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

摘要

class="enumerated" style="list-style-type:decimal">This study assessed the contribution of L-type Ca²⁺ channels and other Ca²⁺ entry pathways to Ca²⁺ store refilling in choroidal arteriolar smooth muscle.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 technique. Cell Ca²⁺ was estimated by fura-2 microfluorimetry.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).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.At -80 mV and in 20 mm TEA, prolonged caffeine exposure produced a transient Ca²⁺-activated Cl⁻ current (ICl(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mm; an ICl(Ca) blocker) and to BAPTA AM, but was abolished by 1 μm nifedipine. This nifedipine-sensitive current reversed at +29 ± 2 mV, which shifted to +7 ± 5 mV in Ca²⁺-free solution. Cyclopiazonic acid (20 μm; an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase) also activated the nifedipine-sensitive sustained current.At -80 mV, a 5 s caffeine exposure emptied Ca²⁺ stores and elicited a transient ICl(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 μm) during refilling reduced the caffeine-activated ICl(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 %).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 μm nifedipine (to 135 ± 22 nm), which reversed on washout.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.