RESUMO
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 fármacosAssuntos
Quinase de Cadeia Leve de Miosina/fisiologia , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Modelos Biológicos , Contração Muscular , Músculo Liso/enzimologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/química , FosforilaçãoRESUMO
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
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
Maturation rates of vascular and visceral smooth muscle (SM) during ovine development were compared by quantifying contractile protein, myosin heavy chain (MHC) isoform contents, and contractile properties of aortas and bladders from female fetal (n = 19) and postnatal (n = 21) sheep. Actin, myosin, and protein contents rose progressively throughout development in both tissues (P = 0.003); however, expression patterns differed. During the last trimester, i. e., 101-130 days (term approximately 145 days), bladder actin and MHC contents were approximately twofold greater (P < 0.04) than those in the aorta. Although the fractional content of 204-kDa SM1 MHC in the bladder decreased from 74 +/- 3% at midgestation to 48 +/- 2% 3 mo postnatal, the aorta exhibited an increase from 30 +/- 2% to 65 +/- 2%. Bladder MHC (MHC-B) migrating at 200 kDa contained only SM2 throughout development. In contrast, 200-kDa MHC in the aorta was predominantly nonmuscle MHC-B at midgestation, which was gradually replaced by SM2 as development progressed. Along with its early expression of SM2, bladder muscle obtained maximal stress generating capacity (1.7 x 10(5) N/m(2)) by term gestation, whereas the aorta exhibited no contractions until after birth. We conclude that whereas aortic SM maturation is delayed until after birth, bladder SM matures biochemically and functionally during prenatal development, thus supporting early requirements for micturition.
Assuntos
Desenvolvimento Muscular , Músculo Liso Vascular/crescimento & desenvolvimento , Músculo Liso/crescimento & desenvolvimento , Bexiga Urinária/crescimento & desenvolvimento , Actinas/análise , Animais , Aorta/química , Aorta/crescimento & desenvolvimento , Proteínas Contráteis/análise , Feminino , Contração Isométrica , Proteínas Musculares/análise , Músculo Liso/química , Músculo Liso/embriologia , Músculo Liso Vascular/química , Músculo Liso Vascular/embriologia , Cadeias Pesadas de Miosina/análise , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/análise , Fosforilação , Ovinos , Bexiga Urinária/química , Bexiga Urinária/embriologiaRESUMO
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
In fetal sheep, umbilical responsiveness to ANG II exceeds systemic vascular responsiveness. Fetal systemic vascular smooth muscle (VSM) exhibits an immature phenotype with decreased contractile protein contents, low 200-kDa myosin heavy chain (MHC) SM2, and significant nonmuscle MHC-B expression, whereas umbilical VSM phenotype is incompletely described. We tested the hypothesis that differences in vascular responsiveness could reflect dissimilarities in VSM phenotype. Actin, MHC, MHC isoforms, and active stresses were compared in strips of femoral arteries and aorta from near-term fetal (n = 12) and adult (n = 12) sheep to those in external and intra-abdominal umbilical arteries. Actin contents in fetal femoral artery and aorta were less (P = 0.006) than in external umbilical artery (7.37 +/- 1.4 and 7.53 +/- 0.7 vs. 21.6 +/- 2.2 microg/mg wet wt, respectively) as were MHC contents (3.17 +/- 0.4 and 2.84 +/- 0. 3 vs. 7.16 +/- 0.7, respectively). Whereas 204- and 200-kDa MHC were expressed equally in fetal systemic arteries, umbilical and adult arteries predominantly expressed the 204-kDa isoform (SM1); only fetal systemic VSM expressed MHC-B. Fetal systemic artery stresses and myosin light chain phosphorylation were less than those in umbilical and adult arteries (P < 0.001). Compared with umbilical and adult arteries, fetal systemic VSM is biochemically and functionally immature and thus umbilical VSM demonstrates precocious maturation resembling adult VSM in protein expression and function.
Assuntos
Artérias/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Actinas/metabolismo , Animais , Aorta/embriologia , Aorta/metabolismo , Artérias/embriologia , Artéria Femoral/embriologia , Artéria Femoral/metabolismo , Contração Muscular , Músculo Liso Vascular/embriologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Leves de Miosina/metabolismo , Fenótipo , Fosforilação , Ovinos , Artérias Umbilicais/embriologia , Artérias Umbilicais/metabolismoRESUMO
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
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
Substantial alterations occur in female reproductive tissues to ensure the successful outcome of and recovery from pregnancy. Although sheep have been widely used to study several aspects of pregnancy, little information is available regarding alterations in myometrial function. We therefore characterized the alterations that occur in ovine myometrial stress-generating capacity and examined mechanisms that might account for these changes. Length-force relations were determined for longitudinal myometrial strips from nonpregnant (n = 6), pregnant (n = 11; 67-140 days gestation), and postpartum (n = 6) ewes. Active stress (force per cross-sectional area) was calculated at optimal length for maximal force as determined from length-force relations. Stimulation by 65 mM KCl resulted in 3.5 times greater stress in strips from late-pregnant vs. nonpregnant ewes, 1.20 +/- 0.16 vs. 0.34 +/- 0.04 x 10(5) N/m2 (+/- SE; P < 0.05), respectively. Responses returned to values seen in strips from nonpregnant ewes within 2 wk postpartum. Increases in stress were not associated with differences in the phosphorylated myosin light-chain fraction or the amount of smooth muscle bundles. Although basal prostacyclin production was 15-fold greater in myometrium from nonpregnant vs. pregnant ewes (222 +/- 28 vs. 14.9 +/- 2.0 pg.mg wet wt-1.h-1), cyclooxygenase inhibition did not potentiate stress responses in strips from nonpregnant animals. However, smooth muscle contents of actin (26.0 +/- 1.8 vs. 19.1 +/- 2.2 micrograms/mg wet wt) and myosin heavy chain (5.5 +/- 0.4 vs. 2.0 +/- 0.3 microgram/mg wet wt) were greater (P < 0.04) in myometrium from late-pregnant vs. nonpregnant ewes. Myometrial growth during ovine pregnancy is associated with reversibly augmented contractile properties that appear to primarily reflect increased cellular contents of contractile proteins.
Assuntos
Miométrio/fisiologia , Período Pós-Parto/fisiologia , Prenhez/fisiologia , Ovinos/fisiologia , Animais , Proteínas Contráteis/metabolismo , Epoprostenol/biossíntese , Feminino , Contração Muscular , Miométrio/anatomia & histologia , Miométrio/citologia , Cadeias Leves de Miosina/metabolismo , Fosforilação , Gravidez , Estresse MecânicoRESUMO
The characteristics of actively growing smooth muscle cells (a variant, SM-3) were compared with those of growth-arrested cells with regard to response of myosin light chain (MLC) phosphorylation. Augmented MLC phosphorylation, in particular diphosphorylation, was observed in actively growing cells when stimulated with 30 microM prostaglandin F2alpha (PGF2alpha). The maximum level of diphosphorylation in growing cells was significantly higher than that in growth-arrested cells. The MLC diphosphorylation was sensitive to protein kinase C down-regulation by phorbol dibutylate and pretreatment by the protein kinase inhibitors, staurosporine (30 nM) and isoquinoline sulphonamide HA1077 (20 microM). The actively growing cells contained larger amounts of protein kinase C than growth-arrested cells. The phosphorylation sites of mono- and diphospho-MLC were determined to be MLC kinase-dependent sites (Thr18, Ser19). The PGF2alpha concentration/response curves of MLC diphosphorylation were shifted to the left and upwards in the presence of the protein phosphatase inhibitor calyculin A. These results suggest that PGF2alpha stimulation of actively growing SM-3 cells augments MLC kinase-dependent MLC diphosphorylation. Protein kinase C is involved indirectly in this reaction, possibly through MLC phosphatase-sensitive regulatory mechanisms.
Assuntos
Músculo Liso Vascular/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Divisão Celular , Linhagem Celular , Galinhas , Dinoprosta/farmacologia , Músculo Liso Vascular/citologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases , CoelhosRESUMO
This review summarizes recent data on the two specific mechanisms of beta-adrenergic relaxation of airway smooth muscle. Beta 2-adrenergic receptor stimulation results in the opening of large-conductance, calcium-activated potassium channels, and an attendant hyperpolarization of the myocyte. Coupling between receptor and channel occurs by phosphorylation-dependent and phosphorylation-independent mechanisms. Inhibition of channel opening by specific peptidyl toxins results in a shift in the dose-dependent relaxation of this tissue by beta-adrenergic hormones. There is also evidence that beta-adrenergic hormones can decrease the calcium sensitivity of contractile elements. This desensitization does not result from the phosphorylation of myosin light chain kinase but may be associated with the activation of a myosin light chain phosphatase.
Assuntos
Relaxamento Muscular/fisiologia , Músculo Liso/fisiologia , Receptores Adrenérgicos beta/fisiologia , Fenômenos Fisiológicos Respiratórios , Animais , Cálcio/fisiologia , Humanos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/fisiologia , Sistema Respiratório/efeitos dos fármacosRESUMO
Adult vascular smooth muscle expresses 204-kD (SM1) and 200-kD (SM2) myosin heavy chain (MHC) isoforms. Fetal vascular smooth muscle expresses another 200-kD isoform, MHC-B, that appears to be developmentally regulated. The ontogeny of expression of these MHC isoforms in vascular and nonvascular smooth muscles is not fully understood and may differ. In the present report we examined the ontogeny of these isoforms in aortic and bladder smooth muscle from male fetal (n = 12, 119-140-d gestation; term 145 +/- 5 d) and neonatal (n = 12, 1-33 d) sheep. Tissues were analyzed for total and soluble protein contents. Actin, MHC, and MHC isoforms were analyzed by SDS-PAGE using 3-20% and 4% polyacrylamide gels, respectively. The expression of the adult and fetal 200-kD MHC isoforms were determined by Western analysis. Between 119 d gestation and 33 d neonatal, age-dependent increases (p < 0.02) occurred in bladder actin (16 +/- 0.8 versus 22 +/- 1.4 micrograms/mg of wet weight), MHC (6.5 +/- 0.2 versus 9.7 +/- 1.1) and both soluble (71 +/- 2.9 versus 92 +/- 6.3) and total protein (78 +/- 3.9 versus 103 +/- 5.5). Aortic smooth muscle actin (8.5 +/- 0.7 versus 17 +/- 1.1), MHC (3.1 +/- 0.4 versus 5.2 +/- 0.5), and soluble (44 +/- 2.3 versus 61 +/- 3.0) and total protein (87 +/- 5.8 versus 108 +/- 3.2) also increased (p < 0.01). Aortic SM1 increased (r = 0.79, p < 0.001) during this time, whereas expression of the 200-kD MHC fell (r = -0.79, p < 0.001). In contrast, bladder SM1 fell (r = -0.88, p < 0.001) as the 200-kD MHC rose (r = 0.88, p < 0.001). The type of 200-kD MHC isoform expressed also differed between tissue types; bladder expressed SM2 and little or no MHC-B throughout this phase of development, whereas fetal aorta appeared to express primarily MHC-B, which decreased as adult SM2 expression rose after birth. Expression of smooth muscle proteins and MHC isoforms are developmentally regulated and tissue-dependent, the latter perhaps reflecting developmental differences in organ growth and/or function.
Assuntos
Músculo Liso Vascular/embriologia , Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Aorta , Bovinos , Masculino , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Ovinos/embriologia , Solubilidade , Bexiga UrináriaRESUMO
In intact smooth muscle, myosin light chain kinase (MLCK) is phosphorylated at its regulatory site by Ca2+/calmodulin-dependent protein kinase II resulting in an increase in the concentration of Ca2+/calmodulin required for half-maximal activation of the enzyme (KCaM). We investigated the physiological significance of MLCK phosphorylation during cycles of contraction and relaxation in tonic (tracheal) and phasic (uterine) smooth muscles. MLCK phosphorylation and dephosphorylation occurred at rates sufficient to modulate the Ca2+ sensitivity of light chain phosphorylation. In contractions of both smooth muscles (though using different sources of activating Ca2+), increases in [Ca2+]i preceded light chain phosphorylation; but, the rate of increase in light chain phosphorylation was significantly greater than the rate of increase in [Ca2+]i. The onset of MLCK phosphorylation with the resultant increase in KCaM coincided with the diminished rate of light chain phosphorylation. During spontaneous contractions of uterine smooth muscle, the Ca2+ transient was characterized by an initial rapid increase, a sustained plateau, and rapid decline. During the sustained phase of the Ca2+ transient, MLCK phosphorylation increased and coincided with dephosphorylation of light chain and relaxation. These results indicate that MLCK is sensitive to small increases in intracellular Ca2+ during the initiation of contraction and that the enzyme subsequently becomes desensitized to Ca2+/calmodulin, thereby limiting the extent of light chain phosphorylation.
Assuntos
Contração Muscular/fisiologia , Músculo Liso/fisiologia , Miométrio/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Traqueia/fisiologia , Animais , Cálcio/metabolismo , Bovinos , Estimulação Elétrica , Ativação Enzimática , Feminino , Humanos , Técnicas In Vitro , Contração Isométrica/fisiologia , Relaxamento Muscular/fisiologia , Músculo Liso/enzimologia , Miométrio/enzimologia , Fosforilação , Traqueia/enzimologiaRESUMO
Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phosphorylation of MLCK is slower than the rates of increase in cytosolic Ca2+ concentrations and phosphorylation of the regulatory light chain of myosin in intact tracheal smooth muscle cells in culture. In permeable cells, increasing the Ca2+ concentration increased the extent of myosin light chain and MLCK phosphorylation. The Ca2+ concentration required for half-maximal phosphorylation was 500 nM for MLCK and 250 nM for myosin light chain. Addition of KN-62 or a synthetic peptide CK II, inhibitors of multifunctional Ca2+/calmodulin-dependent protein kinase II activity, abolished MLCK phosphorylation. Under these conditions, the Ca2+ concentration required for half-maximal light chain phosphorylation decreased to 170 nM. Thus, the Ca2+ concentrations required for MLCK phosphorylation are greater than those required for light chain phosphorylation in smooth muscle cells. Furthermore, phosphorylation of MLCK decreases the Ca2+ sensitivity of light chain phosphorylation. These results can be explained by a regulatory scheme in which calmodulin available for myosin light chain kinase activation is limiting. This is supported by the retention of calmodulin when tracheal smooth muscle cells and tissues are permeabilized in relaxing solution and by the low mobility of rhodamine-calmodulin in intact tracheal smooth muscle cells.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Cálcio/metabolismo , Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Calmodulina/metabolismo , Bovinos , Células Cultivadas , Ácido Egtázico/farmacologia , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Isoquinolinas/farmacologia , Cinética , Microscopia de Fluorescência , Dados de Sequência Molecular , Contração Muscular , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Fragmentos de Peptídeos/farmacologia , Fosforilação , Piperazinas/farmacologia , Rodaminas , Traqueia/efeitos dos fármacos , Traqueia/enzimologia , Traqueia/metabolismoRESUMO
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
Phosphorylation of the regulatory light chain of myosin by the Ca2+/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca2+/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence increases the Ca2+ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca2+ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca2+ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.
Assuntos
Cálcio/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Plaquetas/enzimologia , Plaquetas/fisiologia , Calmodulina/metabolismo , Homeostase , Humanos , Dados de Sequência Molecular , Contração Muscular , Músculo Liso/enzimologia , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/química , FosforilaçãoRESUMO
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 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.