Optical measurement of voltage-dependent Ca2+ influx in frog heart

Sarcolemmal Ca2+ movements in frog ventricular strips were measured by monitoring Ca2+ depletion from the extracellular space with an impermeant Ca indicator dye, antipyrylazo III. Ca2+ depletion was measured as a weighted average of light signals recorded simultaneously at three different wavelengths. This weighting procedure was designed to reduce the motion-induced light scattering and to enhance the Ca2+-related optical signals. Comparison of the time course of Ca2+ depletion signal with that of contraction showed that the rate of Ca2+ depletion was maximal immediately after the upstroke of the action potential but prior to the onset of tension. Peak Ca2+ depletion was reached toward the end of the action potential and amounted to a 10-50 microM decrease in the total extracellular Ca2+ concentration. The reaccumulation of extracellular Ca2+ seen after the action potential was 2-5 sec slower than the relaxation of tension. The rate of Ca2+ depletion had a bell-shaped voltage dependence and was enhanced by epinephrine, suggesting that Ca2+ influx occurred primarily through a slowly inactivating ionic channel. Ca2+ transport through the Na+-Ca2+ exchange system was not significantly altered in the presence of strophanthidin or with decrease of extracellular K+ concentration despite marked potentiation of tension by these agents. Ca2+ depletion measured at the end of a 1-sec clamp pulse had a voltage dependence noticeably different from that of the developed tension. This finding may suggest that a fraction of activator Ca2+ is released from membrane-bound Ca2+ pools in a voltage-dependent manner. Our results show that Ca2+ indicator dyes can be used not only to measure rapid changes in the extracellular Ca2+ concentration during contraction, but also to quantify the contribution of various sarcolemmal Ca2+ transport systems to the generation of tension in cardiac muscle.