Latch-bridge model in smooth muscle: [Ca2+]i can quantitatively predict stress

1990 ◽  
Vol 259 (2) ◽  
pp. C251-C257 ◽  
Author(s):  
C. M. Rembold ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Time Course ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Force Production ◽  
Cross Bridge ◽  
Bridge Model ◽  
Latch State

Ca2+ concentration ([Ca2+])-dependent cross-bridge phosphorylation by myosin light chain kinase is postulated to be the primary regulator of stress development in smooth muscle. A four-state model of cross-bridge function, regulated only by [Ca2+]-dependent changes in myosin kinase activity, has been proposed to explain contraction and the latch state of smooth muscle (high force with reduced cross-bridge cycling and ATP consumption). A key test of this model is to determine whether changes in myoplasmic [Ca2+], per se, can quantitatively predict changes in myosin kinase activity, cross-bridge phosphorylation, and therefore force production. We find that changes in aequorin-estimated myoplasmic [Ca2+] can quantitatively predict the time course of phosphorylation and isometric stress production in response to stimulation with histamine and angiotensin II and during adenosine 3',5'-cyclic monophosphate-mediated relaxation when [Ca2+] is not changing rapidly. These results suggest that changes in myoplasmic [Ca2+] and activation of myosin light chain kinase may be sufficient to explain both contraction and relaxation of agonist stimulated swine carotid arterial smooth muscle.

Regulation of Myosin Light Chain Kinase Activity in Smooth Muscle

1995 ◽  
pp. 139-158 ◽  
Author(s):  
Kristine E. Kamm ◽  
Katherine Luby-Phelps ◽  
Malu G. Tansey ◽  
Patricia J. Gallagher ◽  
James T. Stull
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity

Modulation of Smooth Muscle Myosin Light Chain Kinase Activity by Ca2+/Calmodulin-Dependent, Oligomeric-Type Modifications

Biochemistry ◽  
1995 ◽  
Vol 34 (19) ◽  
pp. 6366-6372 ◽  
Author(s):  
Eduard B. Babiychuk ◽  
Victoria S. Babiychuk ◽  
Apolinary Sobieszek
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Smooth Muscle Myosin ◽  
Muscle Myosin

Regulation of myosin light chain kinase: kinetic mechanism, autophosphorylation, and cooperative activation by Ca2+ and calmodulin

1994 ◽  
Vol 72 (11) ◽  
pp. 1368-1376 ◽  
Author(s):  
Apolinary Sobieszek
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Kinetic Mechanism ◽  
Binding Stoichiometry ◽  
Myosin Interaction ◽  
Cooperative Activation ◽  
Fold Excess

Phosphorylation of the regulatory light chain of myosin catalyzed by myosin light-chain kinase (MLCK) is the key reaction in the regulation of actin–myosin interaction in smooth muscle. It is shown that this reaction is of an ordered type, whereby kinase first binds ATP and then the light chain, and following phosphate transfer, the phosphorylated light chain is released before ADP. The MLCK also phosphorylates itself, and this intramolecular autophosphorylation is Ca2+ and calmodulin (CaM) dependent. It has, however, no pronounced effect on the kinase activity or on its affinity for Ca2+ and CaM. With the aim of understanding the cooperativity of MLCK activation, the activity of the kinase was systematically measured as a function of different ligands involved. In these measurements the isolated light chain and intact filamentous myosin, as well as native actomyosin, were used as substrates. The activation of the kinase by Ca2+ was positively cooperative but only at relatively low CaM levels. The activation by CaM (at saturating Ca2+ levels) was also cooperative, even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between CaM and the kinase. This cooperativity was shown to result from time-dependent changes in the MLCK that take place during incubation with Ca2+ and CaM before addition of ATP in phosphorylation assays. As a result, activity of the kinase as a function of its concentration at constant CaM was biphasic: there was optimum activity at a ratio of 1:1 CaM to kinase and almost complete inhibition of the activity at a three- to six-fold excess of the kinase over CaM. The modification required 10–15 min preincubation (with Ca2+ and CaM) and could be explained by a dimerization of the kinase, demonstrated by the use of a zero-length cross-linker.Key words: kinetic mechanism, autophosphorylation, calcium and calmodulin activation, cooperativity, myosin light chain kinase, smooth muscle.

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

1994 ◽  
Vol 267 (4) ◽  
pp. C1160-C1166 ◽  
Author(s):  
T. M. Butler ◽  
S. R. Narayan ◽  
S. U. Mooers ◽  
M. J. Siegman
Keyword(s):  
Smooth Muscle ◽  
Rate Constant ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Specific Activity ◽  
Energy Requirement ◽  
High Specific Activity ◽  
Cross Bridge ◽  
Muscle Myosin

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.

GTP gamma S-dependent regulation of smooth muscle contractile elements

1992 ◽  
Vol 262 (2) ◽  
pp. C405-C410 ◽  
Author(s):  
Y. Kubota ◽  
M. Nomura ◽  
K. E. Kamm ◽  
M. C. Mumby ◽  
J. T. Stull
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Tracheal Smooth Muscle ◽  
Proportional Increase ◽  
Gamma S ◽  
Muscle Contractile

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) increases the sensitivity of the contractile response to activation by Ca2+ in permeabilized tracheal smooth muscle. Increased tension was associated with a proportional increase in myosin light chain phosphorylation. The site of phosphorylation was determined to be serine-19, which corresponds to the site rapidly phosphorylated by myosin light chain kinase. GTP gamma S did not affect the contraction induced by the protein phosphatase inhibitor okadaic acid but did enhance contraction produced by Ca(2+)-independent myosin light chain kinase. In tracheal homogenates Ca(2+)-dependent myosin light chain kinase activity was not affected by GTP gamma S; however, dephosphorylation of 32P-labeled heavy meromyosin by phosphatase was inhibited. Thus GTP gamma S may increase the Ca2+ sensitivity of contractile elements in tracheal smooth muscle by inhibition of protein phosphatase activity toward myosin light chain.

Cyclic nucleotide dependent relaxation in vascular smooth muscle

1994 ◽  
Vol 72 (11) ◽  
pp. 1380-1385 ◽  
Author(s):  
Nancy L. McDaniel ◽  
Christopher M. Rembold ◽  
Richard A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Cyclic Nucleotide ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Regulatory Light Chain ◽  
Myosin Phosphorylation ◽  
Cross Bridge ◽  
The Relationship

Although not without controversy, the mechanisms inducing contraction of vascular smooth muscle are relatively well defined. There is a stimulus-induced increase in myoplasmic [Ca2+] with activation of myosin light chain kinase by the Ca2+–calmodulin complex, phosphorylation of the 20-kDa regulatory light chain of myosin, with subsequent cross-bridge cycling and force development. Ca2+-dependent phosphorylation of the myosin regulatory light chain appears to be the primary mechanism responsible for regulating stress in vascular smooth muscle. The relationship between myoplasmic [Ca2+] and myosin phosphorylation (i.e., the calcium sensitivity of phosphorylation) is regulated. It is higher with agonist stimulation than in tissues depolarized with high potassium solutions or after skinning procedures. The relationship between myosin phosphorylation and stress appears to be invariant with physiologic stimulation. This suggests that cross-bridge phosphorylation normally determines contraction. The mechanisms of relaxation are less well defined. In the most simple scheme, reduction of myoplasmic [Ca2+] with a fall in myosin light chain kinase activity would suffice to account for dephosphorylation of the regulatory light chain and relaxation. However, other mechanisms have been implicated in cyclic nucleotide dependent relaxation in vascular and other smooth muscle tissues. The current hypotheses of the mechanism of cyclic nucleotide dependent relaxation in vascular smooth muscle are reviewed.Key words: calcium, cyclic adenosine 3′,5′-monophosphate, cyclic guanosine 3′,5′-monophosphate, myosin light chain phosphorylation, vasodilation.

Temporal aspects of excitation-contraction coupling in airway smooth muscle

2001 ◽  
Vol 91 (5) ◽  
pp. 2266-2274 ◽  
Author(s):  
Gary C. Sieck ◽  
Young-Soo Han ◽  
Christina M. Pabelick ◽  
Y. S. Prakash
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Force Generation ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Caged Atp ◽  
Cross Bridge

In airway smooth muscle (ASM), ACh induces propagating intracellular Ca2+ concentration ([Ca2+]i) oscillations (5–30 Hz). We hypothesized that, in ASM, coupling of elevations and reductions in [Ca2+]i to force generation and relaxation (excitation-contraction coupling) is slower than ACh-induced [Ca2+]i oscillations, leading to stable force generation. When we used real-time confocal imaging, the delay between elevated [Ca2+]i and contraction in intact porcine ASM cells was found to be ∼450 ms. In β-escin-permeabilized ASM strips, photolytic release of caged Ca2+ resulted in force generation after ∼800 ms. When calmodulin (CaM) was added, this delay was shortened to ∼500 ms. In the presence of exogenous CaM and 100 μM Ca2+, photolytic release of caged ATP led to force generation after ∼80 ms. These results indicated significant delays due to CaM mobilization and Ca2+-CaM activation of myosin light chain kinase but much shorter delays introduced by myosin light chain kinase-induced phosphorylation of the regulatory myosin light chain MLC20 and cross-bridge recruitment. This was confirmed by prior thiophosphorylation of MLC20, in which force generation occurred ∼50 ms after photolytic release of caged ATP, approximating the delay introduced by cross-bridge recruitment alone. The time required to reach maximum steady-state force was >15 s. Rapid chelation of [Ca2+]i after photolytic release of caged diazo-2 resulted in relaxation after a delay of ∼1.2 s and 50% reduction in force after ∼57 s. We conclude that in ASM cells agonist-induced [Ca2+]i oscillations are temporally and spatially integrated during excitation-contraction coupling, resulting in stable force production.

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