Calcium binding to cardiac myocytes protected from proteolytic enzyme activity.
Excitation-contraction coupling in cardiac muscle is dependent on extracellular calcium and calcium bound to the surface of the myocardial cell. In this study, we examined the physical characteristics of calcium binding to adult guinea pig ventricular myocytes disaggregated mechanically in oxygenated tissue culture medium containing a proteinase inhibitor (aprotinin), and separated from cellular debris by Cytodex beads. Cells prepared in this manner excluded Trypan blue and showed no evidence of spontaneous contraction or contracture. Scatchard plots of calcium binding determined by continuous flow equilibrium dialysis revealed a high-affinity, low-capacity pool, Ka = 65 X 10(3) M-1 and Bt = 1.3 nmol X mg-1 and a low-affinity, high-capacity pool, Ka = 141 M-1 and Bt = 138 nmol X mg-1. The low-affinity pool was not detectable after lanthanum, trypsin or collagenase treatment or in cells prepared without aprotinin in the isolation medium. Both neuraminidase and phospholipase C reduced Bt of the low-affinity pool by one half, but only neuraminidase affected the affinity constant of this pool. Ka was increased to 516.7 M-1, similar to the apparent affinity constant for calcium binding estimated from dP/dtmax measured at several extracellular calcium concentrations (470 M-1). The results suggest that calcium bound to sarcolemmal phospholipids represents the superficial calcium involved in excitation-contraction coupling in the heart.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005260:Female; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007811:Lanthanum / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009439:Neuraminidase / Q000494:pharmacology*; D010740:Phospholipases / Q000494:pharmacology*; D012794:Sialic Acids / Q000032:analysis; D010738:Type C Phospholipases / Q000494:pharmacology*
D012794:Sialic Acids; D007811:Lanthanum; D010740:Phospholipases; D010738:Type C Phospholipases; D009439:Neuraminidase; D002118:Calcium