The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle.

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.

Rapid turnover of myosin light chain phosphate during cross-bridge cycling in smooth muscle.

The rate of phosphatase-mediated dephosphorylation of the regulatory light chain of smooth muscle myosin was determined under nearly steady-state conditions in permeabilized muscles, from the time course of incorporation of 33P-labeled phosphate into the light chain after the photolytic release of [gamma-33P]ATP from high specific activity caged [gamma-33P]ATP. The extent of myosin light chain phosphorylation is unchanged, and, if the kinase and phosphatase reactions are irreversible, the rate constant for the exponential increase in 33P in the light chain is equal to the rate constant for the phosphatase reaction. Under activated conditions (pCa 4.5) at 20 degrees C, the incorporation of 33P into approximately 80% of the phosphorylated light chain is fit by a single exponential with a rate constant of 0.37 s-1. ATP usage due to phosphorylation and dephosphorylation of the light chain is about one-third of the suprabasal energy requirement. The high phosphatase rate constant suggests that dephosphorylation of the light chain is rapid enough to interact with and potentially modify the completion of the cross-bridge cycle.

Cross-bridge cycling at rest and during activation. Turnover of myosin-bound ADP in permeabilized smooth muscle.

Single turnover experiments were performed on myosin-bound ADP by measuring the time course of incorporation of [3H]ADP following rapid formation of [3H]ATP by photolysis of caged [3H]ATP. Permeabilized rabbit portal veins were incubated in a solution at 20 degrees C with 1 mM MgATP, 20 mM phosphocreatine, 1 mg/ml creatine phosphokinase, and containing [14C]ATP and high specific activity caged [3H]ATP. At variable times following a UV flash, the muscle was frozen, nucleotides were extracted, and the ratio 3H:14C in ADP was compared to that in ATP. At rest, the exchange of bound ADP occurred with a rate constant of 0.004 s-1. When the myosin light chain was about 80% thiophosphorylated, and the muscle was generating maximum isometric force, there appeared a fast phase of ADP exchange (44% of the total) which had a rate constant of 0.2 s-1. The change in rate of ADP exchange on myosin is sufficient to explain the measured increase in ATPase activity upon thiophosphorylation of the myosin light chain. A simple analysis of the data suggests that there is a 50-fold increase in the cycling rate of cross-bridges in the muscle upon phosphorylation under isometric conditions. The fraction of ADP exchanged at 10 s following photolytic release of [3H]ATP was found to be approximately linearly related to the degree of thiophosphorylation of the myosin light chain. This supports the idea that phosphorylation of the light chain causes the transition of myosin from the resting (slow ATPase) cycle into the activated (fast ATPase) cycle, and that the fraction of myosin in the fast cycle is directly determined by the degree of light chain phosphorylation. The data are also consistent with the cooperativity model described previously by Vyas et al.

Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle.

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.

Myosin-product complex in the resting state and during relaxation of smooth muscle.

Previous findings suggested that in resting smooth muscle ADP is bound to myosin and that phosphorylation of the myosin, and its subsequent interaction with actin, increases the rate of ADP release. We have now extended these studies to include measurements of bound Pi as well as bound ADP in permeabilized rabbit portal vein. We report that in resting smooth muscle that has been exposed to [3H]ATP and [gamma-32P]ATP, followed by a chase in an unlabeled relaxing solution, the ratio of bound [3H]ADP to bound [32P]Pi is close to unity, and both are released at approximately the same rate. This suggests that myosin exists predominantly with both ADP and Pi bound under resting conditions and that the release of one is quickly followed by the release of the other. In contrast, there is a significant 30% excess of bound Pi over ADP in a muscle during relaxation from an isometric contraction. Under these conditions, while force output is slowly decreasing, both light chain phosphorylation and adenosinetriphosphatase (ATPase) activity have decreased to near-resting values. The time course of relaxation is similar to the time course of Pi release from both the resting and relaxing muscle. We propose that during relaxation the dephosphorylated cross bridges which are bearing force have Pi but not ADP bound and that detachment of the cross bridge (and thus force decay) is limited by Pi release from myosin which occurs at the same rate as in the resting muscle.

Reversible inhibition of acetylcholine contracture of molluscan smooth muscle by heavy metals: correlation to Ca++ and metal content.

The present study examined the effects of three heavy metals on the acetylcholine (ACh) contracture and Ca++ kinetics of the anterior byssus retractor muscle of Mytilus edulis. An isolated tissue bioassay using anterior byssus retractor muscle was prepared according to standard procedures and the isometric tension produced in response to ACh was measured. Ten millimolar Ni++, Co++ or Cd++ reduced the maximum contracture response to ACh in zero-Ca medium in a time-dependent manner. The inhibition was reversed upon restoration of medium containing 10 mM Ca++. The loss (and re-establishment) of contracture response to ACh corresponded to the influx (and efflux) of the heavy metal ions opposite to the direction of Ca++ flow. These results are consistent with the concept that the loss of the ACh contracture response is attributable to the displacement of tissue Ca++ from release sites by heavy metals.

Effect of diazepam on calcium translocation during physiological muscle fatigue.

Stimulation of frog sartorius muscle at 1 Hz leads to an initial positive staircase during the first 120 twitches and is followed by a negative staircase. There is a net calcium influx into two distinct compartments within the muscle during the positive staircase. The two compartments are separated by measuring the calcium extracted from muscles soaked in strontium-Ringer for 15 min and the calcium remaining in the muscle. A net gain of extractable Ca++ (0.32 mumol/g wet wt.) and residual Ca++ (0.18 mumol/g) is observed during positive staircase. A loss in residual Ca++, a gain in extractable Ca++ and a net loss of Ca++ (0.09 mumol/g) to the bathing medium occur during the period preceding physiological muscle fatigue (60 to 120 twitches). Diazepam (EC50, 5.6 X 10(-6) M) causes a marked reduction in the latent period and increases the rate constant 2.6 times the control value for physiological muscle fatigue. A net loss of 0.31 mumol/g of Ca++ to the bathing medium occurs during the interval between 60 and 120 twitches. Diazepam increases net Ca++ efflux 3.5-fold during this interval when compared to control muscles. Diazepam does not affect the Ca++ gained during the positive staircase but accelerates the loss of calcium from the residual and the extractable compartments during the initial phase of physiological muscle fatigue. Physiological muscle fatigue is attributed to an accumulation of calcium in the transverse tubular network and an uncoupling of the muscle action potential from contraction.

Nickel inhibition of calcium release from subsarcolemmal calcium stores of molluscan smooth muscle.

Replacement of calcium in artificial sea water (ASW) by nickel causes a loss of calcium at a single exponential rate (tau = 23.2 min) and the loss of contracture response to acetylcholine (ACh) in the anterior byssus retractor muscle of Mytilus edulis L. The ACh contracture response is lost at two rates; 93% of the ACh contracture response is lost rapidly (tau = 7.43 min) and the remaining 7% of ACh contracture response is lost slowly (tau = 66.6 min). The rapid phase of loss of ACh contracture corresponds to the rapid exponential uptake of nickel (tau = 6.5 min). The loss of ACh contracture response is attributed to the displacement of calcium from membrane release sites by nickel. In O-Ca-Mg ASW, the ACh contracture responses are well maintained over a 35-min period when stimulated by ACh every 5 min. Stimulation by ACh after 1, 20 and 40 min in O-Ca-Mg ASW results in a reduction of ACh contracture response to 15% of the response in Ca ASW. Similar treatment in O-Ca-Ni ASW reduces the ACh contracture response to 4%. Potassium and caffeine contracture responses at 1, 20 and 40 min in O-Ca-Mg ASW are reduced to 25 and 28%. Similar treatment in O-Ca-Ni ASW produces a block of potassium contracture response and reduces caffeine contracture response to 3%. Our 45Ca uptake data demonstrate that calcium influx is not required for the ACh contracture responses of anterior byssus retractor muscle.