L-type and Ca 2+ release channel-dependent hierarchical Ca 2+ signalling in rat portal vein myocytes

Abstract

Ca 2+ signalling events and whole-cell Ca 2+ currents were analyzed in single myocytes from rat portal vein by using a laser scanning confocal microscope combined with the patch-clamp technique. In myocytes in which the intracellular Ca 2+ store was depleted or Ca 2+ release channels were blocked by 10 μM ryanodine, inward Ca 2+ currents induced slow and sustained elevations of [Ca 2+] i. These Ca 2+ responses were suppressed by 1 μM oxodipine and by depolarizations to +120 mV, a potential close to the reversal potential for Ca 2+ ions, suggesting that they reflected Ca 2+ influx through L-type Ca 2+ channels. With functioning intracellular Ca 2+ stores, flash photolysis of caged Ca 2+ gave rise to a small increase in [Ca 2+] i with superimposed Ca 2+ sparks, reflecting the opening of clustered Ca 2+ release channels. Brief Ca 2+ currents in the voltage range from −30 to +10 mV triggered Ca 2+ sparks or macrosparks that did not propagate in the entire line-scan image. Increasing the duration of Ca 2+ current for 100 ms or more allowed the trigger of propagating Ca 2+ waves which originated from the same initiation sites as the caffeine-activated response. Both Ca 2+ sparks and initiation sites of Ca 2+ waves activated by Ca 2+ currents were observed in the vicinity of areas that excluded the Ca 2+ probes, reflecting infoldings of the plasma membrane close to the sarcoplasmic reticulum, as revealed by fluorescent markers. The hierarchy of Ca 2+ signalling events, from submicroscopic fundamental events to elementary events (sparks) and propagated waves, provides an integrated mechanism to regulate vascular tone.