Nifedipine blocks Ca2+ store refilling through a pathway not involving L‐type Ca2+ channels in rabbit arteriolar smooth muscle

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Abstract
1. This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. 2. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing < 10 cells) using the whole-cell perforated patch-clamp tecl-miyue. Cell Ca2+ was estimated by fura-2 microfluorimetry. 3. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). 4. To attempt faster refilling via. L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+](i) transients, but failed to accelerate refilling. 5. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I-Cl(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I-Cl(Ca) blocker) and to BAPTA AM, but was abolished by 1 M nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 muM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. 6. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I-Cl(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 muM) during refilling reduced the caffeine-activated I-Cl(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6%). 7. Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+](i), which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 muM nifedipine (to 135 +/- 22 nM), which reversed on washout. 8. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.