RESUMEN
We have identified dystrophin in highly purified sarcolemmal vesicles isolated from canine and bovine hearts using specific antibodies against the COOH-terminal region of the protein. Bovine cardiac sarcolemma contained a single immunoreactive protein band (Mr. approximately 400,000) whereas the canine cardiac membrane contained a doublet (Mr. approximately 420,000 and approximately 380,000). The higher molecular weight form of canine cardiac dystrophin was more abundant than the lower molecular weight form. These highly purified preparations of the sarcolemmal vesicles should provide a useful tool for structural and functional analysis of the interaction of dystrophin with the plasma membrane.
Asunto(s)
Biomarcadores/análisis , Proteínas Musculares/análisis , Miocardio/análisis , Sarcolema/análisis , Adenosina Trifosfatasas/metabolismo , Animales , Anticuerpos , Complejo Antígeno-Anticuerpo , Calcio/metabolismo , Bovinos , Fraccionamiento Celular , Perros , Distrofina , Ligandos , Peso Molecular , Miocardio/metabolismo , Miocardio/ultraestructura , Sarcolema/metabolismo , Sarcolema/ultraestructura , Sodio/metabolismoRESUMEN
Dystrophin, the protein encoded by the Duchenne muscular dystrophy (DMD) gene, exists in a large oligomeric complex. We show here that four glycoproteins are integral components of the dystrophin complex and that the concentration of one of these is greatly reduced in DMD patients. Thus, the absence of dystrophin may lead to the loss of a dystrophin-associated glycoprotein, and the reduction in this glycoprotein may be one of the first stages of the molecular pathogenesis of muscular dystrophy.
Asunto(s)
Glicoproteínas/deficiencia , Proteínas Musculares/metabolismo , Distrofias Musculares/metabolismo , Animales , Anticuerpos Monoclonales , Membrana Celular/análisis , Cromatografía , Distrofina , Técnica del Anticuerpo Fluorescente , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Humanos , Técnicas de Inmunoadsorción , Sustancias Macromoleculares , Ratones , Ratones Mutantes , Peso Molecular , Proteínas Musculares/análisis , Músculos/análisis , Distrofia Muscular Animal/metabolismo , Conejos , Sarcolema/análisisRESUMEN
Novel proteins unique to either transverse tubules (TS28) or the sarcolemma (SL50) have been identified and characterized, and their in situ distribution in rabbit skeletal muscle has been determined using monoclonal antibodies. TS28, defined by mAb IXE112, was shown to have an apparent relative molecular mass of 28,000 D. Biochemical studies showed that TS28 is a minor membrane protein in isolated transverse tubular vesicles. Immunofluorescence and immunoelectron microscopical studies showed that TS28 is localized to the transverse tubules and in some subsarcolemmal vesicles possibly corresponding to the subgroup of caveolae connecting the transverse tubules with the sarcolemma. In contrast, TS28 is absent from the lateral portion of the sarcolemma. Immunofluorescence studies also showed that TS28 is more densely distributed in type II (fast) than in type I (slow) myofibers. Although TS28 and the 1,4-dihydropyridine receptor are both localized to transverse tubules and subsarcolemmal vesicles, TS28 is not a wheat germ agglutinin (WGA)-binding glycoprotein and does not appear to copurify with the 1,4-dihydropyridine receptor after detergent solubilization of transverse tubular membranes. SL50, defined by mAb IVD31, was shown to have an apparent relative molecular mass of 50,000 D. Biochemical studies showed that SL50 is not related to the 52,000-D (beta subunit) of the dihydropyridine receptor but does bind to WGA-Sepharose. Immunofluorescence labeling imaged by standard and confocal microscopy showed that SL50 is associated with the sarcolemma but apparently absent from the transverse tubules. Immunofluorescence labeling also showed that the density of SL50 in type II (fast) myofibers is indistinguishable from that of type I (slow) myofibers. The functions of TS28 and SL50 are presently unknown. However, the distinct distribution of TS28 to the transverse tubules and subsarcolemmal vesicles as determined by immunocytochemical labeling suggests that TS28 may be directly involved in excitation-contraction coupling. Our results demonstrate that, although transverse tubules are continuous with the sarcolemma, each of these membranes contain one or more unique proteins, thus supporting the idea that they each have a distinct protein composition.
Asunto(s)
Microtúbulos/ultraestructura , Proteínas Musculares/análisis , Músculos/ultraestructura , Sarcolema/ultraestructura , Animales , Anticuerpos Monoclonales , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio , Cromatografía de Afinidad , Diafragma , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Immunoblotting , Microscopía Electrónica , Microtúbulos/análisis , Peso Molecular , Músculos/análisis , Conejos , Receptores Nicotínicos/análisis , Sarcolema/análisisRESUMEN
This paper describes studies on the fatty acid composition of individual phospholipids of the neonatal rat cardiomyocyte as well as in the gas-dissected sarcolemma derived from those cells. There is a sarcolemmal fatty acid asymmetry between the two leaflets of the membrane, which results from an asymmetric phospholipid distribution and particular fatty acid composition of each phospholipid class. The cytoplasmic leaflet is shown to be more unsaturated than the outer one. The phospholipids preferring the inner sarcolemmal leaflet (PE, PS, and PI) are particularly rich in two fatty acids, stearic acid and arachidonic acid. The implications of the data in current models for Ca2+ binding and for disruption of sarcolemma following ischemia and reperfusion damage are discussed.
Asunto(s)
Ácidos Grasos/análisis , Miocardio/citología , Sarcolema/análisis , Animales , Membrana Celular/análisis , Células Cultivadas , Femenino , Miocardio/análisis , Fosfolípidos/análisis , Ratas , Ratas EndogámicasRESUMEN
Two major Ca2(+)-binding glycoproteins Mr 120,000 and 100,000 were isolated from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid -solubilized bovine heart sarcolemma membrane. Peroxidase-conjugated concanavalin A and wheat germ agglutinin lectins bind strongly to the isolated 120- and 100-kDa glycoproteins. Treatment with endoglycosidase F resulted in conversion of the 120-kDa glycoprotein to a form migrating at about 97 kDa. Treatment of the 100-kDa band with endoglycosidase F produced form of about 80 kDa. Endoglycosidase H digestion removes only 5% of the mass of both glycoproteins. the carbohydrate structure of both glycoproteins, is therefore, predicted to be at least 75% complex structure and 25% high mannose or hybrid structure. The 120- and 100-kDa glycoproteins are the major Ca2(+)-binding proteins in the sarcolemma membranes. Intact and endoglycosidase-treated glycoproteins bind 45Ca2+ as analyzed by a 45Ca2+ overlay technique. Using polyclonal antibodies, the 120- and 100-kDa glycoproteins were identified in muscle plasma membranes (ventricles, atria, and uterus smooth muscle). They were, however, not present in non-muscle tissues such as pancreas, liver, and kidney. The 120- and 100-kDa glycoproteins appear to be homologous molecules as judged by their similar V8 protease peptide maps, cross-reactivity with polyclonal antibody, and other physicochemical properties.
Asunto(s)
Proteínas de Unión al Calcio/aislamiento & purificación , Glicoproteínas/aislamiento & purificación , Miocardio/análisis , Sarcolema/análisis , Acetilglucosaminidasa/metabolismo , Animales , Radioisótopos de Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Bovinos , Membrana Celular/análisis , Cromatografía de Afinidad , Concanavalina A/metabolismo , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/metabolismo , Glicósido Hidrolasas/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Peso Molecular , Músculo Liso/análisis , Mapeo Peptídico , Serina Endopeptidasas/metabolismo , Distribución Tisular , Aglutininas del Germen de Trigo/metabolismoRESUMEN
The interaction of the renin-angiotensin system and the sympathetic nervous system in patients with congestive heart failure is not well understood. We tested the hypothesis that angiotensin-converting enzyme inhibitors can resensitize the beta-adrenergic receptor system. Guinea pigs were given captopril, isoproterenol, or both for 2 weeks. At death, cardiac sarcolemmal and light vesicle fractions and intact mononuclear leukocytes were prepared. Captopril treatment led to an up-regulation of cardiac beta 1- but not mononuclear leukocyte beta 2-adrenergic receptors and an increase in isoproterenol-stimulated adenylate cyclase activity in the heart. Animals treated with isoproterenol developed cardiac hypertrophy, had increased plasma norepinephrine levels, and had a decreased number and responsiveness of both cardiac and mononuclear leukocyte beta-adrenergic receptors. Concomitant treatment with captopril attenuated alterations of heart weight, plasma norepinephrine levels, and cardiac beta-receptor density and function. In contrast to its cardiac effects, captopril treatment did not diminish the down-regulation of mononuclear leukocyte beta 2-adrenergic receptors by isoproterenol. Our data suggest that captopril may resensitize the cardiac but not the mononuclear leukocyte beta-adrenergic receptor-adenylate cyclase system after long-term catecholamine exposure.
Asunto(s)
Captopril/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Corazón/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Adenilil Ciclasas/análisis , Animales , AMP Cíclico/análisis , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Cobayas , Insuficiencia Cardíaca/metabolismo , Isoproterenol/uso terapéutico , Leucocitos Mononucleares/análisis , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Miocardio/análisis , Norepinefrina/sangre , Ensayo de Unión Radioligante/métodos , Receptores Adrenérgicos beta/análisis , Sistema Renina-Angiotensina/efectos de los fármacos , Sarcolema/análisis , Sarcolema/efectos de los fármacosRESUMEN
The responsiveness to ouabain of hypertrophied rat hearts has been investigated either in vivo using an isolated Langendorff rat heart perfused at various external calcium concentrations, or in vitro on purified sarcolemma vesicles. (i) The physiological study shows that at 0.25 mM CaCl2, the positive inotropic effect of 10(-5) M ouabain was diminished in hypertrophied hearts (p less than 0.02). At 0.5 mM CaCl2, the drug has no effect in controls, but it has a slight positive inotropic effect in hypertrophied hearts. At 2.50 mM CaCl2, ouabain has a negative inotropic effect accompanied by extrasystoles in controls, but in hypertrophied hearts it still has a positive inotropic effect and is not arrhythmogenic. (ii) After the pretreatment of the hearts with 2.5 mM CaCl2, the responsiveness of the (Na+, K+)-ATPase activity to ouabain was studied: the sarcolemma from hypertrophied heart contains half as many low affinity forms of (Na+, K+)-ATPase for ouabain (35% +/- 6) than in controls (80% +/- 2). Assuming that the low affinity forms are responsible for the toxic effect, these data correlate well with some of the physiological findings and suggest that the diminished toxicity for ouabain in hypertrophied hearts rather reflects a modification of the properties of the (Na+, K+)-ATPases than a change in the myocardial calcium metabolism.
Asunto(s)
Cardiomegalia/fisiopatología , Ouabaína/toxicidad , Animales , Calcio/farmacología , Masculino , Ratas , Ratas Endogámicas , Sarcolema/análisis , Sarcolema/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Cardiac contractile function is dependent on the integrity and function of the sarcolemmal membrane. Swimming exercise training is known to increase cardiac contractile performance. The purpose of the present study was to examine whether a swimming exercise program would alter sarcolemmal enzyme activity, ion flux, and composition in rat hearts. After approximately 11 wk of exercise training, cardiac myosin and actomyosin Ca2+-adenosinetriphosphatase (ATPase) activity was significantly higher in exercised rat hearts than in sedentary control rat hearts. Glycogen content was increased in plantaris and gastrocnemius muscles from exercised animals as was succinic dehydrogenase activity in gastrocnemius muscle of exercised rats in comparison to sedentary rat preparations. Sarcolemmal vesicles were isolated from hearts of exercise-trained and control rats. Sarcolemmal Na+-K+-ATPase and K+-p-nitrophenylphosphatase activities, Na+-Ca2+ exchange, and passive Ca2+ binding did not differ between the two groups. ATP-dependent Ca2+ uptake and 5'-nucleotidase activity were elevated in the cardiac sarcolemmal vesicles isolated from exercised animals compared with sedentary control rats. Sarcolemmal phospholipid composition was not altered by the exercise training. Our results demonstrate that swimming training in rats does not affect most parameters of cardiac sarcolemmal function or composition. However, the elevated sarcolemmal Ca2+ pump activity in exercised rats may help to reduce intracellular Ca2+ and augment cardiac relaxation rates. The enhanced 5'-nucleotidase activity may stimulate adenosine production, which could affect myocardial blood flow. The present results further our knowledge on the subcellular response of the heart to swimming training in the rat.
Asunto(s)
Contracción Miocárdica , Miocardio/análisis , Condicionamiento Físico Animal , Esfuerzo Físico , Sarcolema/análisis , Animales , Glucógeno/análisis , Masculino , Músculos/análisis , Músculos/enzimología , Miocardio/enzimología , Ratas , Ratas Endogámicas , Sarcolema/enzimologíaRESUMEN
Pig heart tissue have been shown to contain 3 different 60,000 Da phosphoproteins. Different purification procedures were used in order to separate them, suggesting that the 3 phosphoproteins differ in their environmental parameters. The 2 major ones appear essentially as peripheral phosphoproteins that are associated with cellular membranes through ionic forces, whereas the third minor phosphoprotein behaves as an integral plasma membrane protein. The three phosphoproteins also differ in their relative amount of phosphorylated serine, threonine and tyrosine residues after in vitro protein kinase assay. Evidence that the 3 phosphoproteins are related arises from the similarity between their respective phosphopeptide maps after partial hydrolysis with proteases, an experiment that also points out relatedness in primary structure between them and the transforming protein of Rous sarcoma virus, pp60v-src. The 3 phosphoproteins, however, do not appear to be immunologically related to pp60v-src since none of them is immunoprecipitated by sera that precipitate pp60v-src. The possibility that the three 60,000 Da phosphoproteins under study represent 3 differentially localized and phosphorylated products of c-src and/or c-src related genes is discussed.
Asunto(s)
Proteínas de la Membrana/aislamiento & purificación , Miocardio/análisis , Fosfoproteínas/aislamiento & purificación , Sarcolema/análisis , Animales , Línea Celular Transformada , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/fisiología , Peso Molecular , Proteína Oncogénica pp60(v-src) , Fosfoproteínas/inmunología , Fosfoproteínas/fisiología , Pruebas de Precipitina , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas pp60(c-src) , Proteínas de los Retroviridae/inmunología , PorcinosRESUMEN
Discontinuous density sucrose gradient centrifugation was used to isolate membrane vesicles from the left ventricle of three normal subjects (one prospective organ donor and two traffic victims whose hearts were obtained 1 hour after death) and nine patients undergoing cardiac transplantation as a consequence of idiopathic dilated cardiomyopathy. Sarcolemma-enriched subcellular fractions, detected in the interface between 8.55% and 25% sucrose, were identified by the increased activity of Na+,K+-ATPase and by enrichment in beta-adrenergic receptor density. The density of beta-adrenergic receptors was lower in vesicles from diseased hearts (610 +/- 71 fmol/mg protein) than in vesicles from normal hearts (1,410 +/- 226 fmol/mg protein; p less than 0.01). alpha 1-Adrenergic receptors were identified in these membrane vesicles by [3H]prazosin binding. Specific binding of [3H]prazosin was about 50% of the total binding at 1 nM, and alpha 1-adrenergic binding sites were saturable at approximately 3 nM. Scatchard analysis revealed 58 +/- 5 fmol/mg protein (KD = 0.90 +/- 0.08 nM) in pathological hearts and 30 +/- 5 fmol/mg protein (KD = 0.90 +/- 0.03 nM) in normal hearts (p less than 0.01). The displacement curve of (-)-norepinephrine in membrane vesicles from normal hearts delineated one subpopulation of alpha 1-adrenergic receptors; the addition of 0.1 mM GTP did not cause right shift. In membrane vesicles from diseased heart, the displacement curve of (-)-norepinephrine disclosed two subpopulations of alpha 1-adrenergic receptors. A right shift that occurred after addition of GTP showed that in this case alpha 1-adrenergic receptors were functionally coupled with GTP-binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Proteínas de Unión al GTP/metabolismo , Miocardio/análisis , Receptores Adrenérgicos alfa/análisis , Sarcolema/análisis , Adulto , Alameticina/farmacología , Unión Competitiva/efectos de los fármacos , ATPasas Transportadoras de Calcio/análisis , Dihidroalprenolol/metabolismo , Ventrículos Cardíacos/análisis , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Miocardio/metabolismo , Norepinefrina/farmacología , Prazosina/metabolismo , Unión Proteica/efectos de los fármacos , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa/efectos de los fármacos , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/análisis , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/análisisRESUMEN
Purification and characterization of Na+ channel protein from mammalian ventricular myocytes has heretofore been complicated by the low concentration of Na+ channels and by the finding that mammalian ventricles contain both tetrodotoxin (TTX)-sensitive channels (TSC), with high affinity for saxitoxin (STX), and TTX-insensitive channels (TIC), with low affinity for STX. Most (perhaps all) of the sodium current for myocardial cell action potentials is carried by TIC. Most, if not all, of the cardiac TSC reside in nerves innervating the heart. To isolate TIC in sufficient quantity for further study, we prepared t-tubular sarcolemmal vesicles from large (sheep) hearts with techniques designated to minimize contamination from nerve plasmalemma. Discontinuous sucrose density gradient centrifugation of these membranes produced membrane vesicles, some of which contained no detectable TSC (range 94-100% TIC, or 0-6% TSC), at a concentration of 200-1500 fmol total sites/mg protein, with yields of 4.0-25.0 mg protein/100 g starting material (ventricle). TTX-insensitive STX-binding sites were solubilized from the membranes by 1% digitonin (and with less stability by Triton X-100). The equilibrium binding constant and dissociation rate coefficient for STX binding to the digitonin-solubilized sites were similar to those of the binding sites for the unsolubilized membranes. Unlabeled TTX competed with [3H]STX for the site with 14 times less affinity than did unlabeled STX. Digitonin-solubilized sites had a half-life for STX binding of about 24 h. Binding could be further stabilized by addition of Mg2+ or Ca2+ and exogenous phospholipid.
Asunto(s)
Ventrículos Cardíacos/análisis , Canales de Sodio/efectos de los fármacos , Animales , Unión Competitiva , Cationes Bivalentes/farmacología , Sistema Libre de Células , Digitonina/farmacología , Ventrículos Cardíacos/metabolismo , Sarcolema/análisis , Saxitoxina/metabolismo , Saxitoxina/farmacología , Ovinos , Canales de Sodio/metabolismo , Solubilidad , Tetrodotoxina/farmacologíaRESUMEN
We studied the subcellular localization of dystrophin in rabbit skeletal muscle. In Western-blot analysis of membrane preparations, dystrophin was associated with the sarcolemmal fraction, as indicated by cholesterol content and co-purification with ouabain-binding activity and beta-adrenergic receptor. Dystrophin was also found with junctional T-tubules, but not with 'free' T-tubules, longitudinal portions or terminal cisternae of the sarcoplasmic reticulum. Dystrophin was not solubilized by high salt solutions, but it was solubilized by low concentrations of detergents (Triton X-100 and deoxycholate), suggesting that it is a peripheral membrane protein.
Asunto(s)
Proteínas de la Membrana/análisis , Proteínas Musculares/análisis , Sarcolema/análisis , Animales , Western Blotting , Colesterol/análisis , Distrofina , Conejos , Retículo Sarcoplasmático/análisis , SolubilidadRESUMEN
Unloaded phosphatidylcholine-cholesterol liposomes enhanced the affinity of adrenoreactive system of the rat heart muscle with the ligands and altered the degree of adrenoreceptors' negative cooperativity. The radioligand analysis with 3H-dihydroalprenolol revealed that the liposomes altered kinetic properties of sarcolemma's receptors as revealed by the enhancement of affinity with the marker, decreasing of maximal specific binding and augmentation of the degree of receptors' negative cooperativity.
Asunto(s)
Liposomas/farmacología , Músculos Papilares/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Animales , Colesterol/farmacología , Colesterol/fisiología , Dihidroalprenolol/farmacocinética , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Contracción Isométrica/efectos de los fármacos , Lípidos de la Membrana/análisis , Lípidos de la Membrana/fisiología , Músculos Papilares/análisis , Músculos Papilares/fisiología , Fosfatidilcolinas/farmacología , Fosfolípidos/análisis , Fosfolípidos/fisiología , Ensayo de Unión Radioligante , Ratas , Receptores Adrenérgicos beta/análisis , Receptores Adrenérgicos beta/fisiología , Sarcolema/análisis , Sarcolema/efectos de los fármacos , Sarcolema/fisiologíaRESUMEN
The murine monoclonal antibody (mAb) CB43, raised against the K-562 erythroleukemia line, reacts with monocytes, tissue macrophages, thymocytes and with all the human lines tested but not with resting lymphocytes, large granular lymphocytes, granulocytes and erythrocytes. However, activated lymphocytes and natural killer cells express the CB43 antigen. Embryonic and fetal fibroblasts are positive, while adult fibroblasts are negative. Proximal convoluted tubules in kidney, epithelial cells in esophagus and breast, and sarcolemma in skeletal muscle are reactive with mAb CB43. This antibody can also bind to isolated guinea pig cardiac myocytes, and, furthermore, can induce a transient inotropic effect on isolated atria. The reactivity with different cell and tissue types and the functional effects of the CB43 mAb were reminiscent of the 4F2/44D7 antibodies, shown previously to block Na+/Ca2+ exchange in heart and skeletal muscle. Co-immunoprecipitation studies with CB43 and 44D7 mAb, using radiolabeled Daudi cells, revealed co-migration of polypeptides of 87 and 38 kDa. However, the epitope recognized by CB43 is not present on the human heavy chain which bears the 44D7/4F2 epitope as demonstrated by the lack of reactivity of CB43 mAb with mouse L cells transfected with the 4F2 heavy chain gene. Thus, CB43 represents a newly described epitope present on the human light chain or dependent on the conformation of the human dimer.
Asunto(s)
Antígenos de Superficie/análisis , Epítopos/análisis , Miocardio/análisis , Sarcolema/análisis , Adulto , Animales , Anticuerpos Monoclonales/fisiología , Reacciones Antígeno-Anticuerpo , Antígenos de Superficie/inmunología , Antígenos de Superficie/aislamiento & purificación , Línea Celular , Niño , Epítopos/inmunología , Epítopos/aislamiento & purificación , Femenino , Cobayas , Atrios Cardíacos/análisis , Humanos , Leucemia Eritroblástica Aguda , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Fracciones Subcelulares/análisis , Linfocitos T/análisis , Distribución TisularRESUMEN
We performed in situ hybridization of myosin heavy-chain (MHC) mRNA on rabbit muscle using a biotin-labeled complementary RNA probe. An 1107-nucleotide fragment from an alpha-cardiac MHC cDNA was used to transcribe an RNA probe 97% similar to slow-twitch and 75% similar to fast-twitch sequences. Serial sections were used to identify slow-twitch fibers in medial gastrocnemius, soleus, and tibialis anterior by immunofluorescence of slow MHC and oxidative capacity by histochemistry. Slow-twitch fibers hybridized by the RNA probe stained heavily after detection with streptavidin-alkaline phosphatase (89% dark and 11% medium density). Fast-oxidative fibers stained intermediately (26% dark, 58% medium, and 16% light) and fast-glycolytic fibers stained lightly (12% medium and 88% light). Biotin-labeled probe and enzymatic detection allowed greater resolution of the subcellular location of the MHC mRNA, a distinct advantage over isotope labeling and autoradiography. A non-uniform distribution of MHC mRNA was recognized within an adult skeletal muscle fiber. High concentrations of MHC mRNA were found under the sarcolemma and between the myofibrils, suggesting the existence of a distribution mechanism. The combination of in situ hybridization and immunocytochemistry allows rapid subcellular localization of both MHC mRNA and its translated protein.
Asunto(s)
Inmunohistoquímica , Músculos/análisis , Miosinas/genética , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Animales , Biotina , Técnica del Anticuerpo Fluorescente , Sondas ARN , Conejos , Ribonucleasa Pancreática/metabolismo , Sarcolema/análisis , Distribución Tisular , Transcripción GenéticaRESUMEN
Phospholipids are believed to play an important role in pathology and physiology of the myocardium. Because of the distinct physico-chemical properties of plasmalogens we studied the plasmalogen content and distribution in the sarcolemma of cultured rat myocytes. Treatment with phospholipase A2 degraded all glycerophospholipids in the outer monolayer. The hydrolysis products were analyzed for plasmalogen content. It is shown that the inner sarcolemmal leaflet is highly enriched in phosphatidylcholine and ethanolamine plasmalogen. This distribution of the plasmalogens might affect bilayer stability and thereby be involved in the destruction of the sarcolemma upon ischemia and reperfusion.
Asunto(s)
Miocardio/análisis , Plasmalógenos/metabolismo , Sarcolema/análisis , Animales , Animales Recién Nacidos , Células Cultivadas , Membrana Dobles de Lípidos/análisis , Miocardio/citología , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Plasmalógenos/análisis , Ratas , Sarcolema/ultraestructuraRESUMEN
We have used fractionation procedures to enrich solubilized cardiac sarcolemma in the Na+-Ca2+ exchange protein. Sarcolemma is extracted with an alkaline medium to remove peripheral proteins and is then solubilized with decylmaltoside. Next, the exchanger is applied to DEAE-Sepharose and eluted with high salt. The DEAE fraction is applied to WGA-agarose, and a small fraction of protein, enriched in the exchanger, can be eluted by changing the detergent to Triton X-100. This fraction is reconstituted into asolectin proteoliposomes for measurement of Na+-Ca2+ exchange activity and gel electrophoresis. The purified fraction has a Na+-Ca2+ exchange activity of 600 nmol Ca2+/mg of protein per s at 10 microM Ca2+ and a purification factor of about 30 as compared with control reconstituted sarcolemmal vesicles. Ca2+-Ca2+ exchange and Na+-Ca2+ exchange activities were both present in the same final reconstituted vesicles indicating that the same protein is responsible for both transport activities. SDS-PAGE reveals two prominent protein bands at 70 and 120 kDa. After mild chymotrypsin treatment (1 microgram/ml), there is no loss of exchange activity, but the 120 kDa band disappears and the 70 kDa band becomes more dense. This suggests that the 70 kDa band is due to an active proteolytic fragment of the 120 kDa protein. Under non-reducing gel conditions, only a single protein band is seen with an apparent molecular weight of 160 kDa. Antibodies to the purified exchanger preparation are able to immunoprecipitate exchange activity and confirm that the 70 kDa protein derives from the 120 kDa protein. We propose that both the 70 and 120 kDa proteins are associated with the Na+-Ca2+ exchanger.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Miocardio/análisis , Animales , Western Blotting , Calcio/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Quimotripsina/metabolismo , Perros , Peso Molecular , Miocardio/metabolismo , Pruebas de Precipitina , Sarcolema/análisis , Intercambiador de Sodio-CalcioRESUMEN
Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).
Asunto(s)
Canales de Calcio , Calcio/fisiología , Miocardio/análisis , omega-Conotoxinas , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Aminoquinolinas , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Bovinos , Técnicas In Vitro , Potenciales de la Membrana , Venenos de Moluscos/farmacología , Proteolípidos , Sarcolema/análisisRESUMEN
The investigation focuses on the phospholipid composition of the sarcolemma of cultured neonatal rat heart cells and on the distribution of the phospholipid classes between the two monolayers of the sarcolemma. The plasma membranes are isolated by 'gas-dissection' technique and 38% of total cellular phospholipid is present in the sarcolemma with the composition: phosphatidylethanolamine (PE) 24.9%, phosphatidylcholine (PC) 52.0%, phosphatidylserine/phosphatidylinositol (PS/PI) 7.2%, sphingomyelin 13.5%. The cholesterol/phospholipid ratio of the sarcolemma is 0.5. The distribution of the phospholipids between inner and outer monolayer is defined with the use of two phospholipases A2, sphingomyelinase C or trinitrobenzene sulfonic acid as lipid membrane probes in whole cells. The probes have access to the entire sarcolemmal surface and do not produce detectable cell lysis. The phospholipid classes are asymmetrically distributed: (1) the negatively charged phospholipids, PS/PI are located exclusively in the inner or cytoplasmic leaflet; (2) 75% of PE is in the inner leaflet; (3) 93% of sphingomyelin is in the outer leaflet; (4) 43% of PC is in the outer leaflet. The predominance of PS/PI and PE at the cytoplasmic sarcolemmal surface is discussed with respect to phospholipid-ionic binding relations between phospholipids and exchange and transport of ions, and the response of the cardiac cell on ischemia-reperfusion.
Asunto(s)
Miocardio/análisis , Fosfolípidos/análisis , Sarcolema/análisis , Animales , Fraccionamiento Celular , Células Cultivadas , Microscopía Electrónica , Miocardio/ultraestructura , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Fosfatidilinositoles/análisis , Fosfatidilserinas/análisis , Fosfolipasas A , Ratas , Esfingomielina Fosfodiesterasa , Esfingomielinas/análisis , Ácido TrinitrobencenosulfónicoRESUMEN
Duchenne muscular dystrophy, a common X-linked recessive human disease, has recently been shown to be caused by the deficiency of a large, low abundance protein called 'dystrophin'. Biochemical techniques have shown dystrophin to be membrane-associated in skeletal muscle, with enrichment of dystrophin in the t-tubules of 'triads'. Other studies using immunohistochemistry on thick (10 micron) sections have shown dystrophin to be located at the periphery of muscle fibres, possibly at the plasma membrane. These results have been interpreted as being either consistent and complementary, or contradictory. To localize dystrophin more precisely relative to these membrane systems we have employed highly sensitive and spatially accurate immuno-gold electron microscopy of ultra-thin (70-100 nm) cryosections. The major distribution of dystrophin was on the cytoplasmic face of the plasma membrane of muscle fibres, and possibly on the contiguous t-tubule membranes. The presented data, taken together with recently accumulated information regarding the primary structure of dystrophin, suggests that dystrophin is a component of the membrane cytoskeleton in myogenic cells. Thus, myofibre necrosis in patients affected with Duchenne muscular dystrophy is likely the result of plasma membrane instability.