THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 17, Issue of June 15, pp. 81574161,1988 Printed in U.S.A.

Muscarinic Stimulation of Calcium Influx and Norepinephrine Release in PC 12 Cells*

(Received for publication, January 28, 1988)

Kazuhide InoueS and James G. KenimerO From the Laboratory of Cellular Physiology, Division of Bacterial Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

Muscarinic cholinergic receptor stimulation evokes catecholamine secretion from some cell types, but the mechanism has not been well characterized. Using phe- ochromocytoma (PC12) cells, we show that the mus- carinic agonist methacholine stimulates 4aCa2+ influx and [‘Hlnorepinephrine release in a dose-dependent manner. Experiments performed in Na+-free medium or with inhibitors of voltage-dependent Ca” channels suggest the involvement of a receptor-activated Ca2+ channel which differs significantly from the voltage- dependent Ca2+ channel involved in nicotinic receptor- stimulated release. Furthermore, both influx and re- lease were inhibited by pertussis toxin (0.6-2.0 ng/ml, 2 1 h) with a dose dependency which paralleled the dose dependency of pertussis toxin-dependent in vivo ADP- ribosylation of a 41-kDa protein. These experiments provide the first evidence that muscarinic stimulation evokes neurotransmitter secretion by opening a recep- tor-activated Ca2+ channel which is controlled by a pertussis toxin-sensitive protein.

Muscarinic as well as nicotinic stimulation has been shown to induce catecholamine secretion in cat adrenal gland (1, 2), rat adrenal medulla (3-5), PC12 cells (6), and other cell types (7-9). The mechanism of nicotinic receptor-stimulated release is thought to involve a receptor-mediated cellular depolariza- tion which activates voltage-sensitive CaZ+ channels resulting in an influx of Caz+ which triggers exocytosis (4,5, 10-13). In contrast, the mechanism of muscarinic receptor-stimulated release has not yet been well characterized (1-6,14). Recently, many investigators have shown that muscarinic stimulation induces phosphoinositide hydrolysis with subsequent forma- tion of both inositol 1,4,5-trisphosphate (Ips)’ (15, 16), which increases cytoplasmic free Ca2+ by mobilizing Ca2+ from in- tracellular stores (17, 18), and l,2-diacylglycerol (15, 16), which activates protein kinase C (19). It has been proposed that muscarinic receptor-evoked secretion of catecholamine is dependent on this Ips-induced CaZ+ mobilization (5,20,21).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’$ Recipient of a National Institutes of Health Fogarty Fellowship and a scholarship of the Ministry of Science and Technology of Japan. Present address: Division of Pharmacology, National Institute of Hygienic Sciences, 18-1 Kamiyoga 1-chome, Setagaya-ku, Tokyo 158, Japan.

dressed. 8 To whom correspondence and reprint requests should be ad-

’ The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; NE, norepinephrine; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul-

phate. fonic acid; PTX, pertussis toxin; IP,, inositol 1,3,4,5-tetrakisphos-

If this mobilization of intracellular Ca2+ were sufficient in itself to evoke release, it would be anticipated that muscarinic receptor-stimulated release could occur in the absence of extracellular Ca” since intracellular Ca2+ mobilization is in- dependent of extracellular Caz+ (20-23). Muscarinic receptor- stimulated catecholamine release, however, does require ex- tracellular Ca2+ (1, 3, 5, and this paper), suggesting the par- ticipation of a mechanism which involves influx of Ca2+. A muscarinic receptor-stimulated influx of Ca2+ has been pre- viously reported (23, 24), but no correlation with release was examined. We, therefore, have investigated muscarinic recep- tor-stimulated NE release and influx of Ca2+ in PC12 cells.

EXPERIMENTAL PROCEDURES

Materiak”PCl2 cells were the gift of Dr. Terry Rogers, Johns Hopkins University School of Medicine, Baltimore, MD. The cells were received in our laboratory at passage 46 and were expanded, and a seed lot was frozen at passage 47 and stored in liquid nitrogen. All experiments described in this manuscript were performed with cells at a passage number between 53 and 68. Cells were cultured in 75- cm2 flasks in Dulbecco’s Modified Eagle’s Medium containing 7% fetal bovine serum (Sterile Systems Inc., Logan, UT), 7% heat- inactivated (56 “C, 40 min) horse serum (M. A. Bioproducts, Walk- ersville, MD), 2 mM L-glutamine (S & S Media Inc., Rockville, MD), and 50 pg/ml gentamicin sulfate (GIBCO, Grand Island, NY) in a humidified atmosphere of 90% air and 10% CO2 at 37 “C. Cells were removed from the flasks for subculture and for plating into assay dishes using a Ca2+/M%+-free solution containing 172 mM NaCl, 5.4 mM KCl, 1 mM NaH2P04, and 5.6 mM glucose, pH 7.4. After approx- imately 5 min in this solution, the cells were detached by gently tapping the side of the flask.

[3H]Norepinephrine [3H-NE] (43.7 Ci/mmol) and 13’P]NAD (24.6 Ci/mmol) were from Du Pont-New England Nuclear. &CaC12 (10-40 mCi/mg) was from Amersham. Collagen (Vitrogen-100) was from Collagen Corp., Palo Alto, CA. CdC12 was from Aldrich. CaC12, NaH2P04, and MgC12 were from Mallinckrodt. Glucose was from Merck. NaCl was from J. T. Baker Chemical Co. KC1 was from Fisher. Pertussis toxin was a gift from Dr. Drusilla Burns, FDA, Bethesda, MD. All other supplies and reagents came from Sigma.

Measurement of pH]NE Rekase”PCl2 cells (1 X lo6) were plated into collagen-coated 35-mm polystyrene dishes and cultured for 2-3 days under the conditions described above. The dishes were then each washed with 1 ml of a balanced salt solution (HEPES-BS) (25) containing 150 mM NaCI, 5 mM KCI, 1.8 mM CaC12, 1.2 mM MgCl2, 1.2 mM NaH2P04, 10 mM glucose, 0.1 mM EDTA, and 25 mM HEPES, pH 7.4, and incubated with [3H]NE (0.125 pCi/dish) in 1 ml of HEPES-BS for 1 h at 37 “C. They were then washed with 1 ml of HEPES-BS and incubated with 1 ml of HEPES-BS for 1 h at room temperature. After washing three times at intervals of 5 min, 300 p~ methacholine or 10 p~ nicotine, with or without other test compounds as indicated, was added in 1 ml of HEPES-BS. After 5 min, the incubation solution was removed and counted in a liquid scintillation spectrometer (Beckman LS 3800). The cells were solubilized in 1 ml of 0.25 N NaOH, the solution was carefully collected, and the radio- activity was counted. In Ca2+-free experiments, cells were washed three times with HEPES-BS as above and then for 20 s with Ca2+- free HEPES-BS, and drugs were added in the Ca2+-free HEPES-BS. In Na+-free experiments, cells were washed three times with Na+-free

8157

8158 Muscarinic-stimulated Neurotransmitter Release TBS (26) containing 5.4 mM KCl, 1.2 mM MgC12, 1.8 mM CaC12, 25 mM glucose, and 170 mM Tris-HC1, pH 7.4, and drugs were added in Na+-free TBS. For a comparative control, agonist-evoked release was determined in TBS buffer containing 150 mM NaC1,5.4 mM KC1, 1.2 mM MgC12, 1.8 mM CaC12, 25 mM glucose, and 20 mM Tris-HC1, pH 7.4.

Measurement of 4sCa2+ Accumulation-PC12 cells were plated in collagen-coated plates as above. “Ca2+ accumulation experiments were performed according to the method of Stallcup (11). Cells were washed and incubated with 1 ml of HEPES-BS for 1 h at 37 “C and then incubated with 1 ml of TBS for 15 min at room temperature. The buffer was then replaced with 1 ml of TBS containing 2 pCi of “CaC12 and 300 pM methacholine or 10 p M nicotine with or without CdC12. Cells were incubated for 2 min at room temperature, then rapidly washed three times with 1 ml of ice-cold TBS. One ml of 0.25 N NaOH was added, and the radioactivity of cell digests was measured as above. Protein concentration was determined by the method of Lowry et al. (27). In Na+-free experiments, cells were washed with Na+-free TBS, and drugs were added in Na+-free TBS.

ADP Ribosylation Assay-Membranes from control PC12 cells and from cells pretreated for 21 h with various concentrations of PTX were prepared by homogenization of the cells with 25 strokes of a Dounce homogenizer in 10 mM Tris hydrochloride, 5 mM MgC12, pH 7.5. The homogenate was centrifuged in an Eppendorf Microfuge at 4 “C for 10 min, and the pellets were resuspended in 50 mM sodium phosphate, pH 7.5. The membranes were incubated with PTX and [32P]NAD as previously described (28) and analyzed by sodium do- decyl sulfate-polyacrylamide gel electrophoresis followed by autora- diography. Size was determined using prestained molecular weight standards (Sigma SDS-7B).

RESULTS

Fig. L4 shows that the muscarinic agonist methacholine evoked [3H]NE release in a dose-dependent manner and that this release was inhibited by the muscarinic antagonist atro- pine. Under the conditions used in these assays, 1-2% of the total [3H]NE radioactivity present in the cells was released during the 5-min test period by unstimulated cells (basal release). Stimulation of muscarinic receptors with 300 p M methacholine resulted in the release of an additional 1-2% of the [3H]NE. Stimulation with nicotine (10 p ~ ) resulted in a significantly greater release (4-10% above basal). The meth- acholine-stimulated release displayed qualitative, as well as quantitative, differences from the nicotine-stimulated release (Fig. 1, B and C). Nicotine-evoked release was dependent on extracellular Ca2+, was inhibited by 0.5 mM Cd2+, 5 mM Coz+, or 1 p~ nicardipine (inhibitors of voltage-dependent Ca2+ channels), and vanished in a Na+-free solution? These results are in accord with the mechanism of nicotinic receptor-evoked release mentioned above. In contrast, methacholine-evoked release was not inhibited by CdZ+, Co2+, nicardipine, or the absence of Na+, but was dependent upon the presence of extracellular Ca2+ in agreement with previous findings (1, 3, 4). These results imply that muscarinic stimulation evokes release by increasing Ca2+ influx through a Ca2+ channel which is very different from the voltage-dependent Ca2+ chan- nel involved in nicotinic receptor-stimulated release. We therefore investigated the effect of methacholine on 45Ca2+ influx into PC12 cells.

Ritchie (10) has reported that nicotinic stimulation of dopamine release in PC12 cells is not inhibited in a sucrose-substituted Na+- free buffer, whereas our results show complete inhibition of nicotinic- stimulated NE release in a Tris-substituted Na+-free buffer. A similar discrepancy between Tris-substituted and sucrose-substituted buffers with respect to the Na+ dependency of nicotine-stimulated NE release has been reported in experiments using bovine adrenal medulla cells (13). The reason for these differences is unknown. We have chosen to use the Tris-substituted preparation because of its reported use in electrophysiological studies (26) and its ability to enhance discrimi- nation between muscarinic- and nicotinic-stimulated responses in our system.

A

- 120, B

100

0

k no

f :: Y 20

- a *

03l Cslree C d C o NlCAR Nelree 300 )IM METHACHOLINE

C

I CCNT Ca free Cd Co NICAR Nelree

1OpM NICOTINE

FIG. 1. Effects on [‘HINE release. A, dose response of metha- choline-evoked [3H]NE release and effects of atropine on the release. Release of [3H]NE during 5 min was calculated as percent of the total radioactivity present in the cells before stimulation. Each point is the mean f S.E. of three determinations with basal (without agonist) release (1.87 * 0.07% per 5 min) subtracted. This experiment was performed on the same day on the same batch of PC12 cells. Data shown are representative of two experiments. B and C, effects of Ca*+-free medium, Cd2+, Co2+, nicardipine, and Na+-free medium on agonist-evoked [‘HINE release. Abbreviations: Cd, 0.5 mM cadmium; Co, 5 mM cobalt; NICAR, 1 p~ nicardipine. The experiments with each treatment in B and C were performed on the same day on the same batch of PC12 cells. The treatments had no effect on basal release. The data are the mean f S.E. of 3-11 determinations with basal values subtracted. Methacholine-stimulated [3H]NE release (net value) was 1.21 f 0.07% per 5 min. Nicotine-stimulated [3H]NE release (net value) was 4.60 f 0.41% per 5 min. Agonist-evoked [3H] NE release is represented as 100% (control). An asterisk indicates a statistically significant difference from control by Student’s t test ( p < 0.05) using raw data.

As shown in Fig. 2A, methacholine-stimulated ‘5Ca2+ ac- cumulation is inhibited by atropine, in agreement with a previous report (23). Fig. 2B shows the effects of 0.5 mM Cd2+ or Na+-free medium on nicotine- and methacholine-stimu- lated 45Ca2+ accumulation. Nicotine-stimulated accumulation was inhibited 50% by Cd” and 80% by Na+-free conditions, whereas methacholine-stimulated accumulation was not sig- nificantly affected by either condition. The CdZ+ insensitivity of methacholine-stimulated “Ca2+ influx suggests that the muscarinic receptor-stimulated Ca2+ channel is not a voltage- dependent Ca2+ channel.

To enhance our study of the effect of methacholine on 45Ca2+ accumulation, we used the Na+-free buffer system to investigate the relationship between [3H]NE secretion and 45Ca2+ accumulation. As shown in Fig. 3A, methacholine stim- ulated [3H]NE release and 45Ca2+ accumulation in a dose-

Muscarinic-stimulated Neurotransmitter Release 8159

cchmxx Alr 100 nM Alr 300 nM

- 300 pM METHACHOLINE -

- ccC.ma Cd 0.5 mM Na Free

FIG. 2. Effects on "Ca2+ accumulation. A, the effects of atro- pine (Atr ) on methacholine-evoked 45CaZ' accumulation. B, effects of 0.5 mM Cd2+ and Na+-free conditions on nicotine-evoked and meth- acholine-evoked "Ca" accumulation. The data are presented as per- centage of control (agonist-stimulated accumulation with basal levels subtracted). Basal level in this experiment was 0.689 f 0.029 nmol/ mg of protein/2 min. Methacholine-stimulated control (net value) was 0.241 f 0.038 nmol/mg of protein/2 min. Nicotine-stimulated control (net value) was 0.856 A 0.030 nmol/mg of protein/2 min. The data are the mean f S.E. of three to six determinations. An asterisk indicates a statistical significant difference from control by Student's t test ( p < 0.05) using raw data.

- 0" 1 10 100 1000 METHACHOLINE CM) XiD] p i 3 5

' 3 : 2 3 : u 1 :s W s g f

- 0 -

0 0 .01 , 1 1 10 100

EXTRACEUULAR Ca (mM)

FIG. 3. A, dose dependency of methacholine-evoked [3H]NE re- lease and "Ca" accumulation in Na+-free conditions. B, extracellular Ca2+ dependency of the [3H]NE release and "Ca2+ accumulation stimulated by 300 p~ methacholine in Na+-free conditions. Release of [3H]NE during 2 min and accumulation of 45Ca2+ at 2 min was measured as in Figs. 1 and 2. Release and accumulation determina- tions were performed on the same day on the same batch of PC12 cells. The data are the mean f S.E. of three determinations.

dependent manner. If Ca2+ influx is a major factor in the stimulation of release, then an increase in the extracellular Ca2+ concentration might be expected to correlate with an increase in release. Indeed this was the case as shown in Fig. 3B. Our results strongly suggest that the muscarinic receptors in PC12 cells are coupled to a unique Ca2+ channel and that muscarinic stimulation evokes Ca2+ influx which triggers NE release.

It has been reported that cardiac muscarinic receptors are coupled to the activation of K+ channels (29-31) and to the inhibition of voltage-dependent Ca2+ channels (32) by PTX- sensitive guanine nucleotide-binding proteins (G-proteins). Norepinephrine and y-aminobutyric acid receptors (33), as well as opiate receptors (34), have also been shown to be coupled to the inhibition of voltage-dependent calcium chan- nels by PTX-sensitive G-proteins. We therefore examined the effect of PTX on our system. The results in Fig. 4A show that PTX inhibited the t3H]NE release and 45Ca2+ accumu- lation stimulated by 300 p~ methacholine in a dose-depend- ent manner. Accumulation of 45Ca2+ was completely blocked by P T X at concentrations greater than 0.5 ng/ml, while [3H] NE release was inhibited by 65%. This PTX-resistant release occurs in the absence of measurable 45Ca2+ influx, but does require the presence of extracellular Ca2+ as previously shown. PTX did not inhibit nicotine-evoked [3H]NE release even at higher concentrations (200-1000 ng/ml, 21 h, data not shown). Furthermore, P T X catalyzed the in vivo ADP-ribo- sylation of a 41-kDa protein with a dose dependency parallel to that of inhibition of 45Ca2' accumulation (Fig. 4B). These results suggest that muscarinic receptors in PC12 cells are coupled, via a PTX-sensitive G-protein, to the stimulation of Ca2+ influx and to the resultant NE release.

We have performed a preliminary screening of a number of drugs which we suspected might be able to provide some additional information as to the mechanism of action of our observed muscarinic-stimulated Ca2+ influx and [3H]NE re- lease. As shown in Table I, dbcAMP (1 mM) had no effect on

O 3

41K

0 0.1 0.2 0.5 1.0 2.0 100

FIG. 4. Effects of pertussis toxin (PTX). A , effects of PTX on methacholine-evoked ["]NE release and "Ca2+ accumulation in Na'-free conditions. Methacholine (300 pM)-evoked [3H]NE release or "Ca" accumulation, with basal values subtracted, is represented as 100%. Experiments were performed individually. Each datum is the mean f S.E. of 5-12 determinations. An asterisk indicates a statistically significant difference from control ( p < 0.05) using raw data. Cells were treated with PTX at each concentration for 21 h. B, ADP-ribosylation of PC12 membranes following pretreatment of cells with PTX as above. Protein contents of samples added in sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 113, 97, 115, 154, 111, 95, and 118 pg of protein/50 pl at 0, 0.1, 0.2, 0.5, 1.0, 2.0, and 100 ng of PTX/ml, respectively.

8160 Muscarinic-stimulated Neurotransmitter Release

TABLE I Effect of various drugs on the stimulation of pH]NE release by 300

y M methacholine or by 10 PM nicotine Experimental conditions are as described in the text. Test drugs

were present during the 5-min test interval. Effect on ['HINE release

Methacholine Nicotine % control

D a g Concentration

Dibutyryl cAMP 1 mM 98.4 f 3.3 48.3 f 0.8 Phorbol 12J3-dibutyrate 1 nM 122 f 20.8 162 f 2.5

10 nM 103 f 27.0 190 f 3.7 Polymixin B 100 yg/ml 101 f 36.8 NT" H-7 100 yM 108 k 17.5 NT TMB-8 1 PM 92.7 f 9.7 NT

10 @M 53.6 f 11.6 NT 100 yM 1.9 f 34.2 NT

Neomycin 0.1 mM 91.5 f 7.4 NT 1 mM 77.3 f 22.0 NT

a NT, not tested.

methacholine-stimulated [3H]NE release, but inhibited nico- tinic-stimulated release by 52%. Phorbol12,13-dibutyrate had no effect on methacholine-stimulated release, but increased nicotine-stimulated release by 62% at 1 nM and 90% at 10 nM. Polymixin B (100 pg/ml) and H-7 (100 p ~ ) each had no effect on methacholine-stimulated release. Treatment with TMB-8 resulted in a dose-dependent inhibition of the re- sponse to methacholine, and neomycin at 1 mM inhibited methacholine-stimulated release by 23%.

DISCUSSION

The PC12 cell line, originally developed by Greene and Tischler (35), has been extensively used in studies investigat- ing the molecular mechanisms involved in neurotransmitter release (6, 10, 11, 23, 25). We report here the results of experiments with these cells which provide new information about the molecular mechanism of action of muscarinic- stimulated neurotransmitter release.

The results presented in Figs. 1 and 2 clearly demonstrate that our PC12 cells contain both muscarinic and nicotinic receptors which are coupled to the stimulation of %a2+ influx and [3H]NE release. Experiments performed under Na+-free conditions or with various inhibitors of voltage-sensitive Ca" channels provide convincing evidence that these two cholin- ergic receptors operate via distinct mechanisms to achieve similar results. Our conclusion that the muscarinic-activated

Ca2+ influx occurs through a channel different from the voltage-sensitive channel activated by nicotine is in agree- ment with Pozzan et al. (23) who reported that Ca2+ influx occurred in PC12 cells through a pathway not inhibitable by verapamil.

The relative importance of muscarinic-stimulated catechol- amine release as compared to nicotinic-stimulated release is varied from species to species (2-5, 7-9). Muscarinic stimu- lation of [3H]NE release in our cells was 3- to 10-fold less than the maximal release seen with nicotine; however, results with both types of agonists were very reproducible. Treatment of PC12 cells with nerve growth factor has been shown to lead to an increase in the number of muscarinic receptors (36) and to an increase in cellular responsiveness to muscarinic ago- nists (23). We are currently evaluating the effect of nerve growth factor treatment on our cells and on the PTX-induced inhibition of NE release and Ca2+ influx.

The truly novel aspect of this report is our data which show that preincubation of PC12 cells with PTX results in an inhibition of the muscarinic stimulation (but not the nicotinic stimulation) of both 4SCa2+ influx and [3H]NE release with a

45

PTX dose dependency which parallels that of the in vivo ADP-ribosylation of a 41-kDa PC12 membrane protein (Fig. 4). Several investigators have previously reported muscarinic- stimulated Caz+ influx into cells (23, 24), and others (29-32) have described the interaction of muscarinic receptors with G-proteins, but we know of no previous report that presents evidence suggesting the involvement of a PTX-sensitive pro- tein in the coupling of muscarinic receptors to the stimulation of a Ca2+ channel. The effect of PTX as reported in this manuscript appears to be in conflict with that of Vicentini et al. (37) who reported that PTX (5-1000 ng/ml; 2-18 h) had no effect on the carbachol-induced rise in intracellular Ca" in PC12 cells when measured by the quin2 method. In their PTX experiment (Table 11, Ref. 37), measurements of intra- cellular calcium were made 30 s after the addition of 0.5 mM carbachol, a t which time a significant proportion of the signal is due to the Ips-induced mobilization of intracellular Ca2+ (23, 37). In our experiments, PTX was shown to completely inhibit methacholine-induced '%a2+ influx measured over a 2-min test interval and to inhibit [3H]NE release by 65%. We would thus suggest that the signal obtained by Vicentini et al. (37) is due to a PTX-insensitive mobilization of intracellular Ca2+ and that it is this Caz+ which is responsible for the PTX- insensitive [3H]NE release seen in our experiments.

G-protein regulation of ion channel permeability has been shown to occur via several different molecular mechanisms. The most well studied coupling mechanism involves G-protein activation of the cAMP cascade which results in the activation of a CAMP-dependent protein kinase and the resultant phos- phorylation of specific regulatory sites on ion channels (38- 40). There is also considerable data that demonstrate G- protein control of channel function via modulation of the hydrolysis of phosphatidylinositol and the resultant genera- tion of the intracellular messengers 1,2-diacylglycerol and Ips (41, 42). Direct G-protein interaction with channels has also been described (30). We have performed several experiments which were aimed at determining the molecular mechanism by which this Ca2+ channel is regulated.

As shown in Table I, incubation of cells with dibutyryl cAMP had no effect on the muscarinic-stimulated [3H]NE release while inhibiting the nicotine-stimulated release by 52%, suggesting that the cAMP cascade is not involved in the muscarinic effect. Phorbol 12,13-dibutyrate, an activator of protein kinase C (5), had no effect on the muscarinic-stimu- lated release, but did facilitate the nicotinic-stimulated re- lease. Two inhibitors of protein kinase C, H-7 (43) and poly- mixin B (44), also had no effect on the muscarinic-stimulated release. These results suggest that protein kinase C is not involved in the muscarinic effect but is involved in the nico- tinic effect. TMB-8, which "antagonizes" intracellular Ca2+ (45), blocked muscarinic-stimulated release, demonstrating the dependence of release on the increase of intracellular Ca". Neomycin, which has been used as an inhibitor of polyphosphoinositide phosphodiesterase to prevent the for- mation of IP3, inhibited the methacholine-stimulated [3H]NE release by 23%, supporting the suggestion that the PTX- resistant release is due to the mobilization of intracellular Ca" by IP3.

It has been reported that Ips (47), inositol 1,3,4,5-tetrakis- phosphate (IP4) (48), or a rise in intracellular Ca2+ concentra- tion (49) can activate receptor-operated Ca2+ channels. Since muscarinic stimulation increases IPS and intracellular Caz+ as mentioned above and since G-proteins have been shown to be involved in phosphoinositide hydrolysis, there is a possi- bility that Ips, IP4, or intracellular Ca2+ could regulate our observed 4sCa2+ influx and subsequent NE release. AS dis-

Muscarinic-stimulated N

cussed above, it has been reported by Vicentini et al. (37) that the muscarinic-stimulated increase of IP3 and resultant rise in intracellular Ca2+ is not sensitive to PTX in PC12 cells. In combination with our data, this would suggest that Ca2+ influx is probably not regulated by IP3, IPI, or by an increase in intracellular Ca2+.

In human platelets, a protein kinase C-activated sodium- proton exchange has been postulated as having a role in the regulation of Ca" influx (50). Since this exchange is reported to be negligible in Na'-free medium (50), it can be excluded from consideration as a possible mechanism to explain our methacholine-induced 45Ca2+ influx. The tendency toward an increase in methacholine-induced 45Ca2+ accumulation in Na+-free medium (Fig. 2B) is possibly explained by the ob- servation (11) that 45Caz+ efflux from PC12 cells is slower in Na+-free medium than in normal saline, probably due to the inhibition of a Na+-Ca2+ exchange mechanism.

In summary, we present new evidence for the mechanism of muscarinic receptor-stimulated NE release which strongly suggests that muscarinic receptors in PC12 cells are coupled to the activation of a CaZ+ channel by a PTX-sensitive protein which we assume to be a G-protein. This CaZ+ channel dis- plays properties which distinguish it from the voltage-sensi- tive Ca2+ channel which is involved in nicotinic-stimulated release in these cells.

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