rMLC Phosphorylation Research Articles

F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs.

1. Dynamic actin reorganization involving actin polymerization and depolymerization may play an important functional role in smooth muscle. 2. This study tested the hypothesis that F-actin stabilization by phalloidin increases tension cost (i.e. ATP hydrolysis rate per unit of isometric force) during Ca2+-induced activation of Triton X-100-permeabilized canine tracheal smooth muscle. 3. Adenosine 5'-triphosphate (ATP) hydrolysis rate was quantified using an enzyme-coupled NADH fluorometric technique, regulatory myosin light chain (rMLC) phosphorylation was measured by Western blot analysis, and maximum unloaded shortening velocity (Vmax) was estimated by interpolation of the force-velocity relationship to zero load during isotonic loading. 4. Maximal activation with 10 microM free Ca2+ induced sustained increases in isometric force, stiffness, and rMLC phosphorylation. However, the increase in ATP hydrolysis rate initially reached peak values, but then declined to steady-state levels above that of the unstimulated muscle. Thus, tension cost decreased throughout steady-state isometric force. 5. Following incubation of permeabilized strips with 50 microM phalloidin for 1 h, the increases in isometric force and stiffness were not sustained despite a sustained increase in rMLC phosphorylation. Also, after an initial decline, tension cost increased throughout activation. Phalloidin had no effect on Vmax during steady-state isometric force or on rMLC phosphorylation. 6. These findings suggest that dynamic reorganization of actin is necessary for optimal energy utilization during contraction of permeabilized airway smooth muscle.

ATP hydrolysis during contraction of permeabilized airway smooth muscle.

This study determined whether the time-dependent decline in the rate of ATP hydrolysis by actomyosin ATPase during sustained isometric force can occur in the absence of a time-dependent decline in regulatory myosin light chain (rMLC) phosphorylation in Triton X-100-permeabilized canine tracheal smooth muscle. Maximal activation with 10 microM Ca(2+) induced sustained increases in isometric force, stiffness, and rMLC phosphorylation; however, the increase in the ATP hydrolysis rate was initially high but then declined to a steady-state level above that of the unstimulated muscle (basal 31.8 +/- 5.8 nmol. cm(-3). s(-1); peak 81.4 +/- 11.3 nmol. cm(-3). s(-1); steady-state 62.2 +/- 9.1 nmol. cm(-3). s(-1)). Activation of strips in which the rMLC was irreversibly and maximally thiophosphorylated with adenosine 5'-O-(3-thiotriphosphate) also induced sustained increases in isometric force and stiffness but a nonsustained increase in ATP hydrolysis rate. There was no significant difference in the peak or steady-state isometric force, stiffness, or ATP hydrolysis rate or in the steady-state maximum unloaded shortening velocity between strips activated by 10 microM Ca(2+) or rMLC thiophosphorylation (0.058 +/- 0.016 and 0.047 +/- 0.011 muscle lengths/s, respectively). Mechanisms other than changes in rMLC phosphorylation contribute to the time-dependent decline in actomyosin ATPase activity during sustained activation of canine tracheal smooth muscle.

CGMP modulation of Ca2+ sensitivity in airway smooth muscle.

A beta-escin-permeabilized canine tracheal smooth muscle preparation was used to test the hypothesis that cGMP decreases Ca2+ sensitivity in airway smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that regulate Ca2+ sensitivity and not by inhibiting Ca2+/calmodulin activation of the contractile proteins. 8-Bromo-cGMP (100 microM) had no effect on the free Ca2+ concentration-response curves generated in the absence of muscarinic receptor stimulation. In the presence of 100 microM ACh plus 10 microM GTP, 8-bromo-cGMP (100 microM) caused a rightward shift of the free Ca2+ concentration-response curve, significantly increasing the EC50 for free Ca2+ from 0.35 +/- 0.03 to 0.75 +/- 0.06 microM; this effect of 8-bromo-cGMP was concentration dependent from 1 to 100 microM. 8-Bromo-cGMP (100 microM) decreased the level of regulatory myosin light chain (rMLC) phosphorylation for a given cytosolic Ca2+ concentration but had no effect on the amount of isometric force produced for a given level of rMLC phosphorylation. These findings suggest that cGMP decreases Ca2+ sensitivity in canine tracheal smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that modulate the relationship between cytosolic Ca2+ concentration and rMLC phosphorylation.

Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle.

We studied in beta-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PDBu) on isometric force at a constant submaximal Ca2+ concentration (i.e., the effect on Ca2+ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca2+ sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19-31) (PSSI), and staurosporine] on PDBu-induced Ca2+ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca2+ sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca2+ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.

Halothane inhibits agonist-induced potentiation of rMLC phosphorylation in permeabilized airway smooth muscle.

Agonist-induced increases in CA2+ sensitivity are mediated in part by mechanisms that increase phosphorylation of the regulatory myosin light chain (rMLC) at constant cytosolic Ca2+ concentration ([Ca2+]i). The current study tested the hypothesis that halothane inhibits acetylcholine (ACh)-induced potentiation of rMLC phosphorylation in beta-escin-permeabilized canine tracheal smooth muscle. ACh plus GTP significantly potentiated the increase in isometric force and rMLC phosphorylation induced by 0.8 microM free Ca2+. However, whereas the potentiation of isometric force was sustained, the potentiation of rMLC phosphorylation was biphasic, peaking at 0.5 min and then declining by approximately 10 min to a steady-state level significantly above that induced by 0.8 microM free Ca2+ alone. This finding suggests that mechanisms in addition to changes in rMLC phosphorylation may mediate ACh-induced Ca2+ sensitization, as has been reported for vascular smooth muscle. Halothane (0.91 +/- 0.10 mM) significantly inhibited ACh plus GTP-induced potentiation of rMLC phosphorylation and isometric force after 2 (peak rMLC phosphorylation) and 15 (steady-state rMLC phosphorylation) min of stimulation. However, the effect of halothane on the potentiation of isometric force was significantly less than that expected from its effect on rMLC phosphorylation (i.e., halothane changed the relationship between rMLC phosphorylation and isometric force). These results demonstrate that halothane inhibits the ACh-induced increase in Ca2+ sensitivity by inhibiting the membrane receptor-coupled mechanisms that increase rMLC phosphorylation at constant submaximal [Ca2+]i. Possible additional effects of halothane on rMLC phosphorylation-independent mechanisms cannot be ruled out.

Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells.

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.