J. Scott Hayes

Cardiac inotropic responses to calcium and forskolin are not altered by prolonged isoproterenol infusion.

Effects of prolonged isoproterenol infusion upon the density of cardiac calcium channels, calcium-mediated contractile responses, and the ability of forskolin to enhance tension development and cyclic AMP accumulation were studied in ventricular muscle preparations from Sprague-Dawley rats. Isoproterenol infusion (400 micrograms/kg per h s.c., 4 days) significantly decreased calcium channel density (Bmax) in cardiac microsomal membranes as quantified by a 32% decrease in specific [3H]nitrendipine binding sites; binding affinity (KD) was unchanged. A 57% decrease of beta-adrenoceptors confirmed homologous down regulation. To examine functional effects of decreased [3H]nitrendipine binding sites, responses to calcium, BAY K8644 and nifedipine were determined in isolated right ventricular strips. Significant decreases in basal developed tension were observed in muscles from isoproterenol-infused rats. However, concentration-dependent increases in contractility in response to CaCl2 or BAY K8644 were comparable, and the negative inotropic effect of nifedipine was unchanged. Whereas isoproterenol infusion was associated with significantly decreased basal cardiac cyclic AMP concentrations, exposure of ventricular strips from either vehicle- or isoproterenol-infused rats to 10 microM forskolin resulted in comparable increases in cyclic AMP and in developed tension. Cumulative, submaximal concentrations of forskolin also produced similar increases in contractility with maximum responses in ventricular strips from vehicle-infused animals attained at 4.4 microM forskolin. Higher concentrations resulted in automaticity. By contrast, ventricle from isoproterenol-infused animals responded to 14.4 microM forskolin with maximal increases in force of contraction.

Phosphorylation of ventricular sarcolemmal membranes does not alter binding properties of nitrendipine

Isoproterenol increased contractility in isolated cat papillary muscles 2-fold with an EC50 of 6.3 X 10(-8) M. Nifedipine (3 X 10(-7) M) reduced contractility in control muscles by 43%; however, inotropic state was restored by isoproterenol with a comparable EC50 of 5 X 10(-8) M. To test the hypothesis that this effect might result from cAMP-dependent phosphorylation of a Ca2+ channel-associated protein, [3H]nitrendipine binding was used to probe the high-affinity 1,4-dihydropyridine site in either phosphorylated or dephosphorylated sarcolemmal vesicles. Kd and Bmax values for binding to phosphorylated sarcolemmal vesicles (0.14 +/- 0.027 nM and 479 +/- 62 fmol/mg protein, respectively) were not significantly different from control values P greater than 0.4). Similarly, dephosphorylation of sarcolemmal vesicles did not alter binding parameters. These data demonstrate that phosphorylation of sarcolemmal vesicles neither alters the binding affinity for [3H]nitrendipine nor promotes an interconversion of dihydropyridine-binding sites from high to low affinity or vice versa. While phosphorylation may regulate the slow Ca2+ channel, this is not reflected as changes in [3H]nitrendipine-binding parameters determined in vitro. Furthermore, the cyclic AMP-dependent phosphorylation state of sarcolemmal proteins does not appear to account for wide variations (more than 100-fold) between Kd values from binding studies and IC50 values determined in pharmacological investigations.

In Vivo Cardiovascular Responses to Isoproterenol, Dopamine, and Tyramine Following Prolonged Infusion of Isoproterenol

The moment-to-moment regulation of cardiac output makes the cardiovascular network a dynamic system. In seconds, performance may be altered radically to increase heart rate and stroke volume to meet the needs of the peripheral, metabolizing tissues. Although the magnitude of contractility may be controlled by fiber length, sympathetic stimulation can enhance inotropic state at any given level of end-diastolic volume or pressure. These regulatory mechanisms perform elegantly in the normal heart, but they are substantially impaired in the failing organ.