IP3-mediated Ca2+ increases do not involve the ryanodine receptor but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells

摘要

Smooth muscle responds to IP3-generating (sarcolemma acting) neurotransmitters and hormones by releasing Ca²⁺ from the sarcoplasmic reticulum (SR) via IP3 receptors (IP3Rs). This release may propagate as Ca²⁺ waves. The Ca²⁺ signal emanating from IP3 generation may be amplified by its activating further Ca²⁺ release from ryanodine receptors (RyRs) in the process of Ca²⁺-induced Ca²⁺ release (CICR). Evidence for this proposal has relied largely on the use of blocking drugs such as ryanodine, tetracaine and dantrolene, reportedly specific inhibitors of RyRs. Here we have examined whether or not Ca²⁺ released via IP3Rs subsequently activates RyRs. In addition, the specificity of the blocking agents has been assessed by determining the extent of their ability to block IP3-mediated Ca²⁺ release under conditions in which RyRs were not activated. IP3-evoked Ca²⁺ release and Ca²⁺ waves did not require or activate RyRs. However, the RyR blocking drugs inhibited IP3-mediated Ca²⁺ signals at concentrations thought to be selective for RyRs. In single colonic smooth muscle cells, voltage clamped in the whole cell configuration, carbachol (CCh) evoked propagating Ca²⁺ waves which were not inhibited by ryanodine when the sarcolemma potential was −70 mV. At −20 mV, at which potential the SR Ca²⁺ content was increased and RyRs activated, ryanodine inhibited the Ca²⁺ waves. Photolysed caged IP3 increased [Ca²⁺]c; ryanodine, by itself, did not reduce the IP3-evoked [Ca²⁺]c increase when the sarcolemma potential was maintained at −70 mV. However, after activation of RyRs by caffeine, in the continued presence of ryanodine, the IP3-evoked [Ca²⁺]c increase was inhibited. In other experiments, RyRs were activated (as evidenced by the occurrence of spontaneous transient outward currents) by depolarizing the sarcolemma to −20 mV and again ryanodine was effective in inhibiting IP3-evoked Ca²⁺ increase. Thus while ineffective by itself, ryanodine inhibited IP3-evoked Ca²⁺ increases, presumably by causing persistent opening of the channel and depleting the SR of Ca²⁺, after RyRs were activated. These experiments establish that IP₃-evoked Ca²⁺ release and Ca²⁺ waves do not activate RyRs; had they done so ryanodine would have inhibited the Ca²⁺ increase. However, under conditions where ryanodine was ineffective against the IP₃-evoked Ca²⁺ transient (i.e. when RyRs were not activated, e.g. at a membrane potential of −70 mV) tetracaine and dantrolene each blocked IP₃-evoked Ca²⁺ increases. The results show that although IP₃-mediated Ca²⁺ release does not activate RyRs, RyR blockers can inhibit IP₃-mediated Ca²⁺ signals.