Measurement of calcium entry and exit in quiescent rat ventricular myocytes

The aim of this work was to obtain the first quantitative measurements of Ca2+ influx and efflux in quiescent cardiac cells. The relationship between free and total Ca2+ was obtained during a caffeine application. This buffering curve was then used to calculate changes of total Ca2+ from measurements of free cytosolic [Ca2+] ([Ca2+](i)) made with Indo-1. The rate of Ca2+ removal from the cytoplasm was calculated by differentiating total Ca2+ with respect to time. The dependence of d(total Ca2+)/dt on [Ca2+](i) was hyperbolic. Inhibition of either Na+-Ca2+ exchange (by addition of 10 mmol l-1 NiCl2 or removal of external Na+) or the sarcolemmal Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) (with carboxyeosin) decreased the calculated efflux. In both cases, the main effect was on the apparent maximum rate (V(max)) with little effect on the Michaelis-Menten constant (K(m)). These results suggest that the Na+-Ca2+ exchange and Ca2+-ATPase have very similar affinities for [Ca2+](i) and that their fractional contributions do not change over the systolic range of [Ca2+](i). Ca2+ influx was quantified in two ways. The first method was to extrapolate the curve relating Ca2+ efflux to [Ca2+](i) to zero [Ca2+](i). This gave a value of 4.49±0.54 μmol l-1 s-1 which was reduced to zero by either removal of external Ca2+ or addition of Ni2+. In other experiments external Ca2+ was removed and the maximum rate of fall of total Ca2+ calculated as 2.53±0.93 μmol l-1 s-1. This approach can be used to provide a quantitative analysis of the control of resting [Ca2+](i).