The contribution of inositol 1,4,5-trisphosphate and ryanodine receptors to agonist-induced Ca²⁺ signaling of airway smooth muscle cells

Abstract

The relative contribution of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and ryanodine receptors (RyRs) to agonist-induced Ca(2+) signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phase-contrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptamine (5-HT) induced Ca(2+) oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an IP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the Ca(2+) oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited Ca(2+) waves generated by the photolytic release of IP(3). However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or IP(3)-induced Ca(2+) oscillations or Ca(2+) wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced Ca(2+) oscillations in a concentration-dependent manner. However, tetracaine did not affect IP(3)-induced Ca(2+) release or wave propagation nor the Ca(2+) content of SMC Ca(2+) stores as evaluated by Ca(2+)-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow Ca(2+) oscillations induced by KCl. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or Ca(2+) oscillations of SMCs were also observed at 37 degrees C. In Ca(2+)-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular Ca(2+) levels indicating that relaxation also resulted from a reduction in Ca(2+) sensitivity. These results indicate that agonist-induced Ca(2+) oscillations in mouse small airway SMCs are primary mediated via IP(3)Rs and that tetracaine induces relaxation by both decreasing Ca(2+) sensitivity and inhibiting agonist-induced Ca(2+) oscillations via an IP(3)-dependent mechanism.

Agonist‐evoked Ca2+ wave progression requires Ca2+ and IP3

“…The drugs blocked Ca 2+ waves leading to the conclusion that RyR are required to progress waves. However, some of these drugs may be less selective than previously assumed and may block IP 3 R at concentrations thought selective for RyR (Bai et al, 2009;MacMillan et al, 2005a;Vites and Pappano, 1992). For example, in avian atria, ruthenium red inhibited the response to IP 3 by an action unrelated to RyR since the response to caffeine was potentiated (Vites and Pappano, 1992).…”

“…For example, in avian atria, ruthenium red inhibited the response to IP 3 by an action unrelated to RyR since the response to caffeine was potentiated (Vites and Pappano, 1992). Ryanodine, dantrolene, tetracaine and procaine also block IP 3 -mediated Ca 2+ release at concentrations which had been thought selective for RyR (Bai et al, 2009;MacMillan et al, 2005a). Dantrolene and tetracaine may block IP 3 R directly (MacMillan et al, 2005a).…”

“…In the absence of precise information on the state of filling of the store, the complex action of ryanodine makes interpretation of its effects on IP 3 -mediated responses difficult. Ryanodine may reduce IP 3 -evoked Ca 2+ release indirectly as a consequence of depletion of the SR of Ca 2+ (Bai et al, 2009;Lamont and Wier, 2004;MacMillan et al, 2005a;McCarron et al, 2003;McCarron et al, 2002). These results question the exclusive use of drugs and pharmacological investigations to determine the mechanism of wave progression.…”

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca2+ Oscillations and Ca2+ Sensitivity

“…The assay was initiated by addition of myosin, and terminated by addition of 2 ml of 1 M perchloric acid and 0.35 M NaH 2 PO 4 ; assay volume was 20 ml. The reaction mixture was filtered with a nitrocellulose membrane (0.45 mm), and the phosphorylated myosin was quantified by measuring the radioactivity emitted by 32 P by Cerenkov counting (18). To ensure that the phosphorylation of the myosin by MLCK was specific for the rMLC, only whole myosin molecules containing the rMLC were used.…”

At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries

“…The bimodal Ca 2þ -dependence of hemichannels resembles the Ca 2þ -dependence of inositol-trisphosphate receptor (IP 3 R) channels, which are Ca 2þ release channels in the endoplasmic reticulum (ER) that play a central role in generating Ca 2þ oscillations in diverse cell types including SMCs. [11][12][13] Based on this similarity, we hypothesized that Cxhemichannels may contribute to Ca 2þ oscillations. Others have suggested ATP release/purinergic signalling may play a role in supporting Ca 2þ oscillations.…”