CA2+ INFLUX THROUGH ATP-GATED CHANNELS INCREMENTS [CA2+]I AND INACTIVATES ICA IN MYOCYTES FROM GUINEA-PIG URINARY-BLADDER

1. Whole-cell patch clamp was combined with microspectrofluometry (Indo-1) to study the effects of bath applied ATP on membrane currents and cytoplasmic Ca2+ concentration ([Ca2+]i) in single smooth muscle cells of the guinea-pig urinary bladder. Experiments were carried out at 22-degrees-C and in 3.6 mM [Ca2+]0. Superimposed K+ currents were reduced by Cs+ dialysis from the patch electrode. 2. At -60 mV, ATP induced an inward current (I(ns,ATP)) that peaked within 0.4 s and then decayed. I(ns,ATP) was activated half-maximally by 1.1-mu-m-ATP and saturated at 50-mu-M-ATP to -1.1 +/- 0.2 nA (mean +/- S.E.M.). At 3.6 mM [Ca2+]0, I(ns,APT) had a reversal potential (E(rev)) of -5 +/- 2 mV. From the shifts in E(rev) during changes in [Na+]0 or [Ca2+]0 we estimated that approximately 7% of I(ns,ATP) is carried by Ca2+ ions. 3. ATP (50-mu-M) increased [Ca2+]i transiently from resting 130 +/- 40 nM to 730 +/- 100 nM. At 22-degrees-C, [Ca2+]i rose at a rate proportional to the instantaneous current amplitude of I(ns,ATP). This relation was lost, however, after warming to 36-degrees-C which increased the peak I(ns,ATP) (Q10 = 1.25) but reduced the peak of the ATP induced [Ca2+]i transient (Q10 = 0.75). We suggest that warming to 36-degrees-C stimulated Ca2+ sequestration and Ca2+ efflux to such a degree that peak [Ca2+]i was attenuated significantly. 4. The contribution of Ca2+ ions to I(ns, ATP) was evaluated from a comparison of the increments in [Ca2+]i due to I(ns,ATP) and due to L-type Ca2+ channel current (I(ca)). For the same increment, I(ns,APT) had to transport 19 times more charge than I(ca). This number suggest that 5.8 +/- 0.8% of I(ns,ATP) is carried by Ca2+ ions which can be translated into a permeability ratio of P(Na):P(Ca) almost-equal-to 1: 1. 5. During bath application of ATP, peak I(Ca) was inhibited by 80 +/- 15%. Inhibition of I(Ca) diminished to 20 +/- 8% after cell dialysis with 40 mM-EGTA, and it was 19 +/- 7% when extracellular Ca2+ had been substituted by Ba2+. These results are in agreement with the hypothesis of 'I(Ca) inactivation by Ca2+'. Depletion of intracellular Ca2+ stores by pre-treatment with 20 mM-caffeine did not attenuate significantly the ATP-induced rise in [Ca2+]i or the ATP-induced inhibition of I(Ca). 6. The ATP-induced [Ca2+]i transients and the reduction of peak I(Ca) recovered along a similar time course. During this period, reduced peak I(Ca) was plotted as a function of elevated [Ca2+]i. This non-equilibrium concentration-effect curve yielded a half-maximal efficacy of 670 +/- 50 nM [Ca2+]i and a Hill coefficient of 3. 7. Since ATP can depolarize and activate Ca2+ influx through L-type Ca2+ channels, Ca2+ transients and membrane potential were investigated under current clamp (K+ electrodes). ATP (50-mu-M) depolarized the membrane to -30 mV only briefly (0.4 s), then, when [Ca2+]i rose at maximal rate, it repolarized to -55 +/- 5 mV which was most likely due to Ca2+ activated K+ conductance. In the absence of ATP action potentials or sustained depolarizations to -20 mV (by current injection) resulted in [Ca2+]i transients smaller than those induced by ATP. The result suggest that Ca2+ influx through ATP-gated non-selective channels determines [Ca2+]i also in non-clamped cells.