Ryanodine reveals multiple contractile and relaxant mechanisms in vascular smooth muscle: Simultaneous measurements of mechanical activity and of cytoplasmic free Ca2+ level with fura-2

Abstract

The effects of ryanodine on changes in cytoplasmic Ca2+ level ([Ca2+](i)) and muscle tension induced by maximum concentrations of phenylephrine (Phe; 1 μM), prostaglandin F(2α) (PGF(2α), 10 μM), caffeine (Caf, 30 mM) and isoprenaline (Iso, 1 μM) were examined in rat aortic strips using fura-2. In normal media, Phe and PGF(2α) produced a phasic contraction, followed by a tonic one. Caf elicited only a transient contraction. When the preparation was treated with 10 μM ryanodine, an increase in [Ca2+](i) was induced accompanied by a nicardipine (1 μM)-resistant contraction which was [Ca2+](O)-dependent. In Ca2+-free solution, the three stimulants elicited transient increases in [Ca2+](i). Transient contractions to Phe and Caf were accompanied by changes in [Ca2+](i). The transient increase in [Ca2+](i) induced by PGF(2α) was not accompanied by a corresponding contraction. Sustained contractions were induced by Phe and PGF(2α) in the absence of external Ca2+, while the increase in [Ca2+](i) was reduced. A larger maximum contraction was induced by PGF(2α) than by Phe. Ryanodine abolished both the Caf- and Phe-induced [Ca2+](i) transient increases and the corresponding contractions, but had no substantial effect on the PGF(2α)-induced [Ca2+](i) transient increase. Ryanodine had no influence on the sustained contractions induced by Phe and PGF(2α). Iso relaxed both sustained contractions most completely, without any detectable change in [Ca2+](i). Treatment of the preparation with ryanodine had no effect on the concentration-response curves for Iso in relaxing the 0.1 μM Phe- or 40 mM K+-induced precontraction. It is suggested that Phe and Caf mobilize Ca2+ from a ryanodine-sensitive Ca2+ store and that PGF(2α) releases Ca2+ from a ryanodine-insensitive Ca2+ store. The former contributes to the transient contraction through a Ca2+-dependent process, while the latter seems not to be directly associated with the contraction. The sustained contraction under Ca2+-free conditions might involve a Ca2+-independent process or a change in the sensitivity of the contractile filaments to Ca2+. In addition to lowering cytoplasmic Ca2+ concentration, it is suggested that Iso counteracts the apparently Ca2+independent process. The ryanodine-sensitive Ca2+ store plays no substantial role in active relaxation by Iso, although it does play a major role in the maintenance of cytoplasmic Ca2+ in a quiescent muscle.