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Modulation of muscarinic receptors on cultured rat cardiocytes by corticosteroids
J Mol Cell Cardiol 17 (Supplement 3) (1985)
IggMODULATION OF MUSCARINIC RECEPTORS ON CULTURED RAT CARDIOCYTES BY CORTICOSTEROIDS: L. Ransn~is, B. Jacobsson, E. Sabler, A. Hjalmarson. Department of Medicine I, University of Gothenburg, Gothenburg, Sweden. Adenylate cyclase is under dual control in cardiocytes, stimulation via beta-adrenergic and inhibition via muscarinic receptors. Previous findings of affinity changes induced by corticosteroids given in-vivo to rats on muscarinic receptor affinity towards agonists are confirmed in a tissue culture system. Exposure of rat cardiocytes in tissue culture to 0.i ~g/ml of hydrocortisone induces an affinity shift of musearinic carbachol binding, measurable both on whole cells and crude membrane preparations. T h e affinity shift is detectable after 6 h exposure to hydrocortisone and consists of a transition of about 25% of the muscarinic receptors from the high(Kd= 7 I~M) towards the super-high(Ka= 0.3 juM) affinity state. T h e affinity shift is ~abolished by inclusion of a protein~ synthesis inhibitor( i0 IIM puromycin) during the steroid exposure while addition of a receptor glycosylation inhibitor, tunicamycin) does not affect the affinity shift. The type of affinity shift is analogous to the shift induced by manganese ions but, in contrast, the shift towards lower affinity induced by guanine nucleotides in the presence of manganese ions is modulated by cortieosteroid exposure. This may be indicative of, either an increased affinity of the M2-muscarinie receptor subtype or a muscarinic receptor subtype interconversion, afte~rsteroid exposure.
2 0 0 R E G U L A T I O N OF CARDIAC ADENYLATE CYCLASE BY GDP A N D GTP. S.E. Harding, P. Harris. Cardiothoracic Institute, 2 Beaumont Street, London W I N 2DX, England. It is well known that GTP increases basal activity of cardiac adenylate cyelase, and supports beta-adrenoeeptor stimulation. The effects of GDP have been more difficult to determine, because GDP is converted to GTP by nucleaside diphosphate kinase (NDK) during the cyclase assay. NDK is associated with the plasma membrane, and uses ATP present in the assay medium to phosphorylate GDP. By lowering the sorcalemmal protein concentration in the medium to 1-2Hg/ml, we have been able to minimise transphosphorylation, so that only 0.1% of an added 1001Jv~ GDP was converted to GTP during the 10 min assay. The low levels of cAMP produced ('~10 fmols/tube) were measured using the acetylated procedure of the NEN RIA kit. GDP was equipotent with GTP in raising basal cyclase activity, but did not support stimulation by isaprenaline. 1001.dv~GDP prevented completely the increase in activity due to 101~A Gpp(NH)p. It is concluded that GTP hydrolysis by the intrinsic GTPase of the Ns-protein may "turn off" activation by hormone plus GTP, but not by GTP alone. We find no evidence that Ns binds tightly the GDP produced by hydrolysis: this is contrary to findings with turkey erythroeyte membranes. If GTP and GDP are freely exchangeable, then the level of beta-receptor stimulation of adenylate cyclase may be regulated by the GTP/GDP ratio in the cytoplasm of the cell. (This work was supported by a grant from the Medical Research Council).
2 0 1 O N E E N A N T I O M E R OF THE P O S I T I V E I N O T R O P I C D I H Y D R O P Y R I D I N E B A Y K 8644 IS A C A L C I U M - A N T A G O N I S T . M. S c h r a m m , M. Bechem, G. F r a n c k o w i a k , R. GroB, G. Thomas. B a y e r P h a r m a R e s e a r c h C e n t e r , D - 5 6 0 0 W u p p e r t a l i, FRG. BAY K 8644 is a n i f e d i p i n e - l i k e d i h y d r o p y r i d i n e , w h i c h e n h a n c e s ins t e a d of d e c r e a s e s C a - i n f l u x t h r o u g h v o l t a g e - s e n s i t i v e C a - c h a n n e l s . We s e p a r a t e d the o p t i c a l i s o m e r s of B A Y K 8644 w i t h a p u r i t y of m o r e t h a n 99.5% and c h a r a c t e r i z e d t h e i r a c t i o n s in r a b b i t a o r t i c p r e p a r a t i o n s . T h e ( - ) - e n a n t i o m e r shows ~he p h a r m a c o l o g i c a l p r o f i l e of the r a e e m i c c o m p o u n d : at 2.7 m m o l / l K- it h~s no e f f e c t s on the tone of a o r t i c rings, b u t it p o t e n t i a t e s the K - - i n d u c e d c o n t r a c t i o n s . U n d e r p a r t l y dep o l a r i z e d c o n d i t i o n s (+ 15 m m o l / l KCI, still s u b t h r e s h o l d for K - - i n ducedQcontractions) it c o n t r a c t s a o r t i c r i n g s d o s e ~ d e p e n d e n t l y (ED
IggMODULATION OF MUSCARINIC RECEPTORS ON CULTURED RAT CARDIOCYTES BY CORTICOSTEROIDS: L. Ransn~is, B. Jacobsson, E. Sabler, A. Hjalmarson. Department of Medicine I, University of Gothenburg, Gothenburg, Sweden. Adenylate cyclase is under dual control in cardiocytes, stimulation via beta-adrenergic and inhibition via muscarinic receptors. Previous findings of affinity changes induced by corticosteroids given in-vivo to rats on muscarinic receptor affinity towards agonists are confirmed in a tissue culture system. Exposure of rat cardiocytes in tissue culture to 0.i ~g/ml of hydrocortisone induces an affinity shift of musearinic carbachol binding, measurable both on whole cells and crude membrane preparations. T h e affinity shift is detectable after 6 h exposure to hydrocortisone and consists of a transition of about 25% of the muscarinic receptors from the high(Kd= 7 I~M) towards the super-high(Ka= 0.3 juM) affinity state. T h e affinity shift is ~abolished by inclusion of a protein~ synthesis inhibitor( i0 IIM puromycin) during the steroid exposure while addition of a receptor glycosylation inhibitor, tunicamycin) does not affect the affinity shift. The type of affinity shift is analogous to the shift induced by manganese ions but, in contrast, the shift towards lower affinity induced by guanine nucleotides in the presence of manganese ions is modulated by cortieosteroid exposure. This may be indicative of, either an increased affinity of the M2-muscarinie receptor subtype or a muscarinic receptor subtype interconversion, afte~rsteroid exposure.
2 0 0 R E G U L A T I O N OF CARDIAC ADENYLATE CYCLASE BY GDP A N D GTP. S.E. Harding, P. Harris. Cardiothoracic Institute, 2 Beaumont Street, London W I N 2DX, England. It is well known that GTP increases basal activity of cardiac adenylate cyelase, and supports beta-adrenoeeptor stimulation. The effects of GDP have been more difficult to determine, because GDP is converted to GTP by nucleaside diphosphate kinase (NDK) during the cyclase assay. NDK is associated with the plasma membrane, and uses ATP present in the assay medium to phosphorylate GDP. By lowering the sorcalemmal protein concentration in the medium to 1-2Hg/ml, we have been able to minimise transphosphorylation, so that only 0.1% of an added 1001Jv~ GDP was converted to GTP during the 10 min assay. The low levels of cAMP produced ('~10 fmols/tube) were measured using the acetylated procedure of the NEN RIA kit. GDP was equipotent with GTP in raising basal cyclase activity, but did not support stimulation by isaprenaline. 1001.dv~GDP prevented completely the increase in activity due to 101~A Gpp(NH)p. It is concluded that GTP hydrolysis by the intrinsic GTPase of the Ns-protein may "turn off" activation by hormone plus GTP, but not by GTP alone. We find no evidence that Ns binds tightly the GDP produced by hydrolysis: this is contrary to findings with turkey erythroeyte membranes. If GTP and GDP are freely exchangeable, then the level of beta-receptor stimulation of adenylate cyclase may be regulated by the GTP/GDP ratio in the cytoplasm of the cell. (This work was supported by a grant from the Medical Research Council).
2 0 1 O N E E N A N T I O M E R OF THE P O S I T I V E I N O T R O P I C D I H Y D R O P Y R I D I N E B A Y K 8644 IS A C A L C I U M - A N T A G O N I S T . M. S c h r a m m , M. Bechem, G. F r a n c k o w i a k , R. GroB, G. Thomas. B a y e r P h a r m a R e s e a r c h C e n t e r , D - 5 6 0 0 W u p p e r t a l i, FRG. BAY K 8644 is a n i f e d i p i n e - l i k e d i h y d r o p y r i d i n e , w h i c h e n h a n c e s ins t e a d of d e c r e a s e s C a - i n f l u x t h r o u g h v o l t a g e - s e n s i t i v e C a - c h a n n e l s . We s e p a r a t e d the o p t i c a l i s o m e r s of B A Y K 8644 w i t h a p u r i t y of m o r e t h a n 99.5% and c h a r a c t e r i z e d t h e i r a c t i o n s in r a b b i t a o r t i c p r e p a r a t i o n s . T h e ( - ) - e n a n t i o m e r shows ~he p h a r m a c o l o g i c a l p r o f i l e of the r a e e m i c c o m p o u n d : at 2.7 m m o l / l K- it h~s no e f f e c t s on the tone of a o r t i c rings, b u t it p o t e n t i a t e s the K - - i n d u c e d c o n t r a c t i o n s . U n d e r p a r t l y dep o l a r i z e d c o n d i t i o n s (+ 15 m m o l / l KCI, still s u b t h r e s h o l d for K - - i n ducedQcontractions) it c o n t r a c t s a o r t i c r i n g s d o s e ~ d e p e n d e n t l y (ED