1. The role of h1-calponin in regulating the contractile properties of smooth muscle was investigated in bladder and vas deferens of mice carrying a targeted mutation in both alleles designed to inactivate the basic calponin gene. These calponin knockout (KO) mice displayed no detectable h1-calponin in their smooth muscles.2. The amplitudes of Ca 2+ sensitization, force and Ca 2+ sensitivity were not significantly different in permeabilized smooth muscle of KO compared with wild-type (WT) mice, nor were the delays in onset and half-times of Ca 2+ sensitization, initiated by flash photolysis of caged GTPyS, different.3. The unloaded shortening velocity (V us ) of thiophosphorylated fibres was significantly (P < 0.05) faster in the smooth muscle of KO than WT animals, but could be slowed by exogenous calponin to approximate WT levels; the concentration dependence of exogenous calponin slowing of V us was proportional to its actomyosin binding in situ.4. Actin expression was reduced by 25-50%, relative to that of myosin heavy chain, in smooth muscle of KO mice, without any change in the relative distribution of the actin isoforms.5. We conclude that the faster V us of smooth muscle of the KO mouse is consistent with, but does not prove without further study, physiological regulation of the crossbridge cycle by calponin. Our results show no detectable role of calponin in the signal transduction of the Ca 2+ -sensitization pathways in smooth muscle.

The differential effects of essential light chain isoforms (LC 17a and LC 17b ) on the mechanical properties of smooth muscle were determined by exchanging recombinant for endogenous LC 17 in permeabilized smooth muscle treated with trifluoperazine (TFP). Co-precipitation with endogenous myosin heavy chain verified that 40 -60% of endogenous LC 17a could be exchanged for recombinant LC 17a or LC 17b . Upon addition of MgATP in Ca 2؉ -free solution, recombinant LC 17 exchange induced slow contractions unaccompanied by regulatory light chain (RLC) phosphorylation only in TFP-treated, but not in untreated, permeabilized smooth muscle; the shortening velocity and rate of force development were approximately 1.5 and 2 times faster, respectively, in response to LC 17a than LC 17b . Additional incubation with recombinant, thiophosphorylated RLC increased the shortening velocity, independent of the LC 17 isoform exchanged. The LC 17 -induced contractions of TFPtreated muscles were abolished by prior addition of nonphosphorylated RLC. We suggest that LC 17 stiffens the lever arm of myosin and, in the absence of regulation by RLC, permits cross-bridge cycling without requiring RLC phosphorylation. Our results are compatible with nonphosphorylated RLC acting as a repressor and with LC 17 isoforms modulating the MgADP affinity and, consequently, rate of cooperative cycling of nonphosphorylated cross-bridges.The markedly different rates of contraction and relaxation in fast, phasic and slow, tonic smooth muscles reflect differences not only at the level of the membrane potential, signal transduction, rates of myosin light chain phosphorylation and dephosphorylation, but also in the kinetic properties of the actomyosin motor. The muscle-specific differences in actomyosin kinetics have been ascribed to the existence of specific myosin isoforms, but their relative contributions to the mechanical properties of smooth muscles have not been unequivocally demonstrated (reviewed in Ref. 1). The expression of the two myosin heavy chain (MHC) 1 isoforms, SM-1 and SM-2 (2) that differ by, respectively, the presence or absence of a 34-amino acid extension of the carboxyl terminus (3), does not correlate with phasic or tonic kinetics of contraction. On the other hand, motility assays show different rates of movement propelled by MHC isoforms that are the products of an alternative splicing mechanism resulting in the presence or absence of a 7-amino acid insert near the nucleotide-binding region of the myosin head (4, 5). Disparate results have been obtained concerning the effects on contractile kinetics of the two LC 17 isoforms, acidic (LC 17a ) and basic (LC 17b ), that differ in 5 of the 9 COOHterminal amino acid residues and are products of a single gene generated by an alternative splicing mechanism (6). The faster kinetics of phasic, smooth muscle in situ (7-9) correlates with the expression of the LC 17a isoform (10, 11), albeit the proportion of myosin heavy chain containing the insert was also variant in these muscles, whereas ...

Ca2+ -sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC d or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPcS elicited Ca 2+ -sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca 2+ -sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP Á RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPcS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.

This review summarizes the role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle and a potential physiological role for telokin. In tonic, compared with phasic, smooth muscles the affinity of cross-bridges in approximately 5 times higher for MgADP and the apparent second-order rate constant for MgATP is approximately 3 times lower. This gives rise to a large population of dephosphorylated cross-bridges in tonic smooth muscle. Such cross-bridges are thought to be major determinants of the different relaxation kinetics of the two types of smooth muscle and contribute to force maintenance at low levels of MLC20 phosphorylation, termed 'catch-like state' (Somlyo & Somlyo 1967) or 'latch' (Dillon et al. 1981). The molecular basis of the different affinities for MgADP and MgATP between tonic and phasic smooth muscle myosin was explored by exchange of essential myosin light chain (LC17) isoforms. In phasic bladder smooth muscle the exchange of LC17b for LC17a caused a significant decrease in the unloaded shortening velocity of non-phosphorylated, slowly cycling cross-bridges, suggesting that the LC17 isoforms contribute to the nucleotide affinity of latch bridges. The role of telokin in Ca(2+)-desensitization in phasic smooth muscle is reviewed. Telokin, the independently expressed C-terminus of myosin light chain kinase, is extensively phosphorylated during forskolin- and 8-br-cGMP-induced relaxation in situ. Telokin accelerated dephosphorylation of the regulatory myosin light chain and relaxed rabbit ileum smooth muscle. The results suggest that telokin contributes to cAMP and/or cGMP kinase-mediated Ca(2+)-desensitization of phasic smooth muscles.

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