RESUMO
The cardiac voltage gated l-type Ca(2+) channel (Cav1.2) constitutes the main entrance gate for Ca(2+) that triggers cardiac contraction. Several studies showed that the distal C-terminus fragment of Cav1.2 α1C subunit (α1C-dCT) is proteolytically cleaved and shuttles between the plasma membrane and the nucleus, which is regulated both developmentally and by Ca(2+). However, the effects of sex and sex hormone 17ß-estradiol (E2, estrogen) on α1C-dCT nuclear translocation are still unexplored. To investigate the sexual disparity in the α1C-dCT nuclear translocation, we first generated an antibody directed against a synthetic peptide (GRRASFHLE) located in α1C-dCT, and used it to probe ventricular myocytes from adult female and male mice. Immunocytochemistry of isolated mouse primary adult ventricular myocytes revealed both nuclear staining and cytosolic punctuate staining around the T-tubules. The ratio of nuclear to cytosolic intensity (Inuc/Icyt) was significantly higher in isolated female cardiomyocytes (1.42±0.05) compared to male cardiomyocytes (1.05±0.02). Western blot analysis of nuclear fraction confirmed these data. Furthermore, we found a significant decrease in nuclear staining intensity of α1C-dCT in both female and male cardiomyocytes upon serum withdrawal for 18h (Inuc/Icyt 1.05±0.02 and 0.89±0.02, respectively). Interestingly, subsequent E2 treatment (10(-8)M) for 8h normalized the intracellular distribution of α1C-dCT in male cardiomyocytes (Inuc/Icyt 1.04±0.02), but not in female cardiomyocytes. Acute treatment of male cardiomyocytes with E2 for 45min revealed a similar effect. This effect of E2 was revised by ICI indicating the involvement of ER in this signaling pathway. Taken together, our results showed that the shuttling of α1C-CT in cardiomyocytes is regulated in a sex-dependent manner, and E2-activated ER may play a role in the nuclear shuttling of α1C-dCT in male cardiomyocytes. This may explain, at least partly, the observed sex differences in the regulation of cardiac Cav1.2 channel activity.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Estradiol/farmacologia , Miócitos Cardíacos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Células Cultivadas , Feminino , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Fatores SexuaisRESUMO
BACKGROUND: Obesity is strongly associated with cardiovascular diseases including systemic hypertension, coronary artery disease and heart failure. Despite several investigations the pathophysiological mechanisms involved remain unclear. We have previously shown that adipose tissue exerts a highly potent activity with an acute depressant effect on cardiomyocytes, thus suggesting direct involvement of adipose tissue in the development of heart dysfunction. OBJECTIVE AND DESIGN: This study investigates the effects of adipocyte factors obtained from subcutaneous adipose tissue on the whole cardiac function by using isolated perfused rat hearts in a Langendorff mode. We recorded changes in coronary flow, developed isovolumetric left ventricular pressure, contraction rate and relaxation rate. RESULTS: We observed a significant decrease in heart contractility parameters as well as in coronary flow within a few seconds of incubation with adipocyte factors. The cardiodepressant effects could not be blocked by the nonselective cyclooxygenase-inhibitor indomethacin. Human adipocytes release tumor necrosis factor-α, interleukin-6 (IL-6) and IL-1ß into extracellular medium. These cytokines were tested for their potential effect but were, however, not responsible for the cardiodepressant effect observed. CONCLUSION: These data indicate that human adipocytes secrete factors with a strong acute depressant effect on cardiac force generation and coronary flow due to contraction of the coronary vessels, thus suggesting a direct role of adipose tissue in the pathogenesis of cardiac dysfunction.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Branco/patologia , Coração/fisiopatologia , Contração Miocárdica , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Obesidade/metabolismo , Adulto , Idoso , Animais , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/patologia , Obesidade/fisiopatologia , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Expression of the non-muscle myosin heavy chain-B (NM-MHC-B, also denoted as the embryonic smooth muscle myosin heavy chain, SMemb) was examined in rat urinary bladder during growth in response to a partial urinary outflow obstruction. Following obstruction, the weight of the urinary bladder increased more than five-fold within 10 days. Immunohistochemistry with a polyclonal antiserum against the C-terminal sequence of NM-MHC-B revealed very few NM-MHC-B immunoreactive cells in the control urinary bladders. In hypertrophic bladders, the number of NM-MHC-B immunoreactive cells markedly increased. The majority of such cells were found in the interstitium surrounding smooth muscle bundles and also in the subserosal and submucosal layers. Western blot analysis showed that the NM-MHC-B expression was transient; the content of NM-MHC-B immunoreactive material had doubled 10 days after obstruction and then declined towards the control level after 6 weeks. Immunohistochemistry revealed co-localization of NM-MHC-B and vimentin within the same cells. NM-MHC-B did not co-localize with smooth muscle actin, suggesting that the source of NM-MHC-B is not a de-differentiated smooth muscle cell or myofibroblast but a non-muscle cell possibly reacting to tissue distension or stress. The NM-MHC-B-positive cells could have a role in the production of extracellular matrix and growth factors or be involved in modulation of spontaneous contractile activity.
Assuntos
Tecido Conjuntivo/metabolismo , Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/biossíntese , Miosinas/metabolismo , Obstrução do Colo da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Bromodesoxiuridina/metabolismo , Divisão Celular , Eletroforese em Gel de Poliacrilamida , Feminino , Expressão Gênica , Hipertrofia/metabolismo , Imuno-Histoquímica , Medições Luminescentes , Proteínas Musculares/metabolismo , Músculo Liso/química , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/imunologia , Miosinas/genética , Miosinas/imunologia , Miosina não Muscular Tipo IIB , Tamanho do Órgão , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Bexiga Urinária/crescimento & desenvolvimento , Bexiga Urinária/imunologia , Bexiga Urinária/patologia , Obstrução do Colo da Bexiga Urinária/patologia , Vimentina/imunologiaRESUMO
A novel calcium channel-associated protein of approximately 700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP-dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the beta(2) subunit of cardiac L-type calcium channels as revealed by coprecipitation experiments using affinity-purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK-encoded protein, which was originally identified in human cell lines of neural crest origin as 700-kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase-polymerase chain reaction. Sequence-directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti-AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back-phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane-associated fraction of pp700 underwent substantial in vivo phosphorylation on beta-adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel beta subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the beta subunit play a physiological role in cardiac beta-adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Membrana/química , Miocárdio/enzimologia , Proteínas de Neoplasias/química , Receptores Adrenérgicos beta/metabolismo , Sequência de Aminoácidos , Animais , Expressão Gênica , Humanos , Técnicas In Vitro , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peso Molecular , Miocárdio/citologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade por Substrato , SuínosRESUMO
Endothelial cells release diffusible substances which modulate myocardial function. Oxygen pressure is one important factor for stimulation and modulation of endothelial function. Here we investigated the effects of a superfusate obtained from hypoxic (pO(2) 40-50 mmHg) porcine endothelial cell culture on human myocardial crossbridge cycling rate. Isometric force development and the rate constant for tension development of demembranated multicellular fibers from the left myocardium of a normal human heart were determined from the low-tension rigor by photolytic release of ATP from caged-ATP. Incubation with hypoxic or normoxic superfusates did not change maximal isometric force development. However, rate constant of tension development of the normal human heart fibers significantly decreased to 43.3% upon incubation with the hypoxic but not normoxic endothelial cell superfusate.
Assuntos
Hipóxia Celular/fisiologia , Endotélio Vascular/metabolismo , Coração/fisiologia , Contração Miocárdica , Miosinas/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Endotélio Vascular/citologia , Humanos , Cinética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Fotólise , SuínosRESUMO
Skeletal muscle contraction of Limulus polyphemus, the horseshoe crab, seemed to be regulated in a dual manner, namely Ca2+ binding to the troponin complex as well phosphorylation of the myosin light chains (MLC) by a Ca2+/calmodulin-dependent myosin light chain kinase. We investigated muscle contraction in Limulus skinned fibers in the presence of Ca2+ and of Ca2+/calmodulin to find out which of the two mechanisms prevails in Limulus skeletal muscle contraction. Although skinned fibers revealed high basal MLC mono- and biphosphorylation levels (0.48 mol phosphate/mol 31 kDa MLC; 0.52 mol phosphate/mol 21 kDa MLC), the muscle fibers were fully relaxed at pCa 8. Upon C2+ or Ca2+/calmodulin activation, the fibers developed force (357+/-78.7 mN/mm2; 338+/-69.7 mN/mm2, respectively) while the MLC phosphorylation remained essentially unchanged. We conclude that Ca2+ activation is the dominant regulatory mechanism in Limulus skeletal muscle contraction.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Cadeias Leves de Miosina/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Caranguejos Ferradura , Fosforilação , Isoformas de Proteínas/metabolismoRESUMO
The intracellular mechanisms underlying the action of the endogenous vasodilators such as NO/EDRF, adenosine, and prostacyclin acting through cGMP and cAMP, respectively, are not well understood. One important action of cyclic nucleotides in smooth muscle relaxation is to lower the cytosolic Ca2+ concentration by enhanced sequestration into the sarcoplasmic reticulum. The present study was undertaken to elucidate the potential role of phosphorylation of phospholamban, the regulator of sarcoplasmic reticulum Ca2+ pump, for the control of coronary vascular tone by NO/EDRF, adenosine, and prostacyclin. Phospholamban was identified in pig coronary artery preparations by immunofluorescence microscopy, Western blotting and in vitro phosphorylation. Segments of pig coronary artery, with either intact or denuded endothelium, were precontracted with prostaglandin F2alpha (PGF2alpha). In endothelium-denuded preparations 3-morpholinosydnonimine (SIN-1), 5'-N-ethylcarboxiamidoadenosine (NECA), and iloprost (ILO) caused both relaxation and phospholamban phosphorylation with the potency: SIN-1 > NECA > ILO. The regulatory myosin light chain was significantly dephosphorylated only by SIN-1. In endothelium-intact pig coronary artery, L-NAME caused additional vasoconstriction and a decrease in phospholamban phosphorylation, while phosphorylation of myosin light chain remained unchanged. An inverse relationship between phospholamban phosphorylation and vessel tone was obtained. Our findings demonstrate significant phospholamban phosphorylation during coronary artery relaxation evoked by NO, prostacyclin, and adenosine receptor activation. Because of the close correlation between phosphorylation of phospholamban and vessel relaxation, we propose that phospholamban phosphorylation is an important mechanism by which endogenous vasodilators, especially endothelial NO/EDRF, control coronary vascular smooth muscle tone.
Assuntos
Adenosina/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Vasos Coronários/fisiologia , Epoprostenol/fisiologia , Óxido Nítrico/fisiologia , Animais , Artérias/metabolismo , Artérias/fisiologia , Vasos Coronários/metabolismo , Técnicas In Vitro , Relaxamento Muscular/fisiologia , Tono Muscular , Cadeias Leves de Miosina/metabolismo , Fosforilação , SuínosRESUMO
The role of myosin isoforms in determining contractile filament velocity in the atrium and ventricle of the pig heart was studied by measuring the motion of fluorescently labeled actin over myosin (in vitro motility assay). A rapid and relatively simple method for purification of myosin from small tissue samples was used. The relative extent of light chain-2 phosphorylation was about 30% in both atrial and ventricular myosin extracts. Although the extracted myosin was not free from contaminating proteins, mainly actin, the mean velocity at optimal pH and 32 degrees C of both atrial (3.3 microns/s) and ventricular (2.3 microns/s) myosin were similar to those obtained using extensively purified myosin. The filament sliding velocities using isolated myosin and actin are lower than those estimated from previously published experiments on skinned fiber preparations, which might reflect an influence on sliding velocity by the filament organization or regulatory proteins in the muscle fiber. However, the ratio between velocities of atrial and ventricular myosin was similar in the motility assay (1.5) and muscle fiber experiments (1.6), which might suggest that these two methods reflect the same fundamental processes in cardiac contraction and that the difference in filament sliding velocity between the atrium and ventricle of the pig heart is determined my their myosin isoforms.
Assuntos
Actinas/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/química , Miosinas/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Átrios do Coração/química , Ventrículos do Coração/química , Concentração de Íons de Hidrogênio , Cinética , Cadeias Leves de Miosina/metabolismo , Miosinas/fisiologia , Fosforilação , SuínosRESUMO
We compared baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in 32Pi-labeled left ventricular myocytes from hearts of 26-wk spontaneously hypertensive rats (SHR) and Wistar-Kyoto controls (WKY). TnI phosphorylation was normalized to myosin light chain 2 phosphorylation, which was invariant. There was no difference in baseline TnI phosphorylation in SHR and WKY, but stimulation with isoproterenol, norepinephrine plus prazosin, forskolin, chloroadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine caused a greater increase in TnI phosphorylation in the SHR than in the WKY. This was observed both in the presence and absence of the phosphatase inhibitor calyculin A; thus the differences in TnI phosphorylation between SHR and WKY are not due to decreased phosphatase activity in the SHR. After stimulation of the beta-adrenergic pathway, phospholamban phosphorylation was not different in SHR and WKY, indicating that the observed differences may be specific for PKA phosphorylation of TnI. The increased PKA-dependent TnI phosphorylation in the SHR resulted in decreased Ca2+ sensitivity of actomyosin adenosinetriphosphatase activity as compared with the WKY. We conclude that increased PKA-dependent TnI phosphorylation in the SHR may contribute to the impaired response to sympathetic stimulation.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Coração/efeitos dos fármacos , Hipertensão/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Troponina I/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Colforsina/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Coração/fisiologia , Ventrículos do Coração , Isoproterenol/farmacologia , Toxinas Marinhas , Cadeias Leves de Miosina/metabolismo , Norepinefrina/farmacologia , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Prazosina/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell-derived smooth muscle cells expressed VSMC-specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC-specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC-specific agonists. Treatment of embryonic stem cell-derived embryoid bodies with retinoic acid and dibutyryl-cyclic adenosine monophosphate (db-cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db-cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RT-PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT-PCR with VSMC-specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db-cAMP-inducible embryonic stem cell model of in vitro vasculogenesis. ES cell-derived cells expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.
Assuntos
Bucladesina/farmacologia , Embrião de Mamíferos/citologia , Músculo Liso Vascular/citologia , Células-Tronco/citologia , Tretinoína/farmacologia , Animais , Sequência de Bases , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/análise , Canais de Potássio/fisiologiaRESUMO
Smooth muscle in megacolon was studied in the lethal spotted mouse and its normal sibling. In megacolon, structural remodeling and a very large increase in total protein content are associated with some changes in the contractile protein isoform composition. 1) There is a higher actin concentration in megacolon, primarily caused by a larger proportion of gamma-isoforms. 2) There was no difference in myosin concentration or in SM1/SM2 heavy chains in megacolon and normal muscle contractile proteins. 3) Only LC17a essential light chain is present in both normal and megacolon. 4) The 25- to 50-kDa 5'-insert occurs in 15-20% of the myosin in normal colon, compared with 5- to 10-fold lower amounts in megacolon. In permeabilized muscles there was no significant difference in unloaded shortening velocity (Vo) with maximal thiophosphorylation of the 20-kDa light chains, nor was there significant difference in the force vs. Ca2+ and force vs. myosin light chain phosphorylation relationships. At approximately 60% myosin light chain phosphorylation after Ca2+ activation, Vo of megacolon was approximately two times faster than Vo of normal muscle. These cellular changes largely account for the higher propulsive velocity of the colon in situ. The distribution of myosin and actin isoforms and the lack of a simple relationship between myosin light chain phosphorylation and Vo point to the operation of additional regulatory processes.
Assuntos
Colo/patologia , Colo/fisiopatologia , Proteínas Contráteis/biossíntese , Megacolo/fisiopatologia , Contração Muscular , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Miosinas/biossíntese , Actinas/biossíntese , Animais , Colo/fisiologia , Heterozigoto , Homozigoto , Hipertrofia , Megacolo/genética , Megacolo/patologia , Camundongos , Camundongos Mutantes , Músculo Liso/fisiologia , Cadeias Pesadas de Miosina/biossíntese , Fosforilação , Valores de ReferênciaRESUMO
Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[3H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha1C forms a major constituent of (+)-[3H]isradipine-labeled LTCCs as 54% of solubilized (+)-[3H]isradipine-binding activity was specifically immunoprecipitated by alpha1C antibodies. Phosphorylation of immunopurified alpha1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta1b- and beta3-subunits. We found beta2- and beta4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. beta1b- and beta3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.
Assuntos
Canais de Cálcio/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais de Cálcio/análise , Humanos , Secreção de Insulina , Insulinoma , Fosforilação , Células Tumorais CultivadasRESUMO
We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC17), and phosphorylation of the 20 kDa MLC (MLC20) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (36.3/35.3/29.4). In vivo expression of MLC17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC20 upon maximal Ca(2+)-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm2 and 5.6 mN/mm2 in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax(appt), extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s-1 (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001).
Assuntos
Processamento Alternativo , Músculo Liso/metabolismo , Miométrio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Adulto , Feminino , Humanos , Isomerismo , Cinética , Músculo Liso/citologia , Músculo Liso/fisiologia , Miométrio/citologia , Miométrio/fisiologia , Cadeias Pesadas de Miosina/genética , Fosforilação , GravidezRESUMO
Human neuroblastoma cells (IMR32) respond to treatment with either dibutyryl-cAMP or nerve factor by acquiring a neuronal phenotype which is accompanied by a marked increase in the density of neuronal (N-type) VDCC currents. Using IMR32 cells as a model for neuronal differentiation, we were interested in examining possible changes in the level of expression of the alpha1B subunit of N-type calcium channels as well as beta subunit isoforms. Upon differentiation with dibutyryl-cAMP and 5-bromo-2-deoxyuridine for 16 days, we observed a dramatic increase in alpha1B protein which initiated between day 8 and 10. Day 10 evidenced maximal expression of alpha1B protein, which was followed by an interval of relatively constant expression of alpha1B (day 12 to day 16). Monitoring beta subunit expression using a pan specific anti-beta antibody (Ab CW20), we observed an increase in expression of a single 82 kDa beta subunit. The predominant 82 kDa beta subunit expressed throughout the course of differentiation was identified as the beta1b isoform using a panel of beta subunit specific antibodies. Of significance, neither the beta2 nor beta3 isoforms were detected in full differentiated IMR32 cells. Contrary to a previous report on the absence of neurotypic expression of VDCC beta subunits in a second model for in vitro differentiation, NGF-treated rat pheochromocytoma cells (PC12 cells) [1], we report the regulated expression of the beta1b protein in differentiated IMR32 cells suggesting a cell specific function for this beta subunit which parallels the acquisition of the neuronal phenotype. The restrictive expression of the beta1b in IMR32 cells may reflect a cell-type specific function that extends beyond its role as an auxiliary subunit of VDCC complexes.
Assuntos
Canais de Cálcio/biossíntese , Neurônios/citologia , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Bromodesoxiuridina/farmacologia , Bucladesina/farmacologia , Canais de Cálcio/química , Diferenciação Celular , Humanos , Dados de Sequência Molecular , Peso Molecular , Neuroblastoma , Prosencéfalo/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50% in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms.
Assuntos
Contração Muscular , Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Leves de Miosina/química , Bexiga Urinária/patologia , Trifosfato de Adenosina/metabolismo , Animais , Hipertrofia/metabolismo , Cinética , Medições Luminescentes , Fotólise , RatosRESUMO
In canine myocardium, the beta-subunit of the L-type Ca2+ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217-222, 1993). We have assessed the identity of the beta-subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca2+ channel beta-subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the beta-subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dtmax. The results imply the involvement of a PKA-mediated phosphorylation of the Ca2+ channel beta-subunit in the adrenergic stimulation of intact canine myocardium.
Assuntos
Canais de Cálcio/metabolismo , Catecolaminas/farmacologia , Miocárdio/metabolismo , Animais , Autorradiografia , Canais de Cálcio/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cães , Feminino , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Masculino , Contração Miocárdica , Norepinefrina/farmacologia , Fosforilação , Conformação Proteica , Reserpina/farmacologiaRESUMO
Most of the patients with congenital heart diseases express the atrial myosin light chain 1 (ALC-1) in the right ventricle. We investigated the functional consequences of ALC-1 expression on the myosin cycling kinetics in the intact sarcomeric structure using multicellular demembranated fibers ("skinned fibers") from the right ventricular infundibulum of patients with Tetralogy of Fallot (TOF), double outlet right ventricle (DORV), and infundibular pulmonary stenosis (IPS), Force-velocity relation was analyzed by the constant-load technique at maximal Ca2+ activation (pCa 4.5). Half-time of tension development (t1/2) was investigated by monitoring contraction initiation upon photolytic release of ATP from caged-ATP in rigor. The patients investigated here expressed between 0 and 27% ALC-1. There was a statistically significant correlation between ALC-l and maximal shortening velocity (Vmax) which rose 1.87-fold from 1.2 muscle length per second (ML/s) to 2.25 ML/s in a normal (0% ALC-1) and diseased (19.9% ALC-1) ventricle. Half-time of tension development decreased 1.85-fold with increasing ALC-1 expression (t1/2) was 0.252 s and 0.136 s at 2 and 18.4% ALC-1, respectively). We conclude that the expression of ALC-1 in the human heart modulates cross-bridge cycling kinetics accelerating shortening velocity and isometric tension production.
Assuntos
Pressão Sanguínea , Cardiopatias Congênitas/fisiopatologia , Coração/fisiopatologia , Contração Miocárdica , Cadeias Leves de Miosina/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Cálcio/farmacologia , Criança , Pré-Escolar , Feminino , Coração/efeitos dos fármacos , Coração/fisiologia , Cardiopatias Congênitas/cirurgia , Ventrículos do Coração , Homeostase , Humanos , Lactente , Cinética , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/fisiopatologia , Contração Miocárdica/efeitos dos fármacos , Cadeias Leves de Miosina/biossíntese , Artéria Pulmonar/fisiopatologia , Estenose da Valva Pulmonar/fisiopatologia , Valores de Referência , Tetralogia de Fallot/fisiopatologiaRESUMO
We investigated the effects of ovarectomy and the steroid hormones estrogen and testosterone on the in vivo expression of heavy (MHC) and light (MLC) chains of myosin in the heart, uterus, and aorta of rats. In the heart, ovarectomy decreased alpha-MHC expression, while both steroid hormones normalized it. Differential steroid hormone effects could be observed on myosin subunit expression of smooth muscle. Testosterone but not estrogen normalized the ovarectomy-induced decreased expression of SM1 and strongly increased the expression of 5'-inserted MHC in the uterus. Estrogen but not testosterone normalized the ovarectomy-induced diminished MLC17a expression. In contrast to the uterus, no steroid hormone effects on myosin subunit expression could be observed in the aorta.
Assuntos
Aorta/metabolismo , Estrogênios/fisiologia , Regulação da Expressão Gênica/fisiologia , Miocárdio/metabolismo , Miométrio/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Leves de Miosina/genética , Testosterona/fisiologia , Sequência de Aminoácidos , Animais , Peso Corporal , Feminino , Dados de Sequência Molecular , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
We investigated the rate of tension development (kappa td) after photolytical release of ATP from P3-1-(2-nitrophenyl)-ethyladenosine-5'-triphosphate ('caged ATP') of atrial and ventricular fibre bundles from pig. Contraction was initiated from high-tension (HT) and low-tension (LT) rigor at maximal Ca2+ activation (pCa 4.5). The kappa td of atrial fibre bundles was 6.8 s-1 from LT and 6.9 s-1 from HT rigor. Rate of tension development of ventricular fibre bundles was significantly lower (P < 0.001) being 1.06 s-1 and 0.94 s-1 from LT and HT rigor, respectively. The kappa td of skinned ventricular fibre bundles incubated in a high [K+], low [Ca2+] (cardioplegic) solution prior to the skinning procedure decreased significantly (P < 0.05) to 0.73 s-1 and 0.63 s-1 from LT and HT rigor, respectively, whereas that of skinned atrial fibre bundles remained at 7.1 s-1 and 6.9 s-1 from LT and HT rigor, respectively. Phosphorylation levels of the myosin light chain 2 isoform in the atrial fibre bundles (ALC-2) was 15.6 +/- 2.7%. The corresponding values for the two ventricular isoforms, VLC-2 and VLC-2*, were 31.2 +/- 0.4% and 25.1 +/- 2.1%, respectively. Phosphorylation levels of fibre bundles incubated in cardioplegic solution prior to skinning were 11.6%, 18.9%, and 15.4% of the ALC-2, VLC-2 and VLC-2*, respectively. The results show that the rate of tension development is more than seven-fold higher in the atrial compared with ventricular fibre bundles. These results correlate with the differences in ATPase activity of the contractile proteins in solution and, most likely, reflect differences in the myosin isoform composition. In ventricular fibre bundles the increased levels of light chain phosphorylation were associated with increased rate of contraction.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Fibras Musculares Esqueléticas/fisiologia , Contração Miocárdica/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Função Atrial , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Técnicas In Vitro , Isoenzimas/metabolismo , Cinética , Fibras Musculares Esqueléticas/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Estimulação Luminosa , Coelhos , Suínos , Função VentricularRESUMO
The amino-terminal domain of the essential myosin light chain (MLC-1) binds to the carboxy terminus of the actin molecule. We studied the functional role of this interaction by two approaches: first, incubation of intact and chemically skinned human heart fibers with synthetic peptide corresponding to the sequences 5 through 14 (P5-14), 5 through 8 (P5-8), and 5 through 10 (P5-10) of the human ventricular MLC-1 (VLC-1) to saturate actin-binding sites, and second, incubation of skinned human heart fibers with a monoclonal antibody (MabVLC-1) raised against the actin-interacting N-terminal domain of human VLC-1 using P5-14 as antigen to deteriorate VLC-1 binding to actin. P5-14 increased isometric tension generation of skinned human heart fibers at both submaximal and maximal Ca2+ activation, the maximal effective peptide dosage being in the nanomolar range. A scrambled peptide of P5-14 with random sequence had no effects up to 10(-8) mol/L, ie, where P5-14 was maximally effective. P5-8 and P5-10 increased isometric force to the same extent as P5-14, but micromolar concentrations were required. Amplitude of isometric twitch contraction, rate of tension development, rate of relaxation, and shortening velocity at near-zero load of electrically driven intact human atrial fibers increased significantly on incubation with P5-14. These alterations were not associated with modulation of intracellular Ca2+ transients as monitored by fura 2 fluorescence measurements. Incubation of skinned human heart fibers with MabVLC-1 increased isometric tension at both submaximal and maximal Ca2+ activation levels, having a maximal effective concentration in the femtomolar range.