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Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.

We tested the prevailing paradigm that relaxation of corpus cavernosum smooth muscle (CCSM) and penile erection depends upon nitric oxide-induced elevation of myoplasmic cGMP and reduced Ca2+-dependent myosin regulatory light chain phosphorylation levels. This hypothesis invokes a reversal of normal activation pathways. Upon stimulation with 250 μM phenylephrine, phosphorylation of the 20 kD myosin regulatory light chains of rabbit or human CCSM increased approximately 4-fold coincident with contraction. Removal of the agonist was followed by a slow reduction in cross-bridge phosphorylation and force to basal levels. The NO donor, sodium nitroprusside elicited a dose-dependent increase in tissue [cGMP] associated with a rapid relaxation in the continued presence of phenylephrine, although cross-bridge phosphorylation remained significantly elevated. Thus the NO-cGMP inhibitory pathway in CCSM is not simply a reversal of excitatory signal transduction mechanisms. An unidentified mechanism contributes to relaxation by decreasing the rate of cross-bridge recruitment through phosphorylation.

We have investigated whether a Ca 2+ -sensitizing substance, the thiadiazinone derivative EMD 53998, can alter the ratio of ATPase activity to force, i.e. the tension cost in skinned fibres of swine cardiac trabecula in which the tension cost was increased by inorganic phosphate. In the presence of 10 mM inorganic phosphate (P i ) and thapsigargin 20 μM, EMD 53998 reduced the energy cost of isometric tension over the entire range of activating Ca 2+ concentrations, resulting in a consistent change in slope (∼ 20% decrease) of the ATPase/force relation. We confirmed that in the absence of added phosphate and at maximal Ca 2+ activation EMD 53998 had little if any effect on tension cost. We had previously reported that the effects of EMD 53998 and P i on calcium sensitivity and maximum isometric tension are mutually antagonistic and our new energy data now support the proposal that EMD 53998 functio lly antagonizes the effects of P i on crossbridges. The decrease in the slope of the relation between ATPase and force caused by EMD 53998 may be interpreted to reflect either a decrease in the rate of ‘detachment’ ( g app ) of crossbridgesor an increase in average force per crossbridge, as predicted by classical crossbridge models. Since the P i release step of the crossbridge cycle is associated with the rate of ‘attachment’ ( f app ) rather than g app , we conclude that the decrease in tension cost with EMD 53998 most likely reflects an increased force per crossbridge. The mechanism of action for this Ca 2+ -sensitizing compound may then involve redistribution of crossbridge states around the P i release step, which may possibly be due to a lowered affinity of crossbridges for phosphate.

Methods for using synthetic peptides to specifically probe the molecular mechanisms for calcium-dependent regulation of contraction in cardiac and smooth permeabilized (or skinned) muscle are described. As examples of the use of these tools, the role of troponin in modulating the cardiac crossbridge cycle and the regulatory action of myosin light chain kinase (MLCK) in smooth muscle in Triton X-100-extracted muscle preparations have been targeted. These "skinned" fibers are functional in terms of contractility but permit precise control of aspects of the "cytoplasmic" environment around the myofilaments, such as calcium and substrate concentration. They also permit the diffusion of peptides into the ′intracellular" compartment. These include peptides derived from the common actin-binding, troponin C-binding sequence of troponin I (the so-called inhibitory sequence, Tnl 104–115) and the calmodulin-binding sequence of MLCK (also known as RS20). The effects of these peptides were monitored in terms of changes in isometric tension and expressed as changes in calcium or calmodulin sensitivity. The calmodulin-binding peptide reduced force at a fixed calcium concentration, indicating decreased calcium sensitivity. This effect was associated with a moderate decrease in myosin light chain phosphorylation and could be reversed with increased calmodulin concentration. We interpret this latter observation to mean that underlying the change in apparent calcium sensitivity is a change in the sensitivity of MLCK to calmodulin. As previously reported, the troponin I-based peptide desensitizes skinned cardiac muscle with respect to calcium by inhibiting the actin activation of the crossbridge cycle. We also discuss the results of recent experiments in which this peptide was used in conjunction with a calcium-sensitizing compound, EMD 53998. These results implicate the phosphate release step as the most likely regulatory step in the crossbridge cycle affected by the peptide and, by extension, troponin I. Peptide studies such as these have provided useful specific insights into the highly complex and multivariable regulatory systems of contraction.

The diazinone derivative EMD 53998 sensitizes skinned myocardial fibers to Ca2+ and enhances maximal calcium-activated force (pCa = 4.5) by approximately 100%; the EC50 is 10 microM in the absence and about 30 microM in the presence of added inorganic phosphate (10 mM). Although concentrations of added phosphate as low as 0.5 mM inhibit force, at high concentrations of EMD 53998 (or = 50 microM), phosphate only inhibits at concentrations exceeding 20 mM. These data suggest that the effects of EMD 53998 and phosphate are mutually antagonistic. Importantly, both EMD 53998 and phosphate had similar effects on force generation in troponin I-depleted (Ca(2+)-independent) skinned fibers, thus demonstrating that these compounds are likely to affect cross-bridges directly and not via the Ca(2+)-regulatory system.

Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca2+ solution (pCa greater than 8), the permeabilized muscle produced 84.8% +/- 20.1% (+/- S.D., n = 98) of the isometric force elicited by high Ca2+ (pCa approximately 4.5) prior to incubation with vanadate. Transfer back to a high Ca2+ solution elicited no additional force (83.2% +/- 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (greater than 90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca2+-dependent regulation. Force then depended on the negative logarithm of Ca2+ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa50, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca2+, but restoration of Ca2+ dependence required incubation with both TnI and TnC. Relaxation at low Ca2+ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20-150 "mu"M), while Ca2+ sensitivity, i.e. the pCa50, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 "mu"M and 150 "mu"M, respectively) were more sensitive to Ca2+ than those reconstituted with cTnC and cTnI (difference in pCa50 approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G104-K-F-K-R-P-P-L-R-R-V-R115-). The four mutants produced by substitution of T for P110, G for P110, G for L111, and G for K105 were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S1 ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca2+ dependence could be restored when gly110sTnI, thr110sTnI or gly111sTnI was used for reconstitution. The mutant gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca2+ concentrations and it caused an increase in Ca2+ sensitivity.

Skinned fibres from porcine ventricles exhibited a higher Ca2+ sensitivity (pCa50, i.e. -log10 Ca2+ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca2+ sensitivity and Ca2+ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca2+ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa50=5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca2+ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca2+ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca2+-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 μM). The Ca2+-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137–148) and, in addition, this effect was antagonized by EMD 53998. These results suggest that EMD 53998 antagonizes the inhibitory action of TnI, possibly by interfering with the interaction of the TnI inhibitory region with actin.

Peptides derived from the sequence of the S1 domain of the myosin heavy chain were tested for their effects on the regulation of cardiac contractility. Basal calcium responsiveness of the contractile apparatus in terms of isometric tension generation and ATPase was determined in chemically demembranated ventricular fibre bundles. Incubation with a series of peptides derived from the peptide sequence around SH thiol group (Cys 707) resulted in a measurable increase in isometric tension and ATPase activity at sub-maximal concentrations of calcium but not at saturating levels of calcium activity, thus demonstrating a “calcium-sensitizing” effect of these peptides. The effects of two of these peptides, S1 687–716 and S1 701–717, are demonstrated to mimic, but importantly were not additive with, the calcium sensitization induced by lowering ATP concentration to 10 μM from 10 mM. This suggests the possibility of a similar mechanism of action underlying both types of sensitization. Because these effects demonstrate tissue specificity, were sensitive with respect to potency to not only amino acid composition but also sequence, and could not be duplicated by a similarly charged, non-homologous peptide, we attribute the effects to be specific to the sequences of these peptides. These data provide further evidence that the sequence between residues 687 and 717 of the S1 domain of the myosin heavy chain influences the calcium responsiveness of the contractile apparatus.

The techniques for permeabilizing (i.e. “skinning”) cells were pioneered by studies on muscle fibers (Filo, Bohr and Ruegg, 1965) and have now been extended to many other cell types. The use of cells with permeabilized membranes has some unique advantages with respect to studying the regulation of cellular processes. The most important of these is the ability to manipulate the internal environment of the cell with a level of sensitivity that is not possible in cells with intact membranes (Knight and Scrutton, 1986).

A new technique for treating skinned cardiac muscle fibers has been developed in which troponin I is extracted, giving rise to unregulated fibers. The effect of the 12-residue troponin I peptide on these fibers indicates that this region of troponin I is solely responsible for muscle relaxation (inhibition of force). Furthermore, troponin I peptide-troponin C reconstituted fibers are stable through several contraction-relaxation cycles indicating the peptide can switch binding sites between actin and troponin C. The troponin I peptide can substitute for the native protein as part of the calcium-sensitive molecular switch that controls muscle regulation.