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Biomedical subjects

C M Hai

Publications and source records attributed to C M Hai.

At least 19 recordsLinked to original sources
PubMed2000

Mechanosensitive modulation of receptor-mediated crossbridge activation and cytoskeletal organization in airway smooth muscle.

Recent findings indicate that mechanical strain (deformation) exerted by the extracellular matrix modulates activation of airway smooth muscle cells. Furthermore, cytoskeletal organization in airway smooth muscle appears to be dynamic, and subject to modulation by receptor activation and mechanical strain. Mechanosensitive modulation of crossbridge activation and cytoskeletal organization may represent intracellular feedback mechanisms that limit the shortening of airway smooth muscle during bronchoconstriction. Recent findings suggest that receptor-mediated signal transduction is the primary target of mechanosensitive modulation. Mechanical strain appears to regulate the number of functional G-proteins and/or phospholipase C enzymes in the cell membrane possibly by membrane trafficking and/or protein translocation. Dense plaques, membrane structures analogous to focal adhesions, appear to be the primary target of cytoskeletal regulation. Mechanical strain and receptor-binding appear to regulate the assembly and phosphorylation of dense plaque proteins in airway smooth muscle cells. Understanding these mechanisms may reveal new pharmacological targets for controlling airway resistance in airway diseases.

Cytoskeleton↗
PubMed2000

Mechanical strain memory in airway smooth muscle.

We investigated the effect of a single rapid stretch on poststretch force and myosin phosphorylation in bovine tracheal smooth muscle. When unstimulated muscle strips were stretched from suboptimal length to optimal length (L(o)), poststretch steady-state force was not significantly different from that of unstretched control at L(o). However, when carbachol-activated muscle strips were stretched from suboptimal length to L(o), poststretch force and myosin phosphorylation were lower than control and significantly correlated with initial length. When poststretch muscle strips were allowed to relax for 1 h and then activated by K(+) depolarization, the developed force remained significantly correlated with initial length. When the same strain was applied in 23 increments to minimize peak stress, poststretch force and myosin phosphorylation increased significantly, approaching the levels expected at L(o). Furthermore, poststretch force development increased after each cycle of contraction and relaxation, approaching the control level after four cycles. These results suggest that activated airway smooth muscle cells can retain relatively precise memory of past strain when they are stretched rapidly with high stress.

Animals↗
PubMed2000

Mechanical signals and mechanosensitive modulation of intracellular [Ca(2+)] in smooth muscle.

We tested the hypothesis that strain is the primary mechanical signal in the mechanosensitive modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle. We found that [Ca(2+)](i) was significantly correlated with muscle length during isotonic shortening against 20% isometric force (F(iso)). When the isotonic load was changed to 50% F(iso), data points from the 20 and 50% F(iso) experiments overlapped in the length-[Ca(2+)](i) relationship. Similarly, data points from the 80% F(iso) experiments clustered near those from the 50% F(iso) experiments. Therefore, despite 2.5- and 4-fold differences in external load, [Ca(2+)](i) did not deviate much from the length-[Ca(2+)](i) relation that fitted the 20% F(iso) data. Maximal inhibition of sarcoplasmic reticular (SR) Ca(2+) uptake by 10 microM cyclopiazonic acid (CPA) did not significantly change [Ca(2+)](i) in carbachol-induced isometric contractions and isotonic shortening. CPA also did not significantly change myosin light-chain phosphorylation or force redevelopment when carbachol-activated muscle strips were quickly released from optimal length (L(o)) to 0.5 L(o). These results are consistent with the hypothesis and suggest that SR Ca(2+) uptake is not the underlying mechanism.

Animals↗
PubMed1999

Mucosal modulation of agonist-induced myosin phosphorylation and contraction in airway smooth muscle.

We investigated the mechanism of mucosal modulation of airway smooth muscle contraction by measuring concentration- and length dependencies of carbachol-induced active stress and myosin phosphorylation in mucosa-intact and mucosa-free bovine tracheal smooth muscle. The concentration dependencies of carbachol-induced active stress in mucosa-intact and mucosa-free smooth muscles were significantly different in maximum but not in half-maximal concentration (EC50). Similar mucosa-dependent difference in maximum was also observed in the concentration dependence of carbachol-induced myosin phosphorylation. As a result, the myosin phosphorylation-active stress relations in mucosa-intact and mucosa-free smooth muscles were not significantly different. Length dependence of carbachol-induced active stress was significant in mucosa-intact smooth muscle, and accompanied by significant length dependence of myosin phosphorylation. These results suggest that the primary effect of mucosal modulation is inhibition of myosin light chain phosphorylation without uncoupling of active stress from myosin phosphorylation in airway smooth muscle.

Animals↗
PubMed1999

Mechanical strain modulates maximal phosphatidylinositol turnover in airway smooth muscle.

Mechanical strain regulates the maximal level of myosin light chain phosphorylation mediated by muscarinic activation in airway smooth muscle. Accordingly, we tested the hypothesis that mechanical strain regulates maximal phosphatidylinositol (PI) turnover (V(max)) coupled to muscarinic receptors in bovine tracheal smooth muscle. We found that PI turnover was not significantly length dependent in unstimulated tissues. However, carbachol-induced PI turnover was linearly dependent on muscle length at both 1 and 100 microM. The observed linear length dependence of PI turnover at maximal carbachol concentration (100 microM) suggests that mechanical strain regulates V(max). When carbachol concentration-PI turnover relationships were measured at optimal length and at 20% optimal length, the results could be explained by changes in V(max) alone. To determine whether the length-dependent step is upstream from heterotrimeric G proteins, we investigated the length dependence of fluoroaluminate-induced PI turnover. The results indicate that fluoroaluminate-induced PI turnover remained significantly length dependent at maximal concentration. These findings together suggest that regulating functional units of G proteins and/or phospholipase C enzymes may be the primary mechanism of mechanosensitive modulation in airway smooth muscle.

Acetylcholine↗
PubMed1998

Length-dependent modulation of smooth muscle activation: effects of agonist, cytochalasin, and temperature.

We tested the hypothesis that mechanical strain modulates agonist sensitivity of smooth muscle by measuring myosin phosphorylation and contractile force in bovine tracheal smooth muscle activated by various concentrations of the muscarinic receptor agonist carbachol and at various muscle lengths. Increasing carbachol concentration by 10,000-fold did not restore myosin phosphorylation levels at shorter muscle lengths to the level at optimal length (Lo). Maximum levels of myosin phosphorylation induced by carbachol at 0.6, 0.8, and 1.0 Lo were similar but became lower at <0.6 Lo. Cytochalasin D significantly attenuated carbachol-induced contraction by 54%. In addition, cytochalasin D treatment induced a parallel downward shift in the length-myosin phosphorylation relation. Lowering temperature from 37 to 23 degrees C did not significantly change the length dependencies of carbachol-induced active force and myosin phosphorylation. These results have led us to conclude that 1) agonist sensitivity and maximum level of activation (as measured by myosin phosphorylation) are targets of length-dependent modulation, 2) actin filaments involved in contraction and length-dependent modulation are distinct in sensitivity to cytochalasin D, and 3) length-dependent modulation is relatively temperature insensitive.

Animals↗
PubMed1997

F-actin disruption attenuates agonist-induced [Ca2+], myosin phosphorylation, and force in smooth muscle.

Cytochalasins B and D (at 10 microM) inhibited stress development induced by 1 microM carbachol in bovine tracheal smooth muscle by 55% and 90%, respectively. Glucose depletion was ineffective in inhibiting carbachol-induced contraction, indicating that inhibition of glucose transport was not the cause. Cytochalasin D-treated smooth muscle cells appeared collapsed, with spiky protrusions from the cell membrane. Deconvolution of fluorescent images of fluorescein isothiocyanate-phalloidin-labeled smooth muscle cells revealed concentrations of actin filaments near the cell periphery, including near the spiky protrusions. Cytochalasin B attenuated carbachol-induced intracellular Ca2+ concentration ([Ca2+]), especially the initial peak intracellular [Ca2+]. Cytochalasin B also attenuated carbachol-induced myosin light chain phosphorylation. However, when the myosin phosphorylation data were plotted against time-matched intracellular [Ca2+] data, the two relationships in control and cytochalasin B-treated smooth muscle were similar, suggesting that the changes in myosin phosphorylation could be explained by the changes in intracellular [Ca2+]. These results suggest that actin filaments in smooth muscle cells are dynamic and may be an integral component of Ca2+ regulation and/or signal transduction in receptor-coupled mechanisms.

Actins↗
PubMed1996

Length-dependent modulation of myosin phosphorylation and contractile force in coronary arterial smooth muscle.

Phosphorylation of the 20,000 dalton myosin light chain (LC) is a central regulatory mechanism of smooth muscle contraction. Our previous findings on airway smooth muscle have led us to hypothesize that length-dependent and length-independent modulation of myosin phosphorylation coexist in smooth muscle. In this study, we tested the general applicability of this hypothesis by investigating the length dependences of myosin phosphorylation and contractile force in bovine coronary arterial smooth muscle. Comparison of the time courses of myosin phosphorylation at optimal (Lo) and preshortened lengths indicated that the initial peak myosin phosphorylation induced by K(+)-depolarization, histamine, and endothelin were all length dependent. Additional experiments focusing on the length dependence of steady-state myosin phosphorylation revealed that the length dependence of K(+)-depolarization-induced steady-state myosin phosphorylation (0.13 mol P(i)/mol LC/Lo) was significant, but smaller than that found in airway smooth muscle. In contrast, the length dependence of endothelin-induced steady-state myosin phosphorylation (0.03 mol P(i)/mol LC/Lo) was insignificant. The different length dependences of depolarization- and endothelin-induced myosin phosphorylation were found to correlate with different length-force relations. The length-force relationships in K+-depolarized and endothelin-activated tissues remained different even when the force was normalized by the maximum value at Lo. Variable length-force relations have been reported but the biochemical basis is not understood. Results from this study suggest that length-dependent modulation of myosin phosphorylation may be an important determinant of length-force relationship in smooth muscle.

Animals↗
PubMed1994

Mechanosensitive modulation of myosin phosphorylation and phosphatidylinositol turnover in smooth muscle.

Myosin light chain phosphorylation and phosphatidylinositol turnover were measured at different muscle lengths in bovine tracheal smooth muscle. The relationship between myosin phosphorylation and muscle length was linear between optimal length (Lo) and 0.1 Lo in both unstimulated and carbachol-activated tissues. However, myosin phosphorylation in carbachol-activated tissues was more sensitive to changes in muscle length. As a result, suprabasal myosin phosphorylation induced by carbachol was significant at Lo but became insignificant at 0.1 Lo. Phosphatidylinositol turnover was assayed by measuring the formation of myo-[3H]inositol phosphates in unstimulated and carbachol-activated tissues using the Li+ method. Pairs of time-matched and length-matched muscle strips were used for control (unstimulated) and activation by carbachol. Phosphatidylinositol turnover in carbachol-activated tissues was more sensitive than that in unstimulated tissues to changing length. As a result, suprabasal phosphatidylinositol turnover induced by carbachol was significant at Lo but became insignificant at 0.1 Lo. These results indicated that myosin phosphorylation and phosphatidylinositol turnover mediated by muscarinic receptor activation were modulated by the mechanical state of smooth muscle.

Animals↗
PubMed1993

Time-dependent uncoupling between myosin phosphorylation and contractile force induced by Ca(2+)-depletion in smooth muscle.

Phosphorylation of the 20,000-Da myosin light chain is an important regulatory mechanism of smooth muscle contraction. In this study, we investigated the uncoupling between carbachol-activated myosin phosphorylation and isometric stress in Ca(2+)-depleted bovine tracheal smooth muscle at low [Ca2+]. In control tissues, lowering extracellular [CaCl2] from 1.6 to 0.1 mM had insignificant effects on carbachol-activated steady-state isometric stress and myosin phosphorylation. In contrast, in Ca(2+)-depleted tissues, lowering [CaCl2]0 from 1.6 to 0.1 mM significantly reduced steady-state isometric stress without significantly changing steady-state myosin phosphorylation, thus uncoupling contractile force from myosin phosphorylation. Time-course data of myosin phosphorylation and isometric stress revealed that isometric stress and myosin phosphorylation were coupled at the beginning of contractions, but then gradually became uncoupled at steady state. We attempted to stabilize the cell membrane and contractile filaments using high [Mg2+]. However, 25 mM [MgSO4] further reduced steady-state isometric stress development at 0.1 mM [CaCl2]0 without significantly changing steady-state myosin phosphorylation. These results indicated that Ca(2+)-depletion induced a time-dependent cellular process which gradually uncouples contractile force from myosin phosphorylation. Furthermore, steady-state isometric stress appeared to be not limited by myosin phosphorylation, but sensitive to the [Ca2+]/[Mg2+] ratio near the cell membrane.

Animals↗
PubMed1993

Effects of substrate and inhibition of oxidative metabolism on contraction and myosin phosphorylation in ASM.

Steady-state active stress in smooth muscle is maintained by cross bridges which undergo continuous cycling and myosin phosphorylation, and the two processes both consume ATP. In this study, we investigated whether energy utilization by cross-bridge cycling and myosin phosphorylation is compartmentalized and examined their relative affinities for ATP in airway smooth muscle. We measured active stress, myosin phosphorylation, O2 consumption, and tissue ATP content in bovine tracheal smooth muscle activated by K+ depolarization when glucose was replaced by pyruvate and when oxidative metabolism was inhibited by hypoxia or uncoupled by 2,4-dinitrophenol. The results indicate that ATP produced from both glycolysis and oxidative metabolism is available to both cross-bridge cycling and myosin phosphorylation. However, steady-state myosin phosphorylation was insensitive to the inhibition of oxidative metabolism by hypoxia and mitochondrial uncoupling when steady-state isometric stress and tissue ATP content were significantly reduced. These results suggest that, relative to actomyosin adenosine 5'-triphosphatase, myosin light chain kinase has a higher affinity for ATP in intact airway smooth muscle. However, peak myosin phosphorylation associated with the initial rapid stress development was sensitive to inhibition of oxidative metabolism, probably reflecting a lower content of intracellular calcium store as a result of metabolic inhibition.

2,4-Dinitrophenol↗
PubMed1993

Fluoroaluminate- and GTP gamma S-induced stress, shortening, and myosin phosphorylation in airway smooth muscle.

GTP-binding proteins in bovine tracheal smooth muscle were activated by fluoroaluminate and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and the sensitivities of fluoroaluminate- and GTP gamma S-induced active stress and myosin phosphorylation to muscle shortening were compared. Relative to the value of myosin phosphorylation at L0, unloaded shortening induced a 63% decrease in fluoroaluminate-activated steady-state myosin phosphorylation, but had no significant effect on GTP gamma S-activated myosin phosphorylation. These results were consistent with the hypothesis that shortening-sensitive and shortening-insensitive signal-transduction pathways coexist in airway smooth muscle. However, unlike myosin phosphorylation, active stress induced by fluoroaluminate was actually less sensitive to shortening. The amount of shortening necessary to reduce active stress to half of that at Lo was 65% in fluoroaluminate-activated tissues, but was only 34% in GTP gamma S-activated tissues. The observation of different sensitivities of fluoroaluminate-activated myosin phosphorylation and active stress suggests that GTP-binding proteins modulate the dependence of active stress on muscle length in smooth muscle.

Aluminum↗
PubMed1992

Adenosine 5'-triphosphate consumption by smooth muscle as predicted by the coupled four-state crossbridge model.

We have proposed a four-state crossbridge model to explain contraction and the latch state in arterial smooth muscle. Ca(2+)-dependent crossbridge phosphorylation was the only postulated regulatory mechanism and the latchbridge (a dephosphorylated, attached crossbridge) was the only novel element in the model. In this study, we used the model to predict rates of ATP consumption by crossbridge phosphorylation (JPhos) and cycling (JCycle) during isometric and isotonic contractions in arterial smooth muscle; then we compared model predictions with experimental data. The model predicted that JPhos and JCycle were similar in magnitude in isometric contractions, and both increased almost linearly with myosin phosphorylation. The predicted relationship between isometric stress and ATP consumption was quasihyperbolic, but approximately linear when myosin phosphorylation was below 35%, in agreement with most of the available data. Muscle shortening increased the predicted values of JCycle up to 3.7-fold depending on shortening velocity and the level of myosin phosphorylation. The predicted maximum work output per ATP was 7.4-7.8 kJ/mol ATP and was relatively insensitive to changes in myosin phosphorylation. The predicted increase in JCycle with shortening was in agreement with available data, but the model prediction that work output per ATP was insensitive to changes in myosin phosphorylation was unexpected and remains to be tested in future experiments.

Adenosine Triphosphate↗
PubMed1992

Agonist-induced myosin phosphorylation during isometric contraction and unloaded shortening in airway smooth muscle.

We measured myosin phosphorylation during isometric contraction at optimal length (Lo) and unloaded shortening induced by K(+)-depolarization, electrical stimulation, carbachol, histamine, and phorbol dibutyrate (PDB) in bovine trachealis. Peak myosin phosphorylation during unloaded shortening was lower than that during isometric contraction in response to all stimuli. The lower peak myosin phosphorylation during unloaded shortening appeared to be a stretch-sensitive response because myosin phosphorylation was either equally low or further reduced during the second unloaded shortening of preshortened tissues. Similar to peak myosin phosphorylation, steady-state myosin phosphorylation was also lower during unloaded shortening in carbachol-induced contractions. However, steady-state phosphorylation during unloaded shortening and isometric contraction were not significantly different in histamine- and PDB-induced contractions. Since the coupling between Ca2+ and myosin phosphorylation was not stretch sensitive, these results suggest the coexistence of stretch-sensitive and stretch-insensitive signal transduction mechanisms in the airway smooth muscle cell membrane, and the stretch-insensitive signal transduction mechanism might involve protein phosphorylation by protein kinase C.

Animals↗
PubMed1991

Length-dependent myosin phosphorylation and contraction of arterial smooth muscle.

Ca2+, calmodulin-dependent myosin light chain phosphorylation is generally considered to be an important regulatory mechanism of smooth muscle contraction. We investigated the length dependence of myosin phosphorylation and active stress induced by K+ depolarization in arterial smooth muscle by measuring the two variables in the swine carotid media held at three steady-state tissue lengths-optimal length for contraction (Lo), 1.5 Lo, and slack length. We found that the length dependence of peak and steady-state myosin phosphorylation with respect to tissue length was different. Peak myosin phosphorylation was highest at Lo but lower at both slack length and 1.5 Lo, whereas steady-state myosin phosphorylation was similar at both Lo and 1.5 Lo, but lower at slack length. Stretching tissues to 1.5 Lo did not significantly change the steady-state myosin phosphorylation induced by K+ depolarization, but releasing tissues to slack length was associated with a 42% decrease in the steady-state myosin phosphorylation induced by K+ depolarization. These data indicated that one or more steps coupling membrane depolarization and Ca(2+)-dependent myosin phosphorylation were length sensitive. Additional data from skinned tissue experiments indicated that the length-sensitive step was not the coupling between Ca2+ and myosin phosphorylation. Therefore, these data together suggest that one or more steps coupling membrane depolarization and the increase in cytosolic Ca2+ concentration are length sensitive.

Animals↗
PubMed1990

Crossbridge phosphorylation and regulation of vascular smooth muscle contraction.

A model is proposed to explain crossbridge regulation in smooth muscle and the latch state (high force with very low crossbridge cycling rates). The model predicts that low levels of [Ca2+] and myosin phosphorylation can induce high values of force. In this analysis we show that reductions in myosin light chain phosphatase activity make stress more proportional to phosphorylation, with abolition of the latch state. This may explain some of the differences in the phosphorylation dependence of force between intact and skinned smooth muscle.

Animals↗