Smooth muscle contraction depends on the state of myosin phosphorylation and hence on the balance of myosin light chain kinase and phosphatase activity. Effects of okadaic acid isolated from black sponge on both enzyme activities and contractility were studied in chemically skinned fibers from guinea pig taenia coli. The toxin strongly inhibits myosin phosphatase and enhances tension development.

1. Clostridium difficile toxin B glucosylates the Ras-related low molecular 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 Ca¥ sensitivity of force and myosin light chain (MLC) phosphorylation in intact intestinal smooth muscle. 2. Small strips of the longitudinal muscle of guinea-pig small intestine were incubated in toxin B (40 ng ml¢) overnight. Carbachol-induced force and intracellular [Ca¥], and, in 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 delayed increase in force (peak 2). The peak of the Ca¥ signal measured with fura_2 preceded peak 1 of force and then declined to a lower suprabasal steady-state level. Peak 2 was not associated with a significant increase in [Ca¥]. Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited. Toxin B had no effect on the Ca¥ transient. 4. In control strips, MLC phosphorylation at peak 2 was 27·7%, which was significantly higher than the resting value (18·6%). The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation. The resting MLC phosphorylation was not significantly different from that of the control strips. 5. The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54% in the control strips. Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca¥ transient and the initial increase in force were not inhibited by toxin B. This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca¥. 6. These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca¥ sensitivity of MLC phosphorylation mediated by Rho proteins.

Caldesmon is known to inhibit actinactivated myosin ATPase activity in solution, to inhibit force production when added to skeletal muscle fibers, and to alter actin movement in the in vitro cell motility assay. (3, 4, 23), while the rate of relaxation may be regulated at apparently basal levels of LC20 phosphorylation (5). Interest in a second regulatory mechanism has been stimulated by the discovery of two actin-binding proteins, caldesmon (6) and calponin (7), which inhibit actin activation of myosin ATPase activity in solution (8-10). Caldesmon is particularly interesting since it is localized in the contractile actomyosin domain of smooth muscle (11), and it has been shown to be able to inhibit the binding of myosin-ATP cross-bridges to actin when added to single skeletal muscle fibers with a resulting inhibition of force production (12). This is an artificial situation, however, since caldesmon is not a component of striated muscle. Our own preliminary work indicated that exogenous caldesmon can inhibit force production. § Furthermore, Katsuyama et al. (14) showed that caldesmon peptides, which presumably displace caldesmon from actin in muscle cells, can activate these cells to produce force. Thus, there is indirect evidence that caldesmon can inhibit contraction in smooth muscle.We have now attempted to show the feasibility that caldesmon is a modulator of force production in smooth muscle. This was done by using chemically skinned chicken gizzard fibers, which were activated by adding a constitutively active fragment of myosin light-chain kinase (I-MLCK) (15). In this way, we were able to phosphorylate the regulatory chains of myosin necessary for activation of contraction without inacThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.tivating the Ca2+/calmodulin-dependent activation pathway by binding caldesmon to calmodulin. This is preferential to thiophosphorylation of myosin, which disrupts the normal phosphorylation-dephosphorylation cycle of myosin. To facilitate diffusion of caldesmon into the fibers, we used a 20-kDa, actin-binding fragment of caldesmon in most of the experiments. This C-terminal fragment (16) inhibits ATP hydrolysis (17) and has been shown to function in the striated muscle system (12). Our results show that caldesmon and the actin-binding fragment of caldesmon shift the relation between force and LC20 phosphorylation toward higher levels of phosphorylation. In contrast, the N-terminal myosinbinding fragment ofcaldesmon and a maleylated derivative of the 20-kDa actin-binding fragment ofcaldesmon had no effect on force production. MATERIALS AND METHODSChicken gizzards were collected at a local farm immediately after sacrifice of the animals and brought to the laboratory in oxygenated, ice-cold physiological saline solution containing 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM CaCl2, 24 mM Hepes (pH 7.4 at 37°C), ...

Calcium-dcpcndent regulation of tension and ATPase activity in pcrmeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadatc to a vanadate-free, low-Ca'+ solution (pCa >8), the pcrmcabilized muscle produced 84.8% f 20.1% (2 S.D.. /1=95) of the isometric force elicited by high Ca" (pCa -4.5) prior to incubation with vanadatc. Transfer back to a hi& Ca'* soludon elicited no additional force (83.2% -t-18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial cxtiaction (>90%) of Troponin I (Tnl). Calcium dependence was restored after incubation with solutions containing either whole cardiac lroponin or a combination of TnI und troponin C subunits. This reversible extraction oftroponin directly demonstrates the role ofTnl in the rcylalion of striated muscle contractility and permits specific substitution of the native TnI with cxogWoualy supplied protein.

The mechanism responsible for the regulation of smooth muscle tone at low levels of myosin light chain (MLC) phosphorylation is still poorly understood. According to one model, so-called latchbridges, which contribute to force maintenance at low levels of MLC phosphorylation, are generated by dephosphorylation of attached and phosphorylated crossbridges. The model predicts that the force generated for a given level of MLC phosphorylation depends on the activity of the MLC phosphatase. We tested this hypothesis by reducing the activity of the phosphatase by at least 80% in two ways: inhibition with okadaic acid and extraction. Under both conditions, higher levels of MLC phosphorylation were required to support a given level of force, suggesting a decreased flux of attached phosphorylated to attached dephosphorylated crossbridges, as predicted by this model. Although, under both conditions, the relationship between force and MLC phosphorylation was shifted to the right, the curves did not superimpose as would have been expected if the phosphatase activity were the only determinant of the coupling between force and phosphorylation. In the extracted fibres, two more proteins, calponin and SM22, were significantly reduced in addition. Therefore, these proteins might be involved in modulating the relationship between force and MLC phosphorylation.