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Home , Carbachol, Muscarinic agonist, Nicotinic agonist, Nicotinic antagonist

The Journal of Neuroscience, February 1989, g(2): 507-512

Nicotinic and Muscarinic Stimulate Rapid Protein Kinase C Translocation in PC1 2 Cells

Robert 0. Messing, Anne M. Stevens, Elizabeth Kiyasu, and Alisa B. Sneade Department of Neurology and the Ernest Gallo Clinic and Research Center, University of California, San Francisco, California 94110

Phosphoinositide hydrolysis, a major mechanism for signal Protein kinase C (PKC) plays a key role in signal transduction transduction in neural cells, generates diacylglycerol, which by hormonesand neurotransmitters that activate phosphoino- can in turn activate protein kinase C (PKC). Although cho- sitide hydrolysis. Breakdown of phosphatidylinositol bisphos- linergic agonists elicit phosphoinositide hydrolysis in neural phate (PIP,) generatesthe intracellular secondmessengers inosi- tissues, little is known about activation of PKC by tol trisphosphate, which mobilizes calcium from intracellular agonists. PKC requires phosphatidylserine for activation, and stores (Berridge and Irvine, 1984), and diacylglycerol, which in intact cells this lipid requirement is satisfied by binding activates PKC by increasing the affinity of the for cal- of the enzyme to cell membranes. Therefore, in intact cells, cium and phospholipid (Nishizuka, 1984). PKC requiresphos- activation of PKC is often associated with a decrease in phatidylserine for activation, and in intact cells this lipid re- cytosolic PKC activity accompanied by an increase in mem- quirement is satisfied by association of the enzyme with cell brane-associated activity. We studied cholinergic-induced membranesduring activation (Kishimoto et al., 1980). Thus, activation of PKC by examining changes in the subcellular tumor-promoting phorbol esters,which potently stimulate the distribution of the enzyme in PC1 2 cells treated with cholin- enzyme by substituting for diacylglycerol (Nishizuka, 1984), ergic . (1 mu) induced large and rapid in- cause a dramatic decreasein cytosolic PKC activity accom- creases in membrane-associated PKC activity; a maximal panied by a large increasein membrane-bound activity (Kraft increase of 460% occurred after 5 set of incubation. Car- and Anderson, 1983). Translocation of PKC activity from cy- bachol-induced PKC translocation was concentration-de- tosolic to membrane fractions of intact cells has also been ob- pendent, with a biphasic dose-response curve yielding ap- served following -mediated PIP, breakdown in some proximate EC,, values of 1 O-6 M and 1 O-4 M for the high- and tissues(Liles et al., 1986; Niedel and Blackshear,1986; TerBush low-affinity components, respectively. Experiments with se- and Holz, 1986) but receptor-mediatedtranslocation has been lective cholinergic agents demonstrated that both muscarin- more difficult to demonstratethan phorbol ester-induced trans- ic and nicotinic receptors are involved in carbachol-induced location (Niedel and Blackshear, 1986). PKC translocation, but the response is predominantly me- Muscarinic cholinergic receptorsare coupled to PIP, hydroly- diated by nicotinic receptor stimulation. Muscarinic-induced sisin brain and in several neural cell lines (Fisher and Agranoff, association of PKC with cell membrane fractions was resis- 1987). Certain neural responsesto muscarinic stimulation ap- tant to extraction by chelators, whereas nicotinic-mediated pear to be mediated by PKC since they can be reproduced by membrane binding was partially reduced by homogenization treatment with phorbol esters.Such responsesinclude inhibition of cells in the presence of EGTA. Omission of calcium from of calcium-activated potassiumcurrents in hippocampus(Ma- the incubation medium or chelation of calcium with EGTA lenka et al., 1986),blockade of the inhibitory effectsof completely blocked muscarinic- and nicotinic-induced trans- and GABA, receptor stimulation in hippocampus (Worley et location. In addition, the calcium al., 1987) and down-regulation of muscarinic receptorsin the reduced the nicotinic response by 60%. These results in- neuroblastomacell line N 1E- 115 (Liles et al., 1986). In N 1E- dicate that carbachol-induced PKC translocation in PC12 l 15, muscarinic stimulation is alsoassociated with translocation cells is due mainly to activation of nicotinic receptors and of PKC activity to the cell membrane (Liles et al., 1986). Al- is modulated by calcium influx through voltage-dependent though nicotinic agonistsstimulate phosphoinositidehydrolysis calcium channels. These studies also suggest that stimu- and PKC associationwith cell membranesin adrenal medullary lation of either nicotinic or muscarinic receptors generates cells (TerBush and Holz, 1986; Eberhard and Holz, 1987) and diacylglycerol, which produces chelator-resistant binding of chick embryo myotubes (Adam0 et al., 1985; Eusebi et al., PKC to cell membranes. 1987) nicotinic-mediated phosphoinositide turnover and PKC activation have not been clearly demonstratedin neural cells. PC 12 is a cell line that allows the study of both nicotinic and Received Sept. 28, 1987; revised June 17, 1988; accepted July 12, 1988. muscarinic modulation of PKC. PC 12 cells expressmuscarinic This work was SUDDOIted bv Clinical Investieator Develomnent Award NSO 115 1 and a grant from the Alcoholic Beverage Medical Research Foundation (to R.O.M.). receptorscoupled to PIP, hydrolysis (Vicentini et al., 1986) and We wish to thank Drs. David A. Greenberg, Ivan Diamond, and Daria Mochley- nicotinic receptors that resembleneuronal nicotinic receptors Rosen for their many helpful suggestions and for reviewing the manuscript. Correspondence should be addressed to Robert 0. Messing, M.D., Building 1, both in their resistanceto cr- blockade(Patrick and Room 101, San Francisco General Hospital, San Francisco, CA 94110. Stallcup, 1977) and in their cross-reactivity with monoclonal Copyright 0 1989 Society for Neuroscience 0270-6474/89/020507-06$02.00/O antibodies to brain nicotinic receptors (Whiting et al., 1987). 508 Messing et al. * Cholinergic-Induced Protein Kinase C Translocation in PC12

PC12 cells treated with nerve growth factor (NGF) develop 400 ,’ morphologic and biochemical characteristics of mature sym- s pathetic neurons (Greene and Tischler, 1976; Dichter et al., E 1977). NGF-differentiated PC12 cells express increased num- \ 350 - -5.5 0 bers of muscarinic receptors (Jumblatt and Tischler, 1982) and are more responsive to nicotinic stimuli (Amy and Bennett, z 300 - 1983). Phorbol esters evoke a variety of responses in PC12 cells, E including neurotransmitter release (Pozzan et al., 1984; Mat- -3 x 250 - thies et al., 1987), inhibition of voltage-dependent calcium up- f take (Di Virgilio et al., 1986; Harris et al., 1986; Messing et al., z 1986) and inhibition of muscarinic-induced phosphoinositide ki 200 - ( hydrolysis (Vicentini et al., 1985a). In addition, phorbol ester Y treatment causes PKC translocation to PC 12 cell membranes a (Matthies et al., 1987). In this report, we use NGF-differentiated 150 ’ I I I I I I PC 12 cells as a model neural system to study cholinergic mod- ulation of PKC. The results provide evidence for both nicotinic- and muscarinic-mediated activation of PKC in neural cells; they also indicate a role for voltage-sensitive calcium channels in mediating the nicotinic response.

Materials and Methods Muterids. DEAE cellulose (DE52) and P8 1 cellulose phosphate paper were purchased from Whatman. [T-~~P]ATP was from Amersham. Phosphatidylserine was purchased from Avanti Polar Lipids. Histone III-S, sn- 1,2 dioleoylglycerol (diC,, ,), carbachol, , leupeptin, phenylmethylsulfonyl fluoride (PMSF), 1,l -dimethyl-4-phenyl pipera- I- zinium iodide (DMPP), , , and nifedipine were obtained from Sigma. NGF (2.5 S) was a gift from Dr. William Mobley. Cell culture. PC12 cells were maintained at 37°C in a humidified I I I I I I ’ iP atmosphere of 90% air and 10% CO, in Dulbecco’s modified Eagle’s O 20 40 60 60 100 120 “300 medium (DMEM) containing 10% horse serum, 5% fetal calf serum, 50 Time (seconds) U/ml penicillin, 50 fig/ml streptomycin, and 2 mM glutamine. Cells were subcultured onto collagen-coated 100 mm plastic dishes (Falcon) Figure 1. Time course of changes in the subcellular distribution of in medium supplemented with 50 @ml of 2.5 S NGF at a plating PKC in PC12 cells stimulated with 1 mM carbachol. Data shown are density of 3 x lo6 cells per dish. Medium was changed 3 and 6 d after mean values + SEM from 3 experiments, expressed as enzyme activity subculturing, and experiments were performed on day 7. present in the cytosolic (0) or particulate (0) fraction of cells per mg of Subcellular fractionation. Cells were rinsed once in DMEM, prein- cell protein. cubated at 37°C for 5 min in 4 ml of DMEM, and incubated at 37°C for various times in 4 ml of fresh DMEM containing indicated drugs. Incubations were terminated by aspirating the medium, rinsing once ence and absence of phosphatidylserine, diC,, ,, and Ca2+. Enzyme ac- with 4 ml of ice-cold buffer containing 250 mM sucrose, 1 mM EDTA. tivity was expressed as picomoles of 12P incorporated per minute per 1 mM EGTA, and 20 mM Tris-HCl (pH 7.5), and scraping the cells from milligram of cell homogenate. Results are reported in these units or as

the dishes in 4 ml of 20 mM Tris-HCl (oH\- 7.5.4”C).I ,, 50 .-&ml leuoeotin.__ I the percent of total PKC activity present in the membrane fraction. and 1 mM PMSF. This process took approximately 25 set to complete. Protein was measured by the Bradford method (Bradford, 1976) with Thereafter, cells were kept on ice for up to 15 min during which time ovalbumin as the standard. Statistical comparisons between mean val- the subcellular distribution of PKC did not change. The samples were ues were made by Student’s t test, and differences were considered then homogenized on ice with 15 strokes of a tight-fitting teflon glass significant where p values i 0.05 were found. homogenizer, sucrose was added to a final concentration of 250 mM, and the cells were homogenized with another 5 strokes. After centrif- Results ugation at 100,000 x g for 60 min at 4”C, the supernatant (cytosol fraction) was applied to a 200 ~1 DE52 column (0.9 cm diameter) equil- PKC activity in unstimulated control PC 12 cells was 336 ? 17 ibrated with buffer A containing 20 mM Tris-HCl (pH 7.5 at 4°C) 1 pmol/min/mg in the cytosol fraction and 77 ? 6 pmol/min/mg mM EDTA. 1 mM EGTA. 12 mM 2-mercautoethanol. and 10% alvcerol. in the membrane fraction (N = 2 1). A similar predominanceof The column was washed with 2 ml of buffer A and eluted batchwise cytosolic PKC activity hasbeen noted in unstimulated cells from with 2.5 ml of buffer A containing 100 mM NaCl. All PKC activity was many other tissues(Nishizuka, 1984). As indicated in Figure 1, found in this fraction. The pellet (crude membrane fraction) was sol- ubilized for 1 hr at 4°C in 4 ml of buffer A containing 1O/o Triton X- 100 treatment of cells with 1 mM carbachol rapidly increasedmem- and centrifuged at 12,000 x g for 10 min to remove insoluble material brane-associatedPKC activity. The effect was maximal by 5 prior to DEAE chromatography, as described for the supematant. set, producing a 4.6-fold increasein membrane-associatedac- PKC assay. The reaction mixture (final volume 100 ~1) contained 50 tivity, and was accompanied by a corresponding decreasein ~1 of sample, 24 fig of phosphatidylserine, 0.8 pg of diC,, I, 1 mM CaCl,, 25 mM MgCl,, 75 fig of histone III-S, and 2 nmol of [+*P]ATP (2-4 cytosolic activity. Total (membrane-associatedplus cytosolic) x lo5 dpm/nmol). Lipids were prepared by sonication in 20 mM Tris- activity in carbachol-stimulated cells was 103 f 2% of total HCl (pH 7.5 at 4°C).The reactionwas started by addingMnCl,, histone, activity in control cells. Enzyme activity in the particulate frac- and [+*P]ATP at 30°C and was stopped after 3 min wiih 30 ~1 of a tion returned to levels equivalent to thosefound in unstimulated solution containina 0.2 M EDTA and 0.2 M ATP. Aliauots of 100 ul cells after 5 min of exposure. were spotted onto z.5 cm2 P-8 1 cellulose phosphate papers. Papers were washed in water (10 ml per paper) 4 x and rinsed once in 95% ethanol The dependenceof translocation on the concentration of car- prior to liquid scintillation counting. PKC activity was measured in bachol is shown in Figure 2. Translocation occurredover a wide triplicate samples as the difference between phosphorylation in the pres- concentration range and the dose-responsecurve demonstrated The Journal of Neuroscience, February 1969, 9(2) 509

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control corb !-llUSC DMPP corb corb atrp mew Figure 2. Stimulation of PKC translocation to PC12 membranes by Figure 3. Effect of cholinergic agonists and antagonists on PKC trans- incubation for 5 set with carbachol. Data shown are mean values k location. Cells were preincubated with or without antagonists (atropine SEM from 3-6 experiments, expressed as the percent of total cellular or mecamylamine) for 5 min and then incubated with indicated antag- PKC activity present in the particulate fraction. onists and agonists for 5 sec. Drugs were used at the following concen- trations:1 mM carbachol(curb), 1 mM muscarine(must), 100 PM DMPP, 100 nM atropine (atrp), and 10 PM mecamylamine (mecu). Data shown 2 phases,with apparent EC,, values of approximately 1O-6 M are mean values k SEM from 3-6 experiments, expressed as the percent for the high-affinity and 1O-4M for the low-affinity component. of total activity present in the particulate fraction. p < 0.00 1 (*) and p < 0.03 (**) relative to control; p < 0.001 (***) and p = 0.05 (****) The high-affinity phasecorresponds to the range of carbachol relative to carbachol. concentrations that produce muscarinic stimulation of phos- phoinositide hydrolysis in PC12 (Vicentini et al., 1986). The low-affinity phaseapproximates the concentrations of carbachol drugs (Pozzan et al., 1986). To determine whether influx of that stimulate nicotinic-mediated 22Na+(Patrick and Stallcup, extracellular calcium is important in cholinergic-induced PKC 1977) and 86Rb+(Robinson and McGee, 1985) uptake in PC 12 translocation, we incubated cells with DMPP or muscarine in cells. the presenceand absenceof calcium, EGTA, or the calcium Since carbachol is both a nicotinic and a muscarinic , channelantagonist, nifedipine. As shown in Figure 4 both mus- and since the concentration-responsecurve shown in Figure 2 carinic- and nicotinic-induced PKC translocation were com- was biphasic, we consideredwhether activation of both cholin- pletely inhibited in calcium-free buffer and in buffer containing ergic receptor subtypesmight be involved in carbachol-induced 3 mM EGTA. Nifedipine (1 MM), which completely blocks cal- translocation of PKC. As indicated in Figure 3, incubation of cium influx through dihydropyridine-sensitive, voltage-depen- cells with 1 mM carbachol for 5 set resulted in a shift of PKC dent calcium channelsin PC 12 cells (Toll, 1982) inhibited max- activity such that 52 f 2% of total activity was associatedwith imal DMPP-induced translocation by approximately 60%. In the particulate fraction. Muscarine stimulated PKC transloca- contrast, the responseto muscarine was unaffected by nifedi- tion maximally at 300 WM, but the responsewas only 24% of pine. Nifedipine alone had no effect on PKC translocation (data that observed with 1 mM carbachol. In contrast, a maximally not shown). effective concentration (100 KM) of the selective Although calcium is required to initiate binding of PKC to DMPP nearly reproduced the responseto carbachol. Atropine cell membranes,binding induced by calcium alone is reversible (100 nM), which completely inhibits muscarinic-stimulated and removed by extraction with calcium-chelating agents(Go- phosphoinositide breakdown in PC12 (Vicentini et al., 1986) palakrishna et al., 1986). Phorbol estersand, to a lesserextent, only slightly reduced the carbachol-stimulatedincrease in mem- diacylglycerol, stabilize membranebinding of the enzyme mak- brane PKC activity. In contrast, the meca- ing binding resistant to removal by chelators (Gopalakrishna et mylamine (1 PM) was far more effective. The predominance of al., 1986). To investigate whether cholinergic agonistscaused a nicotinic effect persisted at later time points as well, since at membrane binding of PKC that was chelator-stable(i.e., due to 2 min of incubation with 1 mM carbachol, 1 PM mecamylamine calcium and diacylglycerol) or chelator-extractable(i.e., due only inhibited PKC translocation by 8 1 f 5%, whereas100 nM atro- to elevated intracellular calcium), we compared cholinergic-in- pine inhibited translocation by only 45 f 8% (N = 3; p < 0.02 ducedPKC translocation in cells homogenizedwith and without for inhibition by mecamylamine relative to inhibition by atro- 1 mM EGTA. As shown in Table 1, muscarine-inducedtrans- pine). Neither atropine nor mecamylaminealone had any effect location was unchangedby homogenization in EGTA, whereas on PKC translocation (data not shown). DMPP-induced translocation was reduced 39% by this treat- PKC activation and binding to cell membranesare modulated ment. Since it was possible that 1 mM EGTA may not have by calcium (May et al., 1985; Wolf et al., 1985; Gopalakrishna chelated all of the calcium releasedduring cell lysis, we also et al., 1986). Nicotinic receptor stimulation depolarizes cells, homogenizedcells in 10 mM EGTA. Homogenization in 10 mM allowing for influx of extracellular calcium through voltage-de- EGTA reduced membrane-associatedPKC activity slightly fur- pendent channels (Stallcup, 1979). Muscarinic stimulation ac- ther in control (7 + 1%) and DMPP-treated cells (19 ? 2%) tivates a calcium influx in PC12 that is not dependent on de- but the difference between these 2 values remained highly sig- polarization and is resistantto organiccalcium channelantagonist nificant @ < 0.007; N = 3). Therefore, both muscarine and 510 Messing et al. * Cholinergic-Induced Protein Kinase C Translocation in PC12

230 Table 1. Membrane binding of PKC in cells homogenized with and without 1 mM EGTA x 210 c .$ Homogenization buffer u 0,190 No EGTA 1 mM EGTA “E DMPP” 41 +- 4 25 I 2 $2.170 a ‘E Muscarine 23 f 1 23 -t 3 None 14 i 1 12 +- 1 E> 150 the percent of total PKC FE Valuesare expressed as activity presentin the particulate n a 130 fraction. E “p < 0.02 for cells homogenizedin EGTA as compared to cells homogenized without EGTA. i 110

90 a bc d 0 b c d control DMPP muscorine observed with muscarine. The influx of calcium through dihy- dropyridine-insensitive calcium channels (Kongsamut and Mil- Figure 4. DMPP- and muscarine-stimulated PKC translocation in the ler, 1986) and through the nicotinic receptor (Stallcup, 1979) presence and absence of calcium, nifedipine, and EGTA. PC12 cells were preincubated for 5 min in buffer containing 120 mM NaCI, 5 mM may explain why nifedipine failed to completely inhibit DMPP- KCl, 1.4 mM CaC&, 0.8 mM MgSO,, 1 mM NaH,PO,, 5 mM glucose, induced translocation. In PC12 cells, muscarinic agonists ele- and 25 mM HEPES (pH 7.4). Cells in condition b were treated with 1 vate intracellular calcium by stimulating calcium influx through PM nifedipine during this period. Cells were then incubated for 5 set in receptor-operated channels and inducing redistribution of cal- the same buffer with agonist alone (a), agonist plus 1 PM nifedipine (b), agonist plus 3 mM EGTA (c), or agonist without calcium (4. Data shown cium from cytoplasmic stores to the cytosol (Pozzan et al., 1986). are mean values + SEM from 3-6 experiments and are expressed as However, the intracellular calcium concentration achieved with enzyme activity present in the particulate fraction per milligram of cell maximal muscarinic stimulation is only approximately one-half protein. Control values (in the absence of agonist) were equivalent in of that achieved by of PC 12 cells (Pozzan et al., calcium-free and calcium containing buffers. p < 0.00 I (*) and p < 0.03 1986). Therefore, nicotinic agonists, by depolarizing cells and (**) relative to control; p < 0.01 (***) relative to DMPP. stimulating calcium influx through voltage-dependent channels, may produce larger increases in intracellular calcium than do muscarinic agonists. Since the association of PKC with mem- DMPP induced chelator-stable association of PKC with PC 12 branes varies with calcium concentration (Akers and Routten- membranes. berg, 1987), the higher concentrations of intracellular calcium achieved with nicotinic receptor stimulation may account for Discussion the greater extent of PKC translocation observed in cells treated Our findings demonstrate that cholinergic stimuli induce rapid with DMPP. and marked increases in PKC activity in membranes of NGF- Although calcium is required for PKC binding to membranes, differentiated PC 12 cells. The effect was evident by 1 set, sug- binding induced by calcium alone is reversible and is removed gesting a role for PKC in early neural responses to cholinergic by calcium chelators (Gopalakrishna et al., 1986). In contrast, signals. We found that PKC translocation occurred via both diacylglycerol, produced by phosphoinositide hydrolysis, sta- muscarinic and nicotinic receptor stimulation. In PC12, nico- bilizes membrane binding of PKC so that binding becomes tinic stimuli induce the release of ACh (Greene and Rein, 1977a), resistant to calcium-chelating agents (Gopalakrishna et al., 1986). but nicotinic-mediated PKC translocation cannot be attributed We found that muscarine-induced translocation of PKC was to ACh release and subsequent muscarinic receptor stimulation, entirely chelator resistant, suggesting that it is mediated by both since a maximally effective concentration of muscarine pro- diacylglycerol and calcium. In contrast, DMPP-induced trans- duced a smaller effect than carbachol and the response to car- location was partially reduced by homogenization in EGTA, bachol was reduced only slightly by atropine. Nicotinic stimuli suggesting that nicotinic-mediated binding of the enzyme to also cause the release of other neurotransmitters, such as cate- membranes involves two components, one mediated by calcium cholamines (Greene and Rein, 1977b), from PC12 cells. How- alone and the other by calcium and diacylglycerol. ever, phosphoinositide breakdown and PKC activation have The finding of chelator-stable binding suggests that nicotinic- not been reported in PC 12 cells exposed to noncholinergic neu- receptor stimulation and the consequent rise in intracellular rotransmitters produced by this cell line. Therefore, it is likely calcium may cause phosphoinositide hydrolysis and diacyl- that PKC translocation was a direct result of nicotinic receptor glycerol formation in PC1 2 cells. Nicotinic-induced phosphoi- activation and was not mediated by released neurotransmitters. nositide hydrolysis has been demonstrated in adrenal chro- Although both nicotinic and muscarinic agonists caused PKC maffin cells, where it is dependent on influx of extracellular translocation, we found the response to nicotinic stimuli to be calcium through voltage-dependent calcium channels (Eberhard much greater. The association of PKC with cell membranes and Holz, 1987). Therefore, in PC12 cells, phosphoinositide requires calcium, and increases in calcium enhance membrane hydrolysis stimulated by depolarization-dependent calcium in- binding of the enzyme (Wolf et al., 1985; Gopalakrishna et al., flux could be responsible for chelator-resistant association of 1986, Akers and Routtenberg, 1987). Calcium influx through PKC with membranes in cells treated with DMPP. dihydropyridine-sensitive calcium channels accounted for much PKC tightly bound to PC12 membranes in the presence of of the difference between nicotinic and muscarinic responses, both calcium and diacylglycerol is likely to be active. The func- since nifedipine inhibited DMPP-induced translocation to levels tional state of PKC loosely bound to membranes by calcium The Journal of Neuroscience, February 1989, 9(2) 511 alone may vary in different tissues. Elevated intracellular cal- References cium may either directly activate PKC (Akers and Routtenberg, Adamo, S., B. M. Zani, C. Nervi, M. I. Senni, M. Molinaro, and F. 1987) or may serve to recruit PKC to the cell membrane, thereby Eusebi (1985) Acetycholine stimulates phosphatidylinositol turn- priming it for subsequent activation by diacylglycerol (May et over at nicotinic receptors of cultured myotubes. FEBS Lett. 190: al., 1985; Wolf et al., 1985). Since the majority of PKC asso- 161-164. Akers, R. F., and A. Routtenberg (1987) Calcium-promoted trans- ciated with membranes after nicotinic stimulation was chelator location of protein kinase C to synaptic membranes: Relation to the sensitive, it is likely that calcium-mediated binding of PKC phosphorylation of an endogenous substrate (protein Fl) involved in plays an important role in normal signal transduction. synaptic plasticity. J. Neurosci. 7: 3976-3983. When we incubated cells in the absence of extracellular cal- Amy, C. M., and E. L. Bennett (1983) Increased sodium ion conduc- cium or in the presence of 3 mM EGTA, muscarine and DMPP t&ce through nicotinic receptor channels in PC12 cells exposed to nerve growth factors. J. Neurosci. 3: 1547-l 553. were unable to stimulate PKC transloction. It is unlikely that Beridge, M. J., and R. F. Irvine (1984) Inositol trisphosphate, a novel this was due to inhibition of drug binding, since ligand binding second messenger in cellular signal transduction. Nature 312: 3 15- to cholinergic receptors can be measured in the absence of cal- 321. cium (Jumblatt and Tischler, 1982; Whiting and Lindstrom, Bradford, M. M. (1976) A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the principle of 1986). Prevention of extracellular calcium influx could explain protein-dye binding. Anal. Biochem. 72: 248-254. the failure of DMPP to induce PKC translocation in cells in- Dichter, M. A., A. S. Tischler, and L. A. Greene (1977) Nerve growth cubated in calcium-free buffer. Incubation of PC 12 cells in cal- factor-induced increase in electrical excitability and acetylcholine sen- cium-free buffer also inhibits muscarinic-stimulated calcium in- sitivity of a rat pheochromocytoma cell line. Nature 268: 50 l-504. Di Virgilio, F., T. Pozzan, C. B. Wollheim, L. M. Vicentini, and J. flux and can reduce muscarinic-induced PIP, hydrolysis and Meldolesi (1986) Tumor promoter phorbol myristate acetate inhib- intracellular calcium mobilization (Vicentini et al., 1985b; Rabe its Ca*+ influx through voltage-gated Ca2+ channels in two secretory et al., 1987). A similar sensitivity of receptor-mediated phos- cell lines, PC12 and RINmSF. J. Biol. Chem. 261: 32-35. phoinositide hydrolysis to external calcium concentration has Eberhard, D. A., and R. W. Holz (1987) Cholinergic stimulation of been noted in other neural tissues (Fisher and Agranoff, 1987). inositol phosphate formation in bovine adrenal chromaffin cells: Dis- tinct nicotinic and muscarinic mechanisms. J. Neurochem. 49: 1634- Muscarinic stimulation of PC1 2 cells incubated in calcium-free 1643. buffer for 1 to 4 min increases intracellular calcium to only 1OO- Eusebi, F., F. Grassi, C. Nervi, C. Caporale, S. Adamo, B. M. Zani, and 140 nM (Vicentini et al., 1985b, Rabe et al., 1987). Calcium at M. Molinaro (1987) Acetylcholine may regulate its own nicotinic these concentrations does not enhance the association of receptor-channel through the C-kinase system. Proc. R. Sot. Lond. 230: 355-365. PKC with rat brain synaptic membranes (Akers and Routten- Fisher, S. K., and B. W. Agranoff (1987) Receptor activation and berg, 1987) or erythrocyte vesicles (Wolf et al., 1985) in vitro. inositol lipid hydrolysis in neural tissues. J. Neurochem. 48: 999- In our experiments, cells were exposed to calcium-free buffer 1017. for only 5 sec. Although this could block calcium influx, it is Gopalakrishna, R., S. H. Barsky, T. P. Thomas, and W. B. Anderson not known whether exposure for 5 set is sufficient to reduce (1986) Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. muscarine-stimulated phosphoinositide hydrolysis. Neverthe- J. Biol. Chem. 261: 16438-16445. less, we speculate that incubation of PC 12 cells in calcium-free Greenberg, M. E., and E. B. Ziff (1984) Stimulation of 3T3 cells induces buffer inhibited extracellular calcium influx and may have re- transcription of the c-fos proto-oncogene. Nature 311: 433-438. duced phosphoinositide hydrolysis, thereby preventing free in- Greenberg, M. E., E. B. Ziff, and L. A. Greene (1986) Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Sci- tracellular calcium from reaching concentrations sufficient to ence 234: 80-83. initiate PKC translocation in response to muscarine. Further Greene, L. A., and G. Rein (1977a) Synthesis, storage and release of work is needed to determine the influence of calcium-free buffer acetylcholine by a noradrenergic pheochromocytoma cell line. Nature on muscarine-induced phosphoinositide hydrolysis after brief 268: 349-35 1. (e.g., 5 set) periods of exposure. Greene, L. A., and G. Rein (1977b) Release of [)H] from a clonal line of pheochromocytoma cells (PC12) by nicotinic The finding of cholinergic-induced PKC translocation sug- cholinergic stimulation. Brain Res. 138: 521-528. gests that PKC is involved in rapid responses of PC 12 cells to Greene, L. A., and A. S. Tischler (1976) Establishment of a norad- cholinergic stimuli. Such responses include neurotransmitter re- renergic clonal line of rat adrenal pheochromocytoma cells which lease (Greene and Rein, 1977b; Rabe et al., 1987) and c-fos respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73: 2424- 2428. proto-oncogene transcription (Greenberg et al., 1986), both of Harris, K. M., S. Kongsamut, and R. J. Miller (1986) Protein kinase which can be induced by treatment with phorbol esters (Green- C mediated regulation of calcium channels in PC-12 pheochromo- berg and Ziff, 1984; Pozzan et al., 1984). We (Messing et al., cytoma cells. Biochem. Biophys. Res. Commun. 134: 1298-1305. 1986) and others (Di Virgilio et al., 1986; Harris et al., 1986) Jumblatt, J. E., and A. S. Tischler (1982) Regulation of muscarinic have found that phorbol esters reduce voltage-dependent cal- liaand binding sites bv nerve growth factor in PC12 phaeochromo- cqtoma cells. Nature i97: 152-l 54. cium influx in PC 12, suggesting that activation of PKC inhibits Kishimoto, A., Y. Takai, T. Mori, U. Kikkawa, and Y. Nishizuka (1980) the function of voltage-dependent calcium channels in this cell Activation of calcium and phospholipid-dependent protein kinase by line. Rane and Dunlap (1986) reported similar findings in dorsal diacylglycerol, its possible relation to phosphatidylinositol turnover. root ganglion cells treated with the PKC activator 1,2-oleoyl- J. Biol. Chem. 2.55: 2273-2276. Kongsamut, S., and R. J. Miller (1986) Nerve growth factor modulates acetylglycerol. These studies, together with our present findings, the drug sensitivity of neurotransmitter release from PC 12 cells. Proc. suggest that ACh may modulate the function of voltage-depen- Natl. Acad. Sci. USA 83: 2243-2247. dent calcium channels via PKC. The stimulatory effects of nico- Kraft, A. S., and W. B. Anderson (1983) Phorbol esters increase the tinic agonists on voltage-dependent calcium channels and PKC amount of Ca2+. phospholipid-dependent protein kinase associated suggest a mechanism whereby PKC could be involved in a neg- with plasma membrane. Nature 301: 621-623. Liles, W. C., D. D. Hunter, K. E. Meier, and N. M. Nathanson (1986) ative-feedback response that inhibits calcium-channel function. Activation of protein kinase C induces rapid internalization and sub- Such a mechanism could be involved in the termination of sequent degradation of muscarinic acetylcholine receptors in neuro- nicotinic-induced calcium signals in neural tissues. blastoma cells. J. Biol. Chem. 261: 5307-5313. 512 Messing et al. * Cholinergic-Induced Protein Kinase C Translocation in PC1 2

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