Calcium uptake by sarcoplasmic reticulum and its modulation by cAMP-dependent phosphorylation in normal and failing human myocardium.
ATP-dependent, oxalate-supported Ca2+ uptake by cardiac sarcoplasmic reticulum was examined in microsomes prepared from left-ventricular free wall myocardium obtained from the explanted failing hearts of transplant recipients with idiopathic dilated cardiomyopathy and the non-failing hearts of kidney donors for whose hearts no suitable recipients were available. There were no significant differences between the two groups with respect to values for Vmax, K0.5 (for Ca2+) or nHill of basal Ca2+ uptake. The stimulation of Ca2+ uptake associated with cAMP-dependent phosphorylation of phospholamban could be reproduced by incubation of microsomes with a monoclonal antibody to phospholamban. Stimulation resulted from a decrease in K0.5, with no changes in Vmax or nHill. The magnitude of stimulation of Ca2+ uptake following incubation with anti-phospholamban monoclonal antibody was identical in preparations from normal and failing hearts. Finally, sarcoplasmic reticulum-associated cGMP-inhibited cAMP phosphodiesterase activity in these preparations was characterised. Measurement of steady-state kinetics and pharmacologic sensitivity indicated that this activity was functionally homogeneous. Preparations from failing and non- failing hearts did not differ with respect to either values for Vmax and Km or susceptibility to inhibition by the cilostamide derivative OPC 3911. These observations indicate that abnormalities in the regulation of intracellular [Ca2+] in failing human myocardium cannot be ascribed to changes in the level or function of the Ca2 + -transporting ATPase, phospholamban or cGMP-inhibited cAMP phosphodiesterase in the sarcoplasmic reticulum.
Key wordsHeart failure sarcoplasmic reticulum Ca2+ uptake Ca2+ ATPase phospholamban phosphodiesterase phosphodiesterase inhibitors cAMP cGMP cilostamide rolipram
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