Inhibition of capacitative calcium entry is not obligatory for relaxation of the mouse anococcygeus by the NO/cyclic GMP signalling pathway.

1. The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. 2. In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic field stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 microM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microM). 3. In single smooth muscle cells, loaded with Fura-2, both carbachol and Tg produced sustained elevations in cytoplasmic calcium levels ([Ca2+]i). SNP inhibited the rise in [Ca2+]i produced by carbachol, an effect attenuated by ODQ. In contrast, neither SNP nor 8-Br-cyclic GMP reduced the elevated [Ca2+]i associated with Tg. 4. In beta-escin skinned preparations, NO had no effect on tone induced by calcium (1 microM in the presence of 100 microM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. 5. The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by refilling the internal stores, since reduction in [Ca2+]i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation.

[Novel mechanisms for increasing Ca2+ sensitivity of contractile apparatus in smooth muscle].

Smooth muscle contraction is primarily regulated not only by changes in cytosolic Ca2+ concentrations ([Ca2+]i) but also by changes in the force/[Ca2+]i ratio. The use of membrane-permeabilization technique facilitated demonstration of an increase in the level of force at constant [Ca2+]i (Ca2+ sensitization). It was clarified that Rho-associated kinase (Rho-kinase) is a novel mediator of Ca2+ sensitization of the smooth muscle contraction, by introducing the recombinant catalytic domain of Rho-kinase into the cytosol of vascular smooth muscle permeabilized with beta-escin. This review article focuses on novel mechanisms, by which activation of receptor-coupled G-protein(s) increases Ca2+ sensitivity of the contractile apparatus in smooth muscle: Rho-kinase and protein kinase C.