Isoforms of myosin in smooth muscle.
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Animals↗
Biomedical subjects
J C Rüegg
Publications and source records attributed to J C Rüegg.
Ca2+-cyclic AMP interaction in chemically skinned smooth muscle.
Using guinea-pig taenia coli smooth muscle that is chemically skinned with Triton X-100 to functionally destroy plasmalemma as well as sarcoplasmic reticulum, we demonstrate that cAMP can enhance the extent as well as the rate of relaxation produced by lowering free [Ca2+] in a skinned fiber bundle precontracted with maximal [Ca2+]. However, these actions of cAMP are strongly dependent on the level of free [Ca2+] in the bathing medium, the effect of cAMP being greatly attenuated at higher [Ca2+]. These data support and further extend the results of our previous study indicating that modulations in [Ca2+] can have a strong influence on the ability of cAMP to produce a direct inhibitory effect on the contractile machinery in smooth muscle.
Animals↗
Calcium sensitivity of myofilaments in cardiac muscle--effect of myosin phosphorylation.
We investigated the influence of the extent of phosphorylation of the myosin P-light-chain on the calcium sensitivity of skinned heart fibres. Treatment of skinned heart fibres with PCM-phosphatase decreased the phosphorylation level of P-light-chain from 0.3 mol P/mol LC-2 and 0.2 mol P/mol LC-2* to 0.16 mol P/mol LC-2 and 0.14 mol P/mol LC-2*. Isometric tension development decreased by 34% at submaximal Ca2+-concentration (pCa 5.5) after incubation with PCM-phosphatase while tension achieved at maximum Ca2+-concentration (pCa 4.3) was not affected. The effect of desensitization on skinned fibres could be reversed by washing out the phosphatase or by addition of myosin light chain kinase.
Actin Cytoskeleton↗
Cyclic GMP-dependent protein kinase relaxes skinned fibers from guinea pig taenia coli but not from chicken gizzard.
The effect of cGMP and cGMP-dependent protein kinase (cG-PK) on contraction and relaxation was studied in skinned smooth muscle fibers from guinea pig taenia coli and chicken gizzard. At a fixed [Ca2+] relaxation was significantly enhanced by activated cG-PK in fibers from guinea pig taenia coli, but not in those from chicken gizzard. The Ca2+-requirement for half maximal tension maintenance was shifted to the right. Relaxation was associated with a decline in phosphorylated myosin light chain-2 from 34% to 25%. Similarly to relaxation activated cG-PK inhibited tension development only in fibers from taenia coli. These results suggest that mammalian and chicken smooth muscle fibers respond differently to cG-PK.
Animals↗
Skinned smooth muscle: calcium-calmodulin activation independent of myosin phosphorylation.
In chemically skinned chicken gizzard smooth muscle fibers investigated shortly after preparation, a contraction may be induced by calcium and calmodulin which is independent of myosin phosphorylation at intermediate Ca2+-concentrations. However, fibers stored for a prolonged period also contract in the absence of exogenous calmodulin and exhibit a close relationship between force development and myosin phosphorylation.
Animals↗
Myosin isoenzymes of vascular smooth and cardiac muscle in the spontaneously hypertensive and normotensive male and female rat: a comparative study.
Cardiac hypertrophy in hypertensive subjects, its biochemical markers, and functional consequences are of great clinical importance but still unclear. We observed a shift of the ventricular isomyosin of adult spontaneously hypertensive (H) rats of both sexes to the V3 form and a decreased myofibrillar ATPase activity in the H animals when compared to normotensive (N) controls. Compared to the male H rats, age-matched female H animals revealed a lower blood pressure, the same or even an elevated magnitude of cardiac hypertrophy, a different ventricular isomyosin pattern, and a higher myofibrillar ATPase activity. In female H rats the V1 and V3 isomyosins were equally distributed (35% V1 and 35% V3), but in male H animals the V3 was predominant (24% V1 and 45% V3). The Ca2+-regulated Mg2+-dependent myofibrillar ATPase of the rat ventricle correlated positively with the amount of V1 when measured at pCa 5 (maximum activation). At submaximum Ca2+-concentrations (pCa 6.9-5.9) the myofibrillar ATPase activities were not changed with the proportion of V1. The cooperativity of the Ca2+-activation of the myofibrillar ATPase increased with increasing amount of V1 (Hill-coefficient 3.7 with 100% V1) and decreased with increased proportion of V3 (Hill-coefficient 1.3 at 45% V3). Two myosin isoenzymes were detected in the aorta of rats, a slow migrating (S2) and a fast migrating (S1) form having both a higher mobility than the ventricular isomyosins. Only one band was observed in the portal vein, which revealed the same mobility as S2. Hypertension did not change the appearance of these vascular smooth muscle isomyosins neither in male nor in female animals.
Animals↗
[At issue: vascular spasms. Calmodulin antagonism to intracellular calcium].
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Calcium Channel Blockers↗
Effects of new inotropic agents on Ca++ sensitivity of contractile proteins.
In cardiac muscle the relationship between contraction and the intracellular free calcium concentration is not unique but may vary over a wide range, as when influenced by new positive inotropic drugs. Modulation of the sensitivity of myofilaments to calcium may be studied in chemically skinned fibers devoid of a functional sarcoplasmic reticulum and in which the ionic composition of the interfilament space can be controlled and influenced at will. At low levels of activation with Ca++ (1 microM), the new nonglycosidic, nonadrenergic cardiotonic drugs sulmazole (AR-L 115 BS) and pimobendane (UD-CG 115 BS) increase calcium-induced contraction of skinned mammalian cardiac fibers by 30% to 50% in concentrations at which they exert a positive inotropic effect in vivo. The effect on skinned fibers is due to an increase in the sensitivity of the myofilaments to calcium and, at least in the case of sulmazole, may be attributed to an increase in calcium affinity of troponin. Calcium-induced contraction of skinned fibers from vertebrate smooth muscle that lacks troponin is not activated by sulmazole and pimobendane. Thus the positive inotropic action of these new cardiotonic drugs might be accounted for in part by their calcium-sensitizing action of myofilaments, although other mechanisms that increase intracellular free calcium may be involved as well.
Actin Cytoskeleton↗
Investigations on calmodulin antagonistic effects of bepridil in intact and skinned fibres of smooth muscle.
Potassium-induced contractions of rat aortic strips are completely inhibited by N-benzyl-beta-(isobutoxymethyl)-N-phenyl-1-pyrrolidine-ethylamine (bepridil, Cordium) 10(-5) mol/l. In contrast, contractions induced by norepinephrine are only partially inhibited (by 40%) by identical doses of bepridil. Since the latter effect could be due to an inhibition of calcium release from intracellular compartments it was investigated how bepridil influences the effect of calcium on functionally isolated contractile structures. Smooth muscle preparations (guinea pig taenia coli and pig coronary artery) were skinned with octoxinol and suspended in adenosine triphosphate salt solution. During long-term incubation (greater than 40 min) of skinned fibres in low-calcium solution (pCa greater than 8) the calmodulin (CaM) is extracted. A calcium-induced contraction occurs only by adding CaM, showing the necessity of CaM for contraction. In the presence of bepridil 10(-4) mol/l the following effects were observed: submaximal contractions induced by calcium (1.1-1.6 mumol/l) were inhibited completely, the effects of calcium (4 mumol/l and 30 mumol/l) were inhibited by 83 +/- 4.5% and 53 +/- 10.5% respectively (mean +/- S.E.M.; n = 5). These effects could be completely antagonized by addition of calmodulin, final concentration 5 mumol/l. Verapamil did not show any inhibitory effects even in high concentrations (up to 10(-3) mol/l). Thus the effects of bepridil on the contractile mechanism in vascular smooth muscle are at least partly mediated through an intracellular mechanism - most probably inhibition of calmodulin.
Animals↗
The anaerobic heart: succinate formation and mechanical performance of cat papillary muscle.
Quiescent or paced, isometrically contracting right ventricular papillary muscles of cat heart were incubated under oxygenated or hypoxic (Po2 less than 10 Torr) conditions in order to investigate lactate and succinate formation. Both compounds were produced and released into the incubation medium during oxygen deficiency. Mechanical performance stimulated synthesis of both compounds under hypoxic as well as oxygenated conditions. Pacing led to the accumulation of large amounts of glutamine both under oxygenated and hypoxic conditions. Hypoxia resulted in a marked depletion of high-energy phosphates and concomitantly mechanical performance was impaired, i.e. developed tension fell rapidly and relaxation rate decreased. Supplying hypoxic, contracting muscles with aspartate (2 mM) resulted in maintenance of muscular function to some extent and led to augmented release of succinate and lactate. The data indicate that anaerobic succinate formation is correlated to the energy requiring processes of the myocardium. Maintenance of myocardial function by the supply of amino acids may be related to their conversion into succinate and to the stimulation of glycolysis.
Alanine↗
Caldesmon-induced inhibition of ATPase activity of actomyosin and contraction of skinned fibres of chicken gizzard smooth muscle.
Caldesmon induces inhibition of MG2+-ATPase activity of actomyosin and relaxation of skinned fibers of chicken gizzard smooth muscle without influencing the level of myosin light chain-1 phosphorylation. Both these effects are reversed by calmodulin at a high molar excess over caldesmon in the presence of Ca2+.
Actomyosin↗
The influence of P-light chain phosphorylation by myosin light chain kinase on the calcium sensitivity of chemically skinned heart fibres.
Phosphorylation of the P-light chain of myosin might be involved in the regulation of cardiac contractility. Thus an enhanced phosphorylation level of the P-light chain catalyzed by Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) increased significantly the Ca2+ sensitivity of chemically skinned ventricular fibre bundles of the pig. This effect was reversible. Whereas force development at submaximal Ca2+ concentration (pCa 5.5) increased by approximately 50% in the presence of MLCK, maximum tension achieved at maximum Ca2+-concentration (pCa 4.3) was not affected.
Animals↗
Modulation of calcium sensitivity in guinea pig taenia coli: skinned fiber studies.
In summary then, skinned fiber experiments have provided evidence showing that the relationship between force development and free calcium ion concentration may be variable. The cyclic-nucleotides c-GMP (cf 38) and c-AMP in particular might be important in modulating calcium sensitivity of skinned fibers possibly via an alteration of myosin phosphorylation. Additionally, the coupling between myosin phosphorylation and the ATPase activity of actomyosin or tension generation may itself be modulated in actomyosin contractile systems. The recognition of the physiological relevance of these modulating mechanisms however must await experiments in which the relationship between force development, free calcium ion concentration and myosin phosphorylation is studied in intact fibers. With the advent of more sophisticated calcium measuring techniques, such information is now available.
Animals↗
Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle.
The contraction induced by a Ca2+-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (Fo), immediate elastic recoil (SE), unloaded shortening velocity (Vus), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca2+ -induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. Fo developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. Vus in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3 +/- 8.9% less than Vus induced by Ca2+ (1.6 X 10(-6) M) in the control fibers. Addition of Ca2+ (1.6 X 10(-6) M) to a contraction induced by MLCK-resulted in small increases in both Fo and Vus. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca2+. We conclude that with respect to actin-myosin interaction, MLCK-and Ca2+ -induced contractions are similar.
Adenosine Triphosphatases↗
Relaxation of skinned coronary arteries depends on the relative concentrations of Ca2+, calmodulin and active cAMP-dependent protein kinase.
Maximally contracted detergent skinned coronary smooth muscle fibres are relaxed by lowering the concentration of free Ca2+. The extent and rate of relaxation depends on the concentration of free Ca2+ and calmodulin (CaM) suggesting that it is the Ca2+ X CaM complex which is responsible for maintaining tension. At a fixed concentration of Ca2+ and CaM further relaxation can be achieved by addition of the catalytic subunit of the cAMP-dependent protein kinase (cAMP-kinase). The extent as well as the relaxation rate depend on the concentration of cAMP-kinase (0.01-0.5 microM) and both are antagonized by high concentrations of Ca2+ and CaM. The Ca2+-requirement for obtaining half maximal concentration is shifted from 1.1 microM to 6.3 microM Ca2+ in the presence of 0.5 microM cAMP-kinase. These data indicate that the response of the contractile apparatus to a change in the free [Ca2+] can be modulated by cAMP-kinase at the level of the contractile proteins. It is further suggested that the tone of coronary smooth muscle is determined by the relative and not by the absolute concentrations of Ca2+, CaM and cAMP-kinase.
Animals↗
The positive inotropic dihydropyridine Bay K 8644 does not affect calcium sensitivity or calcium release of skinned cardiac fibres.
BAY K 8644 is a positive inotropic dihydropyridine which concentration-dependently increased contractile force in guinea-pig atria at 10 to 1000 nmol/l. In chemically skinned cardiac fibres from guinea-pig hearts the substance did not change the tension induced by calcium. Also BAY K 8644 did not release calcium from intracellular myocardial stores like caffeine since it failed to evoke a contractile response in saponin-treated, calcium-loaded cardiac muscle. Thus, the drug exerts its effects neither by sensitizing the contractile apparatus to calcium nor by a caffeine-like action.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
The effect of MgATP on forming and breaking actin-myosin linkages in contracted skinned insect flight muscle fibres.
At neutral pH, fully Ca2+ -activated glycerinated dorsal longitudinal fibre bundles from Lethocerus indicus contract under isometric conditions and respond to release by deactivation, i.e. quick release causes a delayed tension fall. At slightly alkaline pH, the release-induced deactivation becomes a transient phenomenon, i.e. a delayed tension fall is followed by a slow tension recovery. This enabled us to study the effect of MgATP concentration on the phases of deactivation and slow recovery. Reduction of the MgATP concentration slows down the tension response to a quick length change and increases the time constants of the delayed deactivation phase and of the slow recovery phase. The rate constants depend on the ATP concentration according to the Michaelis-Menten law yielding apparent dissociation constants (Km) of 2 mM and 0.09 mM and maximal rate constants of 700 s-1 and 20 s-1 for the deactivation phase (crossbridge detachment) and slow recovery phase (crossbridge reattachment) respectively. The rate of MgATP hydrolysis is also hyperbolically related to the MgATP concentration (Km = 0.14 mM, maximal MgATP turnover rate 1.2 s-1. It is concluded that the effect of MgATP on the deactivation phase, in which crossbridges dissociate strain dependent from the actin, is controlled by at least two mechanisms: (1) fast equilibrium transitions within attached crossbridge states which augment MgATP dissociation from crossbridges with discharged elastic elements; and (2) a crossbridge strain-dependent isomerization of the ternary actin-myosin-MgATP complex which determines crossbridge detachment from the actin.
Actins↗
Phosphatase-mediated modulation of actin-myosin interaction in bovine aortic actomyosin and skinned porcine carotid artery.
Since the Ca2+-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca1+-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca2+ (6.8 X 10(-7) M) and responsiveness to Ca2+ (force attained with 6.8 X 10(-7) Ca2+/force attained with 1.6 X 10(-6) M Ca2+), whereas relaxation was accelerated. Ca2+-dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1-0.9 U/ml). The concentration of Ca2+ (1.1 X 10(-6) M) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca2+-sensitive ATP hydrolysis (39 +/- 0.8 nmole/min/mg actomyosin) nor the extent of phosphorylation (0.68 +/- 0.05 mole PO4/mole light chain) was altered at greater than 5 X 10(-6) M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 microM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ (10(-8) to 1.25 X 10(-5) M). However, significant phosphorylation was present (20-25% of maximum) in the absence of Ca2+-dependent ATPase activity and only 15% of the maximal rate of ATP hydrolysis was expressed until phosphorylation attained 50% of its maximal value. These findings are consistent with the ordered model of myosin phosphorylation suggested by A. Persechini and D. J. Hartshorne [Science (Washington, DC), 213:1383-285, 1961] (36). They also suggest that myosin phosphatase may participate in modulating actin-myosin interactions in vascular smooth muscle.
Actins↗