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Dopaminergic regulation of dopamine release from PC12 cells via a pertussis toxin-sensitive G protein
Neuroscience Letters, 122 (1991) 261-264
261
Elsevier Scientific Publishers Ireland Ltd.
NSL 07513
Dopaminergic regulation of dopamine release from PC12 cells via a pertussis toxin-sensitive G protein Neelima D. Courtney*, A11yn C. Howlett and Thomas C. Westfall Department of Pharmacology, St. Louis University School of Medicine, St. Louis, MO 63104 (U.S.A.) (Received 21 April 1990; Revised version received 10 September 1990; Accepted 30 October 1990)
Key words: Dopamine; D2 receptor; PC12 cell; Pertussis toxin; G protein; Quinpirole Regulation of dopamine (DA) release from PC 12 cells was investigated. Apomorphine and quinpirole, a selective D2 agonist, significantly reduced K+-evoked DA release, and this reduction was reversed by haloperidol. Furthermore, spiroperidol, a selective D2 antagonist, and haloperidol, a nonselective DA antagonist, enhanced the K+-evoked DA release. Pertussis toxin treatment of the cells abolished the quinpirole-induced reduction of K+-evoked DA release. Also, the haloperidol-induced enhancement of K+-evoked DA release was not seen in pertussis toxin treated cells. These results, therefore, suggest the presence of D2 receptors on PC 12 cells which result in the modulation of K ÷-evoked DA release via a pertussis toxinsensitive G protein.
It is well established that dopamine (DA) is an important neurotransmitter/neuromodulator in specific regions of the central [1, 5, 6, 8, 23] and peripheral nervous systems [10, 11, 18]. Dopamine receptors have been classified into two subtypes, Dl and D2 receptors [11, 16, 19], based mainly on the selective affinities for various DA agonists and antagonists. In the central nervous system, the release of the neurotransmitter can be decreased by activation of autoreceptors that may be of the D2 subtype. Previous studies have suggested that DE agonists inhibit the nicotinic-induced secretion of catecholamines from chromaffin cells isolated from the adrenal medulla [2, 20]. Furthermore, Quik et al. have shown the existence of high-affinity [3H]spiperone (a selective D2 antagonist) binding sites in adrenal medulla [21]. PCI2 cells are a transformed cell line derived originally from adrenal medullary cells which synthesize and store primarily dopamine and, to a lesser extent, norepinephrine [12]. Also, the vesicular contents can be released by depolarization induced by KCI [13] and activation of nicotinic acetylcholine receptors [3, 22]. Since PC12 cells are derived from adrenal chromaffin cells, we reasoned that PC12 cells may express D2 receptors. The purpose of the
*Current address: VA Medical Center, Research Service (151E), Portland Division, 3710 SW US Veterans Hospital Road, Portland, OR 97201, U.S.A. Correspondence: T. C. Westfall, Department of Pharmacology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, U.S.A.
present study was to determine if D2 receptors on PC 12 cells modulate the release of dopamine in a manner similar to the dopamine autoreceptor regulation of release in the central nervous system. If so, this study will provide a clear system to study DA autoregulation in peripheral tissue. PC 12 cells were a gift of Dr. Eugene Johnson, Department of Pharmacology, Washington University, St. Louis MO. All experiments were carried out with passage numbers between 39 and 55. Cells were maintained in Dulbecco's modified Eagle's (DME) medium supplemented with 10% fetal calf serum and 5% heat-inactivated horse serum in a 100% humidified atmosphere with approximately 8% CO2 at 37°C. Cells were split at weekly intervals with a media change in mid-week. Cells (1-2 x 106) were plated on 35 mm wells 16 h prior to the release experiments. For all release experiments, serum-containing media were removed and replaced with Gey's balanced salt solution having the following composition (mM): NaC1 129, KCI 5.07, CaCl2 2.48, MgCl2 1.03, glucose 11 and N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid 15 (pH 7.4). The Gey's solution was then removed after a 15-min adaptation period and the cells were incubated at 37°C in fresh Gey's solution containing either no drug or an appropriate agonist and/or antagonist. At the end of 2 min, the Gey's solution was transferred into tubes containing perchloric acid (PCA) (final concentration 0.02 N) with 0.05% cysteine for the measurement of the released DA. Cellular DA was determined after cell lysis by 1 N PCA with 1%
262
cysteine. DA was analyzed by HPLC using a reversephase column and an electrochemical detector [24]. The results are expressed as percent of K +-stimulated DA release. Basal release of DA from PCI2 cells was about 24% of the total cellular DA content. (Total cellular content= 12.5+2.4 nmol DA/mg of protein, n=7.) In the presence of nicotine, there was a two-fold increase above basal DA release. K+-evoked DA release was approximately 2-fold greater than that evoked by nicotine. Similar results were observed by Baizer and Weiner [3]. Apomorphine significantly inhibited the K+-evoked DA release (Fig. 1A). In order to characterize the pharmacology of this response, various Dl or D 2 agonists or antagonists were examined. Quinpirole, a selective D2 agonist, inhibited the K +-evoked DA release in a dose-dependent manner (Fig. I B) having a reduction of 48% seen in the presence of 10/aM quinpirole. Neither apomorphine nor quinpirole at this concentration affected basal release. The DI agonist, SKF 38393, failed to reduce the K+-evoked DA release (data not shown). The inhibition in response to both apomorphine and quinpirole was reversed by a
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nonselective DA antagonist haloperidol, and also by a D2 antagonist spiroperidol (Fig. I), whereas SCH 23390, a D1 antagonist, failed to reverse the quinpirole induced inhibition (data not shown). These results suggest the presence of D2 receptors on PC12 cells which modulate the depolarization-induced release of DA. Both haloperidol and spiroperidol significantly enhanced the K+-evoked release of DA (Fig. 2). None of the antagonists tested affected basal DA release (data not shown). Haloperidol at a concentration of 10 #M doubled the K +-evoked DA release. A similar enhancement by haloperidol was also seen with the nicotineevoked DA release (data not shown). The K +-evoked release of DA was increased by approximately 48% by 1 #M spiroperidol. There was no further increase of the K +-evoked DA release by greater spiroperidol concentrations. Interestingly, the enhancement seen with haloperidol was greater than the enhancement seen with spiroperidol. Perhaps this difference in enhancement could be explained on the basis of the action of haloperidol on adrenergic as well as dopaminergic receptors. A Dl antagonist, SCH 23390, failed to enhance the K+-evoked DA release (data not shown). The enhancement of the
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Fig. l. Effects of apomorphine (AP 1 #M) and quinpirole (Q) on 56 mM K+-evoked DA release. These are results from a typical experiment with each data point being the mean-F S.E.M. of triplicate measurements. At least 3 such experiments were performed (BSL = basal), K = K ÷, H L = 1 0 #M haloperiodol, S P = I #M spiroperidol. Q I = 1 #M Q, Q I 0 = 1 0 #M Q. *P<0.005 when compared with basal; **P < 0.05 when compared with K +-stimulated release.
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Fig. 2. Effects of haloperidol (HL 10/~M) and spiroperidol (SP 1 pM) on 56 m M K+-evoked DA release. These are results from a typical experiment with each data point being the mean _4-S.E.M. of triplicate measurements. At least 3 such experiments were performed. BSL = basal, SPl = l #M SP, SP10= 10 #M SP, K = K +. *P<0.005 when compared with K +-stimulated DA release.
263
K +- and nicotine-induced release of DA by the DA antagonists is consistent with blockade of D2 receptors expected to be activated by the released DA. Similar haloperidol-induced disinhibition has been seen in adrenal chromaffin cells by Artalego et al. [2, 20]. In order to probe the mechanism for the D2-induced inhibition of DA release in PC12 cells, we studied the effect of pertussis toxin on this inhibition. Activation of striatal synthesis modulating DA autoreceptors by apomorphine [4], and DA-induced inhibition of the firing of dopaminergic neurons in the substantia nigra [15] could be blocked by pertussis toxin. These findings suggest that at least some D2 receptor subtypes function by interaction with the pertussis toxin-sensitive G proteins, Gi or Go [9]. It is known, for instance, that pertussis toxin catalyzes the transfer of ADP-ribose to the (alpha) subunit of the trimeric Gi and Go proteins, thereby inactivating them [7, 17].
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Fig. 3. Effects of pertussis toxin on 56 m M K +- and Nic-evoked DA release. Cells were incubated with pertussis toxin (50 ng/ml) for 16 h prior to the release experiments. ADP-ribosylation was measured as described previously [14]. These are results from a typical experiment with each data point being the m e a n _ S . E . M , of triplicate measurements. At least 3 such experiments were performed. The inset represents an autoradiogram of S D S - P A G E of ADP-ribosylated membranes. Lanes 1 and 2 are from cells treated with pertussis toxin (50 ng/ml) for 16 h. Lanes 3 and 4 are from untreated cells. Membranes were incubated in the absence (1, 3) or presence (2, 4) of pertussis toxin. BSL=basal, P = w i t h pertussis toxin, Q=Quinpirole, K = K ÷, Nic = Nicotine, *P < 0.05 when compared with untreated cells (p - ) .
ADP-ribosylation measurement of PC 12 membranes treated with pertussis toxin revealed a band in 39 to 41 apparent molecular weight region after SDS-PAGE (Fig. 3, inset, lane 4). This band was not observable after in vitro ADP-ribosylation and SDS-PAGE of the membranes from the cells pretreated with pertussis toxin for 16 h (Fig. 3, lane 2). We did these experiments because we wanted to demonstrate (1) the presence of pertussis toxin-sensitive G proteins in PC12 cell membranes; (2) that the pretreatment of PC12 cells with pertussis toxin results in in situ ADP-ribosylation of the pertussis toxinsensitive G proteins. The reduced levels of in vitro ADPribosylation of the membranes from the cells pretreated with pertussis toxin are interpreted as due to prior in situ ADP-ribosylation. Pretreatment of PC12 cells with pertussis toxin produced an enhancement (30%) of the K+-evoked DA release when compared with K+-evoked DA release of untreated cells (Fig. 3A). Similar increases in the nicotinic-evoked DA release were also observed in pertussis toxin-treated cells (Fig. 3A). Furthermore, the haloperidol-induced enhancement of K+-induced release was not seen in pertussis toxin-treated cells (data not shown). More importantly, quinpirole failed to decrease K +evoked DA release in pertussis toxin treated cells, indicating the involvement of a pertussis toxin-sensitive G protein in the quinpirole-induced inhibition of K +evoked DA release (Fig. 3B). There appeared to be an interexperimental difference in the effect of pertussis toxin on the K+-evoked release of dopamine as seen in Fig. 3A,B. The subtle changes in culture media and/or changes in passage number may account for these interexperimental differences. Pertussis toxin treatment affected neither DA content (data not shown) nor the basal DA release. The mean of basal DA release from three such experiments was not significantly different with or without pertussis toxin treatment. These results therefore show that both haloperidol and pertussis toxin can enhance the evoked release of DA. While haloperidol is known to act at the level of the D2 receptor, pertussis toxin is known to act at a step beyond the receptor, namely at the level of the G protein. This pertussis toxin effect suggests a close association between the releasemodulating D2 receptor and a pertussis toxin-sensitive G protein. Taken together, the above observations strongly suggest the presence of D2 receptors on the PC12 cells and that activation of these receptors modulates the nicotine and K +-depolarization-evoked release of DA. Furthermore, these release-modulating D 2 receptors seemed to be coupled to a pertussis toxin-sensitive G protein. This work was supported in part by DA 02668; NS 07254; DA 03690 and NS 00867
264 1 Anden, N.E., Rubenson, A., Fuxe, K. and H6kfelt, T., Evidence for dopamine receptor stimulation by apomorphine, J. Pharm. Pharmacol., 19 (1967) 627~i29. 2 Artalejo, A.R., Garcia, A.G., Montiel, C. and Sanchez-Garcia, P., A dopaminergic receptor modulates catecholamine release from the cat adrenal gland, J. Physiol., 362 (1985) 359-368, 3 Baizer, L. and Weiner, N., Regulation of dopamine release from PC 12 pheochromocytoma cell culture during stimulation with elevated potassium or carbachol, J. Neurochem., 44 (1985) 495-501. 4 Bean, A.J., Shepard, P.D., Bunney, B.S., Nestler, E.S. and Roth, R.H., The effects of pertussis toxin on autoreceptor-mediated inhibition of dopamine synthesis in the rat striatum., Mol. Pharmacol., 34 (1988) 715-718. 5 Bunney, B.S., Waiters, J.R., Roth, R.H. and Aghajanian, G.K., Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity, J. Pharmacol. Exp. Ther., 185 (1973) 560-571. 6 Bunney, B.S., and Aghajanian, G.K. D-Amphetamine-induced inhibition of central dopaminergic neurons: mediation by a striatonigral feedback loop, Science, 192 (1976) 391-393. 7 Codina, J., Hildebrandt, J., Ivengar, R., Birnbaumer, L., Sekura, R.D. and Manclark, C., Pertussis toxin substrate, the putative N~ component of adenyl cyclases, is an (alpha-beta) heterodimer regulated by guanine nucleotide and magnesium, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 4276-4280. 8 Farnebo. L. and Hamberger, B., Drug-induced changes in the release of 3H monoamines from field stimulated rat brain slices, Acta Physiol. Scand., 371 (1971) 35-44. 9 Gilman, A.G., G proteins; transducers of receptor-generated signals, Ann. Rev. Biochem., 56 (1987) 615649. 10 Goldberg, L.I., Volkman, P.H. and Kohli, J.D., A comparison of the vascular dopamine receptor with other dopamine receptors, Annu. Rev. Pharmacol. Toxic. 18 (1978) 57--79. l 1 Goldberg, L.I. and Kohli, J.D., Peripheral dopamine receptors: A classification based on potency series and specific antagonism, Trends Pharmacol. Sci., 4 (1983) 64q56. 12 Green, L.A. and Tischler, A.S., Establishment of noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond
13
14
15
16 17
18 19 20
21
22
23
24
to nerve growth factor, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2424~2428. Green, L.A. and Rein, G., Short-term regulation of catecholamine synthesis in an NGF responsive clonal line of rat pheochromocytoma cells, J. Neurochem., 30 (1978) 549-555. Howlett, A.C., Qualy, J.M. and Khachatrian, L.L., Involvement of G~ in the inhibition of adenylate cyclase by cannabimimetic drugs, Mol. Pharmacol., 29 (1986) 307 313. Innis, R.B. and Aghajanian, G.K., Pertussis toxin blocks autoreceptor-mediated inhibition of dopaminergic neurons in rat substantia nigra, Brain Res., 411 (1987) 139-143. Kebabian, J. and Calne, D.B., Multiple receptors for dopamine, Nature, 277 (1979) 93-96. Katada, T. and Ui, M., Direct modification of the membrane adenylate cyclase system by islet-activating proteins due to ADP-ribosylation of a membrane protein, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3129-3133. Lackovic, Z. and Kohli, J.D., Evidence that dopamine is a neurotransmitter in peripheral tissues, Life Sci., 32 (1983) 1665 1674. Langer, S.Z., Presynaptic regulation of the release of catecholamines, Pharmacol. Rev., 32 (1980) 337-367. Montiel, C., Artalejo, A.R., Bermejo, P.M. and Sanchez-Garcia, P., A dopaminergic receptor in adrenal medulla as a possible site of action for the droperidol-evoked hypertensive response, Anesthesiology, 65 (1986)474-479. Quik, M., Bergeron, L. Mount, H. and Phillie, J., Dopamine D2 receptor binding in adrenal medulla: characterization using (3H) spiperone, Biochem. Pharmacol., 36 (1987) 3707-3713. Richie, A.K., Catecholamine secretion in rat pheochromocytoma cell line: two pathways for calcium entry, J. Physiol., 286 (1979) 541 561. Snyder, S.H., Taylor, K.M., Coyle, J.T. and Meyerhoff, J.L., The role of brain dopamine in behavioral regulation and the actions of psychotropic drugs, Am. J. Psychiat., 127 (l 970) 199-207. Voight, M., Wang, R.Y. and Westfall, T.C., Cholecystokinin octapeptides alter the release of endogenous dopamine from the rat nucleus accumbens in vitro, J. Pharmacol. Exp. Ther., 237 (1986) 147 153.
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Elsevier Scientific Publishers Ireland Ltd.
NSL 07513
Dopaminergic regulation of dopamine release from PC12 cells via a pertussis toxin-sensitive G protein Neelima D. Courtney*, A11yn C. Howlett and Thomas C. Westfall Department of Pharmacology, St. Louis University School of Medicine, St. Louis, MO 63104 (U.S.A.) (Received 21 April 1990; Revised version received 10 September 1990; Accepted 30 October 1990)
Key words: Dopamine; D2 receptor; PC12 cell; Pertussis toxin; G protein; Quinpirole Regulation of dopamine (DA) release from PC 12 cells was investigated. Apomorphine and quinpirole, a selective D2 agonist, significantly reduced K+-evoked DA release, and this reduction was reversed by haloperidol. Furthermore, spiroperidol, a selective D2 antagonist, and haloperidol, a nonselective DA antagonist, enhanced the K+-evoked DA release. Pertussis toxin treatment of the cells abolished the quinpirole-induced reduction of K+-evoked DA release. Also, the haloperidol-induced enhancement of K+-evoked DA release was not seen in pertussis toxin treated cells. These results, therefore, suggest the presence of D2 receptors on PC 12 cells which result in the modulation of K ÷-evoked DA release via a pertussis toxinsensitive G protein.
It is well established that dopamine (DA) is an important neurotransmitter/neuromodulator in specific regions of the central [1, 5, 6, 8, 23] and peripheral nervous systems [10, 11, 18]. Dopamine receptors have been classified into two subtypes, Dl and D2 receptors [11, 16, 19], based mainly on the selective affinities for various DA agonists and antagonists. In the central nervous system, the release of the neurotransmitter can be decreased by activation of autoreceptors that may be of the D2 subtype. Previous studies have suggested that DE agonists inhibit the nicotinic-induced secretion of catecholamines from chromaffin cells isolated from the adrenal medulla [2, 20]. Furthermore, Quik et al. have shown the existence of high-affinity [3H]spiperone (a selective D2 antagonist) binding sites in adrenal medulla [21]. PCI2 cells are a transformed cell line derived originally from adrenal medullary cells which synthesize and store primarily dopamine and, to a lesser extent, norepinephrine [12]. Also, the vesicular contents can be released by depolarization induced by KCI [13] and activation of nicotinic acetylcholine receptors [3, 22]. Since PC12 cells are derived from adrenal chromaffin cells, we reasoned that PC12 cells may express D2 receptors. The purpose of the
*Current address: VA Medical Center, Research Service (151E), Portland Division, 3710 SW US Veterans Hospital Road, Portland, OR 97201, U.S.A. Correspondence: T. C. Westfall, Department of Pharmacology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, U.S.A.
present study was to determine if D2 receptors on PC 12 cells modulate the release of dopamine in a manner similar to the dopamine autoreceptor regulation of release in the central nervous system. If so, this study will provide a clear system to study DA autoregulation in peripheral tissue. PC 12 cells were a gift of Dr. Eugene Johnson, Department of Pharmacology, Washington University, St. Louis MO. All experiments were carried out with passage numbers between 39 and 55. Cells were maintained in Dulbecco's modified Eagle's (DME) medium supplemented with 10% fetal calf serum and 5% heat-inactivated horse serum in a 100% humidified atmosphere with approximately 8% CO2 at 37°C. Cells were split at weekly intervals with a media change in mid-week. Cells (1-2 x 106) were plated on 35 mm wells 16 h prior to the release experiments. For all release experiments, serum-containing media were removed and replaced with Gey's balanced salt solution having the following composition (mM): NaC1 129, KCI 5.07, CaCl2 2.48, MgCl2 1.03, glucose 11 and N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid 15 (pH 7.4). The Gey's solution was then removed after a 15-min adaptation period and the cells were incubated at 37°C in fresh Gey's solution containing either no drug or an appropriate agonist and/or antagonist. At the end of 2 min, the Gey's solution was transferred into tubes containing perchloric acid (PCA) (final concentration 0.02 N) with 0.05% cysteine for the measurement of the released DA. Cellular DA was determined after cell lysis by 1 N PCA with 1%
262
cysteine. DA was analyzed by HPLC using a reversephase column and an electrochemical detector [24]. The results are expressed as percent of K +-stimulated DA release. Basal release of DA from PCI2 cells was about 24% of the total cellular DA content. (Total cellular content= 12.5+2.4 nmol DA/mg of protein, n=7.) In the presence of nicotine, there was a two-fold increase above basal DA release. K+-evoked DA release was approximately 2-fold greater than that evoked by nicotine. Similar results were observed by Baizer and Weiner [3]. Apomorphine significantly inhibited the K+-evoked DA release (Fig. 1A). In order to characterize the pharmacology of this response, various Dl or D 2 agonists or antagonists were examined. Quinpirole, a selective D2 agonist, inhibited the K +-evoked DA release in a dose-dependent manner (Fig. I B) having a reduction of 48% seen in the presence of 10/aM quinpirole. Neither apomorphine nor quinpirole at this concentration affected basal release. The DI agonist, SKF 38393, failed to reduce the K+-evoked DA release (data not shown). The inhibition in response to both apomorphine and quinpirole was reversed by a
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nonselective DA antagonist haloperidol, and also by a D2 antagonist spiroperidol (Fig. I), whereas SCH 23390, a D1 antagonist, failed to reverse the quinpirole induced inhibition (data not shown). These results suggest the presence of D2 receptors on PC12 cells which modulate the depolarization-induced release of DA. Both haloperidol and spiroperidol significantly enhanced the K+-evoked release of DA (Fig. 2). None of the antagonists tested affected basal DA release (data not shown). Haloperidol at a concentration of 10 #M doubled the K +-evoked DA release. A similar enhancement by haloperidol was also seen with the nicotineevoked DA release (data not shown). The K +-evoked release of DA was increased by approximately 48% by 1 #M spiroperidol. There was no further increase of the K +-evoked DA release by greater spiroperidol concentrations. Interestingly, the enhancement seen with haloperidol was greater than the enhancement seen with spiroperidol. Perhaps this difference in enhancement could be explained on the basis of the action of haloperidol on adrenergic as well as dopaminergic receptors. A Dl antagonist, SCH 23390, failed to enhance the K+-evoked DA release (data not shown). The enhancement of the
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Fig. l. Effects of apomorphine (AP 1 #M) and quinpirole (Q) on 56 mM K+-evoked DA release. These are results from a typical experiment with each data point being the mean-F S.E.M. of triplicate measurements. At least 3 such experiments were performed (BSL = basal), K = K ÷, H L = 1 0 #M haloperiodol, S P = I #M spiroperidol. Q I = 1 #M Q, Q I 0 = 1 0 #M Q. *P<0.005 when compared with basal; **P < 0.05 when compared with K +-stimulated release.
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263
K +- and nicotine-induced release of DA by the DA antagonists is consistent with blockade of D2 receptors expected to be activated by the released DA. Similar haloperidol-induced disinhibition has been seen in adrenal chromaffin cells by Artalego et al. [2, 20]. In order to probe the mechanism for the D2-induced inhibition of DA release in PC12 cells, we studied the effect of pertussis toxin on this inhibition. Activation of striatal synthesis modulating DA autoreceptors by apomorphine [4], and DA-induced inhibition of the firing of dopaminergic neurons in the substantia nigra [15] could be blocked by pertussis toxin. These findings suggest that at least some D2 receptor subtypes function by interaction with the pertussis toxin-sensitive G proteins, Gi or Go [9]. It is known, for instance, that pertussis toxin catalyzes the transfer of ADP-ribose to the (alpha) subunit of the trimeric Gi and Go proteins, thereby inactivating them [7, 17].
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K (p+)
Fig. 3. Effects of pertussis toxin on 56 m M K +- and Nic-evoked DA release. Cells were incubated with pertussis toxin (50 ng/ml) for 16 h prior to the release experiments. ADP-ribosylation was measured as described previously [14]. These are results from a typical experiment with each data point being the m e a n _ S . E . M , of triplicate measurements. At least 3 such experiments were performed. The inset represents an autoradiogram of S D S - P A G E of ADP-ribosylated membranes. Lanes 1 and 2 are from cells treated with pertussis toxin (50 ng/ml) for 16 h. Lanes 3 and 4 are from untreated cells. Membranes were incubated in the absence (1, 3) or presence (2, 4) of pertussis toxin. BSL=basal, P = w i t h pertussis toxin, Q=Quinpirole, K = K ÷, Nic = Nicotine, *P < 0.05 when compared with untreated cells (p - ) .
ADP-ribosylation measurement of PC 12 membranes treated with pertussis toxin revealed a band in 39 to 41 apparent molecular weight region after SDS-PAGE (Fig. 3, inset, lane 4). This band was not observable after in vitro ADP-ribosylation and SDS-PAGE of the membranes from the cells pretreated with pertussis toxin for 16 h (Fig. 3, lane 2). We did these experiments because we wanted to demonstrate (1) the presence of pertussis toxin-sensitive G proteins in PC12 cell membranes; (2) that the pretreatment of PC12 cells with pertussis toxin results in in situ ADP-ribosylation of the pertussis toxinsensitive G proteins. The reduced levels of in vitro ADPribosylation of the membranes from the cells pretreated with pertussis toxin are interpreted as due to prior in situ ADP-ribosylation. Pretreatment of PC12 cells with pertussis toxin produced an enhancement (30%) of the K+-evoked DA release when compared with K+-evoked DA release of untreated cells (Fig. 3A). Similar increases in the nicotinic-evoked DA release were also observed in pertussis toxin-treated cells (Fig. 3A). Furthermore, the haloperidol-induced enhancement of K+-induced release was not seen in pertussis toxin-treated cells (data not shown). More importantly, quinpirole failed to decrease K +evoked DA release in pertussis toxin treated cells, indicating the involvement of a pertussis toxin-sensitive G protein in the quinpirole-induced inhibition of K +evoked DA release (Fig. 3B). There appeared to be an interexperimental difference in the effect of pertussis toxin on the K+-evoked release of dopamine as seen in Fig. 3A,B. The subtle changes in culture media and/or changes in passage number may account for these interexperimental differences. Pertussis toxin treatment affected neither DA content (data not shown) nor the basal DA release. The mean of basal DA release from three such experiments was not significantly different with or without pertussis toxin treatment. These results therefore show that both haloperidol and pertussis toxin can enhance the evoked release of DA. While haloperidol is known to act at the level of the D2 receptor, pertussis toxin is known to act at a step beyond the receptor, namely at the level of the G protein. This pertussis toxin effect suggests a close association between the releasemodulating D2 receptor and a pertussis toxin-sensitive G protein. Taken together, the above observations strongly suggest the presence of D2 receptors on the PC12 cells and that activation of these receptors modulates the nicotine and K +-depolarization-evoked release of DA. Furthermore, these release-modulating D 2 receptors seemed to be coupled to a pertussis toxin-sensitive G protein. This work was supported in part by DA 02668; NS 07254; DA 03690 and NS 00867
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