C. Zeugner

The dependence of unloaded shortening velocity on Ca++, calmodulin, and duration of contraction in "chemically skinned" smooth muscle.

Unloaded shortening velocity, a mechanical parameter associated with the rate of cross-bridge cycling, was investigated in chemically skinned guinea pig taenia coli and hog carotid artery. Shortening velocity was measured by the technique described by Edman, whereby large length steps are rapidly imposed on the muscle and the time under unloaded conditions is determined from the isometric myograms. Shortening velocity determined in this manner was similar to Vmax from the Hill force-velocity relations reported for both living and skinned taenia coli, and, in the case of carotid artery, was at least as large as that reported for living muscle. The behavior of shortening velocity was qualitatively similar for both preparations. Shortening velocity was strongly temperature dependent, with a Q]o of approximately 3.6. Shortening velocity was found to be dependent on both the Ca++ and calmodulin concentration. In contrast to the dependence of isometric force on Ca++-calmodulin, shortening velocity could be increased further by the addition of Ca++ and/or calmodulin under conditions when isometric force was maximized. Incubation with ATP-7S, which presumably maximizes the phosphorylahon of myosin, did not increase shortening velocity beyond the maximum value obtained in the presence of Ca++- calmodulin alone. The development of shortening velocity after exposure to high Ca++ solution was found to precede that of isometric force. The steady state value tended to be slightly lower than the maximum shortening velocity, the largest difference observed being less than 1.5-fold. Thus, whereas both isometric force and shortening velocity are dependent on the Ca++-calmodulin concentration in skinned smooth muscle, the dependencies are not identical, differing with respect to temporal development and concentration. These differences may underlie the decline in velocity with maintained isometric force observed in living smooth muscle.

Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate

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 > 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca22+ (pCa ∼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 (>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.

The positive inotropic calcium sensitizer EMD 53998 antagonizes phosphate action on cross-bridges in cardiac skinned fibers

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.

Inhibition of TnI-TnC interaction and contraction of skinned muscle fibres by the synthetic peptide TnI (104- 115) *

Circular dichroism was used to study the induction of helix in TnC or TnI-TnC by the TnI peptide [104–115] at various Ca2+ concentrations. The increase in negative ellipticity and pCa2+ values for the peptide-TnC complex, indicates that binding of the peptide to TnC, induces a small helical conformational change in TnC. This results in an increase in the Ca2+ binding constant and the pCa50 value required to induce 50% of Ca2+-dependent helix in TnC. The introduction of the peptide to a preformed mixture of TnI-TnC resulted in an increase in negative ellipticity and a decrease in the pCa50 and the apparent Ca2+ binding constant towards the values obtained for the TnI peptide-TnC complex and away from those of TnI-TnC. This demonstrates that the TnI peptide can successfully compete with TnI for TnC and thereby inhibit the TnI-TnC interaction. The addition of the TnI peptide to skinned rabbit psoas or porcine cardiac fibres resulted in the inhibition of the force development and a decrease in the pCa50 values required for 50% Ca2+ activation. The magnitude of the inhibition of tension development and the shift in the Ca2+ sensitivity for skinned cardiac muscle fibres was approximately half that observed with skeletal muscle fibres. In view of the CD findings, these skinned fibre results can be accounted for by the peptide inhibiting the TnI interaction with TnC. However, it is possible that the TnI peptide also has a direct inhibitory effect on TM-actin. Mastoparan, another TnC binding peptide, also inhibited the tension development in skinned skeletal and cardiac muscle fibres, but was much less efficient than the TnI peptide.

Skinned coronary smooth muscle: Calmodulin, calcium antagonists, and cAMP influence contractility

SummaryThe effects of Ca2+, calmodulin, cAMP, the catalytic subunit of cAMP-dependent protein kinase (CSU) and some Ca2+ antagonists were studied in chemically (Triton X-100) skinned coronary smooth muscle. Calmodulin increased the Ca2+ responsiveness of the muscle fiber as indicated by the reduction in the threshold as well as the half-maximal activating Ca2+ concentration. Trifluoroperazine, a calmodulin antagonist, inhibited Ca2+-calmodulin-induced contraction. Both cAMP anCSU were effective inhibitors of contraction induced at an intermediate Ca2+ concentration. Fendiline, a Ca2+-antagonist, at 2×10−4 M produced a significant inhibitory effect, which was reduced by increasing the Ca2+ concentration. From other Ca2+ antagonists tested, W-7, but not D600 and verapamil, produced some inhibitory effect. The data indicate that the responses of skinned coronary smooth muscle to Ca2+, calmodulin and cAMP are similar to those obtained with other skinned smooth muscles. Furthermore, skinned fiber preparation can serve as a useful tool to investigate possible direct effects of drugs on the activating and regulatory systems in smooth muscle.

A calmodulin-binding peptide relaxes skinned muscle from guinea-pig taenia coli

During smooth muscle activation the calcium calmodulin complex interacts with myosin light chain kinase (MLCK) whereby activating it. A synthetic peptide analogue (RS20) corresponding to the calmodulin recognition sequence of MLCK has been synthesized and previously found to inhibit the calmodulin stimulated light chain kinase activity. Here we studied the effect of this peptide on skinned fibers from guinea pig taenia coli. Maximal contractions induced by 30 μM Ca2+ at 0.1 μM calmodulin could be completely relaxed by the peptide at 1 μM. The inhibitory effect was accompanied by partial dephosphorylation only of the regulatory myosin light chain. Relaxation could be reversed by addition of calmodulin which also increased the extent of light chain phosphorylation.The calmodulin concentration required for reversing the inhibition depended on the concentration of the inhibitory peptide suggesting that the peptide competed with MLCK for the calmodulin binding site. As the calcium-calmodulin-peptide mixture constitutes a calmodulin buffer, our results suggest, that the peptide is a calmodulin antagonist unique in terms of its potency and that less than nanomolar concentrations of free calmodulin may be required for inducing smooth muscle contractions.

Myosin and Troponin Peptides Affect Calcium Sensitivity of Skinned Muscle Fibres

It is well known that the force developed by the contractile system depends on the calcium ion concentration in the medium surrounding the myofilaments as well as on the Ca++-sensitivity or the calcium responsiveness of the contractile apparatus (Ruegg 1988). We have studied the effect of peptides derived from troponin-I and the 20kDa domain of myosin subfragment-1 which affect the calcium sensitivity of the contractile system. These effects are of particular interest in the case of cardiac muscle, where the relationship between intracellular calcium concentration and force may vary over a wide range. For instance, hearts that are subjected to long periods of ischemia may develop a contracture even at basal levels of free calcium (Allshire et al. 1987, Allen and Orchard 1987). This is probably due to a potentiation of ATPase and force generation occurring at low levels of ATP (Winegrad 1979). Indeed, Guth and Potter (1987) showed, that attached crossbridges increase the calcium responsiveness of skinned fibres at low ATP-concentrations, when some crossbridges are in the nucleotide-free state. Under these conditions, the contractile system is activated, and even potentiated at very low calcium ion concentrations which normally do not elicit a contraction (cf. Weber and Murray 1973). In vitro, such a potentiation also occurs in the presence of excess of myosin subfragment-1, S1, indicating that there is cooperativity in the binding of S-1 to actin (Greene and Eisenberg 1980). Here we propose as a working hypothesis that it may be the actin-binding region of subfragment-1 around the SH-1 group which by, interacting with actin, is capable of “turning on” actin, so that calcium responsiveness increases.

Ca2+-cyclic AMP interaction in chemically skinned smooth muscle

Using guinea-pig taenia coli smooth muscle that is chemically skinned with Triton X-100 to functionally destroy plasmalemma as well as sarcoplasmic reticulum, we demonstrate that cAMP can enhance the extent as well as the rate of relaxation produced by lowering free [Ca2+] in a skinned fiber bundle precontracted with maximal [Ca2+]. However, these actions of cAMP are strongly dependent on the level of free [Ca2+] in the bathing medium, the effect of cAMP being greatly attenuated at higher [Ca2+]. These data support and further extend the results of our previous study indicating that modulations in [Ca2+] can have a strong influence on the ability of cAMP to produce a direct inhibitory effect on the contractile machinery in smooth muscle.