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Increased muscarinic receptor binding in heart membranes by an inhibitor of protein kinase C
Volume 242, number 1, 175-177
FEB 06619
December 1988
Increased muscarinic receptor binding in heart membranes by an inhibitor of protein kinase C Tony L. Creazzo and Robert W. Wrenn
Medical College of Georgia, Department of Anatomy, Augusta, GA 30912-2000, USA Received 22 September 1988 The number of muscarinic acetylcholine receptors (MAChRs), as detected by binding of [3H]quinuclidinyl benzilate (QNB), was investigated under conditions which promote protein phosphorylation. Incubation of a crude heart membrane preparation in the presence of ATP/Mg2+ reduced MAChR number by 50~. Incubation with polymyxin B, an inhibitor of protein kinase C, blocked the effect of ATP/Mgz+ and increased MAChR number by 74%. Muscarinic receptor; Phosphorylation; Acetylcholine; (Heart)
1. I N T R O D U C T I O N P r o l o n g e d e x p o s u r e o f intact h e a r t cells t o c h o l i n e r g i c a g o n i s t s results in a b i p h a s i c r e d u c t i o n in t h e n u m b e r o f m u s c a r i n i c a c e t y l c h o l i n e recept o r s ( M A C h R s ) d e t e c t a b l e b y r a d i o l a b e l e d ant a g o n i s t s [1]. A t least p a r t o f the a p p a r e n t r e d u c t i o n o f M A C h R s is d u e t o persistent b i n d i n g o f t h e u n l a b e l e d agonist. T h e loss o f d e t e c t a b l e M A C h R s c o r r e l a t e s with d e c r e a s e d sensitivity o f the heart to cholinergic agonists. This p h e n o m e n o n o f M A C h R d e s e n s i t i z a t i o n is p o o r l y u n d e r s t o o d . O n e p o s s i b l e m e c h a n i s m is t h a t desens i t i z a t i o n results f r o m p h o s p h o r y l a t i o n o f either t h e M A C h R o r a closely a s s o c i a t e d p r o t e i n . I n supp o r t o f this h y p o t h e s i s it has b e e n r e p o r t e d t h a t in vitro conditions which favor protein phosphorylat i o n result in a loss o f M A C h R s [ 2 - 4 ] . Recently, K w a t r a a n d H o s e y [5] h a v e f o u n d t h a t t r e a t m e n t o f h e a r t s with t h e a g o n i s t c a r b a c h o l leads to 1 0 - 1 2 - f o l d increases in p h o s p h o r y l a t i o n o f the M A C h R . In this r e p o r t we s h o w t h a t p o l y m y x i n B ( P M B ) , a selective i n h i b i t o r o f p r o t e i n kinase C
Correspondence address: T.L. Creazzo, Department of Anatomy, Medical College of Georgia, Augusta, GA 30912-2000, USA
[6], p r e v e n t s the loss o f M A C h R s w h i c h results from incubation of crude heart membrane preparations under conditions which favor protein phosphorylation.
2. M A T E R I A L S A N D M E T H O D S Fresh adult chicken hearts were a gift from Amick's Farm, Batesburg, SC. l-[3H]Quinuclidinyl benzilate (QNB) was purchased from Amersham/Searle. Atropine sulfate, adenine nucleotides and protein kinase A inhibitor were purchased from Sigma. Polymyxin B was purchased from United States Biochemical Corporation.
2.1. Tissuepreparation A crude membrane preparation was prepared in the following manner. The ventricles were coursely chopped, washed and homogenized with a Polytron in 100 mg tissue/ml of 50 mM Tris-HC1, pH 7.4 (buffer). The homogenate was centrifuged at 1000 x g for 10 min and the pellet discarded. The supernate was centrifuged at 21000 x g for 30 rain. The supernate was discarded and the pellet resuspended in buffer to yield approx. 2 mg protein per ml. All steps were carried out at 4°C. The preparation was frozen at -80°C until used. 2.2. Assay of ffHIQNB binding [3H]QNB binding was assayed and analyzed according to Cl;eazzo and Hartzell [7] except that non-specific binding was determined using 10/~M atropine sulfate. Assays were carried out overnight at room temperature in triplicate and terminated by filtration. Each assay tube contained 2 ml of buffer with
Published by Elsevier Science Publishers B.V. (BiomedicalDivision) 00145793/88/$3.50 © 1988 Federation of European Biochemical Societies
175
Volume 242, number 1
FEBS LETTERS
December 1988 2O
500 pM [3H]QNB (970/0 saturation) and 50/zl of homogenate (50-100/zg protein). Other additions are noted in the figure legends. Data from multiple experiments are expressed as
A
means _+SE.
15 D
3. RESULTS
tJ
To test whether MAChR number is affected by conditions which would favor phosphorylation by protein kinases membranes were incubated in the presence of various adenine nucleotides (fig. 1). Incubation with adenine nucleotides in the absence of Mg 2+ produced either no effect or a slight elevation of MAChR binding to [3H]QNB. In the presence of Mg 2+, ATP reduced the apparent number of MAChRs by 50%. In contrast, ADP and AMP reduced MAChRs only slightly when in the presence of Mg2+. Similarly, the nonhydrolyzable ATP analog, AMP-PNP, had little effect. Scatchard analysis showed that the nucleotides or Mg2+ had no effect on [3H]QNBbinding affinity (Kd -- 11 pM, not shown). These results suggest that protein phosphorylation reduces the number of MAChRs that can be detected by [3H]QNB. To test further the possibility that phosphorylation reduces MAChR-binding sites, membranes were incubated with PMB, a specific inhibitor of protein kinase C (PKC) [61. Fig.2A shows that PMB by itself increased the number of MAChRs in a dose-dependent fashion. In the presence of ATP and Mg 2+, PMB similarly increased MAChR sites
t.
I00
E 0 t_.) till l O&
50
2
3
I ATP
o/
o., i
i
i
0
50 PM8 (wM)
1O0
200
"~ 150
10(1
g 50
I1.
PM8
PKA-I
Fig.2. (A) PMB dose response. (Open squares) Incubation with PMB only; (filled squares) incubation with PMB plus 5 mM ATP and MgCI2. (B) Effect of incubation of 100/~M PMB (n = 3) and 10 units/ml cAMP-dependent protein kinase inhibitor (PKA-I; n = 2) and 5 mM ATP and MgCI2. The results were normalized to controls containing 5 mM ATP and Mg~+ without inhibitors.
4. DISCUSSION AOP
AMP
AMP-PNP
Fig.l. Effect of adenine nucleotides with and without Mg2+ on [3HIQNB binding. (Open bars) Effects of 5 mM ATP, AI~P and AMP and 1 mM AMP-PNP; (filled bars) effects of nucleotides plus 5 mM MgC12. The results were normalized to controls containing either no additions or 5 mM MgC12. The number of experiments is given above each bar.
176
10
in a dose-dependent manner though not as effectively as with PMB alone. Whether or not in the presence of ATP and Mg 2+, the maximum number of detectable MAChR-binding sites in the presence of 100/~M PMB was about 18 pM. Fig.2B indicates a 74% mean increase in the number of MAChRs with 100/~M PMB in the presence of ATP and Mg2+. In contrast, an inhibitor of cAMP-dependent protein kinase [8] reduced MAChRs by 14%.
150 2
¢4-
The results demonstrate that incubation of crude ventricle membranes with PMB, a specific inhibitor of PKC [6], substantially increases the number of MAChRs detectable with [3H]QNB. Secondly, PMB inhibited the reduction of MAChRs that occurred in the presence of
Volume 242, number 1
FEBS LETTERS
A T P / M g 2÷. The cAMP-dependent protein kinase inhibitor (Walsh Inhibitor) [8], however, caused a slight reduction in M A C h R number. These results suggest two possible mechanisms. The first is that P K C phosphorylation of the M A C h R prevents binding of cholinergic ligands. The second possibility is that P K C phosphorylation activates a protein phosphatase which catalyzes dephosphorylation o f the M A C h R . This in turn would cause release of persistently bound acetylcholine, thereby allowing detection by [3H]QNB. Under this scenario the observed loss of MAChRs in the presence of added A T P / M g 2÷ (fig. 1) m a y be due to some other protein kinase activity. The data presented here do not distinguish between these two possible mechanisms. T h o u g h PKC activity is Ca 2÷ dependent it was not necessary to add Ca 2+ in order to observe M A C h R loss in A T P / M g 2+. The addition of 200/zM Ca 2÷ to the assay did not enhance M A C h R loss over A T P / M g 2÷ alone (not shown). This is not surprising since contaminating Ca 2÷ is probably 10-20/zM or more. The Km for Ca 2÷ stimulated P K C phosphorylation o f histone H I is 35/~M which decreases to 5 - 1 2 / z M in the presence of phospholipid cofactors [9]. Also, the presence of other m e m b r a n e lipid co factors, such as diglycerides, lowers the Km for Ca 2÷ to levels below 1/~M [10-11]. As already indicated by this discussion we cannot exclude the possibility that the reduction of MAChRs, observed in the presence o f A T P / M g 2÷, was due to a kinase other t h a n PKC. It is interesting that PMB by itself increases M A C h R binding without the addition of A T P / M g 2÷. One explanation is that P M B inhibits phosphorylation o f the M A C h R by the particulate P K C and A T P / M g 2+ already present in the crude m e m b r a n e preparation. This is a reasonable assumption if the Km for A T P / M g 2+ phosphoryla-
December 1988
tion with P K C is in the low micromolar range; such as, for example, the Km for P K C phosphorylation o f histone H1 (4.4/zM) [12]. The data discussed in this report support the hypothesis that PKC-catalyzed protein phosphorylation plays a m a j o r role in M A C h R desensitization a n d / o r down regulation. Consistent with this hypothesis is a recent report indicating that P K C inhibits M A C h R - m o d u l a t e d Ca 2÷ release and entry in h u m a n salivary cells [13].
Acknowledgements: The authors gratefully acknowledge the technical assistance of Mr Greg Oblak in performing the studies described herein. This work was supported by NIH grants R29 HL39039 and PO1 HL36059. REFERENCES [1] Galper, J.B., Dziekan, L.C., O'Hara, D.S. and Smith, T.W. (1982) J. Biol. Chem. 257, 10344-10356. [2] Burgoyne, R.D. (1980) FEBS Lett. 122, 288-292. [3] Burgoyne, R.D. (1983) J. Neurochem. 40, 324-331. [4] Ho, A.K.S. and Wong, J.H. (1985) Biochem. Biophys. Res. Commun. 133, 1193-1200. [5] Kwatra, M.M. and Hosey, M.M. (1986) J. Biol. Chem. 261, 12429-12432. [6] Wrenn, W. and Wooten, M. (1984) Life Sci. 35,267-276. [71 Creazzo, T.L. and Hartzell, H.C. (1985) J. Neurochem. 45, 710-718. [8] Walsh, D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer, D.H. and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985. [9] Wise, B.C., Raynor, R.L. and Kuo, J.F. (1982) J. Biol. Chem. 257, 8481-8488. [10] Kishimoto, A., Takal, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1979) Biochem. Biophys. Res. Commun. 91, 1218-1224. [11] Kaibuchi, K., Takai, Y. and Nishizuka, Y. (1981) J. Biol. Chem. 255, 2273-2276. [12] Wise, B.C., Glass, D.B., Jen Chou, C.-H., Raynor, R.L., Katoh, N., Schatzman, R.C., Turner, R.S., Kibler, R.F. and Kuo, J.F. (1982) J. Biol. Chem. 257, 8489-8495. [131 He, X., Wu, X. and Baum, B.J. (1988) Biochem. Biophys. Res. Commun. 152, 1062-1069.
177
FEB 06619
December 1988
Increased muscarinic receptor binding in heart membranes by an inhibitor of protein kinase C Tony L. Creazzo and Robert W. Wrenn
Medical College of Georgia, Department of Anatomy, Augusta, GA 30912-2000, USA Received 22 September 1988 The number of muscarinic acetylcholine receptors (MAChRs), as detected by binding of [3H]quinuclidinyl benzilate (QNB), was investigated under conditions which promote protein phosphorylation. Incubation of a crude heart membrane preparation in the presence of ATP/Mg2+ reduced MAChR number by 50~. Incubation with polymyxin B, an inhibitor of protein kinase C, blocked the effect of ATP/Mgz+ and increased MAChR number by 74%. Muscarinic receptor; Phosphorylation; Acetylcholine; (Heart)
1. I N T R O D U C T I O N P r o l o n g e d e x p o s u r e o f intact h e a r t cells t o c h o l i n e r g i c a g o n i s t s results in a b i p h a s i c r e d u c t i o n in t h e n u m b e r o f m u s c a r i n i c a c e t y l c h o l i n e recept o r s ( M A C h R s ) d e t e c t a b l e b y r a d i o l a b e l e d ant a g o n i s t s [1]. A t least p a r t o f the a p p a r e n t r e d u c t i o n o f M A C h R s is d u e t o persistent b i n d i n g o f t h e u n l a b e l e d agonist. T h e loss o f d e t e c t a b l e M A C h R s c o r r e l a t e s with d e c r e a s e d sensitivity o f the heart to cholinergic agonists. This p h e n o m e n o n o f M A C h R d e s e n s i t i z a t i o n is p o o r l y u n d e r s t o o d . O n e p o s s i b l e m e c h a n i s m is t h a t desens i t i z a t i o n results f r o m p h o s p h o r y l a t i o n o f either t h e M A C h R o r a closely a s s o c i a t e d p r o t e i n . I n supp o r t o f this h y p o t h e s i s it has b e e n r e p o r t e d t h a t in vitro conditions which favor protein phosphorylat i o n result in a loss o f M A C h R s [ 2 - 4 ] . Recently, K w a t r a a n d H o s e y [5] h a v e f o u n d t h a t t r e a t m e n t o f h e a r t s with t h e a g o n i s t c a r b a c h o l leads to 1 0 - 1 2 - f o l d increases in p h o s p h o r y l a t i o n o f the M A C h R . In this r e p o r t we s h o w t h a t p o l y m y x i n B ( P M B ) , a selective i n h i b i t o r o f p r o t e i n kinase C
Correspondence address: T.L. Creazzo, Department of Anatomy, Medical College of Georgia, Augusta, GA 30912-2000, USA
[6], p r e v e n t s the loss o f M A C h R s w h i c h results from incubation of crude heart membrane preparations under conditions which favor protein phosphorylation.
2. M A T E R I A L S A N D M E T H O D S Fresh adult chicken hearts were a gift from Amick's Farm, Batesburg, SC. l-[3H]Quinuclidinyl benzilate (QNB) was purchased from Amersham/Searle. Atropine sulfate, adenine nucleotides and protein kinase A inhibitor were purchased from Sigma. Polymyxin B was purchased from United States Biochemical Corporation.
2.1. Tissuepreparation A crude membrane preparation was prepared in the following manner. The ventricles were coursely chopped, washed and homogenized with a Polytron in 100 mg tissue/ml of 50 mM Tris-HC1, pH 7.4 (buffer). The homogenate was centrifuged at 1000 x g for 10 min and the pellet discarded. The supernate was centrifuged at 21000 x g for 30 rain. The supernate was discarded and the pellet resuspended in buffer to yield approx. 2 mg protein per ml. All steps were carried out at 4°C. The preparation was frozen at -80°C until used. 2.2. Assay of ffHIQNB binding [3H]QNB binding was assayed and analyzed according to Cl;eazzo and Hartzell [7] except that non-specific binding was determined using 10/~M atropine sulfate. Assays were carried out overnight at room temperature in triplicate and terminated by filtration. Each assay tube contained 2 ml of buffer with
Published by Elsevier Science Publishers B.V. (BiomedicalDivision) 00145793/88/$3.50 © 1988 Federation of European Biochemical Societies
175
Volume 242, number 1
FEBS LETTERS
December 1988 2O
500 pM [3H]QNB (970/0 saturation) and 50/zl of homogenate (50-100/zg protein). Other additions are noted in the figure legends. Data from multiple experiments are expressed as
A
means _+SE.
15 D
3. RESULTS
tJ
To test whether MAChR number is affected by conditions which would favor phosphorylation by protein kinases membranes were incubated in the presence of various adenine nucleotides (fig. 1). Incubation with adenine nucleotides in the absence of Mg 2+ produced either no effect or a slight elevation of MAChR binding to [3H]QNB. In the presence of Mg 2+, ATP reduced the apparent number of MAChRs by 50%. In contrast, ADP and AMP reduced MAChRs only slightly when in the presence of Mg2+. Similarly, the nonhydrolyzable ATP analog, AMP-PNP, had little effect. Scatchard analysis showed that the nucleotides or Mg2+ had no effect on [3H]QNBbinding affinity (Kd -- 11 pM, not shown). These results suggest that protein phosphorylation reduces the number of MAChRs that can be detected by [3H]QNB. To test further the possibility that phosphorylation reduces MAChR-binding sites, membranes were incubated with PMB, a specific inhibitor of protein kinase C (PKC) [61. Fig.2A shows that PMB by itself increased the number of MAChRs in a dose-dependent fashion. In the presence of ATP and Mg 2+, PMB similarly increased MAChR sites
t.
I00
E 0 t_.) till l O&
50
2
3
I ATP
o/
o., i
i
i
0
50 PM8 (wM)
1O0
200
"~ 150
10(1
g 50
I1.
PM8
PKA-I
Fig.2. (A) PMB dose response. (Open squares) Incubation with PMB only; (filled squares) incubation with PMB plus 5 mM ATP and MgCI2. (B) Effect of incubation of 100/~M PMB (n = 3) and 10 units/ml cAMP-dependent protein kinase inhibitor (PKA-I; n = 2) and 5 mM ATP and MgCI2. The results were normalized to controls containing 5 mM ATP and Mg~+ without inhibitors.
4. DISCUSSION AOP
AMP
AMP-PNP
Fig.l. Effect of adenine nucleotides with and without Mg2+ on [3HIQNB binding. (Open bars) Effects of 5 mM ATP, AI~P and AMP and 1 mM AMP-PNP; (filled bars) effects of nucleotides plus 5 mM MgC12. The results were normalized to controls containing either no additions or 5 mM MgC12. The number of experiments is given above each bar.
176
10
in a dose-dependent manner though not as effectively as with PMB alone. Whether or not in the presence of ATP and Mg 2+, the maximum number of detectable MAChR-binding sites in the presence of 100/~M PMB was about 18 pM. Fig.2B indicates a 74% mean increase in the number of MAChRs with 100/~M PMB in the presence of ATP and Mg2+. In contrast, an inhibitor of cAMP-dependent protein kinase [8] reduced MAChRs by 14%.
150 2
¢4-
The results demonstrate that incubation of crude ventricle membranes with PMB, a specific inhibitor of PKC [6], substantially increases the number of MAChRs detectable with [3H]QNB. Secondly, PMB inhibited the reduction of MAChRs that occurred in the presence of
Volume 242, number 1
FEBS LETTERS
A T P / M g 2÷. The cAMP-dependent protein kinase inhibitor (Walsh Inhibitor) [8], however, caused a slight reduction in M A C h R number. These results suggest two possible mechanisms. The first is that P K C phosphorylation of the M A C h R prevents binding of cholinergic ligands. The second possibility is that P K C phosphorylation activates a protein phosphatase which catalyzes dephosphorylation o f the M A C h R . This in turn would cause release of persistently bound acetylcholine, thereby allowing detection by [3H]QNB. Under this scenario the observed loss of MAChRs in the presence of added A T P / M g 2÷ (fig. 1) m a y be due to some other protein kinase activity. The data presented here do not distinguish between these two possible mechanisms. T h o u g h PKC activity is Ca 2÷ dependent it was not necessary to add Ca 2+ in order to observe M A C h R loss in A T P / M g 2+. The addition of 200/zM Ca 2÷ to the assay did not enhance M A C h R loss over A T P / M g 2÷ alone (not shown). This is not surprising since contaminating Ca 2÷ is probably 10-20/zM or more. The Km for Ca 2÷ stimulated P K C phosphorylation o f histone H I is 35/~M which decreases to 5 - 1 2 / z M in the presence of phospholipid cofactors [9]. Also, the presence of other m e m b r a n e lipid co factors, such as diglycerides, lowers the Km for Ca 2÷ to levels below 1/~M [10-11]. As already indicated by this discussion we cannot exclude the possibility that the reduction of MAChRs, observed in the presence o f A T P / M g 2÷, was due to a kinase other t h a n PKC. It is interesting that PMB by itself increases M A C h R binding without the addition of A T P / M g 2÷. One explanation is that P M B inhibits phosphorylation o f the M A C h R by the particulate P K C and A T P / M g 2+ already present in the crude m e m b r a n e preparation. This is a reasonable assumption if the Km for A T P / M g 2+ phosphoryla-
December 1988
tion with P K C is in the low micromolar range; such as, for example, the Km for P K C phosphorylation o f histone H1 (4.4/zM) [12]. The data discussed in this report support the hypothesis that PKC-catalyzed protein phosphorylation plays a m a j o r role in M A C h R desensitization a n d / o r down regulation. Consistent with this hypothesis is a recent report indicating that P K C inhibits M A C h R - m o d u l a t e d Ca 2÷ release and entry in h u m a n salivary cells [13].
Acknowledgements: The authors gratefully acknowledge the technical assistance of Mr Greg Oblak in performing the studies described herein. This work was supported by NIH grants R29 HL39039 and PO1 HL36059. REFERENCES [1] Galper, J.B., Dziekan, L.C., O'Hara, D.S. and Smith, T.W. (1982) J. Biol. Chem. 257, 10344-10356. [2] Burgoyne, R.D. (1980) FEBS Lett. 122, 288-292. [3] Burgoyne, R.D. (1983) J. Neurochem. 40, 324-331. [4] Ho, A.K.S. and Wong, J.H. (1985) Biochem. Biophys. Res. Commun. 133, 1193-1200. [5] Kwatra, M.M. and Hosey, M.M. (1986) J. Biol. Chem. 261, 12429-12432. [6] Wrenn, W. and Wooten, M. (1984) Life Sci. 35,267-276. [71 Creazzo, T.L. and Hartzell, H.C. (1985) J. Neurochem. 45, 710-718. [8] Walsh, D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer, D.H. and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985. [9] Wise, B.C., Raynor, R.L. and Kuo, J.F. (1982) J. Biol. Chem. 257, 8481-8488. [10] Kishimoto, A., Takal, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1979) Biochem. Biophys. Res. Commun. 91, 1218-1224. [11] Kaibuchi, K., Takai, Y. and Nishizuka, Y. (1981) J. Biol. Chem. 255, 2273-2276. [12] Wise, B.C., Glass, D.B., Jen Chou, C.-H., Raynor, R.L., Katoh, N., Schatzman, R.C., Turner, R.S., Kibler, R.F. and Kuo, J.F. (1982) J. Biol. Chem. 257, 8489-8495. [131 He, X., Wu, X. and Baum, B.J. (1988) Biochem. Biophys. Res. Commun. 152, 1062-1069.
177