Role of superficial calcium binding sites in the inotropic response of isoproterenol and ouabain
Creators: name: Fawzi, Ahmad B.
Publisher: name: University of British Columbia
Year: 2010

description: Mammalian myocardial contractility is believed to be regulated by the amount of calcium contained in a highly labile superficial calcium pool. The purpose of the first part of this study was to determine the role of such sites in the positive inotropic effect of isoproterenol. Lanthanum, an ion known to be restricted to the extracellular space and which displaces the superficially-bound calcium, was selected as a tool for this investigation. In Langendorff preparations of the guinea pig heart, lanthanum decreased the basal contractility index (+dP/dtmax) in a concentration-dependent fashion (0.05-3 µM) and blocked the inotropic response of isoproterenol in a non-competitive manner (0.25-3 µM). Three µM lanthanum: 1) reduced basal contractility and the maximum response to isoproterenol by 97 and 95%, respectively; 2) had no significant effect (p&gt;0.05) on basal and isoproterenol-induced cyclic AMP levels; and 3) had no effect on the kd of [³H]nitrendipine binding, but reduced the Bmax by 31%. While 1 µM lanthanum reduced basal contractility and the maximum response to isoproterenol by 90 and 70%, respectively, it had no effect on [³H]nitrendipine binding. These results suggest that the effects of such low concentrations of lanthanum (≤3 µM) are not related to a direct action on the calcium channels and are not mediated by an inhibition of isoproterenol stimulation suggest that superficially-bound calcium is required for the inotropic response of isoproterenol. The purpose of the second part of this study was to elucidate the biochemical nature of the superficial calcium binding sites, the sialic acids in particular, in the inotropic response of cardiotonic agents. To determine the role of the glycocalyx residues of sialic acids in excitation-contraction coupling and the inotropic response to cardiotonic agents, I studied the effect of removal of the sialic acids following neuraminidase treatment on the response to ouabain, isoproterenol, calcium and reduced extracellular sodium in Langendorff preparations of adult guinea pig hearts. Neuraminidase treatment (0.01 U/ml, 1 h) reduced the magnitude of the positive inotropic response to 2.5x10⁻⁷M ouabain and the maximum response to 5x10⁻⁷ M ouabain by about 46 and 30%, respectively, but did not prevent ouabain toxicity. Neuraminidase treatment did not affect the contractility produced by calcium concentration alterations up to 5 mM calcium or the positive inotropic effect produced by lowering external sodium to as low as 80 mM. The inotropic response to as high as 10⁻⁸ M isoproterenol was also not affected. The contractility response developed to calcium concentrations greater than 5 mM and to 5x10⁻⁸ M isoproterenol were significantly reduced (p&lt;0.05) by neuraminidase treatment. The content of sialic acids in neuraminidase-treated hearts used in the above concentration-response reduced by 70.7, 66.1, 65.6 and 66.2%, respectively. Neuraminidase treatment had no effect on basal (Na⁺-K⁺)ATPase and Mg²⁺ -ATPase activities of (Na⁺-K⁺)ATPase-containing membrane preparations of the guinea pig left ventricle. Neuraminidase treatment neither influenced the sensitivity of the enzyme (Na⁺-K⁺)ATPase to ouabain inhibition nor did it affect the characteristics of [³H]ouabain binding to the preparation. These results suggest that the sialic acids of the glycocalyx in the guinea pig left ventricle play an important role in part of the inotropic response to subtoxic concentrations of ouabain.