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A selective effect of protein kinase C activation on pirenzepine high-affinity muscarinic receptors in a neuronal clone
European Journal of Pharmacology, 129 (1986) 201-202
201
Elsevier
EJP 022RC
Rapid communication
A selective effect of protein kinase C activation on pirenzepine high-affinity muscarinic receptors in a neuronal clone Wi S. Lai and E s a m E. E 1 - F a k a h a n y * Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, U.S.A. Received 11 August 1986, accepted 12 August 1986
In many biological systems, one of the major functions of protein kinase C appears to be related to a negative feedback control of neurotransmitter and hormone receptors (Nishizuka, 1986). Tumor promotor phorbol esters are potent activators of protein kinase C both in vivo and in vitro (Castagna et al., 1982). Thus, this class of compounds has been employed as a tool to explore the functions of protein kinase C. Recently, it has been shown in our laboratory (Lai and ElFakahany, manuscript in preparation), and in others (Kanba et al., 1986), that phorbol esters inhibit muscarinic receptor-mediated increase in cyclic G M P synthesis in mouse neuroblastoma cells (clone NIE-115), a response which is believed to involve activation of the M 1 muscarinic receptor subtype (Cioffi and EI-Fakahany, in press; Kanba et al., 1986). On the other hand, phorbol esters have no effect on cellular responses mediated by the M 2 receptor conformation (Kanba et al., 1986). It has also been demonstrated that phorbol esters are very effective in translocating cytosolic protein kinase C to an active cell membrane-bound form in this neuronal clone (Liles et al., 1986). Therefore, we decided to investigate whether activation of protein kinase C by phorbol esters in these cells might preferentially affect the binding of pirenzepine, a selective antagonist of M1 muscarinic receptors. In control cells, pirenzepine displayed binding to high-affinity (M~) and low-affinity (M2)
muscarinic receptor conformations, as detected by its competition for the specific binding of 0.2 nM [3H]N-methylscopolamine ([3H]NMS). The highaffinity binding sites of pirenzepine represented 40.6 + 10.6% (mean + S.E.M.) of the total muscarinic receptor population (table 1). On the TABLE 1 Effects of PMA on pirenzepine binding in mouse neuroblastoma cells a. Mouse neuroblastoma cells (clone N1E-115, passages 11 and 12) were grown as described previously (Cioffi and E1-Fakahany, in press). Cells were incubated in monolayer in serum free tissue culture medium in presence and in absence of 100 nM PMA for 60 rain at 37°C. After harvesting and washing the cells with an isoosmotic salt buffer solution (Cioffi and El-Fakahany, in press), intact cells ( - 1 mg protein) were incubated with 0.2 nM [3H]NMS in the presence of increasing concentrations of pirenzepine (1 nM-10 p,M). Each competition curve contained 17 concentrations of displacer. Incubations were in a final volume of 1 ml of buffer for 90 min at 15°C. The binding assay was terminated by filtration through glass fiber G F / B filters and the competition curves were analyzed by iterative nonlinear least-squares regression as described previously (Cioffi and El-Fakahany, in press). K H and K L are the equilibrium dissociation constants of the high- and low-affinity sites of pirenzepine, respectively, and the numbers in parentheses represent their relative densities, n is the number of independent experiments where a certain receptor model was preferred, divided by the total number of experiments performed. Best fit n Control 2-site b 5 / 6 PMA
1-site
4/5
K H (p,M) 0.06_+ 0.02 (40.6 _+10.6%) -
(0%) * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
K L (/tM) 0.49_+ 0.17 (59.4 _ 10.6%) 0.29 _+ 0.02
(lOO%)
a Data are shown as the means_+S.E.M, b P < 0 . 0 1 as compared to a one-site fit.
202 o t h e r hand, t r e a t m e n t of n e u r o b l a s t o m a cells with 100 n M of p h o r b o l - 1 2 - m y r i s t a t e - 1 3 - a c e t a t e ( P M A ) for 60 min at 3 7 ° C a b o l i s h e d the ability of p i r e n z e p i n e to recognize m u l t i p l e receptor conformations. Thus, c o m p u t e r - a s s i s t e d analysis of c o m p e t i t i o n curves of [ 3 H ] N M S b i n d i n g by pirenzep i n e in intact cells resulted in a single affinity state for p i r e n z e p i n e in P M A - t r e a t e d cells (table 1). It is n o t e w o r t h y that the e q u i l i b r i u m dissocia t i o n c o n s t a n t of pirenzepine a c c o r d i n g to this one-site r e c e p t o r m o d e l is not significantly different (P > 0.05) from that of the low-affinity site in u n t r e a t e d cells (table 1). Meanwhile, P M A d i d not cause any significant changes in the specific b i n d ing of 0.2 n M [ 3 H ] N M S (8.73 + 0.55 f m o l / m g p r o t e i n a n d 7.80 + 0.51 f m o l / m g p r o t e i n in control a n d treated cells, respectively). O u r present d a t a represent the first r e p o r t of a selective a n d differential effect of p r o t e i n kinase C activation on m u s c a r i n i c r e c e p t o r subtypes. These results suggest that P M A t r e a t m e n t has resulted in a t r a n s f o r m a t i o n of high-affinity p i r e n z e p i n e b i n d ing sites to a low-affinity state, w i t h o u t reducing the total r e c e p t o r p o p u l a t i o n labeled b y the n o n selective ligand [ 3 H ] N M S . Such a decrease in the relative d e n s i t y of p i r e n z e p i n e high-affinity sites might underlie the i n h i b i t o r y effect of P M A on m u s c a r i n i c M t r e c e p t o r - m e d i a t e d s t i m u l a t i o n of cyclic G M P synthesis in mouse n e u r o b l a s t o m a N1E-115 cells.
Acknowledgements This work was supported by a contract from the U.S. Army Research Office (DAAG-29-85-K-0123). Esam E. EI-Fakahany is a recipient of a Research Career Development Award from the National Institutes of Health (AG-00344). The authors would like to thank Ms. Renee Powe for her excellent typing and Mr. Michael Gentry for his assistance.
References Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y. Nishizuka, 1982, Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumorpromoting phorbol esters, J. Biol. Chem. 257, 7847. Cioffi, C.L. and E.E. E1-Fakahany, Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: Selective loss of agonist low-affinity and pirenzepine high-affinity binding sites, J. Pharmacol. Exp. Ther. (in press). Kanba, S., K.S. Kanba and E. Richelson, 1986, The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibits muscarinic (M 1) receptor-mediated inositol phosphate release and cyclic GMP formation in murine neuroblastoma cells (clone N1E-115), European J. Pharmacol. 125, 155. Liles, W.C., D.D. Hunter, K.E. Meier and N.M. Nathanson, 1986, Activation of protein kinase C induces rapid internalization and subsequent degradation of muscarinic acetylcholine receptors in neuroblastoma cells, J. Biol. Chem. 261, 5307. Nishizuka, Y., 1986, Studies and perspectives of protein kinase C, Science 233, 305.
201
Elsevier
EJP 022RC
Rapid communication
A selective effect of protein kinase C activation on pirenzepine high-affinity muscarinic receptors in a neuronal clone Wi S. Lai and E s a m E. E 1 - F a k a h a n y * Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, U.S.A. Received 11 August 1986, accepted 12 August 1986
In many biological systems, one of the major functions of protein kinase C appears to be related to a negative feedback control of neurotransmitter and hormone receptors (Nishizuka, 1986). Tumor promotor phorbol esters are potent activators of protein kinase C both in vivo and in vitro (Castagna et al., 1982). Thus, this class of compounds has been employed as a tool to explore the functions of protein kinase C. Recently, it has been shown in our laboratory (Lai and ElFakahany, manuscript in preparation), and in others (Kanba et al., 1986), that phorbol esters inhibit muscarinic receptor-mediated increase in cyclic G M P synthesis in mouse neuroblastoma cells (clone NIE-115), a response which is believed to involve activation of the M 1 muscarinic receptor subtype (Cioffi and EI-Fakahany, in press; Kanba et al., 1986). On the other hand, phorbol esters have no effect on cellular responses mediated by the M 2 receptor conformation (Kanba et al., 1986). It has also been demonstrated that phorbol esters are very effective in translocating cytosolic protein kinase C to an active cell membrane-bound form in this neuronal clone (Liles et al., 1986). Therefore, we decided to investigate whether activation of protein kinase C by phorbol esters in these cells might preferentially affect the binding of pirenzepine, a selective antagonist of M1 muscarinic receptors. In control cells, pirenzepine displayed binding to high-affinity (M~) and low-affinity (M2)
muscarinic receptor conformations, as detected by its competition for the specific binding of 0.2 nM [3H]N-methylscopolamine ([3H]NMS). The highaffinity binding sites of pirenzepine represented 40.6 + 10.6% (mean + S.E.M.) of the total muscarinic receptor population (table 1). On the TABLE 1 Effects of PMA on pirenzepine binding in mouse neuroblastoma cells a. Mouse neuroblastoma cells (clone N1E-115, passages 11 and 12) were grown as described previously (Cioffi and E1-Fakahany, in press). Cells were incubated in monolayer in serum free tissue culture medium in presence and in absence of 100 nM PMA for 60 rain at 37°C. After harvesting and washing the cells with an isoosmotic salt buffer solution (Cioffi and El-Fakahany, in press), intact cells ( - 1 mg protein) were incubated with 0.2 nM [3H]NMS in the presence of increasing concentrations of pirenzepine (1 nM-10 p,M). Each competition curve contained 17 concentrations of displacer. Incubations were in a final volume of 1 ml of buffer for 90 min at 15°C. The binding assay was terminated by filtration through glass fiber G F / B filters and the competition curves were analyzed by iterative nonlinear least-squares regression as described previously (Cioffi and El-Fakahany, in press). K H and K L are the equilibrium dissociation constants of the high- and low-affinity sites of pirenzepine, respectively, and the numbers in parentheses represent their relative densities, n is the number of independent experiments where a certain receptor model was preferred, divided by the total number of experiments performed. Best fit n Control 2-site b 5 / 6 PMA
1-site
4/5
K H (p,M) 0.06_+ 0.02 (40.6 _+10.6%) -
(0%) * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
K L (/tM) 0.49_+ 0.17 (59.4 _ 10.6%) 0.29 _+ 0.02
(lOO%)
a Data are shown as the means_+S.E.M, b P < 0 . 0 1 as compared to a one-site fit.
202 o t h e r hand, t r e a t m e n t of n e u r o b l a s t o m a cells with 100 n M of p h o r b o l - 1 2 - m y r i s t a t e - 1 3 - a c e t a t e ( P M A ) for 60 min at 3 7 ° C a b o l i s h e d the ability of p i r e n z e p i n e to recognize m u l t i p l e receptor conformations. Thus, c o m p u t e r - a s s i s t e d analysis of c o m p e t i t i o n curves of [ 3 H ] N M S b i n d i n g by pirenzep i n e in intact cells resulted in a single affinity state for p i r e n z e p i n e in P M A - t r e a t e d cells (table 1). It is n o t e w o r t h y that the e q u i l i b r i u m dissocia t i o n c o n s t a n t of pirenzepine a c c o r d i n g to this one-site r e c e p t o r m o d e l is not significantly different (P > 0.05) from that of the low-affinity site in u n t r e a t e d cells (table 1). Meanwhile, P M A d i d not cause any significant changes in the specific b i n d ing of 0.2 n M [ 3 H ] N M S (8.73 + 0.55 f m o l / m g p r o t e i n a n d 7.80 + 0.51 f m o l / m g p r o t e i n in control a n d treated cells, respectively). O u r present d a t a represent the first r e p o r t of a selective a n d differential effect of p r o t e i n kinase C activation on m u s c a r i n i c r e c e p t o r subtypes. These results suggest that P M A t r e a t m e n t has resulted in a t r a n s f o r m a t i o n of high-affinity p i r e n z e p i n e b i n d ing sites to a low-affinity state, w i t h o u t reducing the total r e c e p t o r p o p u l a t i o n labeled b y the n o n selective ligand [ 3 H ] N M S . Such a decrease in the relative d e n s i t y of p i r e n z e p i n e high-affinity sites might underlie the i n h i b i t o r y effect of P M A on m u s c a r i n i c M t r e c e p t o r - m e d i a t e d s t i m u l a t i o n of cyclic G M P synthesis in mouse n e u r o b l a s t o m a N1E-115 cells.
Acknowledgements This work was supported by a contract from the U.S. Army Research Office (DAAG-29-85-K-0123). Esam E. EI-Fakahany is a recipient of a Research Career Development Award from the National Institutes of Health (AG-00344). The authors would like to thank Ms. Renee Powe for her excellent typing and Mr. Michael Gentry for his assistance.
References Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y. Nishizuka, 1982, Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumorpromoting phorbol esters, J. Biol. Chem. 257, 7847. Cioffi, C.L. and E.E. E1-Fakahany, Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: Selective loss of agonist low-affinity and pirenzepine high-affinity binding sites, J. Pharmacol. Exp. Ther. (in press). Kanba, S., K.S. Kanba and E. Richelson, 1986, The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibits muscarinic (M 1) receptor-mediated inositol phosphate release and cyclic GMP formation in murine neuroblastoma cells (clone N1E-115), European J. Pharmacol. 125, 155. Liles, W.C., D.D. Hunter, K.E. Meier and N.M. Nathanson, 1986, Activation of protein kinase C induces rapid internalization and subsequent degradation of muscarinic acetylcholine receptors in neuroblastoma cells, J. Biol. Chem. 261, 5307. Nishizuka, Y., 1986, Studies and perspectives of protein kinase C, Science 233, 305.