ABSTRACT-The effects of exoenzyme C3 of Clostridium botulinum on Ca"-and drug-induced tension developments were investigated in j3-escin skinned smooth muscle of guinea pig ileum to test the involvement of a small G-protein in the regulation of myofilament Ca" sensitivity. C3 is known to ADP-ribosylate the rho p21 family of small G-proteins. Treatment with C3 (0.35 tug/ml, for 30 min) shifted the pCa-tension curve rightward along the Ca 21 concentration axis, indicating a decrease in Ca 21 sensitivity of the contractile elements. The inhibitory effect of C3 was not preserved after treatment with GDP(3S (1 mM), an antagonist of GTP for the binding to G-proteins. Stimulation of muscarinic receptors with carbachol (CCh, 100 ttM) shifted the pCa-tension curve leftward, indicating Ca 21 sensitization of tension development. The Ca2+-sensitizing effect of CCh was not observed after C3 treatment. When GTPrS (10 ttM), an activator of G-proteins, was applied at a plateau of tension development produced by a moderate concentration of Ca2+, further increase in tension was elicited and the effect of GTPrS was inhibited by C3 treatment. The results suggest the possible involvement of a rho p21-like small G-protein in the regulation of Ca 21 sensitivity of smooth muscle myofilaments. Keywords:Carbachol, Ca 2+-sensitization, Smooth muscle, GTP-binding protein, ContractionThe primary mechanism underlying smooth muscle contraction is phosphorylation of the 20-kD myosin light chain (MLC20) by MLC20 kinase that is activated by Ca 2+-calmodulin (1). It is well known that various receptor agonists can produce a greater tension development than high KCl solution even if cytosolic Ca 21 concentration ([Ca 2+];) is increased to the same level in intact smooth muscle (2, 3). This phenomenon was clearly demonstrated in [Ca 2+];-clamped, permeabilized smooth muscle preparations (4, 5). The greater tension development is associated with an increase in MLC20 phosphorylation (6-8), and thus an increase in the ratio of MLC20 kinase activity to MLC phosphatase activity has been suggested (9, 10). Heterotrimetric G-proteins have been suggested to serve as transmembrane signal transducers for receptors by which the Ca2+-sensitizing effect is mediated (5, 7), but signaling pathways responsible for the receptor/G-protein-mediated Ca 2+-sensitizing effect have not been clarified yet. Recent studies indicate that C3 and EDIN, exoenzymes of Clostridium botulinum and Staphylococcus aureus, respectively, selectively ADP-ribosylate the rho p21 family of a superfamily of ras p21 and its related small G-proteins (11 -13). Hirata et al. (14) found that EDIN and C3 blocked GTPTS-induced Ca 21 sensitization of tension development and caused ADP-ribosylation of a rho p21-like protein in saponin-skinned arterial smooth muscle, and they suggested involvement of a member of the rho p21 family, rho A p21, in the Ca 21 sensitization. Current knowledge suggests that it would be attractive to test the possibility that Ca" -sensitizing effects of agonists can be blo...

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ABSTRACT-To characterize the calcium (Ca")-releasing effects of histamine and GTPrS, the drug-in duced tension developments were measured in (3-escin-treated skinned longitudinal smooth muscle of guinea pig ileum. Intracelluar Ca2+ stores were loaded with Ca" by incubating the muscle for 10 min in a Ca" containing solution. Histamine (10-100 pM), applied after Ca" -loading, produced a transient rise in ten sion. The effect of histamine was not preserved after treatment with 20 mM caffeine, a Ca2+-store releaser. The effect of histamine was potentiated by GTP; inhibited by GDP(3S, an antagonist of GTP for binding to G-proteins; or heparin, an antagonist of inositol 1,4,5-trisphosphate (IP3) for binding to its receptor; and mimicked by IP3. When GTPrS (20 ttM) was applied and continued to be present for 15 min, a transient rise in tension followed by a small, sustained rise in tension was elicited. The effect of GTPrS was completely inhibited by GDP(3S. The initial, transient component of the biphasic GTPrS response was abolished or markedly inhibited after treatment with caffeine, heparin or the calcium ionophore A23187. The present results suggest that histamine and GTPrS cause a release of Ca 21 from caffeine-sensitive stores which is mediated by IP3 formed through a G-protein-coupled mechanism. The GTPrS-induced Ca 2+ release is not considered to involve such an IP3-independent process as described in chemically-skinned arterial muscle.

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The effects of trimebutine on Ca 2+ release and modulation of Ca 21 sensitivity of contractile elements induced by carbachol (CCh) were investigated using a tension measuring method in p-escin-treated skinned smooth muscle of the longitudinal muscle layer of guinea pig ileum. Trimebutine (10-100 1tM) con centration-dependently inhibited tension development brought about by Ca 2+ release from intracellular stores induced by CCh (10 1M), but did not affect those induced by inositol 1,4,5-trisphosphate (IP3, 25 tiM) or caffeine (5 mM). The inhibitory effect was reversible. Trimebutine (100 pM) neither altered the Ca 21 sensitivity of the contractile elements nor affected the effects of GTPrS (50 pM) and CCh (100 pM) in poten tiating Ca 21 sensitivity of the contractile elements after the Ca 21 storage function had been eliminated by A23187. These results suggest that trimebutine inhibits CCh-induced Ca 2+ release by acting at some point during the coupling of muscarinic receptors through a G-protein to phospholipase C and thus reducing the accumulation of IP3.