RESUMO
Contraction and myosin light-chain phosphorylation were measured in electrically stimulated tracheal smooth muscle. Latencies for the onset of force, stiffness, and light-chain phosphorylation were 500 milliseconds. Myosin light chain was phosphorylated from 0.04 to 0.80 mole of phosphate per mole of light chain with a pseudo-first-order rate of 1.1 per second with no evidence of an ordered or negatively cooperative process. Following the period of latency, stiffness increased with phosphorylation and both increased more rapidly than isometric force. The linear relation between stiffness and phosphorylation during activation suggests independent attachment of each myosin head upon phosphorylation.
Assuntos
Contração Muscular , Músculo Liso/fisiologia , Miosinas/metabolismo , Animais , Carbacol/farmacologia , Estimulação Elétrica , Técnicas In Vitro , Cinética , Contração Muscular/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina , Fosforilação , Proteínas Quinases/metabolismo , Traqueia/fisiologiaRESUMO
Smooth muscle contraction is initiated primarily by an increase in intracellular Ca2+, Ca(2+)-dependent activation of myosin light chain kinase, and phosphorylation of myosin light chain. In this investigation, we identified pregnancy-associated alterations in myosin light chain phosphorylation, force of contraction, and content of contractile proteins in human myometrium. Steady-state levels of myosin light chain phosphorylation and contractile stress were correlated positively in both tissues, but the myometrial strips from pregnant women developed more stress at any given level of myosin light chain phosphorylation. During spontaneous contractions and during conditions that favor maximal generation of stress, the rate and extent of myosin light chain phosphorylation were attenuated in myometrial strips from pregnant women. The content of myosin and actin per milligram of protein and per tissue cross-sectional area was similar between myometrium of nonpregnant and pregnant women. Although cell size was significantly increased in tissues obtained from pregnant women, the amounts of contractile proteins per cellular cross-sectional area were similar. In addition, myosin light chain kinase and phosphatase activities were similar in the two tissues. The content of caldesmon was significantly increased in myometrium of pregnant women, whereas that of calponin (a smooth muscle-specific protein associated with the thin filaments) was not different. We conclude that adaptations of human myometrium during pregnancy include (a) cellular mechanisms that preclude the development of high levels of myosin light chain phosphorylation during contraction and (b) an increase in the stress generating capacity for any given level of myosin light chain phosphorylation.
Assuntos
Miométrio/enzimologia , Miosinas/metabolismo , Gravidez/fisiologia , Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Feminino , Humanos , Proteínas dos Microfilamentos/metabolismo , Contração Muscular , Miométrio/citologia , Miométrio/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , CalponinasRESUMO
Although regulation of angiotensin II receptor (AT) binding in vascular and uterine smooth muscle is similar in nonpregnant animals, studies suggest it may differ during pregnancy. We, therefore, examined binding characteristics of myometrial AT receptors in nulliparous (n = 7), pregnant (n = 24, 110-139 d of gestation), and postpartum (n = 21, 5 to > or = 130 d) sheep and compared this to vascular receptor binding. We also determined if changes in myometrial binding reflect alterations in receptor subtype. By using plasma membrane preparations from myometrium and medial layer of abdominal aorta, we determined receptor density and affinity employing radioligand binding; myometrial AT receptor subtypes were assessed by inhibiting [125I]-ANG II binding with subtype-specific antagonists. Compared to nulliparous ewes, myometrial AT receptor density fell approximately 90% during pregnancy (1,486 +/- 167 vs. 130 +/- 16 fmol/mg protein) and returned to nulliparous values > or = 4 wk postpartum; vascular binding was unchanged. Nulliparous myometrium expressed predominantly AT2 receptors (AT1/AT2 congruent to 15%/85%), whereas AT1 receptors predominated during pregnancy (AT1/AT2 congruent to 80%/20%). By 5 d postpartum AT1/AT2 congruent to 40%/60%, and > 4 wk postpartum AT2 receptors again predominated (AT1/AT2 congruent to 15%/85%). In studies of ANG II-induced force generation, myometrium from pregnant ewes (n = 10) demonstrated dose-dependent increases in force (P < 0.001), which were inhibited with an AT1 receptor antagonist. Postpartum myometrial responses were less at doses > or = 10(-9) M (P < 0.05) and unaffected by AT2 receptor antagonists. Vascular and myometrial AT receptor binding are differentially regulated during ovine pregnancy, the latter primarily reflecting decreases in AT2 receptor expression. This is the first description of reversible changes in AT receptor subtype in adult mammals.
Assuntos
Músculo Liso/metabolismo , Miométrio/metabolismo , Prenhez/metabolismo , Receptores de Angiotensina/classificação , Receptores de Angiotensina/metabolismo , Angiotensina II/antagonistas & inibidores , Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Aorta/metabolismo , Ligação Competitiva , Membrana Celular/metabolismo , Feminino , Técnicas In Vitro , Contração Muscular , Músculo Liso Vascular/metabolismo , Período Pós-Parto , Gravidez , OvinosRESUMO
During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers or from endothelial cells (eNOS) may relax vascular smooth muscle contributing to functional hyperemia. To examine the relative importance of these pathways, smooth muscle myosin regulatory light chain (smRLC) phosphorylation was assessed as an index of vascular tone in isolated extensor digitorum longus (EDL) muscles from C57, nNOS(-/-), and eNOS(-/-) mice. The smRLC phosphorylation (in mol phosphate per mol smRLC) in C57 resting muscles (0.12 +/- 0.04) was increased 3.7-fold (0.44 +/- 0.03) by phenylephrine (PE). Reversal of this increase with electrical stimulation (to 0.19 +/- 0.03; P < 0.05) was partially blocked by N(omega)-nitro-l-arginine (NLA). In nNOS(-/-) EDL, the PE-induced increase in smRLC phosphorylation (0.10 +/- 0.02 to 0.49 +/- 0.04) was partially decreased by stimulation (0.25 +/- 0.04). In eNOS(-/-) EDL, the control value for smRLC was increased (0.24 +/- 0.04), and PE-induced smRLC phosphorylation (0.36 +/- 0.06) was decreased by stimulation even in the presence of NLA (to 0.20 +/- 0.02; P < 0.05). These results suggest that in addition to NO-independent mechanisms, NO derived from both nNOS and eNOS plays a role in the integrative vascular response of contracting skeletal muscle.
Assuntos
Fibras Musculares de Contração Rápida/fisiologia , Músculo Liso Vascular/fisiologia , Óxido Nítrico/metabolismo , Vasodilatação/fisiologia , Animais , Western Blotting , Genótipo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Cadeias Leves de Miosina/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Fenilefrina/farmacologia , Fosforilação , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
Nitric oxide (NO) from Ca(2+)-dependent neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers may modulate vascular tone by a cGMP-dependent pathway similar to NO derived from NOS in endothelial cells (eNOS). In isolated fast-twitch extensor digitorum longus (EDL) muscles from control mice, cGMP formation increased approximately 166% with electrical stimulation (30 Hz, 15 s). cGMP levels were not altered in slow-twitch soleus muscles. The NOS inhibitor N(omega)-nitro-l-arginine abolished the contraction-induced increase in cGMP content in EDL muscles, and the NO donor sodium nitroprusside (SNP) increased cGMP content approximately 167% in noncontracting EDL muscles. SNP treatment but not electrical stimulation increased cGMP formation in muscles from nNOS(-/-) mice. cGMP formation in control and stimulated EDL muscles from eNOS(-/-) mice was less than that obtained with similarly treated muscles from control mice. Arteriolar relaxation in contracting fast-twitch mouse cremaster muscle was attenuated in muscles from mice lacking either nNOS or eNOS. These findings suggest that increases in cGMP and NO-dependent vascular relaxation in contracting fast-twitch skeletal muscle may require both nNOS and eNOS.
Assuntos
GMP Cíclico/biossíntese , Contração Muscular/fisiologia , Músculo Esquelético/irrigação sanguínea , Óxido Nítrico Sintase/metabolismo , Animais , Vasos Sanguíneos/fisiologia , Western Blotting , Estimulação Elétrica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo IIIRESUMO
Whereas there is much evidence in support of a role for prostaglandins (PG) in the parturitional process, it has not been demonstrated unequivocally that PGs are the physiological uterotonins involved in the induction of the myometrial contractions of spontaneous labor in women. This study was conducted to evaluate the contractile responsiveness of human myometrial tissue in vitro to PGs and to compare this response with that of other uterotonins, viz. oxytocin and endothelin-1. We found that treatment of uterine smooth muscle strips obtained from nonpregnant and pregnant women with PGE2 (10(-8)-10(-6) M) caused a biphasic response characterized by an initial single contraction of increased amplitude and duration, followed by relaxation and a long period (10-15 min) of quiescence. Conversely, PGE2 acted in rat myometrium to cause a monophasic response of increased contractile frequency and force. Whereas uterine smooth muscle from nonpregnant women was responsive to PGF2 alpha, the contractile responsiveness of myometrium from pregnant women was weak. This weak response to PGF2 alpha was found in myometrium of women in labor and in myometrium of women not in labor. 15-Methyl-PGF2 alpha evoked a small response in myometrium from pregnant women. Under identical in vitro conditions, PGF2 alpha (10(-8)-10(-6) M) and 15-methyl-PGF2 alpha (10(-6) M) caused sustained contractions in human vascular smooth muscle tissues (fetal aorta and arterial smooth muscle from chorionic vessels). Similarly, oxytocin and endothelin-1 (in myometrium from pregnant women) were effective in stimulating the force and frequency of myometrial contraction in vitro. We conclude that the myometrium of pregnant women, as evaluated in vitro, is refractory to the contractile effects of PGE2 and PGF2 alpha.
Assuntos
Dinoprosta/fisiologia , Dinoprostona/fisiologia , Endotelinas/fisiologia , Ocitocina/fisiologia , Gravidez/fisiologia , Contração Uterina/fisiologia , Análise de Variância , Animais , Feminino , Humanos , Técnicas In Vitro , RatosRESUMO
Carotid arteries from control and deoxycorticosterone acetate (DOCA) hypertensive swine were examined for alterations in structure and in contractile properties. Vessels were excised 7 weeks after subcutaneous implantation of the steroid and subsequent elevation in mean arterial pressure from 102 to 133 mm Hg. The carotid media was 1.8 times thicker in arteries from hypertensive animals than in arteries from control animals. This enlargement was associated with an increase in muscle mass, as the fraction of the media composed of smooth muscle cells remained unchanged. Maximal active stress induced by several agonists normalized for cell cross-sectional area was unaltered. No change was observed in sensitivity or maximal response to norepinephrine, histamine, or KCl depolarization. Isotonic shortening rates were also comparable, as was the time course of shortening velocity to a constant afterload during tonic contractions. It is concluded that an enlargement of the carotid media develops in this model of hypertension. However, this response is not associated with detectable alterations in contractile system function.
Assuntos
Artérias Carótidas/fisiopatologia , Hipertensão/fisiopatologia , Animais , Fenômenos Biomecânicos , Artérias Carótidas/patologia , Desoxicorticosterona , Hipertensão/induzido quimicamente , Hipertensão/patologia , Hipertrofia , Técnicas In Vitro , Masculino , Potássio/farmacologia , Suínos , Vasoconstrição/efeitos dos fármacosRESUMO
For many years the simple view was held that contractile force in smooth muscle was proportional to cytosolic Ca2+ concentrations ([Ca2+]i). With the discovery that phosphorylation of myosin light chain by Ca2+/calmodulin-dependent myosin light chain kinase initiated contraction, regulation of the contractile elements developed more complex properties. Molecular and biochemical investigations have identified important domains of myosin light chain kinase: light chain binding sites, catalytic core, pseudosubstrate prototope, and calmodulin-binding domain. New protein phosphatase inhibitors such as okadaic acid and calyculin A should help in the identification of the physiologically important phosphatase and potential modes of regulation. The proposal of an attached, dephosphorylated myosin cross bridge (latch bridge) that can maintain force has evoked considerable controversy about the detailed functions of the myosin phosphorylation system. The latch bridge has been defined by a model based on physiological properties but has not been identified biochemically. Thin-filament proteins have been proposed as secondary sites of regulation of contractile elements, but additional studies are needed to establish physiological roles. Changes in the Ca2+ sensitivity of smooth muscle contractile elements with different modes of cellular stimulation may be related to inactivation of myosin light chain kinase or activation of protein phosphatase activities. Thus, contractile elements in smooth muscle cells are not dependent solely on [Ca2+]i but use additional regulatory mechanisms. The immediate challenge is to define their relative importance and to describe molecular-biochemical properties that provide insights into proposed physiological functions.
Assuntos
Contração Muscular , Proteínas Musculares/fisiologia , Músculo Liso Vascular/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência MolecularRESUMO
Phosphorylation of myosin light chain kinase by a Ca(2+)-dependent protein kinase increases the concentration of Ca2+/calmodulin required for half-maximal activation. The Ca2+ concentrations required for myosin light chain kinase phosphorylation in permeable smooth muscle are similar to those required for myosin light chain phosphorylation. Both GTP gamma S and carbachol increase the Ca2+ sensitivity of myosin light chain kinase phosphorylation as well as light chain phosphorylation. It is proposed that a similar G-protein mediated mechanism regulates the Ca(2+)-dependent phosphorylation of these two contractile proteins in smooth muscle.
Assuntos
Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Cálcio/metabolismo , Carbacol/farmacologia , Bovinos , Permeabilidade da Membrana Celular , Escina/farmacologia , Técnicas In Vitro , Miosinas/metabolismo , FosforilaçãoRESUMO
Smooth muscle cell calponin (h1 or basic isoform) is an actin-binding protein that inhibits actomyosin MgATPase activity and is abundantly expressed in differentiated smooth muscle. Western blots showed bovine tracheal (BT) smooth muscle cells in culture expressed only 2 +/- 1% (n = 8) of the amount of calponin in tissues, while NIH-3T3 fibroblasts expressed none. We tested the hypothesis that introduction of calponin to cultured BT and 3T3 cells would inhibit cytoskeletal activities associated with cell proliferation. To achieve high-efficiency expression, an adenovirus encoding the CMV-calponin construct (Adv-CaP) was generated by homologous recombination in 293 cells. With greater than 90% of BT and 3T3 cells infected with Adv-CaP, calponin expression (32 and 11 microg/mg total protein, respectively) was similar to that in smooth muscle tissues (51 microg/mg). Cells were infected with Adv-CaP for 48 h, replated at low density and proliferation rates were assessed by cell density and [3H]thymidine incorporation. Cell growth and DNA synthesis by Adv-CaP-infected cells were inhibited to one-third control values for both BT and 3T3 cells. Expressed calponin was localized primarily on stress fibers in both cell types. Calponin may act at the cytoskeletal level to retard signaling pathways that normally lead to tight coupling between cell shape and DNA synthesis.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Músculo Liso/citologia , Músculo Liso/fisiologia , Células 3T3 , Actinas/análise , Adenoviridae , Animais , Bovinos , Divisão Celular , Células Cultivadas , Vetores Genéticos , Cinética , Camundongos , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/biossíntese , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Traqueia , Transfecção , CalponinasRESUMO
Tracheal smooth muscle precontracted with carbachol relaxes upon the addition of 3 microM okadaic acid. Although cytosolic Ca2+ concentrations decrease, myosin light chain remains highly phosphorylated (50%). In smooth muscle treated with carbachol alone or carbachol plus okadaic acid 32P is incorporated into a single peptide on myosin light chain which corresponds to the site phosphorylated by myosin light chain kinase. Treatment with okadaic acid alone does not result in myosin light chain phosphorylation or tension development. These results suggest that a cellular mechanism other than myosin light chain phosphorylation can regulate contractile tension.
Assuntos
Éteres Cíclicos/farmacologia , Músculo Liso/efeitos dos fármacos , Miosinas/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Animais , Cálcio/metabolismo , Carbacol/farmacologia , Bovinos , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Músculo Liso/metabolismo , Ácido Okadáico , Fosfoproteínas/fisiologia , Fosforilação , Traqueia/efeitos dos fármacosRESUMO
Nitric oxide generated by neuronal nitric oxide synthase in contracting skeletal muscle fibers may regulate vascular relaxation via a cGMP-mediated pathway. Neuronal nitric oxide synthase content is greatly reduced in skeletal muscles from mdx mice. cGMP formation increased in contracting extensor digitorum longus muscles in vitro from C57 control, but not mdx mice. The increase in cGMP content was abolished with NG-nitro-L-arginine. Sodium nitroprusside treatment increased cGMP levels in muscles from both C57 and mdx mice. Skeletal muscle contractions also inhibited phenylephrine-induced phosphorylation of smooth muscle myosin regulatory light chain. Arteriolar dilation was attenuated in contracting muscles from mdx but not C57 mice. NO generated in contracting skeletal muscle may contribute to vasodilation in response to exercise.
Assuntos
GMP Cíclico/metabolismo , Músculo Esquelético/fisiologia , Músculo Liso Vascular/metabolismo , Miosinas/metabolismo , Óxido Nítrico/metabolismo , Animais , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Esforço Físico , VasodilataçãoRESUMO
PURPOSE: To assess the mechanical ability of the inferior oblique neurofibrovascular bundle (NFVB) to act as an ancillary origin for the inferior oblique muscle after anterior transposition. METHODS: Stress-strain relations and Young's modulus of elasticity, a measure of tissue stiffness, were determined for the NFVB in vitro, in situ, and in vivo in dynamic and static conditions. For comparison, similar studies were performed in vitro on the superior oblique tendon (SOT). RESULTS: Young's moduli for NFVB in situ (6.3 MPa [megapascals]) and in vivo (11.8 MPa) were approximately 2 and 4 times greater (P < 0.05), respectively, than those of isolated NFVB in vitro at 5% to 10% dynamic strain (3 MPa). In dynamic conditions, Young's moduli in vitro for the NFVB and the SOT were similar. CONCLUSIONS: The NFVB is a biomaterial that has stiffness properties similar to the SOT. Within the range of forces typical of normal eye movements (79 to 393 mN), the NFVB alone can tolerate forces of 98 mN at 0% to 10% strain and 393 mN at 15% to 20% strain, based on dynamic in vitro analysis. The greater measured stiffness in situ and in vivo suggest that the NFVB in the intact orbit potentially has a resting strain of 15% to 20%, and additional tissues in parallel with the NFVB also contribute to total stiffness. These data support the hypothesis that the NFVB, acting alone or in concert with adjacent orbital tissues, may form an ancillary origin for the inferior oblique muscle after anterior transposition.
Assuntos
Fibras Nervosas/fisiologia , Músculos Oculomotores/inervação , Nervo Oculomotor/fisiologia , Tecido Conjuntivo/anatomia & histologia , Tecido Conjuntivo/fisiologia , Elasticidade , Movimentos Oculares , Humanos , Junção Neuromuscular/anatomia & histologia , Junção Neuromuscular/fisiologia , Nervo Oculomotor/anatomia & histologia , Órbita/anatomia & histologia , Tendões/anatomia & histologia , Tendões/fisiologiaRESUMO
OBJECTIVES: To characterize changes in myometrial contractile proteins and myosin heavy chain (MHC) isoforms during ovine fetal and neonatal development and after pregnancy. We hypothesized that ovine myometrium demonstrates progressive cellular differentiation and maturation which begins in utero and extends into the postnatal period, and that pregnancy causes further cellular alterations. METHODS: Myometrium was obtained from female fetal (72- to 140-days of gestation, n = 19; term = approximately 145 days), postnatal (1 day to 3 months, n = 25), and parous noncycling nonpregnant (n = 9) sheep to measure total and soluble proteins, actin, MHC, and MHC isoforms. Contractile proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and expression of 200-kD MHC isoforms were determined with Western immunoblots. RESULTS: The contents of total and soluble proteins and actin and total myosin gradually increase (P <.003) during ovine development. Although the contribution of smooth-muscle 204-kD MHC increased (P <.001) from 23 +/- 8% of total MHC at <100 days of gestation to 75 +/- 2% 3 to 4 months postnatally, the 200-kD species fell proportionately. Before birth, MHC-B, a fetal isoform, is the predominant 200-kD protein; postnatally, it is replaced by SM2, demonstrating a switch from a synthetic to a mature contractile smooth-muscle phenotype. Pregnancy is associated with further increases in actin contents and redistribution of the contents of the 204-kD and SM2 MHC isoforms. CONCLUSIONS: Although the fetal and postnatal uterus has no known functional demand, ovine myometrial differentiation and maturation begin in the midtrimester and continue throughout the postnatal period. Thus, changes in smooth-muscle phenotype occur prenatally, as evidenced by a switch from MHC-B to SM2, which may signal completion of organ development and preparation for adult function. Pregnancy results in further modifications in myometrial proteins.
Assuntos
Desenvolvimento Muscular , Músculo Liso/crescimento & desenvolvimento , Paridade , Útero/crescimento & desenvolvimento , Actinas/análise , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Proteínas Musculares/análise , Músculo Liso/química , Músculo Liso/embriologia , Cadeias Pesadas de Miosina/análise , Miosinas/análise , Fenótipo , Gravidez , Ovinos , Útero/química , Útero/embriologiaRESUMO
Ca2+ is a primary second messenger that binds to an intracellular receptor protein, calmodulin. Increases in cytosolic Ca2+ concentration mediated by activation of cell surface receptors result in the formation of a Ca2+ calmodulin complex that regulates many Ca2+-dependent cellular processes. In smooth muscle, Ca2+/calmodulin activates myosin light chain kinase, which phosphorylates the regulatory light chain of myosin. This phosphorylation reaction increases the actin-activated MgATPase activity of myosin and is associated with increases in contractile properties, including force, stiffness, and maximal shortening velocity. These biochemical and biomechanical responses occur rapidly (seconds) in response to physiological stimulation involving neurotransmitter activation of smooth muscle cells. Thus, the Ca2+-dependent phosphorylation of the myosin light chain is a primary event in activation of smooth muscle contraction.
Assuntos
Cálcio/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Sistemas do Segundo Mensageiro , Actinas/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Calmodulina/metabolismo , Humanos , FosforilaçãoRESUMO
Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C and the multifunctional calmodulin-dependent protein kinase II. Since phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labelled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A, perhaps by calmodulin-dependent protein kinase II, may play a role in reported desensitization of contractile elements in smooth muscle to activation by Ca2+.
Assuntos
Cálcio/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Músculo Liso/enzimologia , FosforilaçãoAssuntos
Músculo Liso/metabolismo , Miosinas/metabolismo , Sistemas do Segundo Mensageiro , Cálcio/fisiologia , Carbacol/farmacologia , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Contração Muscular , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Traqueia/citologiaRESUMO
Rapid, coordinated contractions of tracheal smooth muscle were elicited by either direct electrical depolarization of muscle cells or treatment with tetraethylammonium which produced spontaneous phasic contractile activity. Both types of contraction were blocked by the calcium channel antagonist verapamil, indicating that these contractions are supported primarily by calcium of extracellular origin. With direct electrical stimulation, force was biphasic and phosphate content of the phosphorylatable light chain (P-light chain) of myosin increased rapidly (approximately 2.5 s) from 0.1 to 0.4 mol phosphate/mol P-light chain, then decreased to levels above resting values. Phosphorylation increased more rapidly than force. Under conditions of spontaneous activity, phasic contractions occurred above a level of basal tone significantly greater than resting force, and minimum values of phosphorylation measured at the base of contraction were significantly greater than those observed in the resting muscle. Phosphorylation oscillated with force (from 0.2 to 0.4 mol phosphate/mol P-light chain) and peak values occurred during the rising phase of contraction. Time courses of phosphorylation and force showed evidence of a prolonged state of activation of myosin following dephosphorylation. These results suggest that phosphorylation and dephosphorylation of myosin P-light chain are sufficiently rapid to participate in regulation of contractility during phasic mechanical activity.
Assuntos
Contração Muscular , Miosinas/metabolismo , Traqueia/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Bovinos , Estimulação Elétrica , Fosforilação , Compostos de Tetraetilamônio/farmacologiaRESUMO
Contraction strength was measured in ear artery and aorta of the rabbit and coronary artery of the pig during replacement of chloride with anions of the lyotrophic series. Contractions induced by depolarization with elevated external potassium were potentiated in the presence of foreign anions, while those induced by noradrenaline were affected only at higher concentrations of the drug. Drug induced contractions in calcium-free solutions were not affected by the anions. In the presence of nitrate and excess K the change of membrane potential per tenfold change in (K)o was 56mV in chloride and 50 mV in nitrate solution. Enhancement of contraction strength by nitrate was not associated with alterations in membrane potential. Stimulation of aorta with 29.5 mM K resulted in a larger net uptake of 45Ca in nitrate than in chloride. K-stimulated 45Ca efflux was greater in the ear artery in NO3 solution than Cl solution while 45Ca efflux induced by noradrenaline was not effected by anion replacement. These results suggest that in these arteries anions potentiate contraction by enhancing the influx of external calcium, and do not modify the release of internal stores of calcium.
Assuntos
Artérias/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Nitratos/farmacologia , Animais , Cálcio/administração & dosagem , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Músculo Liso/metabolismo , Nitratos/administração & dosagem , Potássio/administração & dosagem , CoelhosRESUMO
Transients in myoplasmic [Ca2+] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K+ -stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca2+ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an improved estimate of activation rates.