Calmodulin stimulation of smooth muscle plasmalemmal vesicle Ca2+ uptake: direct or indirect effect?
Current evidence suggests that Ca(2+)-calmodulin-dependent protein kinase(s) may be involved in the regulation of smooth muscle contractility. The plasmalemmal Ca2+ pump plays an important role in smooth muscle contractility, and this pump is stimulated by calmodulin. However, it is not known whether this is due to direct activation, or calmodulin-dependent protein phosphorylation, or both. We tested these hypotheses using a plasma membrane vesicle preparation from porcine antral smooth muscle. Inclusion of calmodulin in the Ca(2+)-uptake assay decreased the free Ca2+ concentration at which Ca2+ uptake is 50% of the maximal rate (1.5 +/- 0.21 to 0.84 +/- 0.06 microM) of the Ca2+ pump for Ca2+, without changing maximal rate of Ca2+ uptake (Vmax) (8.0 +/- 1.1 to 8.4 +/- 0.7 nmol.min-1.mg-1). In contrast, prephosphorylation in the absence of Ca(2+)-calmodulin increased Ca(2+)-uptake rates at both low (pCa 6.0) and high Ca2+ concentration (pCa 5.0), suggesting an increase in Vmax. Further phosphorylation in the presence of Ca(2+)-calmodulin was not associated with any further increases in the Ca(2+)-uptake rates at pCa 6.0. However, inclusion of calmodulin in the Ca(2+)-uptake assays stimulated the rates (pCa 6.0) of both unphosphorylated and prephosphorylated vesicles to a similar extent. These findings suggest that the stimulation of the smooth muscle plasmalemmal Ca2+ pump by calmodulin is predominantly due to a direct effect via a mechanism distinct from that by plasma membrane phosphorylation.
Zhang
C
C
Department of Physiology and Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0576.
Paul
R J
RJ
Kranias
E G
EG
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D013552:Swine
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; C061167:RS 20; D000255:Adenosine Triphosphate; D002118:Calcium