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Influence of aldosterone and its antagonists on rat cardiac function
2239 P.fr.139 ]
Physiological role of the new ouabain receptor protein (31.5 KD) from cat cardiac muscle, using moneclonal AB against the protein Fujino, S., Togashi, K., Nakai, T., Satoh, K., Kado, T. and Fujino *, M. Dept. of Pharmacology, Hokkaido Inst. of Pharmaceutical Sciences, Otaru, 047-02 and * Dept. of Pharmacology, Sapporo Medical College, Sapporo, Japan
We recently reported that a new glycoprotein of 31.5 KD, which has a high affinity for ouabain and is independent of (Na+-K +) ATPase, was ~olub'dized from transverse tubule membrane-junctional SR complexes ('ITM-JSR) in cat cardiac ventricle muscles (Fujino et al., 1989). To clarify the mechanism of ouabain potentiation, physiolcr~cal role of the protein was studied using a monoclonal antibody (AB) against the protein. Monoclonal hybridoma cell lines secreting AB against the 31.5 KD protein from cat cardiac TI'M-JSR were isolated. The AB was purified using protein A affmity chromatography. 1. The specificity of the AB was established by it's ability to coimmunoprecipitate the 31.5 KD protein. The AB revealed a single immunoprecipitating component of molecular weight of 31.5 KD from CHAPS extracts of non-reducing condition. 2. Kitten papillary muscles driven electrically (0.1 Hz) were immersed in a Tyrode solution containing the AB for 60 rain, and then washed out with Tyrode solution. Thirty rain after the AB removal, both twitch and K contracture were inhibited without changes of resting- and action -potentials and caffeine contracture. 3. The kitten papillary muscles were immersed in a Tyrode solution containing 5 mM 14C-phenylglyoxal (PGO), a ar~inirte-specific modifying reagent, for 10 min and then washed out with Tyrode solution. Sixty min after the PGO removal, the twitch was markedly inhibited without change of the action potential. At this point, C-PGO binding protein was solubilized from TI'M-JSR in the muscles by means of Concanavalin A affmity chromatography. The SDS PAGE-profile showed that molecular size of the protein is 31.5 KD under a non-reducing condition. The results indicate that the same protein of 31.5 KD was ~ e receptor for not only ouabain but also PGO. Antagonistic effects of ouabain and PGO on the E-C coupling process in cardiac muscles were recognized. 4. This AB cross-reacted with the PGO binding gl3coprotein (31.5 KD), which was obt?~aed using a similar procedure from skeletal muscle TTM-JSR of frog and has been considered a key protein in the excitation contraction (E-C) link in skeletal muscles (Fujino et al., 1986). Conclusion: Results suggest that the new ouabain receptor protein of 31.5 KD would be ~ key protein in the E-C coupling process in the cardiac muscles.
Reference 1) S. Fujino et al.; Experientia 45, 467 (1989). 2) M. Fujino et al.; Proc. Int. Union Physiol. Sci. 16, 207 (1986)2). P.fr.140 [
Influence of aldosterone and its antagonists on rat cardiac function Moreau, D., Chardigny, J.-M. and Pelletier *, B. Laboratoire de Physiologie, Facult~ de M~decine, F-21033 Dijon cedex and * IxdToratories SEARLE, 52 rue Marcel Dassauh, F-92514 Boulogne-Billancourt cedex, France
Aldosterone antagonists are used in hypertension therapy for over approximatively 25 years. But only a few studies have been reported about the cardiac effects of these pharmacological products. On the other hand, the influence of
72.~40 aldosterone on the myocardium has not been yet clearly considered. Therefore, in the present work, the direct cardiac effects of aldosterone and two antagonists, spironolactone and potassium canrenoate were examined in isolated perfused rat hear~ Hearts from Sprague-Dawley male rats (IFFA CREDO) weighing 342 4-6 g (n--47) ,.vere perfused by atrial channel with a modified Krebs-Henseleit saline solution. The saline contained 1,2 propanediol (1~ vol) as vehicle for aidosterone and spironolactone and glucose (5.5 m m o H - l ) was the only exogenous substrate. The vehicle did not affect the functional parameters of hearts. The dose-response relation of rat hearts (n--5) was established by ir~reasing the aldosterone concentration ~t~ the perfusion fluid front 10-13 to 10-s moll-!. Aldosterone significatively decreased the coronary flow (EDs0 - 1.52 10 -9 moll -1) and increased the aortic flow (EDso ~ 7.89 10 -11 mol.l-l). Remaining rat hearts were perfused for 90 min. Aldosterone (10 -s'¢° mo1.1-11 was added to the perfusion fluid from the 30th to the 60th min of atrial perfusion. Some of the hearts received one of both antagonists studied from the 20th to the 70th rain of perfusion. The concentration of those antagonists was 10-5 m o l l - i . The addition of aidosterone (10 -s moll -1) in the perfusion fluid resulted in a significant decrease in the coronary flow ( - 4 ~ 1 and a marked increased in both aortic flow ( + 21~;) and cardiac output ( + 11~;) as well as stroke volume ( + 8~). Spironolactone inhibited the coronary flow decrease and limited both aortic flow and cardiac output increase ( + 3~; and + 2~ respectively, P < 0.005). In the opposite, potassium canrenoate abolished both aortic and cardiac output increase, but the coronary flow breakdown was greater ( - 11~, P < 0.005). We concluded that aldosterone presented a positive inotropic effect on the rat myccardium and a vasoconstrictive influeace on coronary arteries. Spironolactone inhibited both these effects and acted as an aldosterone antagonist in the rat heart. Con t,he other hand, potassium canrenoate only inhibited the inotropic effcc*,s of aldosterone, but enhanced its vasoconstrictive eff~t. These differences between both aldosterone antagonists studied could be due either to different m~hanisms in the action of aldosterone on vasomuscular cells and myocytes, as illustrated by the difference between the dose-r~,t.'~,onse relation, or to different specific effects of both antagonists considered. Further studies are needed to precise the mechanis--,~_~involved and consider clinical implications. Supported by grant from the Searle Fondation for Hypertension Research. P.fr.141 [
Influence of adenosine receptor agonists and antagonists on cAMP-dependent protein kinase activity and contractility in guinea-pig papillary muscles Stein, B., Danielsen, W., Neumann, J., Schmitz, W., Scholz, H. and Starbatty, J. Abteilung Allgemeine Pharmakologie, Universitiits-Krankenhaus Eppendorf, MartinistraBe 32, D.2000 Hamburg 20, F.R.G. In ventricular cardiac muscle adenosine exerts negative inotropic effects in the presence of cAMP-increasing agents while adenosine alone has no negative inotropic or even a weak positive inotropic effect (Schrader et aI., 1977). The mechanism of the negative inotropic effect is still a matter of controversy. A decrease in cellular cAMP level due to an inhibition of the adenylate cyclase has been reported (Schrader et al., 19771 while others found no change in the cellular cAMP content (Schmitz et al., 1985). Therefore, we investigated whether the effects of the A:-adenosine receptor (AR) agonist (-)-Ne-phenyfisopropyladenosine (R-PIA) and the A2-AR agonist 5'-N-ethylcarboxamidadeno-sine (NECAi in the presence of isoprenaline (ISO) on force of contraction (FC) in guinea-pig papillar muscles are ac~companied by a corresponding reduction in cAMP-dependent protein kinase (PKA) activity. Th~ influence of the selective A~- and A2-AR antagonists 1,3-dipropyi-8-cyclopentylxanthine (DPCPX) and 9-chloro-2(2-furanyl)-5,6-dihy&-o-l,2,4-triazolo[l,5-c] quinazolin-5-imine (CGS 15943A) on the effects of R-PIA and NECA on PICA-activity and FC were studied. In electrically driven (1Hz) guinea-pig papillary muscles the PKA activity ratio as defined as enzyme activity ratio in the absence and presence of 5 Itmol/l cAMP was determined according to Keely et al. (1975). All experiments were performed in the presence of adenosine deaminase (1/~g/nd) to exclude interference from endogenous adenosine. ISO at a concentration of 0.01/tmol/l increased FC to about 330 + 29~$. Additionally applied R-PIA or NECA (1/tmol/1 each) reduced FC to 241 + 29% and 217 + 7~ respectively (n -- 6-8, p 0.051. In the same preparations ISO increased
Physiological role of the new ouabain receptor protein (31.5 KD) from cat cardiac muscle, using moneclonal AB against the protein Fujino, S., Togashi, K., Nakai, T., Satoh, K., Kado, T. and Fujino *, M. Dept. of Pharmacology, Hokkaido Inst. of Pharmaceutical Sciences, Otaru, 047-02 and * Dept. of Pharmacology, Sapporo Medical College, Sapporo, Japan
We recently reported that a new glycoprotein of 31.5 KD, which has a high affinity for ouabain and is independent of (Na+-K +) ATPase, was ~olub'dized from transverse tubule membrane-junctional SR complexes ('ITM-JSR) in cat cardiac ventricle muscles (Fujino et al., 1989). To clarify the mechanism of ouabain potentiation, physiolcr~cal role of the protein was studied using a monoclonal antibody (AB) against the protein. Monoclonal hybridoma cell lines secreting AB against the 31.5 KD protein from cat cardiac TI'M-JSR were isolated. The AB was purified using protein A affmity chromatography. 1. The specificity of the AB was established by it's ability to coimmunoprecipitate the 31.5 KD protein. The AB revealed a single immunoprecipitating component of molecular weight of 31.5 KD from CHAPS extracts of non-reducing condition. 2. Kitten papillary muscles driven electrically (0.1 Hz) were immersed in a Tyrode solution containing the AB for 60 rain, and then washed out with Tyrode solution. Thirty rain after the AB removal, both twitch and K contracture were inhibited without changes of resting- and action -potentials and caffeine contracture. 3. The kitten papillary muscles were immersed in a Tyrode solution containing 5 mM 14C-phenylglyoxal (PGO), a ar~inirte-specific modifying reagent, for 10 min and then washed out with Tyrode solution. Sixty min after the PGO removal, the twitch was markedly inhibited without change of the action potential. At this point, C-PGO binding protein was solubilized from TI'M-JSR in the muscles by means of Concanavalin A affmity chromatography. The SDS PAGE-profile showed that molecular size of the protein is 31.5 KD under a non-reducing condition. The results indicate that the same protein of 31.5 KD was ~ e receptor for not only ouabain but also PGO. Antagonistic effects of ouabain and PGO on the E-C coupling process in cardiac muscles were recognized. 4. This AB cross-reacted with the PGO binding gl3coprotein (31.5 KD), which was obt?~aed using a similar procedure from skeletal muscle TTM-JSR of frog and has been considered a key protein in the excitation contraction (E-C) link in skeletal muscles (Fujino et al., 1986). Conclusion: Results suggest that the new ouabain receptor protein of 31.5 KD would be ~ key protein in the E-C coupling process in the cardiac muscles.
Reference 1) S. Fujino et al.; Experientia 45, 467 (1989). 2) M. Fujino et al.; Proc. Int. Union Physiol. Sci. 16, 207 (1986)2). P.fr.140 [
Influence of aldosterone and its antagonists on rat cardiac function Moreau, D., Chardigny, J.-M. and Pelletier *, B. Laboratoire de Physiologie, Facult~ de M~decine, F-21033 Dijon cedex and * IxdToratories SEARLE, 52 rue Marcel Dassauh, F-92514 Boulogne-Billancourt cedex, France
Aldosterone antagonists are used in hypertension therapy for over approximatively 25 years. But only a few studies have been reported about the cardiac effects of these pharmacological products. On the other hand, the influence of
72.~40 aldosterone on the myocardium has not been yet clearly considered. Therefore, in the present work, the direct cardiac effects of aldosterone and two antagonists, spironolactone and potassium canrenoate were examined in isolated perfused rat hear~ Hearts from Sprague-Dawley male rats (IFFA CREDO) weighing 342 4-6 g (n--47) ,.vere perfused by atrial channel with a modified Krebs-Henseleit saline solution. The saline contained 1,2 propanediol (1~ vol) as vehicle for aidosterone and spironolactone and glucose (5.5 m m o H - l ) was the only exogenous substrate. The vehicle did not affect the functional parameters of hearts. The dose-response relation of rat hearts (n--5) was established by ir~reasing the aldosterone concentration ~t~ the perfusion fluid front 10-13 to 10-s moll-!. Aldosterone significatively decreased the coronary flow (EDs0 - 1.52 10 -9 moll -1) and increased the aortic flow (EDso ~ 7.89 10 -11 mol.l-l). Remaining rat hearts were perfused for 90 min. Aldosterone (10 -s'¢° mo1.1-11 was added to the perfusion fluid from the 30th to the 60th min of atrial perfusion. Some of the hearts received one of both antagonists studied from the 20th to the 70th rain of perfusion. The concentration of those antagonists was 10-5 m o l l - i . The addition of aidosterone (10 -s moll -1) in the perfusion fluid resulted in a significant decrease in the coronary flow ( - 4 ~ 1 and a marked increased in both aortic flow ( + 21~;) and cardiac output ( + 11~;) as well as stroke volume ( + 8~). Spironolactone inhibited the coronary flow decrease and limited both aortic flow and cardiac output increase ( + 3~; and + 2~ respectively, P < 0.005). In the opposite, potassium canrenoate abolished both aortic and cardiac output increase, but the coronary flow breakdown was greater ( - 11~, P < 0.005). We concluded that aldosterone presented a positive inotropic effect on the rat myccardium and a vasoconstrictive influeace on coronary arteries. Spironolactone inhibited both these effects and acted as an aldosterone antagonist in the rat heart. Con t,he other hand, potassium canrenoate only inhibited the inotropic effcc*,s of aldosterone, but enhanced its vasoconstrictive eff~t. These differences between both aldosterone antagonists studied could be due either to different m~hanisms in the action of aldosterone on vasomuscular cells and myocytes, as illustrated by the difference between the dose-r~,t.'~,onse relation, or to different specific effects of both antagonists considered. Further studies are needed to precise the mechanis--,~_~involved and consider clinical implications. Supported by grant from the Searle Fondation for Hypertension Research. P.fr.141 [
Influence of adenosine receptor agonists and antagonists on cAMP-dependent protein kinase activity and contractility in guinea-pig papillary muscles Stein, B., Danielsen, W., Neumann, J., Schmitz, W., Scholz, H. and Starbatty, J. Abteilung Allgemeine Pharmakologie, Universitiits-Krankenhaus Eppendorf, MartinistraBe 32, D.2000 Hamburg 20, F.R.G. In ventricular cardiac muscle adenosine exerts negative inotropic effects in the presence of cAMP-increasing agents while adenosine alone has no negative inotropic or even a weak positive inotropic effect (Schrader et aI., 1977). The mechanism of the negative inotropic effect is still a matter of controversy. A decrease in cellular cAMP level due to an inhibition of the adenylate cyclase has been reported (Schrader et al., 19771 while others found no change in the cellular cAMP content (Schmitz et al., 1985). Therefore, we investigated whether the effects of the A:-adenosine receptor (AR) agonist (-)-Ne-phenyfisopropyladenosine (R-PIA) and the A2-AR agonist 5'-N-ethylcarboxamidadeno-sine (NECAi in the presence of isoprenaline (ISO) on force of contraction (FC) in guinea-pig papillar muscles are ac~companied by a corresponding reduction in cAMP-dependent protein kinase (PKA) activity. Th~ influence of the selective A~- and A2-AR antagonists 1,3-dipropyi-8-cyclopentylxanthine (DPCPX) and 9-chloro-2(2-furanyl)-5,6-dihy&-o-l,2,4-triazolo[l,5-c] quinazolin-5-imine (CGS 15943A) on the effects of R-PIA and NECA on PICA-activity and FC were studied. In electrically driven (1Hz) guinea-pig papillary muscles the PKA activity ratio as defined as enzyme activity ratio in the absence and presence of 5 Itmol/l cAMP was determined according to Keely et al. (1975). All experiments were performed in the presence of adenosine deaminase (1/~g/nd) to exclude interference from endogenous adenosine. ISO at a concentration of 0.01/tmol/l increased FC to about 330 + 29~$. Additionally applied R-PIA or NECA (1/tmol/1 each) reduced FC to 241 + 29% and 217 + 7~ respectively (n -- 6-8, p 0.051. In the same preparations ISO increased