CLOSTRIDIUM-DIFFICILE TOXIN-B INHIBITS CARBACHOL-INDUCED FORCE AND MYOSIN LIGHT-CHAIN PHOSPHORYLATION IN GUINEA-PIG SMOOTH-MUSCLE - ROLE OFRHO-PROTEINS

1. Clostridium difficile toxin B glucosylates the Ras-related low mole cular mass GTPases of the Rho subfamily thereby inactivating them. In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol-induced increase in the Ca2+ sen sitivity of force and myosin light chain (MLC) phosphorylation in inta ct intestinal smooth muscle. 2. Small strips of the longitudinal muscl e of guinea-pig small intestine were incubated in toxin B (40 ng ml(-1 )) overnight. Carbachol-induced force and intracellular [Ca2+], and, i n a separate series, force and MLC phosphorylation, were determined. 3 . Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delaye d increase in force (peak 2). The peak of the C2+ signal measured with fura-2 preceded peak 1 of force and then declined to a lower suprabas al steady-state level. Peak 2 was not associated with a significant in crease in [Ca2+]. Toxin B nearly completely inhibited peak 2 while pea k 1 was not significantly inhibited. Toxin B had no effect on the Ca2 transient. 4. In control strips, MLC phosphorylation at peak 2 was 27 .7 %, which was significantly higher than the resting value (18.6%). T he inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosph orylation. The resting MLC phosphorylation was not significantly diffe rent from that of the control strips. 5. The initial increase in MLC p hosphorylation determined 3 s after exposure to carbachol was 54 % in the control strips. Toxin B also inhibited this initial phosphorylatio n peak despite the fact that the Ca2+ transient and the initial increa se in force were not inhibited by toxin B. This suggests that Rho prot eins play an important role in setting the balance between MLC phospho rylation and dephosphorylation reactions even at high levels of intrac ellular Ca2+. 6. These findings are consistent with the hypothesis tha t the delayed rise in force elicited by carbachol is due to an increas e in the Ca2+ sensitivity; of MLC phosphorylation mediated by Rho prot eins.