Increase in calcium sensitivity of cardiac myofibrils contributes to the cardiotonic action of sulmazole.

The aim of this study was to investigate whether increasing calcium sensitivity of myofibrils plays a role in the positive inotropic activity of the cardiotonic agent sulmazole. We studied the effects of the stereoisomers of sulmazole on cardiac contractility in vivo and in vitro, arterial blood pressure, cardiac (Na-K)ATPase activity, cAMP/cGMP-phosphodiesterase activity of cardiac and smooth muscle tissue and calcium sensitivity of skinned myocardial fibres. Both stereoisomers of sulmazole were equipotent vasodilators in vivo and this can be explained by their equipotent cAMP- and cGMP-phosphodiesterase inhibitory activities in smooth muscle tissue. However, (+)sulmazole was a much stronger positive inotropic agent than (-)sulmazole in vivo and in vitro. This difference in inotropic activity cannot be explained by cAMP- or cGMP-phosphodiesterase inhibition or (Na-K)ATPase inhibition in cardiac tissue. Only (+)sulmazole produced a dose-dependent increase in calcium sensitivity of skinned myocardial fibres. Therefore, the calcium sensitizing effect on myofibrils evoked by (+)sulmazole might be responsible for the difference in inotropic activity observed between the stereoisomers of sulmazole.

Calcium-requirement for activation of skinned vascular smooth muscle from spontaneously hypertensive (SHRSP) and normotensive control rats.

The aim of this study was to clarify whether the increased vascular tone in spontaneous hypertension of rats is due to a change of the calcium-sensitivity of the contractile proteins themselves. In skinned rat tail artery rings from SHRSP and WKY rats the calcium-requirement for half maximal activation (3.6 X 10(-6)M Ca++ for both rat strains) as well as relaxation half times (1.45 +/- 0.43 min, SHRSP and 1.63 +/- 0.48 min, WKY) were found to be identical. The extent of myosin phosphorylation in the contracted and in the relaxed state did not differ between SHRSP and WKY. It is concluded that changes at the level of the contractile proteins are not involved in the increased vascular tone of SHRSP essential hypertension.

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle.

The contraction induced by a Ca2+-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (Fo), immediate elastic recoil (SE), unloaded shortening velocity (Vus), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca2+ -induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. Fo developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. Vus in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3 +/- 8.9% less than Vus induced by Ca2+ (1.6 X 10(-6) M) in the control fibers. Addition of Ca2+ (1.6 X 10(-6) M) to a contraction induced by MLCK-resulted in small increases in both Fo and Vus. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca2+. We conclude that with respect to actin-myosin interaction, MLCK-and Ca2+ -induced contractions are similar.

Effect of Ca2+-independent myosin light chain kinase on different skinned smooth muscle fibers.

In various skinned smooth muscle fiber preparations, (porcine carotid artery, rat tail artery, chicken gizzard and Taenia coli from guinea pig) a Ca2+-independent myosin light chain kinase (MLCK) initiated a contraction in absence of Ca2+. While the Ca2+ insensitive MLCK was effective on the vertebrate smooth muscles it did not act on the invertebrate skinned skeletal muscle preparation from Limulus and anterior byssus retractor muscle from Mytilus edulis. The results indicate that in vertebrate smooth muscles phosphorylation is sufficient for activation and that there is no obligatory role for an additional mechanism in initiation of contraction.

Cyclic-AMP mediated relaxation of chemically skinned fibers of smooth muscle.

Smooth muscle from guinea pig taenia coli was chemically skinned with Triton X-100 and stored in ATP-salt solution containing 50% glycerol at -20 degrees C. Fiber bundles were relaxed at Ca2+-concentrations below 10(-7) M, but contracted at 10(-6) M Ca2+. The isometric tension developed could be partly relaxed by the addition of c-AMP (in the presence of NaF), and it could also be inhibited following preincubation with the catalytic subunit of c-AMP dependent protein kinase. The inhibitory effect was much more pronounced at intermediate Ca2+-concentrations (e.g. 10(-6)) than at concentrations producing a maximum contraction, suggesting that Ca-sensitivity had been lowered. Sodium fluoride which was required to potentiate the c-AMP effects was found to have a slight relaxing effect per se. The c-AMP effect may be mediated through activation of cyclic AMP-dependent kinase, producing, phosphorylation of the myosin light chain kinase which, according to Adelstein et al. (1978), may result in a net dephosphorylation of the myosin light chains and a concomittant inhibition of the contractile response.

Phosphorylation of smooth muscle myosin and myosin light chains.

The most popular theory to account for the regulation of the contractile activity of smooth muscle, at the contractile protein level, is based on the phosphorylation and dephosphorylation of the myosin molecule. The enzymes involved are a myosin light chain kinase and a phosphatase, respectively. In this communication a method is given for the purification of the kinase. Using the purified kinase in combination with calmodulin, the pH dependence and rates of P transfer were examined. An Arrhenius plot of phosphorylation rates indicated that Q10 is approximately 2. The rates of P transfer to myosin light chains at 25 C and 37 C were about 15 and 34 mumol.min-1.mg-1 kinase, respectively. It is shown also that the rate of phosphorylation of isolated myosin light chains is significantly faster than the rate obtained when whole myosin is used as the phosphate acceptor, the latter being at least an order of magnitude slower. This difference in rates was not due entirely to the difference in physical states of the two substrates since at an increased ionic strength, where myosin was soluble, the rate of phosphorylation of the light chain fraction was still considerably faster than the rate of phosphorylation of whole myosin.

A Ca2+-sensitive myosin light chain kinase, regulating pig carotid smooth muscle actomyosin ATPase.

In this paper the correlation between phosphate incorporation into the regulatory light chain of myosin by a Ca2+-dependent myosin light chain kinase, and the Ca2+-sensitive ATPase activity and superprecipitation behaviour of arterial actomyosin, is described.

Effects of trifluoperazine on smooth muscle actomyosin. Short communication.

Trifluoperazine (TFP) inhibits superprecipitation and ATPase activity of smooth muscle actomyosin. This effect appears not to be due to the inhibitory effect of TFP on the Ca++-dependent modulator and the myosin light chain kinase, which are known to be cofactors required for activation of smooth muscle actomyosin.

Calcium-sensitivity of pig-carotid-actomyosin ATPase in relation to phosphorylation of the regulatory light chain.

Ca2+-dependent phosphorylation of the 20000-Mr regulatory light chain was found to be a necessary condition for the Ca2+-sensitivity of the Mg2+-dependent ATPase activity and superprecipitation of pig carotid actomyosin. Actin-myosin interaction independent of phosphorylation and Ca2+ (ATPase activity and superprecipitation) were demonstrated in aged actomyosin preparations and in preparations from which the regulatory light chains were removed by papain digestion.

Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K+- and Ca2+-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.