Hirasaki, A

Agonist-induced increases in CA2+ sensitivity are mediated in part by mechanisms that increase phosphorylation of the regulatory myosin light chain (rMLC) at constant cytosolic Ca2+ concentration ([Ca2+]i). The current study tested the hypothesis that halothane inhibits acetylcholine (ACh)-induced potentiation of rMLC phosphorylation in beta-escin-permeabilized canine tracheal smooth muscle. ACh plus GTP significantly potentiated the increase in isometric force and rMLC phosphorylation induced by 0.8 microM free Ca2+. However, whereas the potentiation of isometric force was sustained, the potentiation of rMLC phosphorylation was biphasic, peaking at 0.5 min and then declining by approximately 10 min to a steady-state level significantly above that induced by 0.8 microM free Ca2+ alone. This finding suggests that mechanisms in addition to changes in rMLC phosphorylation may mediate ACh-induced Ca2+ sensitization, as has been reported for vascular smooth muscle. Halothane (0.91 +/- 0.10 mM) significantly inhibited ACh plus GTP-induced potentiation of rMLC phosphorylation and isometric force after 2 (peak rMLC phosphorylation) and 15 (steady-state rMLC phosphorylation) min of stimulation. However, the effect of halothane on the potentiation of isometric force was significantly less than that expected from its effect on rMLC phosphorylation (i.e., halothane changed the relationship between rMLC phosphorylation and isometric force). These results demonstrate that halothane inhibits the ACh-induced increase in Ca2+ sensitivity by inhibiting the membrane receptor-coupled mechanisms that increase rMLC phosphorylation at constant submaximal [Ca2+]i. Possible additional effects of halothane on rMLC phosphorylation-independent mechanisms cannot be ruled out.

This study used a beta-escin-permeabilized canine tracheal smooth muscle preparation to test the hypothesis that the volatile anesthetic halothane decreases myofilament Ca2+ sensitivity by inhibiting the membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity and not by inhibiting Ca(2+)-calmodulin activation of the contractile proteins. Acetylcholine (ACh) caused a GTP-dependent increase in force at constant submaximal cytosolic Ca2+ concentration. ACh, guanosine-5'-O-(3-thiotriphosphate), and the protein kinase C agonist 12,13-phorbol dibutyrate each significantly decreased the concentration of free Ca2+ producing a half-maximal response from 0.77 +/- 0.09 microM (Ca2+ alone) to 0.16 +/- 0.01, 0.19 +/- 0.02, and 0.37 +/- 0.03 microM, respectively, demonstrating an increase in myofilament Ca2+ sensitivity. Halothane (0.92 +/- 0.12 mM) had no effect on the free Ca2+ concentration-response curves generated by Ca2+ alone. However, in the presence of 3 microM ACh plus 10 microM GTP to maximally activate muscarinic receptors, halothane significantly increased the EC50 for free Ca2+ from 0.17 +/- 0.01 microM to 0.38 +/- 0.03 microM. These findings suggest that halothane decreases myofilament Ca2+ sensitivity in beta-escin-permeabilized canine tracheal smooth muscle by inhibiting the membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity.