Calcium-dependent stress maintenance without myosin phosphorylation in skinned smooth muscle

Stress development depended on calcium-stimulated myosin phosphorylation in an arterial smooth muscle preparation in which the concentration of calcium was controlled. However, developed stress was maintained at a concentration of calcium that did not support phosphorylation. These results, in conjunction with other evidence, suggest that the interaction of two regulatory mechanisms with different calcium sensitivities regulate both stress and the rate and energetics of contraction.     

Inhibition of myosin light chain kinase by p21-activated kinase

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation.     

Autoregulation of enzymes by pseudosubstrate prototopes: myosin light chain kinase

The myosin light chain kinase requires calmodulin for activation. Tryptic cleavage of the enzyme generates an inactive 64-kilodalton (kD) fragment that can be further cleaved to form a constitutively active, calmodulin-independent, 61-kD fragment. Microsequencing and amino acid analysis of purified peptides after proteolysis of the 61- and 64-kD fragments were used to determine the amino-terminal and carboxyl-terminal sequences of the 64-kD fragment. Cleavage within the calmodulin-binding region at Arg505 generates the catalytically inactive 64-kD fragment, which is incapable of binding calmodulin. Further digestion removes a carboxyl-terminal fragment, including the pseudosubstrate sequence Ser484-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met- Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg505 and results in a calmodulin-independent 61-kD fragment. Both the 61- and 64-kD fragments have the same primary amino-terminal sequences. These results provide direct support for the concept that the pseudosubstrate structure binds the active site and that the role of calmodulin is to modulate this interaction. Pseudosubstrates may be utilized in analogous ways by other allosterically regulated enzymes.     

Myosin light chain phosphorylation does not modulate cross-bridge cycling rate in mouse skeletal muscle

An attempt was made to determine whether phosphorylation of the myosin light chain represents a thick filament-associated mechanism for modulating the rate of cross-bridge cycling in mouse skeletal muscle. When the degree of light chain phosphorylation was varied independently of tetanus duration, there was no correlation of phosphorylation with cross-bridge turnover rate, as measured by the shortening velocity of the muscle. It is concluded that in intact skeletal muscle phosphorylation of the myosin light chain does not in itself modulate cross-bridge cycling rate and that previously reported changes in cycling rate were due to other factors that may vary with tetanus duration.     

Phosphorylation of smooth muscle myosin: evidence for cooperativity between the myosin heads

The relationship between the actin-activated adenosinetriphosphatase activity of smooth muscle myosin and the extent of myosin light chain phosphorylation is nonlinear. It is suggested that the phosphorylation of the two heads of smooth muscle myosin is an ordered process and that the two heads are influenced by cooperative interactions.     

Catecholamine and dibutyryl cyclic AMP effects on myosin adenosine triphosphatase in cultured rat heart cells

Catecholamines and dibutyryl adenosine 3', 5'-monophosphate (dibutyryl cyclic AMP) increase the activity of myosin adenosine triphosphatase in cultured rat heart cells. Dichloroisoproterenol, an inhibitor of the beta receptor of the catecholamines, inhibits the action of the catecholamines but not of cyclic AMP.     

Continued expression of neonatal myosin heavy chain in adult dystrophic skeletal muscle

The expression of myosin heavy chain isoforms was examined in normal and dystrophic chicken muscle with a monoclonal antibody specific for neonatal myosin. Adult dystrophic muscle continued to contain neonatal myosin long after it disappeared from adult normal muscle. A new technique involving western blotting and peptide mapping demonstrated that the immunoreactive myosin in adult dystrophic muscle was identical to that found in neonatal normal muscle. Immunocytochemistry revealed that all fibers in the dystrophic muscle failed to repress neonatal myosin heavy chain. These studies suggest that muscular dystrophy inhibits the myosin gene switching that normally occurs during muscle maturation.     

Phosphorylation of myosin light chains in mouse fast-twitch muscle associated with reduced actomyosin turnover rate

Phosphorylation of the 18,000-dalton light chains of the fast-twitch myosin in mouse extensor digitorum longus muscles was correlated with reduction in the rate of the actomyosin adenosinetriphosphatase in vivo, but neither of these changes occurred in the soleus muscle. These results suggest that actomyosin interactions can be down-regulated by a reversible covalent modification of myosin light chains, that a mechanism for thick-filament regulation occurs in vertebrate skeletal muscle, and that the expression of this regulation may be limited to a specific fiber type.     

Localization of calcium pump activity in smooth muscle

A microsomal fraction isolated from longitudinal smooth muscle of guinea pig ileum actively sequesters calcium ion in the presence of magnesium and adenosine triphosphate in a fashion previously described for microsomes of the rabbit aorta. This activity in guinea pig ileum appears to be associated primarily with the plasma membrane as is found in the red cell. By contrast the uptake of calcium in aortic smooth muscle appears to be associated to an appreciable extent with intracellular membranes, possibly analogous to the sarcoplasmic reticulum of skeletal muscle.     

Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum

The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.     

Regulation of calcium concentration in voltage-clamped smooth muscle cells

The regulation of intracellular calcium concentration in single smooth muscle cells was investigated by simultaneously monitoring electrical events at the surface membrane and calcium concentration in the cytosol. Cytosolic calcium concentration rose rapidly during an action potential or during a voltage-clamp pulse that elicited calcium current; a train of voltage-clamp pulses caused further increases in the calcium concentration up to a limit of approximately 1 microM. The decline of the calcium concentration back to resting levels occurred at rates that varied with the calcium concentration in an apparently saturable manner. Moreover, the rate of decline at any given calcium concentration was enhanced after a higher, more prolonged increase of calcium. The process responsible for this enhancement persisted for many seconds after the calcium concentration returned to resting levels. Thus, the magnitude and duration of a calcium transient appear to regulate the subsequent calcium removal.     

Effects of dibutyryl cyclic AMP on restoration of function of damaged sciatic nerve in rats

In two experiments, the sciatic nerve of rats was either crushed or hemisected, and N(6),O(2)-dibutyryl adenosine 3',5'-monophosphate or saline was injected intramuscularly near the site of the lesion. In both types of nerve damage, the sensorimotor functions of animals treated with N(6),O(2)-dibutyryl adenosine 3',5'-monophosphate returned earlier than did those of saline-treated control animals.     

Cooperative critical thermal transition of potassium accumulation in smooth muscle

The steady-state levels of potassium and sodium of taenia coli of guinea are critically affected by varying temperature in the narrow range 12 degrees to degrees C. For the accumulation of both cations the critical temperature, T(c), is 13.8 degrees C the presence of millimolar external potassium. The value of T(c), decreases 10.0 degrees C when the external potassium is raised to 10 millimolar. Since, at a fixed Temperature, the potassium accumulation follows a cooperative mechanism, the results are compared with the quantitative predictions of this approach. The itical thermal transition behavior can be described in terms of the cooperative cumulation process.     

Effects of lairage after transport on post mortem muscle glycolysis,protein phosphorylation and lamb meat quality

The objective of this study was to investigate the effect of lairage after transport on post mortem muscle glycolysis, protein phosphorylation and lamb meat quality. Two preslaughter animal treatments, transport for 3 h and lairage for 0 h (T3L0) and transport for 3 h and then lairage for 12 h (T3L12), were compared with a control treatment of 0 h transport and 0 h lairage. Data obtained showed that preslaughter transport had a significant effect on lamb meat quality. Loins from lambs of the T3L0 treatment showed higher (P=0.026) pH_{24 h} and higher (P=0.021) pH_{48 h} values, but lower (P<0.001) drip loss and lower (P<0.05) glycolytic potential at 0 h post mortem than those of the T3L12 and control groups. Muscle samples of the T3L0 group showed higher (P=0.046) shear force and lower (P=0.005) b* value than those of the T3L12 group. Muscle glycogen concentration at 0, 2, 4 h post mortem were lower (P<0.05) in the T3L0 group than in control. No significant difference (P>0.05) in most meat quality parameters was determined between the T3L12 group and control, showing lairage for 12 h allowed lambs to recover from the effects of transport for 3 h and resulted in similar meat quality characteristics compared to no transport. Lairage after transport did not affect most meat quality indices in comparison with control, but increased the meat drip loss and b* value of lambs possibly through decreasing glycogen concentration and glycolytic potential.     

Antagonistic adrenergic-muscarinic regulation of M current in smooth muscle cells

The beta-adrenergic agonist isoproterenol and analogs of adenosine 3',5'-monophosphate (cAMP) induced a potassium current, M current, in freshly dissociated gastric smooth muscle cells. Muscarinic agonists suppress this current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. Thus, M current can be induced by an agent and regulated in antagonistic fashion by beta-adrenergic and muscarinic systems.     

Contraction of granulation tissue in vitro: similarity to smooth muscle

Strips of granulation tissue from three different experimental models contract in vitro when treated with substances that induce contraction of smooth muscle. Because the fibroblasts in such tissues have some ultrastructural features typical of smooth muscle, our findings indicate that fibroblasts are able to modulate toward a cell type that is morphologically and functionally close to smooth muscle.     

Cooperative control of potassium accumulation by ouabain in vascular smooth muscle

The effects of ouabain on potassium accumulation were studied in the dog carotid artery. It was confirmed that vascular smooth muscle lost potassium in the presence of ouabain greater than 10(-9) molar. This effect could be reversed by systematically increasing potassium in the external medium. The action of ouabain on ion accumulation was represented quantitatively with the application of a recent biophysical approach.     

Regional changes in calcium underlying contraction of single smooth muscle cells

The role of calcium in regulating the contractile state of smooth muscle has been investigated by measuring calcium and contraction in single smooth muscle cells with the calcium-sensitive dye fura-2 and the digital imaging microscope. The concentration of free calcium in the cytoplasm increased after stimulation of the cells by depolarization with high potassium or by application of carbachol. Changes in calcium always preceded contraction. The increase in calcium induced by these stimuli was limited to less than 1 microM. Calcium within the nucleus was also subject to a limitation of its rise during contraction. Intranuclear calcium rose from 200 nM at rest to no more than 300 nM while cytoplasmic calcium rose to over 700 nM. These apparent ceilings for both cytoplasmic and intranuclear calcium may result either from negative feedback of calcium on cytoplasmic and nuclear calcium channel gating mechanisms, respectively, or from the presence of calcium pumps that are strongly activated at the calcium ceilings.