal response was subtracted

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from all data and then the residual net stimulated response expressed as a percentage of that generated in the presence of histamine alone (% maximum). Data are the mean + S.E.M. of n = 6 - 1 2 determinations from 3 separate cell preparations. Single asterisk, significantly reduced compared to that in the presence of histamine alone ( P < 0.001). Double asterisks, significantly increased from that in the presence of histamine a l o n e ( P < 0 . 0 1 ) . . _

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rosporine (86_+ 5% of the response in the presence of histamine alone; P < 0.01) but still amounted to only a partial inhibition at concentrations as high as 1 /zM staurosporine (48 + 3%; Fig. 4). In contrast to the PDB-mediated inhibition the inhibitory action of staurosporine was not prevented by prior exposure to the protein kinase C inhibitor Ro 31-8220. The [3H]inositol phosphate accumulation being 61 + 4% of the response to histamine alone, in the presence of staurosporine (100 nM) compared to 76 + 3% in the presence of both staurosporine (100 nM) and Ro 31-8220 (10 /,tM) (n = 6 from three separate experiments), While staurosporine was unable to prevent PDB-inhibition of histamine-activated phospholipase C it was able to prevent other PDB stimulated responses in these cells, Incubation with PDB (100 nM for 15 min) increased the rate of [3H]effiux from cells preloaded with [3H]noradrenaline; this rose from 1.9 + 0.2% cell content in 15 min in unstimulated cells to 3.7 + 0.2% in the presence of PDB ( P < 0.0001). Preincubation with staurosporine (100

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PDB cone. (nM) Fig. 3. The effect of staurosporine on the PDB-inhibition of hi stimulated accumulation of [3H]inositol phosphates. Adrenal n7 cells, loaded with [3H]inositol, were preincubated for 20 min indicated concentration of staurosporine 0 ([3), 0.1 nM (11), 1 t 10 nM ( • ) or 100 nM ( ~ ) prior to the addition of increasing c¢ tions of phorbol 12,13-dibutyrate for a further 10 rain. Cells w stimulated for 15 min with 10 /.tM histamine and the total [3E phosphates were analysed as described in Materials and methods experiment the mean basal response was subtracted from all data the residual net stimulated response expressed as a percentage generated in the presence of histamine alone (% maximum). Dat mean+S.E.M, of n = 6-12 determination from 3-4 separate ce rations.

SJ. Bunn. H.I. Saunder,s / European Journal o[ Pharmacology - Molecular Pharmacology Section 290 (1995) 227-230

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kinase A and C a Z ' / c a l m o d u l i n - d e p e n d e n t protein II-selective inhibition on histamine and CaZ~-stim phospholipase C activity in these cells. H89, a I: kinase A selective inhibitor (Chijiwa et al., 1990), d inhibit either response. In the presence of 30 /xM 1-t~ highest concentration tested, the accumulation of [ ositol phosphates to 15 min stimulation with histami /.tM) or Ca -z- (470 nM) was 92 + 5% and 113 + 7% respective control responses. (n = 9 from three sc experiments). High concentrations of calmodulin antagonist W7 ited phospholipase C activity stimulated by 470 nM (100 /zM W7 reducing the response to 51 + 5% ( p occurring in the presence of Ca 2' alone < 0.001) lower concentrations of W7 were without effect from two separate experiments). To further investig; possible involvement of C a " ' / c a l m o d u l i n - d e p e n d e ] tein kinase I1 in the possible regulation of phospht

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Staurosporine cone. (nM) Fig. 4. The effect of staurosporine on histamine or CaZ'-stimulated [3H]inositol phosphates accumulation. Intact ( I ) or o~-toxin permeabilized ( [ ] ) adrenal medullary cells loaded with [3H]inositol, were preincubated for 20 min with increasing concentrations of staurosporine. Cells were then stimulated for 15 min with histamine (11,10 /.t,M) or, in the case of permeabilized cells, by increasing the concentration of free Ca"" (~.470 nM). The stimulation was terminated and total [3H]inositol phosphates extracted and analysed as described in Materials and methods. In each experiment the mean basal response was subtracted from all data and then the residual net stimulated response expressed as a percentage of that generated in the presence of stimulator alone (~, maximum). Data are the mean +_S.E.M. of n = 6 - 1 2 determination from 3-4 separate cell preparations.

resulted in a 388 + 2 3 % increase in total [3H]inositol phosphate accumulation compared to ceils incubated with a Ca-" "-free buffer for the same period of time (n = 9 from three separate experiments). Staurosporine caused a concentration-dependent inhibition of this Ca2+-stimulated phospholipase C-activity with statistically significant inhibition of the response to 470 nM Ca 2' occurring at staurosporine concentration of 10 nM or above (Fig. 4). Interestingly, in contrast to histamine stimulated phospholipase C response the Ca" +-stimulated response was only inhibited by the highest concentration of PDB tested. Thus while 10 nM PDB was without effect 100 nM PDB reduced the response to 61 -t- 3% of that generated in the presence of 470 nM Ca 2+ alone (n = 9 from 3 separate experiments), 3.3. Effect o f protein kinase A and Ca 2 +/calmodulindependent protein kinase H inhibition on phospholipase C activity In addition to the broad-spectrum protein kinase inhibitor staurosporine we examined the effect of protein

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Fig. 5. The effect of staurosporine on inositol phospholipid histamine stimulated adrenal medullary cells. Adrenal medull preloaded with [~H]inositol were preincubated for 20 min in the or absence of staurosporine (100 nM) and then stimulated for 15 or without histamine (10 /,tM). The inositol phospholipids were and deacylated as described in Materials and methods and the glycerophosphoinositols separated into glycerophophoinosit~ glycerophosphoinositol phosphate (GPIP) and glycerophosphoinc phosphate (GPIP z) fractions by simple anion exchange chroma Data are the mean+ S.E.M. of 9 determinations from three experiments and is presented as a percentage of the relevant glyl phoinositol level recovered from cells stimulated with buffer ak absence of staurosporine. Basal (open columns), basal in the pr staurosporine (shaded columns), histamine-stimulated (eros columns) and histamine in the presence of staurosporine (filled Asterisk, P <0.01 compared to the levels recovered from ce]

lated with histamine alone.

S.J. Bunn. ILl. Saunders / European Journal of Pharmacology - Molecular Pharmacology Section 290 (1995) 227-236

C-activity the effect of the pseudosubstrate inhibitory peptide (o~Ca2"/calmodulin-dependent protein kinase 11273_302, Malinow et al., 1989) was examined in cells rendered permeable to large molecules with digitonin. In digitonin permeabilized ceils a 15 min incubation with 4 7 ( 1 nM Ca 2+ increased the level of [3H]inositol phosphates to 293 + 14% of that generated in a Ca2'-free buffer, o~Ca2 ÷/caimodulin-dependent protein kinase 11273_302 (51) p,M) caused a small reduction in this response (81 ± 5% of that occurring in the presence of 470 nM Ca 2+ alone, P < 0.02, n = 8 from two separate experiments),

3.4. Effect of staurosporine on inositol phospholipid lel,eL~ In addition to inhibiting the generation of [3H]inositol phosphates staurosporine was found to reduce the level of inositol phospholipids (as measured from analysis of the dcacylation products) in cells stimulated with histamine, Following a 15 min incubation the levels of glycerophosphoinositol, glycerophosphoinositol phosphate and glycerophosphoinositol bisphosphate (the deacylation products of phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, respectively) recovered from cells stimulated with histamine (10 /.tM were not significantly different from those of cells incubated with buffer alone (Fig. 5). Staurosporine (100 nM) did not reduce the level of glycerophosphoinositol, glycerophosphoinositol phosphate or glycerophosphoinositol bisphosphate in unstimulated cells but significantly reduced the level of all three deacylation products in cells stimulated with histamine (Fig. 5).

4. Discussion We have previously demonstrated that histamine stimulation of phospholipase C activity in these cells is mediated exclusively through the activation of histamine H~ receptors (Wan et al., 1989). The observation that the response to a near maximally effective concentration of histamine (10 t-tM) was markedly attenuated by pretreating the cells with protein kinase C-activating phorbol ester (PDB) is in agreement with previous reports (Wan et al., 1989; Jones et al., 1990; Boarder and Challiss, 1992). The demonstration that this inhibition also occurred after protein kinase C activation with the synthetic diacylglycerol DOG is important because in some systems phorbol esters may activate distinct protein kinase C isoforms or indeed have actions in addition to protein kinase C-stimulation (lssandou and Rozengurt, 1989; Strulovici et al., 1989; Goode and Hart, 1990; Zoukhri et al., 1993). The phorboi ester or DOG-mediated inhibitions reported here are consistent with a signal-transduction pathway in which protein kinase C mediates a negative feedback onto receptor-

stimulated phospholipase C activity, as proposed in other ceil-types (Watson and Lapetina, 1985; Brown 1987; Vicentini et al., 1985). In rat basophilic RB cells this phorboi ester induced inhibition is me exclusively by the o~ and e protein kinase C isc (Ozawa et al., 1993). While the isozyme(s) responsi this inhibition in the chromaffin cell has not been fied it should be noted, however, that both the a protein kinase C isozymes are present in these (Paviovic-Surjancev et al., 1993). The observatic inhibition of protein phosphatase activity with okada also attenuates histamine stimulated phospholipase tivity supports the proposal that this inhibition in protein kinase mediated phosphorylation. PDB or DOG inhibition of phospholipase C provides evidence that the pathway for protein ki~ feedback inhibition exists in adrenal medullary c does not, however, indicate whether feedback occt lowing endogenous protein kinase C activation diacylglycerol generated in response to receptor s tion of phospholipasc C. The operation of such e nous feedback has been demonstrated in a num tissues (King and Rittenhouse, 1989, Bazan et al., Iriuchijima and Mori, 1990) where the inclusiol protein kinase C inhibitor enhanced receptor-stir phospholipase C activity. In bovine adrenal medulla: the selective protein kinase C inhibitor Ro 31-82~ vented the PDB-inhibition of the histamine-stimul~ sponse and also caused a small but significant incr phospholipase C activity in response to histamin~ (Fig. 2). This latter observation indicates that duri tamine stimulation of these cells protein kinase C fe inhibition does indeed occur, although probably to more limited extent than in some other cell-types ([ and Challiss, 1992). A surprising observation from the current stuq that the effects of Ro 31-8220 were not reproduced broad-spectrum protein kinase inhibitor stauro,, Staurosporine not only failed to prevent PDB-inhib histamine-stimulated phospholipase C activity but trast to Ro 31-8220 it was a potent inhibitor histamine response alone (Fig. 2 and Fig. 3). The fa staurosporine to prevent PDB inhibition of the hi~ response is unlikely to be due to an inactivity protein kinase C in these cells, since staurosporine other PDB stimulated events such as protein phosl: tion and [3H]release from cells preloaded with [ adrenaline (see Results). Similarly, it is unlikely 1 contrasting effects of Ro 31-8220 and staurosporine attributed to differential activities towards different kinase C isozymes. While Ro 31-8220 is slightl effective against the 'conventional' than 'novel' kinase C isozymes (IC5o values of 5 nM and against ot and e, respectively) compared to staurc (IC50 values 28 nM and 25 nM against o~ and ~, tively) this is unlikely to be a significant factor

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concentrations of these drugs used in this current study (Wilkinson et al., 1993). Thus the contrast between the actions of Ro 31-8220 and staurosporine probably result from staurosporine having an action in addition to inhibiting protein kinase C. Histaminergic activation of phospholipase C, when measured at prolonged times such as the 15 min used here, is almost completely dependent on the presence and thus presumed influx of extracellular ('a -~' (Bunn et al., 1995). Thus staurosporine could potentially inhibit the histaminergic phospholipase C response by interfering with Ca 2 '-influx across the cell membrane. This would seem an unlikely explanation; however, because staurosporine also inhibited phospholipase C activity stimulated by directly increasing the Ca 2" concentration in or-toxin pcrmeabilizcd cells (thus by-passing the influx mechanisms). Staurosporinc being a potent broad-spectrum protein kinasc inhibitor (Ruegg and Burgess, 1989) would be expected to inhibit protein kinases in addition to protein kinase C. The possible involvement of protein kinase A or Ca-~+/calmodulin-dcpendent protein kinase I1 in the staurosporine-mediatcd attentuation of phospholipase C has been determined. The lack of effect of H89 indicates that protein kinase A is unlikely to have mediated this action, Similarly, these data do not strongly support a role for Ca2"/calmodulin-dependent protein kinase 11 bccausc while the calmodulin antagonist W7 did inhibit Ca-"'stimulated phospholipase C activity the concentration required was markedly higher than that reported to inhibit Ca2+/calmodulin-dependent protein kinase I1 in other systerns (Leblanc et al., 1992, Saville and Houslay, 1994). Furthermore the Ca-'~/calmodulin-dependent protein kinase II pseudosubstrate inhibitor, o~Ca-"~/calmodulin-dependent protein kinase IIz73 ~.z- caused only a very small reduction in the Ca -~'--stimulated phospholipase C response in digitonin permeabilized cells. In parallel experiments we have found that the same concentration of otCaZ*/calmodulin-dependent protein kinase 11273_302(50 /zM) almost fully inhibited Ca-~'/calmodulin stimulated tyrosine hydroxylase phosphorylation in digitonin permeabilized chromaffin cells (unpublished data), an event probably mediated by Ca-"/calmodulin-dependent protein kinase II (Bunn et al., 1994, tlaycock, 1993). In addition to serine-directed protein kinases staurosporine has been documented to inhibit tyrosine kinase activity in some cells (Augustine et al., 1991; Yamada et al., 1992). While this is of potential interest because phospholipase (?y is activated through tyrosine phosphorylation (Berridge 1993) there is currently no evidence that stimulation of G-protein coupled receptors, such as the histamine H~ histaminergic receptor examined here, enhances tyrosine kinase activity in the adrenal medullary cells. Thus while the involvement of a tyrosine kinase can not be excluded the availablc data do not provide strong support for the suggestion that staurosporinc inhibition of phospholipase C activity is the result of a staurosporinc

action against a protein kinase other than, or in addit protein kinasc C. Staurosporinc could cause an apparent inhibiti phospholipase C activity by reducing the level phospholipasc C substrate phosphatidylinositol 4 phosphate. This was evaluated by determining the Ic' glycerophosphoinositoi, glycerophosphoinositol ph~ and glycerophosphoinositol bisphosphate (the deac'. products of phosphatidyinositol, phosphatidyinosi phosphatc and phosphatidylinositol 4,5-bisphosphal tracted from cells stimulated in the presence or abse staurosporinc (100 nM). Firstly, it should bc nott after 15 min stimulation with a near maximally cf concentration of histamine (10 /xM) the levels of g] phosphoinositol, glyccrophosphoinositol phosphat glycerophosphoinositol bisphosphate were not signif altered from those in unstimulatcd cells (Fig. 5). Tt activities of the enzymes involved in inositol phosp biosynthesis were sufficient to maintain the sup phosphatidylinositol 4,5-bisphosphate even under cant and maintained phospholipasc C activity. In th cncc of staurosporine, however, histamine stimulat suited in a significant reduction in thc level of al glycerophosphoinositols (Fig. 5). These data supp suggestion that staurosporine may inhibit phospholi activity by reducing the level of available substrate ever, the site at which this inhibition occurs is nf rcsolvcd. Inhibition of the phosphoinositidc kina.~ unlikely to bc thc primary mechanism involved Ithe level of glycerophosphoinositol as well as glyce~ phoinositol phosphate and glycerophosphoinositol b phate was significantly reduced. Interestingly, Sm Mooberry (1992) reported that staurosporinc did hibit phosphoinositide kinase. Whatever the precise mechanism involved the data indicate that the original observation, i.e. th~ rosporine was unable to reduce PDB-inhibition histamine-stimulated phospholipase C response, m~ from staurosporine-mediated inhibition of a step u 1 in the inositol phospholipid cycle of that inhibi protein kinase C. In summary these data demonstr~ activation of protein kinase C by PDB or DOG histamine stimulation of phospholipase C activity in adrenal medullary cells. While this inhibition can vented by pretreating the cells with the protein ki specific inhibitor Ro 31-8220 it is potentiated broad-spectrum kinase inhibitor staurosporine. This ently paradoxical action of staurosporine may be at1 to its ability to reduce the availability of the phospE C substrate phosphatidylinositol 4,5-bisphosphate. the precise cellular locus at which this action is n can not as yet be identified these data clearly dem, that two widely used protein kinase C inhibitor: rosporine and Ro 31-8221), have quite different ef! phospholipase C activity in a well characterised c turc system.

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Acknowledgements Dr G. Lawton of Roche Products Ltd. is acknowledged for his generous gift of Ro 31-8220. We arc grateful to Drs P e t e r Dunkley, David Powis and Michael Boarder for their help and advice in preparing the manuscript and to Leone Austin for preparing the cell cultures. This work was supported by a n N H and MRC (Australia) project grant to S J B w h o w a s a n R. Douglas Wright F e l l o w .

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