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Alterations of β-receptor-adenylyl cyclase coupling by long-term ATP depletion in cultured rat cardiomyocytes
European Journal of Pharmacology - Moh,cular Pharmacolo,W Section, 208 ( 1991 ) 261 - 264 (0/ 1991 Elsevier Science Publishers B.V. All rights reserved 0922-41(16/91/$113.511
261
E J P M O L 8{1{1~'~6
Short communication
Alterations of ,8-receptor-adenylyl cyclase coupling by long-term ATP depletion in cultured rat cardiomyocytes B e r n h a r d W a g e n k n e c h t and C l a u d i a B e u t t l e r Medizinische Klinik 1, Klinikum (;ro.~shadern, Uni~ el:fftb't Miinchen, IVS000 Munich 70. (;ermat~y Received II1 July 1991, revised MS received 7 August 1991, accepted 20 August 1991
In cultured rat heart muscle cells, reversible long-term ATP depiction induces a decrease in fl-adrcnoccptor density and a fall in isoproterenol- as well as forskolin-stimulated cAMP formation. However, isoproterenol-stimulatcd adenylyl cyclase activity in membrane preparations is not reduced after ATP depletion. These results suggest that the decreased responsiveness to catccholamines during myocardial ischemia cannot be explained by alterations of thc /3-adrenoceptor-adenylyl cyclase system alone. ATP depletion; Deoxyglucosc; Noradrenaline:/3-Adrcnoccptor; Adenylyl cyclase, Cardiomyocytes
I. Introduction Acute myocardiai ischemia induces in intact heart preparations an early sensitization of the/3-adrenoceptor-adenylyl cyclase system (Maisel et al., 1987; Strasser et al., 19901 followed by a late decline of adenylyl cyclase activity (Vatner et al., 1988; Wolff et al., 1989; Strasser et al., 19901 and a decrease of the activity of G~ (Susanni et al., 1989). As inhibition of oxidative phosphor3,1ation by cyanide results also in a receptorspecific sensitization and in an abolition of catecholamine-induced descnsitizahon, tile ios5 of high-energy phosphates seems to play a key role in the regulation of ,8-adrcnoceptor-adenylyl cyclase durip.g ischemia (Strasser et al., 19901. In cultured heart muscle cells, however, ATP depletion by hypoxia or metabolic inhibition yields conflicting results. The number of /3-adrenoceptor binding sites is decreased (Marsh and Sweeney, 1989; Rocha-Singh et al., 19891 or incrcased (Buja et al., 1985; Thandroyen et al., 19891 with higher (Rocha-Singh et al., 1989: Thandroyen et al., 19891 or lower (Marsh and Sweeney, 19891 isoproterenol-stimu~ lated adenylyl cyclase activity. Therefore, the aim of the present study was to reinvestigate the long-term effect of two important aspects of myocardial ischemia - - loss of high-ener~' phosphates and elevated ca~.echolamine levels - - on /~-adrenoceptor-adenylyl cy-
Correspondence to: Dr. lBernhard Wagenknech:, Kreiskrankenhaus Fi.irstenfetdbruck, Interne Ableihmg, Dachauer S~.iasse 33. W-g0S{~ Fiirstenfeldbruck, German.',;. Tel. 08i 41/09-1 : Fax 08141/993{)4.
clase coupling. Cultured neonatal rat heart muscle cells were exposed for 24 h to the glycolytic inhibitor deoxyglucose in the presence or absence of noradrenaline.
2. Materials and methods The materials and methods have been described in dct.'Jil previously (Reithmann and Werdan, 1989; Reithmann et al.. 198o; Werdan and E r d m a n n !089) and are outlined here briefly.
2.1. Preparatic, n and culture o f cardiac muscle cel.ls 30-!0{1 hearts from 1- to 3-day-o!d neonatal rats were excised under sterile conditions and disaggregated at 3 7 ° C in a ~rypsin (0.12q~)-coHagenase ((l,(13C~)-salt solution (Ca r'-. Mg2+-free). The cell suspension was seeded into Nunclon plastic culture dishes ~1.()--...d × it." celts/era-) m CMRL 1415 ATM medium supplemented with 1()Ok fetal calf serum, 109; horse serum and 0.02 m g / m l gentamicin. After 24 h, serumcontaining medium was replaced by serum-free culture medium (CMRL 1415 ATM medium + 2 × 10 -~ M ;r~c,,l;r~ . . . . . . *t. . . . . . c + 4 x i6 7 M iron. . . . . . . . .q-. . .11"~ . . - 7 R,I . . . .rl~ .a,~a.~u~a,~ut saturated transferrin + 4 x 10 -7 M bovine serum albumin) supplemented with 211 mM Hepes, pH = 7.4. After 12 h i:; serum-free medium, the incubation solution was changed for a further 24 h to serum-free medium (2.5 mM glucose, 1 mM ascorbic acid)± 5 mM deoxyglucose in the presence or absence of 10 -a M
262
(-)-noradrenaline. After the culture period, cell-bound ( - ) - n o r a d r e n a l i n e was removcd by thorough washing of the cells in control medium (washing period: 2 h, 37 ° C).
2. 6. Statistical analysi~ Statistical significances were calculated according to variance analysis and Student's t-test for unpaired observations; P _< 0.05.
2.2. Receptor binding assay Specific [ 3 H ] ( - ) - C G P binding under equilibrium conditions was determined in Nunclon plastic culture dishes: (1-80) × 104 cpm [ 3 H ] ( - ) - C G P 12177/dish (1.5 x 10- t~ M to 9.7 x 10 -'~ M); specific activity 49 C i / m m o l (Amersham-Buchler, Braunschweig, Germany); temper',.ture 37 o C; incubation period 60 min; incubation volume 2.1 ml. Nonspecific binding was determined in the presence of 10 -5 M ( - ) - t i m o l o l and yielded about 1(1% of total binding at the KD of [ ~ H ] ( - ) - C G P 12177. The apparent affinity constant (K D) for [ ~ H ] ( - ) - C G P 12177 was derived from linear Scatchard plots.
3. Results
Long-term (24 h) ATP depletion in cultured neonatal rat heart muscle cells with 5 mM deoxyglucose reduces cell ATP content reversibly from 21.0 + 0.6
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Basal and agonist-stimulated cAMP formation was measured in heart muscle cells cultured in plastic flasks. 10 -6 M isoprenaline or 10 -5 M forskolin with 0.5 m m o l / l IBMX (3-isobutyl-l-methylxanthine) were added for an incubation period of 3 min after preequilibration of the cells in serum-free culture medium with 0.5 m m o l / l IBMX for 12 rain. The cells were lysed by the addition of ! ml 5% TCA for 1 h at 4 ° C and the samples were adjusted to pH 7 with 2 N NaOH. cAMP was isolated by sequential chromatography and determined with a c A M P assay kit (Amersham-Buchler) as previously described (Reithmann and Werdan, t989; Reithmann et al., 1989).
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2.4. Adenylyl cyclase assay in crude heart cell membranes E
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For preparation of crude heart muscle cell membranes, cells were scraped from the plastic culture flask and homogenized in ice-cold buffer A (10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride). The homogenates were centrifuged at 40,000 x g for 30 min at 4 °C, resuspended with a syringe needle in buffer A and frozen in liquid nitrogen. Adenylyl cyclase activity was determined as described in detail in Reithmann and Werdan (1989) and Reithmann et al, (1989).
2.5. A TP content Cells were rinsed with ice-cold calcium (1.8 mM)sorbitol (280 mM) solution and extracted on ice with perchloric acid (8%) for 15 min, ATP content of neutralized PCA extracts was determined by bioluminescence using luciferin/luciferase and checked by reverse-phase HPLC. Cell protein was measured according to the method of Lowry (Reithmann et al., 1989).
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Fig. 1. Effect of A T P depletion ( d e o ~ g l u c o s e ) - + n o r a d r e n a l i n e on specific [ 3 H ] ( - ) - C G P 12177 binding a n d isoproterenol- a n d forskolin-stimulated cAMP-formation. Heart muscle cells were incubated for 24 h in the presence of synthetic medium = control (C). 5 mM deox2,.'glucose (DG), l0 -~' M noradrenaline (NA) or 5 mM deo~glucose~- 10 - 6 M noradrenaline ( D G + N A ) . (A) Specific [ 3 H } ( - ) - C G P 12177 binding ( 6 . 1 × 1 0 - m M) was determined as described in Materials a n d methods. The values are given as mean_+ S.E.M.: n = 24-39. The difference in binding between C a n d D G , NA and D G + N A is significant: P_< 0.05. (B) lsoproterenol (10 - 6 M)- and (C) forskolin (10 -5 M)-stimulated cAMP-formation was measured in intact heart muscle cells as described in Materials a n d methods. D a t a are given as mean + S.E.M.: n = 8-15. The difference in c A M P formation between C a n d DG, N A a o d D G + N A is significant: P < 0.05.
263 n m o l / m g p r o t e i n to 1 2 . 9 + 0 , 6 n m o l / m g p r o t e i n ( m e a n + S.E.M.). A f t e r a n additional 24 h i n c u b a t i o n in culture m e d i u m w i t h o u t m e t a b o l i c inhibitors, cell A T P c o n t e n t r e t u r n s to control values (20.4 + 1.2 n m o l / m g p r o t e i n ; m e a n + S.E.M.). Fig. 1 shows the effect o f 5 m M deoxyglucose with o r w i t h o u t i0 -~' M n o r a d r e n a l i n e o n / 3 - a d r e n o c e p t o r density (specific C G P binding, fig. 1A) a n d c A M P - f o r m a t i o n (fig, 1B a n d C) in intact cells. 10 --6 M n o r a d r e n a l i n e desensitizes t h e /3-receptor-adenylyl cyclase system with a d e c r e a s e in specific [ 3 H ] ( - ) - C G P 12177 b i n d i n g to 5 9 . 8 + 1.7% a n d a fall in i s o p r o t e r e n o l - s t i n m l a t e d c A M P f o r m a t i o n to 41.7 + 3.2% a n d f o r s k o l i n - s t i m u l a t e d c A M P formation to 37.2 + 2.7%. T h e glycoiytic i n h i b i t o r deoxyglucose by itself r e d u c e s specific [ 3 H ] ( - ) - C G P 12177 b i n d i n g to 76.5 + 3.1% w i t h o u t any c h a n g e in affinity (K D control: 2 . 2 x 10 - t ° M; K D deoxyglucose: 2.5 x 1 0 - tn M; m e a n ; n = 2). I s o p r o t e r e n o l - s t i m u l a t e d c A M P f o r m a t i o n is 57.4 + 6.6% a n d f o r s k o l i n - s t i m u l a t e d c A M P f o r m a t i o n is 18.9 + 3.5% of control. In deoxyglucose-treated cells, n o r a d r e n a l i n e f u r t h e r r e d u c e s i s o p r o t e r e n o l - as well as f o r s k o l i n - s t i m u l a t e d c A M P f o r m a t i o n w i t h o u t a statistically significant d e c r e a s e in C G P binding. In c o n t r a s t to t h e results o b t a i n e d in i n t a c t cells, isopro~erenol (10 -4 M ) - s t i m u l a t e d adenylyl cyclase activity ( p m o l c A M P " 20 min - ~ - m g p r o t e i n - ~) in c r u d e m e m b r a n e p r e p a r a t i o n s of rat h e a r t muscle cells is r e d u c e d a f t e r n o r a d r e n a l i n e b u t n o t a f t e r deoxyglucose p r e t r e a t m e n t (control: 104.9_+ 17.9; nora d r e n a l i n e : 50.0 .+_ 13.4; deoxyglucose: 96.8 +__ 21.0; m e a n _+ S.D.; n = 3).
4. Discussion T h e i m p o r t a n c e of i s c h e r n i a - i n d u c e d A T P d e p l e t i o n for the a l t e r a t i o n s in t h e /3-receptor-adenylyl cyclase system is still controversial. U s i n g a well-defined m o d e l system of c u l t u r e d n e o n a t a l rat h e a r t muscle cells, which lacks s e c o n d a r y i n f l u e n c e s by t h e n e r v o u s syst e m , we show t h a t reversible l o n g - t e r m A T P d e p l e t i o n d e c r e a s e s / 3 - a d r e n o c e p t o r density a n d i s o p r o t e r e n o l as well as f o r s k o l i n - s t i m u l a t e d c A M P f o r m a t i o n . T h e s e d a t a are in good a g r e e m e n t with results o b t a i n e d in earlier s t u d i e s d u r i n g long periods of hypoxia ( M a r s h a n d Sweeney, 1989; T h a n d r o y e n et al., 1989). However, t h e d e c r e a s e in r e c e p t o r - m e d i a t e d adenylyl cyclase s t i m u l a t i o n a f t e r A T P d e p l e t i o n is only o b s e r v e d in intact cells, b u t n o t in m e m b r a n e p r e p a r a t i o n s . T h i s suggests t h a t t h e d e c r e a s e d c a t e c h o l a m i n e responsiveness of A T P - d e p l e t e d i n t a c t h e a r t muscle cells is n o t d u e to d a m a g e o r a l t e r a t i o n s of t h e adenylyl cyclase system itself. T o s i m u l a t e t h e c a t e c h o l a m i n e excess d u r i n g myocardial ischemia, t h e effect o f n o r a d r e n a l i n e in A T P d e p l e t e d h e a r t muscle cells was investigated. Nor-
a d r e n a l i n e f u r t h e r r e d u c e s r e c e p t o r - d e p e n d e n t (isop r o t e r e n o l s t i m u l a t i o n ) as well as r e c e p t o r - i n d e p e n d e n t (forskolin s t i m u l a t i o n ) c A M P f o r m a t i o n in A T P d e p l e t e d h e a r t cells. In conclusion, the data p r e s e n t e d h e r e show t h a t long periods of A T P d e p l e t i o n can i m p a i r / 3 - r e c e p t o r - a d e n y l y l cyclase signalling. T h e diff e r e n t results o 9 t a i n e d in intact cells a n d m e m b r a n e p r e p a r a t i o n s suggcst t h a t besides direct a l t e r a t i o n s of the / 3 - a d r e n o c e o t o r - G - p r o t e i n - a d e n y l y l cyclase axis - as i n d u c e d by c a t e c h o l a m i n e s - - also c h a n g e s in the i n t r a c e l l u l a r e n v i r o n m e n t , e.g. c h a n g e s in cellular subs t r a t e levels or ionic h o m e o s t a s i s have to be considered.
Acknowledgements We would like to ihank Ms. Renate Reng and Mrs. tteike Maier for their excellent technical assistance, Dr~ H. Metzger, Iloechst AG, Frankfurt a.M., Germany, for generously providing forskolin and Professor K. Werdan and Dr. C. Reithmann for their critical comments. The data shown in this paper are part of the thesis of C.B. This work was supported by the Deutsche Forschungsgemeinschaft and the Friedrich-Baur-Stiftung.
References Buja, M.L., K.H. Muntz, T. Rosenbaum, Z. Haghani, D.K. Buja, A. Sen. K.R. Chien and J.T. Willerson, 1985. Characterization of a potentially reversible increase in /3-adrenergic receptors in isolated, neonatal rat cardiac myocytes with impaired energy metabolism, Circ. Res. 57, 640. Maisel, A.S., H.J. Motulsky. M.G. Ziegler and P.A. lnsel, 1987, Ischemia- and agonist-induced changes in a- and /3-adrenergic receptor traffic in guinea pig hearts, Am. J, Physiol. 253, HI 159. Marsh, J.D. and K.A. Sweeney, 1989,/3-Adrenergic receptor regulation during hypoxia in intact cultured heart cells, Am. J. Physiol. 256, H275. Reithmann, C. and K. Werdan. 1989, Noradrenaline-induced desensitization in cultured heart cells as a model for the defects of the adenylate cyclase system in severe heart failure, Naunyn-Schmied. Arch. Pharmacol. 339. 138. Reithmann, C., P. Gierschik, D. Sidiropoulos, K. Werdan and K.H. Jakobs, 1989, Mechanism of noradrenaline-indvced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitor, G-protein a-subunits, Eur. J. Pharmacol. 172, 211. Roeha-Singh. K,, N. Honbo and J.S, Karliner, 1989, Independent effects of hypoxia and glucose deprivation on the /3-adrenoceptor-adenylate eyclase system in cuhured neonatal ventricular myocytes. J. Am. Coll. Cardiol. 13, 142A. Strasser, R.H.. J. Krimmer, R. Braun-Dullaeus, R. Marquetant and W. Kiibler, 1990, Dual sensitization of the adrenereSc system in early myocardial ischemia: independent regulation of the /3adrenergic receptors and the adenylyl cyclase, J. Mol. Cell. Cardiol. 22, 1405. Susanni, E.E., T.W. Manders, D.R. Knight, D,E. Vamer, S.F. Vatner and C.L Homcy. 1989, One hour ~,ff myocardial ischemia decreases the activity of the stimulatory guanine-nueleotide regulatory protein Gs, Circ. Res. 65, 1145. Thandroyen, F.T., K. Muntz, 1". Rosenbaum, B. Ziman, J.T. Wilierson and L.M. Buja, 1989,/3-Receptor-adenylate cyclase coupling
264 in hypoxic neonatal rat xentricular m~ocytcs+ Am. J. Physiol, 256, [ t 1209, Valner, D.E., D.R. KnighI, Y.T, Shen. J.X. Thomas, ('.J. [tomcy and S.F. Valner, IroNs, ()no htll.lr o! myocardial i>,chcnlia in cclnscious dogs increases ~-adrcnergic receptOlS, bnt dcCl'C[Ises adenyI:Jte cyclasc activity, J. Mol. ('ell. ('ardiol. 211, 75.
Werdun, K. and U. Erdmanm 1989, Preparation and culture of embryonic and neonatal heart muscle cells: nmdificalion of trimsport activity, Methods Enzynml, 173. 634. Wolff, A.A.. D.K. Ilines and J.S. Karlincr, 1989, Refined membrane preparations mask ischemic fall in myocardial /iLreceptor density, Am. J. Physiol. 257, 1111132.
261
E J P M O L 8{1{1~'~6
Short communication
Alterations of ,8-receptor-adenylyl cyclase coupling by long-term ATP depletion in cultured rat cardiomyocytes B e r n h a r d W a g e n k n e c h t and C l a u d i a B e u t t l e r Medizinische Klinik 1, Klinikum (;ro.~shadern, Uni~ el:fftb't Miinchen, IVS000 Munich 70. (;ermat~y Received II1 July 1991, revised MS received 7 August 1991, accepted 20 August 1991
In cultured rat heart muscle cells, reversible long-term ATP depiction induces a decrease in fl-adrcnoccptor density and a fall in isoproterenol- as well as forskolin-stimulated cAMP formation. However, isoproterenol-stimulatcd adenylyl cyclase activity in membrane preparations is not reduced after ATP depletion. These results suggest that the decreased responsiveness to catccholamines during myocardial ischemia cannot be explained by alterations of thc /3-adrenoceptor-adenylyl cyclase system alone. ATP depletion; Deoxyglucosc; Noradrenaline:/3-Adrcnoccptor; Adenylyl cyclase, Cardiomyocytes
I. Introduction Acute myocardiai ischemia induces in intact heart preparations an early sensitization of the/3-adrenoceptor-adenylyl cyclase system (Maisel et al., 1987; Strasser et al., 19901 followed by a late decline of adenylyl cyclase activity (Vatner et al., 1988; Wolff et al., 1989; Strasser et al., 19901 and a decrease of the activity of G~ (Susanni et al., 1989). As inhibition of oxidative phosphor3,1ation by cyanide results also in a receptorspecific sensitization and in an abolition of catecholamine-induced descnsitizahon, tile ios5 of high-energy phosphates seems to play a key role in the regulation of ,8-adrcnoceptor-adenylyl cyclase durip.g ischemia (Strasser et al., 19901. In cultured heart muscle cells, however, ATP depletion by hypoxia or metabolic inhibition yields conflicting results. The number of /3-adrenoceptor binding sites is decreased (Marsh and Sweeney, 1989; Rocha-Singh et al., 19891 or incrcased (Buja et al., 1985; Thandroyen et al., 19891 with higher (Rocha-Singh et al., 1989: Thandroyen et al., 19891 or lower (Marsh and Sweeney, 19891 isoproterenol-stimu~ lated adenylyl cyclase activity. Therefore, the aim of the present study was to reinvestigate the long-term effect of two important aspects of myocardial ischemia - - loss of high-ener~' phosphates and elevated ca~.echolamine levels - - on /~-adrenoceptor-adenylyl cy-
Correspondence to: Dr. lBernhard Wagenknech:, Kreiskrankenhaus Fi.irstenfetdbruck, Interne Ableihmg, Dachauer S~.iasse 33. W-g0S{~ Fiirstenfeldbruck, German.',;. Tel. 08i 41/09-1 : Fax 08141/993{)4.
clase coupling. Cultured neonatal rat heart muscle cells were exposed for 24 h to the glycolytic inhibitor deoxyglucose in the presence or absence of noradrenaline.
2. Materials and methods The materials and methods have been described in dct.'Jil previously (Reithmann and Werdan, 1989; Reithmann et al.. 198o; Werdan and E r d m a n n !089) and are outlined here briefly.
2.1. Preparatic, n and culture o f cardiac muscle cel.ls 30-!0{1 hearts from 1- to 3-day-o!d neonatal rats were excised under sterile conditions and disaggregated at 3 7 ° C in a ~rypsin (0.12q~)-coHagenase ((l,(13C~)-salt solution (Ca r'-. Mg2+-free). The cell suspension was seeded into Nunclon plastic culture dishes ~1.()--...d × it." celts/era-) m CMRL 1415 ATM medium supplemented with 1()Ok fetal calf serum, 109; horse serum and 0.02 m g / m l gentamicin. After 24 h, serumcontaining medium was replaced by serum-free culture medium (CMRL 1415 ATM medium + 2 × 10 -~ M ;r~c,,l;r~ . . . . . . *t. . . . . . c + 4 x i6 7 M iron. . . . . . . . .q-. . .11"~ . . - 7 R,I . . . .rl~ .a,~a.~u~a,~ut saturated transferrin + 4 x 10 -7 M bovine serum albumin) supplemented with 211 mM Hepes, pH = 7.4. After 12 h i:; serum-free medium, the incubation solution was changed for a further 24 h to serum-free medium (2.5 mM glucose, 1 mM ascorbic acid)± 5 mM deoxyglucose in the presence or absence of 10 -a M
262
(-)-noradrenaline. After the culture period, cell-bound ( - ) - n o r a d r e n a l i n e was removcd by thorough washing of the cells in control medium (washing period: 2 h, 37 ° C).
2. 6. Statistical analysi~ Statistical significances were calculated according to variance analysis and Student's t-test for unpaired observations; P _< 0.05.
2.2. Receptor binding assay Specific [ 3 H ] ( - ) - C G P binding under equilibrium conditions was determined in Nunclon plastic culture dishes: (1-80) × 104 cpm [ 3 H ] ( - ) - C G P 12177/dish (1.5 x 10- t~ M to 9.7 x 10 -'~ M); specific activity 49 C i / m m o l (Amersham-Buchler, Braunschweig, Germany); temper',.ture 37 o C; incubation period 60 min; incubation volume 2.1 ml. Nonspecific binding was determined in the presence of 10 -5 M ( - ) - t i m o l o l and yielded about 1(1% of total binding at the KD of [ ~ H ] ( - ) - C G P 12177. The apparent affinity constant (K D) for [ ~ H ] ( - ) - C G P 12177 was derived from linear Scatchard plots.
3. Results
Long-term (24 h) ATP depletion in cultured neonatal rat heart muscle cells with 5 mM deoxyglucose reduces cell ATP content reversibly from 21.0 + 0.6
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9_.3. Cyclic AMP formation assay •~
Basal and agonist-stimulated cAMP formation was measured in heart muscle cells cultured in plastic flasks. 10 -6 M isoprenaline or 10 -5 M forskolin with 0.5 m m o l / l IBMX (3-isobutyl-l-methylxanthine) were added for an incubation period of 3 min after preequilibration of the cells in serum-free culture medium with 0.5 m m o l / l IBMX for 12 rain. The cells were lysed by the addition of ! ml 5% TCA for 1 h at 4 ° C and the samples were adjusted to pH 7 with 2 N NaOH. cAMP was isolated by sequential chromatography and determined with a c A M P assay kit (Amersham-Buchler) as previously described (Reithmann and Werdan, t989; Reithmann et al., 1989).
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2.4. Adenylyl cyclase assay in crude heart cell membranes E
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For preparation of crude heart muscle cell membranes, cells were scraped from the plastic culture flask and homogenized in ice-cold buffer A (10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride). The homogenates were centrifuged at 40,000 x g for 30 min at 4 °C, resuspended with a syringe needle in buffer A and frozen in liquid nitrogen. Adenylyl cyclase activity was determined as described in detail in Reithmann and Werdan (1989) and Reithmann et al, (1989).
2.5. A TP content Cells were rinsed with ice-cold calcium (1.8 mM)sorbitol (280 mM) solution and extracted on ice with perchloric acid (8%) for 15 min, ATP content of neutralized PCA extracts was determined by bioluminescence using luciferin/luciferase and checked by reverse-phase HPLC. Cell protein was measured according to the method of Lowry (Reithmann et al., 1989).
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DG+NA
Fig. 1. Effect of A T P depletion ( d e o ~ g l u c o s e ) - + n o r a d r e n a l i n e on specific [ 3 H ] ( - ) - C G P 12177 binding a n d isoproterenol- a n d forskolin-stimulated cAMP-formation. Heart muscle cells were incubated for 24 h in the presence of synthetic medium = control (C). 5 mM deox2,.'glucose (DG), l0 -~' M noradrenaline (NA) or 5 mM deo~glucose~- 10 - 6 M noradrenaline ( D G + N A ) . (A) Specific [ 3 H } ( - ) - C G P 12177 binding ( 6 . 1 × 1 0 - m M) was determined as described in Materials a n d methods. The values are given as mean_+ S.E.M.: n = 24-39. The difference in binding between C a n d D G , NA and D G + N A is significant: P_< 0.05. (B) lsoproterenol (10 - 6 M)- and (C) forskolin (10 -5 M)-stimulated cAMP-formation was measured in intact heart muscle cells as described in Materials a n d methods. D a t a are given as mean + S.E.M.: n = 8-15. The difference in c A M P formation between C a n d DG, N A a o d D G + N A is significant: P < 0.05.
263 n m o l / m g p r o t e i n to 1 2 . 9 + 0 , 6 n m o l / m g p r o t e i n ( m e a n + S.E.M.). A f t e r a n additional 24 h i n c u b a t i o n in culture m e d i u m w i t h o u t m e t a b o l i c inhibitors, cell A T P c o n t e n t r e t u r n s to control values (20.4 + 1.2 n m o l / m g p r o t e i n ; m e a n + S.E.M.). Fig. 1 shows the effect o f 5 m M deoxyglucose with o r w i t h o u t i0 -~' M n o r a d r e n a l i n e o n / 3 - a d r e n o c e p t o r density (specific C G P binding, fig. 1A) a n d c A M P - f o r m a t i o n (fig, 1B a n d C) in intact cells. 10 --6 M n o r a d r e n a l i n e desensitizes t h e /3-receptor-adenylyl cyclase system with a d e c r e a s e in specific [ 3 H ] ( - ) - C G P 12177 b i n d i n g to 5 9 . 8 + 1.7% a n d a fall in i s o p r o t e r e n o l - s t i n m l a t e d c A M P f o r m a t i o n to 41.7 + 3.2% a n d f o r s k o l i n - s t i m u l a t e d c A M P formation to 37.2 + 2.7%. T h e glycoiytic i n h i b i t o r deoxyglucose by itself r e d u c e s specific [ 3 H ] ( - ) - C G P 12177 b i n d i n g to 76.5 + 3.1% w i t h o u t any c h a n g e in affinity (K D control: 2 . 2 x 10 - t ° M; K D deoxyglucose: 2.5 x 1 0 - tn M; m e a n ; n = 2). I s o p r o t e r e n o l - s t i m u l a t e d c A M P f o r m a t i o n is 57.4 + 6.6% a n d f o r s k o l i n - s t i m u l a t e d c A M P f o r m a t i o n is 18.9 + 3.5% of control. In deoxyglucose-treated cells, n o r a d r e n a l i n e f u r t h e r r e d u c e s i s o p r o t e r e n o l - as well as f o r s k o l i n - s t i m u l a t e d c A M P f o r m a t i o n w i t h o u t a statistically significant d e c r e a s e in C G P binding. In c o n t r a s t to t h e results o b t a i n e d in i n t a c t cells, isopro~erenol (10 -4 M ) - s t i m u l a t e d adenylyl cyclase activity ( p m o l c A M P " 20 min - ~ - m g p r o t e i n - ~) in c r u d e m e m b r a n e p r e p a r a t i o n s of rat h e a r t muscle cells is r e d u c e d a f t e r n o r a d r e n a l i n e b u t n o t a f t e r deoxyglucose p r e t r e a t m e n t (control: 104.9_+ 17.9; nora d r e n a l i n e : 50.0 .+_ 13.4; deoxyglucose: 96.8 +__ 21.0; m e a n _+ S.D.; n = 3).
4. Discussion T h e i m p o r t a n c e of i s c h e r n i a - i n d u c e d A T P d e p l e t i o n for the a l t e r a t i o n s in t h e /3-receptor-adenylyl cyclase system is still controversial. U s i n g a well-defined m o d e l system of c u l t u r e d n e o n a t a l rat h e a r t muscle cells, which lacks s e c o n d a r y i n f l u e n c e s by t h e n e r v o u s syst e m , we show t h a t reversible l o n g - t e r m A T P d e p l e t i o n d e c r e a s e s / 3 - a d r e n o c e p t o r density a n d i s o p r o t e r e n o l as well as f o r s k o l i n - s t i m u l a t e d c A M P f o r m a t i o n . T h e s e d a t a are in good a g r e e m e n t with results o b t a i n e d in earlier s t u d i e s d u r i n g long periods of hypoxia ( M a r s h a n d Sweeney, 1989; T h a n d r o y e n et al., 1989). However, t h e d e c r e a s e in r e c e p t o r - m e d i a t e d adenylyl cyclase s t i m u l a t i o n a f t e r A T P d e p l e t i o n is only o b s e r v e d in intact cells, b u t n o t in m e m b r a n e p r e p a r a t i o n s . T h i s suggests t h a t t h e d e c r e a s e d c a t e c h o l a m i n e responsiveness of A T P - d e p l e t e d i n t a c t h e a r t muscle cells is n o t d u e to d a m a g e o r a l t e r a t i o n s of t h e adenylyl cyclase system itself. T o s i m u l a t e t h e c a t e c h o l a m i n e excess d u r i n g myocardial ischemia, t h e effect o f n o r a d r e n a l i n e in A T P d e p l e t e d h e a r t muscle cells was investigated. Nor-
a d r e n a l i n e f u r t h e r r e d u c e s r e c e p t o r - d e p e n d e n t (isop r o t e r e n o l s t i m u l a t i o n ) as well as r e c e p t o r - i n d e p e n d e n t (forskolin s t i m u l a t i o n ) c A M P f o r m a t i o n in A T P d e p l e t e d h e a r t cells. In conclusion, the data p r e s e n t e d h e r e show t h a t long periods of A T P d e p l e t i o n can i m p a i r / 3 - r e c e p t o r - a d e n y l y l cyclase signalling. T h e diff e r e n t results o 9 t a i n e d in intact cells a n d m e m b r a n e p r e p a r a t i o n s suggcst t h a t besides direct a l t e r a t i o n s of the / 3 - a d r e n o c e o t o r - G - p r o t e i n - a d e n y l y l cyclase axis - as i n d u c e d by c a t e c h o l a m i n e s - - also c h a n g e s in the i n t r a c e l l u l a r e n v i r o n m e n t , e.g. c h a n g e s in cellular subs t r a t e levels or ionic h o m e o s t a s i s have to be considered.
Acknowledgements We would like to ihank Ms. Renate Reng and Mrs. tteike Maier for their excellent technical assistance, Dr~ H. Metzger, Iloechst AG, Frankfurt a.M., Germany, for generously providing forskolin and Professor K. Werdan and Dr. C. Reithmann for their critical comments. The data shown in this paper are part of the thesis of C.B. This work was supported by the Deutsche Forschungsgemeinschaft and the Friedrich-Baur-Stiftung.
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