Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta‐adrenergic–stimulated cardiac Ca²⁺‐ATPase

Abstract

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase, with an estimated IC-50 of 2.5 X 10(-8) M. The present studies were initiated to evaluate the mechanism of inhibition of Ca2+-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca2+-ATPase indicated alteration of Vmax and Km by Plictran (1 and 5 X 10(-8) M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased Vmax of both ATP- and Ca2+-dependent enzyme activities. However, the Km of enzyme was decreased only for Ca2+. Plictran inhibited isoproterenol-stimulated Ca2+-ATPase activity by altering both Vmax and Km of ATP as well as Ca2+-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca2+. Preincubation of enzyme with 15 microM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 X 10(-7) M propranolol and 5 X 10(-8) M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 X 10(-5) M coenzyme A in combination with 5 X 10(-8) M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca2+-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.

Inhibition of Ca2+ transport associated with cAMP-dependent protein phosphorylation in rat cardiac sarcoplasmic reticulum by triorganotins

“…Phosphorylation of this protein results in stimulation of CaZ+-ATPase and Ca2+ transport by the SR (Scheme 1; LaRaia and Morkin 1974; Tada et al 1975;Wray and Gray 1977;Kranias et al 1980a). Our previous studies indicated that plictran, one of the triorganotins, inhibited [~-adrenergic stimulation of the calcium pump in cardiac SR (Sahib and Desaiah 1986). Initially, we have investigated whether 45Ca uptake and Ca2+-ATPase in cardiac SR are affected following treatment with these organotins.…”

In Vitro and In Vivo Inhibition of Rat Brain Nitric Oxide Synthase Activity by Phencyclidine

“…Analyses of arginine, NADPH, and Ca 2+ activation kinetics were performed to understand the type of inhibition of NOS activity by PCP (0.5 mM). Double-reciprocal plots of kinetic data were constructed according to the method of Lineweaver and Burk (1934) as described previously (Sahib and Desaiah 1986). The data obtained as a function of arginine, NADPH, and Ca 2+ were subjected to regression analysis and linear regression lines were plotted for best straight line ® t. Kinetic constants were calculated and presented in ® gure legends.…”