CROSS-SIGNALING BETWEEN L-TYPE CA2+ CHANNELS AND RYANODINE RECEPTORS IN RAT VENTRICULAR MYOCYTES

Calcium-mediated cross-signaling between the dihydropyridine (DHP) rec eptor, ryanodine receptor, and Na+-Ca2+ exchanger was examined in sing le rat ventricular myocytes where the diffusion distance of Ca2+ was l imited to <50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca2+-indicator dye, Fura-2, or 2 mM Fu ra-2 plus 14 mM EGTA decreased the magnitude of I-Ca-triggered intrace llular Ca2+ transients (Ca-i-transients) from 500 to 20-100 nM and com pletely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximate to 140 mu M). Inactivation kinetics of I-Ca in highly Ca2+-buffered cells wa s retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were de pleted by caffeine applied 500 ms before activation of I-Ca, while ina ctivation was accelerated if caffeine-induced release coincided with t he activation of I-Ca. Quantitative analysis of these data indicate th at the rate of inactivation of I-Ca was linearly related to SR Ca2+-re lease and reduced by >67% when release was absent. Thapsigargin, aboli shing SR release, suppressed the effect of caffeine on the inactivatio n kinetics of I-Ca. Caffeine-triggered Ca2+-release, in the absence of Ca2+ entry through the Ca2+ channel (using Ba2+ as a charge carrier), caused rapid inactivation of the slowly decaying Ba2+ current. Since Ba2+ does not release Ca2+ but binds to Fura-2, it was possible to cal ibrate the fluorescence signals in terms of equivalent cation charge. Using this procedure, the amplification factor of I-Ca-induced Ca2+ re lease was found to be 17.6+/-1.1 (n=4). The Na+-Ca2+ exchange current, activated by caffeine-induced Ca2+ release, was measured consistently in myocytes dialyzed with 0.2 but not with 2 mM Fura-P. Our results q uantify Ca2+ signaling in cardiomyocytes and suggest the existence of a Ca2+ microdomain which includes the DHP/ryanodine receptors complex, but excludes the Na+-Ca2+ exchanger. This microdomain appears to be f airly inaccessible to high concentrations of Ca2+ buffers.