Voltage‐dependent calcium currents and cytosolic calcium in equine airway myocytes.

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Abstract
1. The relationship between voltage‐dependent calcium channel current (I(Ca)) and cytosolic free calcium concentration ([Ca2+]i) was studied in fura‐2 AM‐loaded equine tracheal myocytes at 35 degrees C and 1.8 mM Ca2+ using the nystatin patch clamp method. The average cytosolic calcium buffering constant was 77 +/‐ 3 (n = 14), and the endogenous calcium buffering constant component is likely to be between 15 and 50. 2. I(Ca) did not evoke significant calcium‐induced calcium release (CICR) since (i)[Ca2+]i scaled with the integrated I(Ca) over the full voltage range of evoked calcium currents, (ii) increases in [Ca2+]i associated with I(Ca) were consistent with cytoplasmic buffering of calcium ions entering through voltage‐dependent calcium channels (VDCCs) only, (iii) there was a fixed instantaneous relationship between transmembrane calcium flux (J(Ca)) and the change in cytosolic free calcium concentration (delta [Ca2+]i) during I(Ca), (iv) caffeine (8 mM) triggered 8‐fold higher calcium transients than I(Ca), and (v) I(Ca) evoked following release of intracellular calcium by caffeine resulted in an equivalent delta[Ca2+]i‐J(Ca) relationship. 3. The time constant (T) for the decay in [Ca2+]i was 8.6 +/‐ 1.5 s (n = 8) for single steps and 8.6 +/‐ 1.1 s (n = 13) following multiple steps that increased [Ca2+]i to much higher levels. Following application of caffeine (8 mM), however, [Ca2+]i decay was enhanced (T = 2.0 +/‐ 0.2 s, n = 3). The rate of [Ca2+]i decay was not voltage dependent, was not decreased in the absence of extracellular Na+ ions, and no pump current was detected. 4. We conclude that under near physiological conditions, neither CICR nor Na(+)‐Ca2+ exchange play a substantial role in the regulation of I(Ca)‐induced increases in [Ca2+]i, and that, even following release of intracellular calcium by caffeine, Na(+)‐Ca2+ exchange does not play an appreciable role in the removal of calcium ions from the cytosol.