Phosphorylation of myosin light chains in perfused rat heart. Effect of adrenaline and increased cytoplasmic calcium ions.

1. A method was developed for the isolation of essentially pure myosin light chains from perfused rat heart. The phosphorylation of the P-light chains was estimated by hydrolysis and measurement of phosphate released, by electrophoresis in 8 M-urea and by 32P incorporation in perfusion with [32P]Pi. 2. In control perfusions there was 0.5-0.6 mol of phosphate/mol of P-light chain. This was not changed by perfusion with 5 microM-adrenaline for 10-40s. Perfusion for 1 min with medium containing 7.5 mM-CaCl2, or for 30s with medium containing 118 mM-KCl, also did not change the phosphorylation of P-light chains. 3. It is concluded that phosphorylation of P-light chains is not important in mediating the action of inotropic agents in the heart.     

Selective phosphorylation of myosin light chain in intact skeletal muscle

The phosphorylation of myosin light chain was quantitated in fast and slow chicken skeletal muscles and in frog sartorius and semitendinosus muscles. The phosphate content of light chain was determined either as moles [³²P]phosphate per mole of light chain in ³²P-labeled muscles or as percentage phosphorylated light chain of the total P-light chain, measured by densitometry after separating the phospho and dephospho forms of P-light chain with two-dimensional gel electrophoresis. Both methods revealed that the percentage of total P-light chain which was phosphorylated did not exceed 50% either in maximally tetanized or caffeine-contracted skeletal muscle. This suggests that one of the two P-light chains is selectively phosphorylated in skeletal muscle.     

Different phosphorylated forms of myosin in contracting tracheal smooth muscle

Calmodulin-dependent myosin light chain kinase phosphorylates two light chain subunits on each myosin molecule. We have developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin in smooth muscle. Four protein bands were separated in tissue extracts by nondenaturing polyacrylamide gel electrophoresis in the presence of pyrophosphate. Immunoblots demonstrated that three forms (designated M, MP, and MP2) reacted with rabbit antisera prepared against the purified phosphorylatable light chain (P-light chain) from bovine tracheal smooth muscle. Evidence was obtained that M, MP, and MP2 represented nonphosphorylated, monophosphorylated, and diphosphorylated myosin, respectively, and that the other protein band was probably filamin. The formation of different phosphorylated forms of myosin was measured in bovine trachealis strips neurally stimulated from 1.0 to 3.5 s and quick-frozen. There was no detectable MP or MP2 in unstimulated muscles; the extent of P-light chain phosphorylation measured directly was 0.02 +/- 0.01 mol of phosphate/mol of P-light chain. After 2.5-s stimulation, maximal values of 0.63 +/- 0.06 mol of phosphate/mol of P-light chain and 0.40 +/- 0.06 MP2/myosintotal were obtained. During continuous neural stimulation from 1.0 to 3.5 s, the relationship between the extent of P-light chain phosphorylation (measured directly or calculated) and the relative amount of MP2 is consistent with a random phosphorylation process.     

Myocardial functional correlates of cardiac myosin light chain 2 phosphorylation

In vitro and in situ studies have proposed a potentiation of submaximal force production after myosin light chain 2 (P-light chain) phosphorylation in mammalian striated muscle. The purpose of this study was to ascertain the relationship between the augmentation in left ventricular pressure development and cardiac myosin P-light chain phosphorylation at different times during and after submaximal treadmill exercise involving adult female Sprague-Dawley rats. In vivo hemodynamic measurements were monitored with an indwelling high-fidelity solid-state pressure transducer. Exercise heart rate, peak left ventricular (LV) pressure, and rate of LV pressure development/relaxation (LV +/- dP/dt) were significantly elevated compared with a normal sedentary group (P less than 0.001). Peak LV pressure remained significantly elevated throughout 20 min of postexercise recovery (P less than 0.01), and heart rate, LV end-diastolic pressure, and LV +/- dP/dt returned rapidly to preexercise values. Corresponding to these in vivo hemodynamic changes, increased levels of P-light chain phosphorylation were observed during both exercise (16%, P less than 0.01) and subsequent recovery periods (14%, P less than 0.02) compared with the NC group. A quasi-temporal relationship was observed between postexercise peak LV pressure potentiation and P-light chain phosphorylation. These results demonstrate that cardiac myosin P-light chain phosphorylation is associated, in part, with the augmentation of peak LV pressure observed during both exercise and recovery.     

The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers

We have studied the effect of myosin P-light chain phosphorylation on the isometric tension generated by skinned fibers from rabbit psoas muscle at 0.6 and 10 microM Ca2+. At the lower Ca2+ concentration, which produced 10-20% of the maximal isometric tension obtained at 10 microM Ca2+, addition of purified myosin light chain resulted in a 50% increase in isometric tension which correlated with an increase in P-light chain phosphorylation from 0.10 to 0.80 mol of phosphate/mol of P-light chain. Addition of a phosphoprotein phosphatase reversed the isometric tension response and dephosphorylated P-light chain. At the higher Ca2+ concentration, P-light chain phosphorylation was found to have little effect on isometric tension. Fibers prepared and stored at -20 degrees C in a buffer containing MgATP, KF, and potassium phosphate incorporated 0.80 mol of phosphate/mol of P-light chain. Addition of phosphoprotein phosphatase to these fibers incubated at 0.6 microM Ca2+ caused a reduction in isometric tension and dephosphorylation of the P-light chain. There was no difference before and after phosphorylation of P-light chain in the normalized force-velocity relationship for fibers at the lower Ca2+ concentration, and the extrapolated maximum shortening velocity was 2.2 fiber lengths/s. Our results suggest that in vertebrate skeletal muscle, P-light chain phosphorylation increases the force level at submaximal Ca2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament.     

Phosphorylation of rodent cardiac myosin light chain 2: effects of exercise

The purpose of this study was to ascertain whether the degree of cardiac myosin light chain 2 (P-light chain) phosphorylation occurs as a function of changes in cardiovascular functional state as induced by 1) treadmill exercise (20-27 m/min, 0% grade for 20, 30, 45 min) (phase I) and 2) pharmacological intervention (phase II) in adult female Sprague-Dawley rats. It was hypothesized that cardiac myosin phosphorylation is regulated in accordance with time-dependent sustained elevations in myocardial work demands requiring alterations in either heart rate or left ventricular pressure development. Exercise heart rates (HR) and double products (HR x DP) were equivalent among the three exercise groups and were significantly elevated in comparison with the normal-rest (NR) group (P less than 0.05). In phase II, isoproterenol elicited higher HR, although the atenolol group exhibited a marked reduction in HR, mean arterial pressure, and double product relative to NR (P less than 0.05). Percent myosin P-light chain phosphorylation exhibited both a HR- and a work load-dependent modulation in P-light chain levels (-9% to +23% change) in the two phases of the study (P less than 0.05). These data are consistent with the view that the above responses are associated with modulations in intracellular calcium concentrations commensurate with the alterations in HR and left ventricular pressure. Also, elevations in P-light chain phosphorylation could serve to augment left ventricular pressure development under these functional states.     

Myosin light chain phosphorylation and contractile performance of human skeletal muscle

Twitch tension and maximal unloaded velocity of human knee extensor muscles were studied under conditions of low phosphate content of the phosphorylatable light chains (P-light chains) of myosin and elevated phosphate content, following a 10-s maximal voluntary isometric contraction (MVC). After the MVC, twitch tension was significantly potentiated, with greater potentiation observed at a shorter muscle length (p less than 0.05). The MVC was associated with at least a twofold increase in phosphate content of the fast (LC2F) and two slow (LC2S and LC2S') P-light chains, but this increase was unrelated to muscle length. No significant differences in knee extension velocity were observed between conditions where P-light chains had low or elevated phosphate content. Positive but nonsignificant correlations were noted between the extent of twitch potentiation and phosphate content of individual P-light chains as well as the percentage of type II muscle fibres in vastus lateralis muscle. No significant relationships were determined for myosin light chain kinase activity and either P-light chain phosphorylation or type II fibre percentage. These data suggest that, unlike other mammalian fast muscles, P-light chain phosphorylation of mixed human muscles is not strongly associated with altered contractile performance.     

Myosin light-chain phosphatase.

1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.     

Length-dependence of isometric twitch tension potentiation and myosin phosphorylation in mouse skeletal muscle

The effect of changes in muscle length on post-tetanic isometric twitch tension potentiation and myosin P-light chain phosphorylation-was studied at 23°C in the mouse extensor digitorum longus muscle. The length-tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L₀. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L₀ to a maximum of 0.3 at 1.2 L_0. The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post-tetanus. P-light chain phosphorylation remains constant between 30 s and 3 min post-tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P-light chain phosphor-ylation. Net myosin phosphorylalion measured after a 1.5 s tetanus at 1.23 L₀ is 35% less than that obtained under the same conditions at L_0. Thus, contraction-induced phosphorylation of P-light chain decreases with increased muscle length and post-tetanic potentiation at a constant level of P-light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca²⁺ ion transient as the muscle is lengthened.     

A comparative study of the myosin light chain kinases from myoblast and muscle sources. Studies on the kinases from proliferative rat myoblasts in culture, rat thigh muscle, and rabbit skeletal muscle.

Myosin light chain kinases have been isolated from rat thigh and rabbit skeletal muscle and cultured rat myoblasts. From these preparations, two types of kinases can be distinguished: calcium-dependent and calcium-independent. Both types of kinases can phosphorylate isolated P-light chains of myosin from several sources (skeletal muscle, cardiac muscle, and platelet). Data are shown which support the phosphorylation of the same site on the non-muscle P-light chains by both types of kinases. The rates of these reactins are, however, different for the two types of kinases. Kinetic analysis of the myoblast kinase shows differing affinities for various P-light chains (non-muscle greater than cardiac greater than skeletal). In the proliferative rat myoblast, phosphorylation of myosin is a prerequisite for actin activation of the myosin ATPase activity.     

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein phosphorylation during spontaneous contraction of smooth muscle

The relationship between spontaneous contraction and protein phosphorylation of rat uterine smooth muscle was studied. Myometrial strips from estrogen-dominated rats were incubated in [³²P]orthophosphate and then frozen at various levels of isometric tension. Proteins were separated by gel electrophoresis and the incorporation of ³²P was measured. Contraction was associated with the phosphorylation of one major protein (20,000 M_r). This phosphorylation preceded maximal tension development and dephosphorylation preceded complete spontaneous relaxation. Two-dimensional gel electrophoresis indicates that the 20,000-M_r protein is the myosin light chain which has been implicated in the regulation of smooth muscle contraction.     

Purification and properties of myosin light-chain kinase from fast skeletal muscle.

1. A procedure is described for the isolation of myosin light-chain kinase from rabbit fast skeletal muscle as a homogeneous protein. 2. Myosin light-chain kinase is a monomeric enzyme of mol.wt. 77000. Under some conditions of storage it is converted into components of mol.wts. about 50000 and 30000 that possess enzymic activity. 3. The enzyme is clearly different in structure and properties from any other protein kinase so far isolated from muscle. 4. The enzyme is highly specific for the P-light chain (18000-20000-dalton light chain) of myosin and requires Ca2+ for activity. 5. The P-light chain is phosphorylated at a similar rate whether isolated or associated with the rest of the myosin molecule. 6. The effects of pH, bivalent cation and other nucleotides on the enzymic activity are described. 7. The role of the phosphorylation of the P-light chain of myosin in muscle function is discussed.     

Stretch-induced myosin light chain phosphorylation in rat uterus

Stretching of rat uterine strips induced phosphorylation of the 20,000-Da light chain of myosin to the same extent as was observed in strips contracted by carbachol or oxytocin. Stretching also reversed the partial dephosphorylation of light chain caused by treatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for 1 min. However, complete dephosphorylation of the light chain with 50-min EGTA-treatment could not be reversed by stretch. When stretched uterine strips containing light chain with a phosphate content greater than 0.75 mol/mol were quick-released, active force developed. On the other hand, when the phosphate content of light chain was reduced to less than 0.25 mol/mol, quick-release of the stretched strips did not produce active force. It is shown that Ca2+ mobilized from intracellular sources is involved in stretch-induced phosphorylation. The data indicate that myosin light chain phosphorylation is a prerequisite for active force development in smooth muscle.     

Myosin light chain phosphorylation in intact human muscle

The phosphate content of the fast (LC2F) and two slow (LC2S and LC2S1) phosphorylatable light chains (P-light chains) in myosin isolated from biopsy samples of rested human vastus lateralis muscle averaged 0.21, 0.28 and 0.25 mol of phosphate per mol of P-light chain, respectively. Following a 10 s maximal contraction, phosphate content was increased by almost 2-fold in the fast and two slow P-light chains. After prolonged, moderate cycling activity phosphate content was only slightly increased in the three P-light chains. These data suggest that, unlike animal skeletal muscle, myosin light chain kinase and phosphatase activities are similar in human fast and slow muscle fibres.     

Chronic low frequency stimulation reduces myosin phosphorylation in rabbit fast twitch muscle.

The effect of 1-12 days of electrical stimulation (10 Hz) on the ability to phosphorylate the P-light chain of myosin was studied in rabbit tibialis anterior muscle. Myosin phosphorylation was induced by exposure of the stimulated muscle and that of the contralateral leg to a single conditioning stimulus train (5 Hz) for 25 s via the motor nerve. Isometric tension was measured as were the myosin light chain composition and the activities of the enzymes responsible for phosphorylation and dephosphorylation. A computer simulation of the potential effect of a stimulation-induced disruption of Ca2+ metabolism on phosphorylation was also performed. Chronic stimulation for as little as 1 day eliminated light chain phosphorylation and reduced the myosin light chain kinase activity by approximately 36%. Conversely, phosphatase activity and light chain composition were unaffected. The model demonstrated that a slight depression in the magnitude of the Ca2+ transient could potentially attenuate phosphorylation. The data suggest that phosphorylation of myosin is extremely sensitive to prolonged muscle activity. Furthermore, it appears more likely that this sensitivity is related to regulation of intracellular free Ca2+ than to the other elements of the calmodulin-dependent system for myosin phosphorylation examined.     

Stretch activates myosin light chain kinase in arterial smooth muscle

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.     

Myosin phosphorylation, twitch potentiation, and fatigue in human skeletal muscle

Twitch tension and phosphate incorporation into the phosphorylatable light chains (P-light chains) of myosin were studied during a 10-min recovery period following a 10- or 60-s maximal voluntary isometric contraction (MVC) in 18 subjects. Analysis of muscle biopsy samples obtained before, immediately after, 1 min, and 10 min following the 10-s MVC revealed that the 10-s MVC produced a modest but transient metabolic displacement from rest, a 35% decrease in phosphocreatine, and a threefold elevation in lactate concentration. Immediately after the 60-s MVC, ATP was decreased by 20%, phosphocreatine decreased by 84%, and lactate was elevated by 15-fold. Lactate remained elevated over the 10-min recovery period. Twitch force was maximally potentiated following the 10-s MVC and declined to rest by 10 min of recovery. Twitch force was 0.66 of rest value immediately after the 60-s MVC, then increased over the next 4 min to reach a potentiated value 21% greater than rest, before declining. Significant phosphate incorporation into P-light chains was observed immediately after both contractions, but dephosphorylation to rest values at the end of recovery was only noted for the 60-s condition. These results demonstrate an inconsistent relationship between twitch tension enhancement and P-light chain phosphorylation in the in vivo human model.     

Effect of vanadate on force and myosin light chain phosphorylation in skinned aortic smooth muscle.

The effects of vanadate were examined on Ca2+-activated force and phosphorylation of 20-kDa myosin light chain in membrane-permeabilized rabbit aortic smooth muscle strips. Addition of vanadate during maximum contraction reduced the force in a dose-dependent manner, and inhibited it almost completely at 1 mM. Two-dimensional polyacrylamide gel electrophoretic analyses revealed that vanadate also reduced the phosphorylation of 20- kDa myosin light chain in a dose-dependent manner from approximately 50% in the absence of vanadate to approximately 20% in the presence of 1 mM vanadate. The effects of 1 mM vanadate on purified myosin light chain kinase and phosphatase were then examined using purified myosin as substrate, and it was found that vanadate neither inhibited myosin light chain kinase nor activated myosin light chain phosphatase. These results indicate that the reduction in the 20-kDa myosin light chain phosphorylation level by vanadate may be effected through its inhibition of the force generation in skinned smooth muscle strip, as evidenced by the finding that vanadate eliminated the enhancement of myosin light chain kinase activity brought about by the interaction between purified myosin and actin.     

Phosphorylation of myosin light chain by protease activated kinase I

Protease activated kinase I from rabbit reticulocytes has been shown to phosphorylate the P-light chain of myosin light chains isolated from rabbit skeletal muscle. The enzyme is not activated by Ca²⁺ and calmodulin or phospholipids. Protease activated kinase I is not inhibited by trifluoperazine at concentrations up to 200 μM or by the antibody to the Ca²⁺, calmodulin-dependent myosin light chain kinase from rabbit skeletal muscle. Two-dimensional peptide mapping of chymotryptic digests of myosin P-light chain show the site phosphorylated by the protease activated kinase is different from that phosphorylated by the Ca²⁺, calmodulin-dependent myosin light chain kinase.