RESUMEN
Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Potenciales de Acción/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Ouabaína/administración & dosificación , Ouabaína/farmacología , Aminoimidazol Carboxamida/administración & dosificación , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inyecciones Intraperitoneales , Masculino , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/metabolismo , Ratas , Ratas Wistar , Ribonucleótidos/administración & dosificación , Ribonucleótidos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadRESUMEN
The third author's name should read: V. V. Matchkov.
RESUMEN
Dysferlin protein plays a key role in the multimolecular complex responsible for the maintenance of sarcolemma integrity and skeletal muscle cell functioning. We studied the membrane distribution of nicotinic acetylcholine receptors and α2 isoform of Na,K-ATPase in motor endplates of m. soleus in dysferlin-deficient Bla/J mice (a dysferlinopathy model). Endplates of Bla/J mice were characterized by increased area (without changes in fragmentation degree) and reduced density of the membrane distribution of nicotinic acetylcholine receptors in comparison with the corresponding parameters in control С57Bl/6 mice. The density of the membrane distribution of α2 isoform of Na,K-ATPase was also reduced, but the level of the corresponding mRNA remained unchanged. It can be hypothesized that abnormal membrane localization of α2 isoform of Na,K-ATPase results from adaptive skeletal muscle remodeling under conditions of chronic motor dysfunction.
Asunto(s)
Membrana Celular/metabolismo , Músculo Esquelético/metabolismo , Isoformas de Proteínas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Disferlina/genética , Disferlina/metabolismo , Masculino , Ratones , Isoformas de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Methyl-ß-cyclodextrin (0.1 mM) reduced resting potential of muscle fibers and abolished local endplate membrane hyperpolarization in rat diaphragm. This effect was associated with selective reduction of electrogenic activity of α2-isoform of Na,K-ATPase without changes in the level of intracellular acetylcholine. Experiments with cholesterol marker filipin showed that methyl-ß-cyclodextrin in this dose induced cholesterol translocation from lipid rafts to liquid phase of the membrane without its release into extracellular space. This modification of lipid rafts by methyl-ß-cyclodextrin presumably impaired the mechanism maintaining electrogenesis in endplates mediated by modulation of Na,K-ATPase by non-quantum acetylcholine. Cholesterol can serve as a molecular component of this mechanism.
Asunto(s)
Colesterol/metabolismo , Diafragma/efectos de los fármacos , Diafragma/metabolismo , beta-Ciclodextrinas/farmacología , Acetilcolina/metabolismo , Animales , Masculino , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , ATPasa Intercambiadora de Sodio-Potasio/metabolismoAsunto(s)
Factor de Crecimiento Epidérmico/farmacología , Ganglios Espinales/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Canales de Sodio/metabolismo , Animales , Células Cultivadas , Ganglios Espinales/citología , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/citología , Ratas , Ratas WistarRESUMEN
Rats were chronically treated with nicotine via subcutaneous injections up to a dose 6 mg/kg/day during 2-3 weeks. After this period, resting membrane potential and action potentials of muscle fibres as well as isometric twitch and tetanic (20 s(-1) and 50(-1)) contractions of isolated rat diaphragm were studied. To estimate electrogenic contribution of the alpha2 isoform of the Na+, K(+)-ATPase ouabain in concentration 1 microM was used. Chronic nicotine exposure induced depolarization of resting membrane potential of 2.2 +/- 0.6 mV (p < 0.01). In rats chronically exposed to nicotine, electrogenic contribution of the Na+, K(+)-ATPase alpha2 isoform was twofold lesser than in control animals (3.7 +/- 0.6 mV and 6.4 +/- 0.6 mV, respectively, p < 0.01). Chronic nicotine exposure did not affect force of twitch and tetanic contractions in response to direct or indirect stimulation. A decrease in the twitch contraction time as well as in the rise time of tetanic contractions was observed. Fatigue dynamics was unchanged. The results suggest that chronic nicotine exposure leads to decrease of the Na+, K(+)-ATPase alpha2 isoform electrogenic activity, and as a consequence to damage of the rat diaphragm muscle electogenesis.
Asunto(s)
Diafragma/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Nicotina/administración & dosificación , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Potenciales de Acción/efectos de los fármacos , Animales , Diafragma/enzimología , Diafragma/fisiología , Masculino , Ouabaína/farmacología , RatasRESUMEN
The data have been obtained that confirm the identity of the electrogenic mechanism of hyperpolarization by nanomolar concentrations of cholinergic ligands in the extrasynaptic region and endogenous nonquantal acetylcholine in the synaptic region of a skeletal muscle fiber. In both cases, this mechanism is realized through the involvement of the alpha2 isoform of Na, K-ATPase and operates in the absence of Na+ entry through membrane channels. At the same time, there are peculiar properties which take place under functional disorders. Thus, the effectiveness of this mechanism in the synaptic region selectively increases under rat hindlimb unloading and decreases in case of dystrophin deficit in mdx mice. The last fact suggests that dystrophin is a molecular component that is essential for the functioning of the electrogenic mechanism of local hyperpolarization of the end-plate membrane.
Asunto(s)
Distrofina/deficiencia , Fibras Musculares Esqueléticas/fisiología , Unión Neuromuscular/fisiología , Membranas Sinápticas/fisiología , Acetilcolina/fisiología , Animales , Distrofina/genética , Fenómenos Electrofisiológicos , Suspensión Trasera , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratas , Ratas Wistar , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/fisiologíaRESUMEN
Some of the electrophysiological parameters of m. soleus of rat and Mongolian gerbil, and Ca ions content in fiber myoplasm were compared in different periods of gravitational unloading simulated by tail-suspension. No difference was found between the control animals as for membrane potential at rest, electrogenic activities of Na-K-ATPase and its isoforms, and input resistance of m. soleus fibers. At the same time, unlike rats, gerbils exhibited a substantial Ca decrease in myoplasm. From day one to 14 of gravitational unloading the pace of electrophysiological changes in gerbil's m. soleus was noticeably slower than of rat's, whereas Ca ions depositing in myoplasm was observed in both species already at the beginning ofsuspension. Analysis of the results suggests that adaptive changes in m. soleus of Mongolian gerbil and rat during simulated gravitational unloading are fundamentally different due to, probably, peculiar water-electrolyte metabolism, type of locomotion, and other factors which are still unclear.
Asunto(s)
Calcio/metabolismo , Fibras Musculares Esqueléticas/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Ingravidez , Animales , Calcio/análisis , Gerbillinae , Masculino , Potenciales de la Membrana/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/enzimología , Ouabaína/farmacología , Ratas , Ratas Wistar , Simulación de IngravidezRESUMEN
Nicotine in concentration 100 nM which corresponds to concentration of nicotine circulating in tobacco smokes induced hyperpolarization by approximately 4 mV of muscle fibres of the rat isolated diaphragm, as well as an increase in amplitude and acceleration of action potentials. Similar hyperpolarization was induced by nicotine and acetylcholine in the rat soleus muscle. In this muscle, the hyperpolarization developed more slowly than in diaphragm revealing initial slight depolarization. Non-competitive blocker of open channel of nicotinic acetylcholine receptor, proadifen, abolished nicotine- or acetylcholine-induced depolarization but not the hyperpolarization. In the diaphragm, the hyperpolarization was blocked by specific inhibitors of the Na,K-ATPase, ouabain (50 nM) or marinobufagenin (2 nM) suggesting an involvement of the Na,K-ATPase. Estimation of elecrogenic contributions of isoforms of the Na,K-ATPase showed that the hyperpolarization was due to an increase in electrogenic contribution of alpha 2 isoform without change in contribution of alpha 1 isoform. Nicotine did not affect parameters of muscle contractions in response to direct stimulation.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Bufanólidos/farmacología , Cardiotónicos/farmacología , Masculino , Ouabaína/farmacología , Isoformas de Proteínas/metabolismo , Ratas , Ratas Wistar , Vasoconstrictores/farmacologíaRESUMEN
After three days of hind limb unloading, the depolarization of muscle fibers from -71.0 +/- 0.5 mV to -66.8 +/- 0.7 mV as well as a decrease in muscle excitability and a trend to fatigue acceleration were observed. After hind limb unloading, the electrogenic contribution of the ouabain-sensitive alpha2 isoform of Na,K-ATPase, tested as depolarization due to the administration of 1 microM ouabain, decreased from 6.2 +/- 0.6 to 0.5 +/- 0.8 mV. The contribution of the ouabain-resistant alpha1 isoform, estimated as additional depolarization after the administration of 500 microM ouabain, decreased from 4.6 +/- 0.6 to 2.6 +/- 0.6 mV. After hind limb unloading, the fluorescence intensity of single muscle fibers loaded with Fluo-4-AM increased more than four times, indicating an increase in intracellular Ca2+ concentration. The effect was prevented by local delivery of nifedipine, which blocks L-type Ca2+ channels. These data suggest the existence of a selective mechanism of suppression of the alpha2-pump electrogenic contribution, which led to the depolarization of soleus muscle fibers after 3 days of hind limb unloading. The depolarization in turn may activate L-type Ca2+ channels, resulting in intracellular Ca2+ accumulation.
Asunto(s)
Calcio/metabolismo , Músculo Esquelético/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/fisiología , Cationes Bivalentes , Técnicas In Vitro , Isoenzimas/fisiología , Masculino , Potenciales de la Membrana , Fibras Musculares Esqueléticas/metabolismo , Nifedipino/farmacología , Ratas , Ratas Wistar , IngravidezRESUMEN
The contractile properties of the postural rat soleus muscle at the early stage of the gravitational unloading (3-day rat hindlimb suspension) have been studied using different modes of muscle contraction (twitch and tetanic contraction of the isolated muscle, Ca-induced contraction of isolated skinned fibers). A significant enhancement of the twitch maximal tension of unloaded muscles without changes in time-dependent characteristics was observed, although the half-relaxation time tended to increase. The fiber diameter did not change (42.37 +/- 0.76 vs 43.43 +/- 1.15 microm in controls). The Ca-induced maximal isometric tension in unloaded soleus was significantly decreased (32.1 +/- 1.05 vs 37.6 +/- 1.52 mg in controls, p < 0.05). The maximal specific tension was respectively decreased (23.14 +/- 0.77 vs 27.6 +/- 2.36 kN/m in controls). The pCa50 in unloaded muscle decreased from 6.05 +/- 0.02 in controls to 5.97 +/- 0.02 (p < 0.05), indicating the loss of myofibrillar calcium sensitivity. The analysis with the calcium probe Fluo-4AM demonstrated that the intracellular [Ca2+] was sufficiently increased after hindlimb suspension. At the same time, the relative content of titin and nebulin did not change.
Asunto(s)
Contracción Muscular , Músculo Esquelético/fisiología , Animales , Calcio/metabolismo , Proteínas del Citoesqueleto/metabolismo , Técnicas In Vitro , Masculino , Fibras Musculares Esqueléticas/fisiología , Relajación Muscular , Ratas , Ratas Wistar , Sarcómeros/metabolismo , IngravidezRESUMEN
It was shown that the specific inhibitors of Na+, K(+)-ATPase ouabain and marinobufagenin increased the contraction of an isolated rat diaphragm (positive inotropic effect) by up to approximately 15% in a dose-dependent manner with EC50 = 1.2 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The results indicate the involvement of the ouabain-sensitive alpha 2 isoform of Na+, K(+)-ATPase. The analysis of ouabain-resting membrane potential dose-response relationships in the presence and absence of hyperpolarizing concentration of acetylcholine (100 nM) suggests the existence of two pools of alpha 2 Na+, K(+)-ATPase with different affinities for ouabain. The pool with a higher ouabain affinity is involved in the hyperpolarizing effect of acetylcholine and, presumably, in the positive inotropic effect of ouabain, which might be a mechanism of regulation of muscle efficiency by circulating endogenous inhibitors of Na+, K(+)-ATPase.
Asunto(s)
Bufanólidos/farmacología , Diafragma/efectos de los fármacos , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Diafragma/fisiología , Técnicas In Vitro , Isoenzimas/antagonistas & inhibidores , Isoenzimas/fisiología , Masculino , Potenciales de la Membrana , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Estimulación QuímicaRESUMEN
The interaction between the nicotinic acetylcholine receptor and Na+,K(+)-ATPase described previously was further studied in isolated rat diaphragm and in a membrane preparation of Torpedo californica electric organ. Three specific agonists of the nicotinic receptor: acetylcholine, nicotine and carbamylcholine (100 nmol/L each), all hyperpolarized the non-synaptic membranes of muscle fibers by up to 4 mV. Competitive antagonists of nicotinic acetylcholine receptor, d-tubocurarine (2 mcmol/L) or alpha-bungarotoxin (5 nmol/L) completely blocked the acetylcholine-induced hyperpolarization indicating that the effect requires binding of the agonists to their specific sites. The noncompetitive antagonist, proadifen (5 mcmol/L), exerted no effect on the amplitude of hyperpolarized but decreased K0.5 for this effect from 28.3 +/- 3.6 nmol/L to 7.1 +/- 2.3 nmol/L. Involvement of the Na+,K(+)-ATPase was suggested by data demonstrating that three specific Na+,K(+)-ATPase inhibitors: ouabain, digoxin or marinobufagenin (100 nmol/L each), all inhibit the hyperpolarizing effect of acetylcholine. Acetylcholine did not affectation either the catalytic activity of the Na+,K(+)-ATPase purified from sheep kidney or the transport activity of the Na+,K(+)-ATPase in the rat erythrocytes, i. e. in preparations not containing acetylcholine receptors. Hence, acetylcholine does not directly affect the Na+,K(+)-ATPase. In a Torpedo membrane preparation, ouabain (< or = 100 nmol/L) increased the binding of the fluorescent ligand: Dansyl-C6-choline (DCC). No ouabain effect was observed either when the agonist binding sites of the receptor were occupied by 2 mmol/L carbamylcholine, or in the absence Mg2+, when the binding of ouabain to the Na+,K(+)-ATPase is negligible. These results indicate that ouabain only affects specific DCC binding and only when bound to the Na+,K(+)-ATPase. The data obtained suggest that, in two different systems, the interaction between the nicotinic acetylcholine receptor and the Na+,K(+)-ATPase specifically involve the ligand binding sites of these two proteins.
Asunto(s)
Órgano Eléctrico/fisiología , Músculo Esquelético/fisiología , Receptores Nicotínicos/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Acetilcolina/farmacología , Animales , Transporte Biológico , Bufanólidos/farmacología , Catálisis , Membrana Celular/fisiología , Compuestos de Dansilo/química , Diafragma/fisiología , Digoxina/farmacología , Órgano Eléctrico/ultraestructura , Eritrocitos/metabolismo , Ligandos , Magnesio/fisiología , Masculino , Potenciales de la Membrana , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Ouabaína/farmacología , Compuestos de Amonio Cuaternario/química , Ratas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , TorpedoRESUMEN
Effects of Na+,K(+)-ATPase inhibitor: marinobufagenin, on contractile and electric characteristics of isolated rat diaphragm were studied for the first time. Marinobufagenin induced dose-dependent (EC50 = 0.3 +/- 0.1 nM) increase in the contraction force (positive inotropic effect). At 1-2 nM, it slowed down the fatigue induced by continuous direct stimulation (2/s) of the muscle. Marinobufagenin at the same concentrations did not affect resting membrane potential or parameters of action potentials of muscle fibers, while at 10 and 20 nM it induced hyperpolarization by approximately 2 mV. Marinobufagenin blocked dose-dependently (IC50 = 2.9 +/- 2.0 nM) hyperpolarizing effect of acetylcholine (100 nM) mediated by increase in electrogenic contribution of alpha2 isoform of the Na+,K(+)-ATPase. This result suggests a capability of marinobufagenin to inhibit this isoform of the Na+,K(+)-ATPase. Possible mechanisms of marinobufagenin effects in skeletal muscle are discussed.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Bufanólidos/farmacología , Contracción Muscular/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Vasoconstrictores/farmacología , Acetilcolina/farmacología , Animales , Bufanólidos/metabolismo , Diafragma/efectos de los fármacos , Diafragma/fisiología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/fisiología , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
In experiments with isolated neuromuscular preparation of the rat diaphragm, selective blockade of alpha2 isoform of the Na,K-ATPase with ouabain (1 mcmol/L) induced steady depolarization of muscle fibers that reached a maximum of 4 mV, a decrease in amplitude of muscle fiber action potential, and prolonged raising and decline phases of the action potential. At the same time, the force, time to peak, and half relaxation time of the isometric muscle twitch were increased, as well as the area under the contraction curve. During continuous fatiguing stimulation (2/s), a more pronounced decline of contraction speed was observed in presence of ouabain; dynamics of the half-relaxation time remaining unchanged. It is suggested that blockade of alpha2 isoform of the Na,K-ATPase impairs excitation-contraction coupling resulting in a delay of Ca2+ release from sarcoplasmic reticulum. The increase in contraction force seems to result from a mechanism similar to that of positive inotropic effect of cardiac glycosides in heart muscle. Physiological significance of the skeletal muscle alpha2 isoform of the Na,K-ATPase in regulation of Ca2+ and Na+ concentrations near triadic junctions and in regulatory processes involving the Na,K-ATPase endogenous modulators or transmitter acetylcholine is discussed.
Asunto(s)
Diafragma/fisiología , Inhibidores Enzimáticos/administración & dosificación , Contracción Muscular/efectos de los fármacos , Ouabaína/administración & dosificación , Animales , Masculino , Contracción Muscular/fisiología , Ratas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
Acetylcholine (ACh) hyperpolarized the rat diaphragm muscle fibers by 4.5 +/- 0.8 mV (K0.5 = = 36 +/- 6 nmol/l). The AC-induced hyperpolarization was blocked by d-tubocurarine and ouabain in nanomolar concentrations. This effect of ACh was not observed in cultured C2C12 muscle cells and in Xenopus oocytes with expressed embryonic mouse muscle nicotinic acetylcholine receptors (nAChR) or with neuronal alpha 4 beta 2 nAChR. In membrane preparations from the Torpedo californica electric organ, containing both nAChR and Na, K-ATPase, 10 nmol/l ouabain modulated the binding kinetics of the cholinergic ligand dansyl-C6-choline to the nAChR. These results suggest that in-sensitive alpha 2 isoform) and nAChR in a state with high affinity to Ach and d-tubocurarine may form a functional complex in which binding of ACh to nAchR is coupled to activation of the Na, K-ATPase.
Asunto(s)
Músculo Esquelético/fisiología , Receptores Nicotínicos/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Acetilcolina/farmacología , Animales , Células Cultivadas , Colinérgicos/farmacología , Diafragma/efectos de los fármacos , Diafragma/fisiología , Órgano Eléctrico/efectos de los fármacos , Órgano Eléctrico/metabolismo , Órgano Eléctrico/ultraestructura , Técnicas In Vitro , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Membranas/efectos de los fármacos , Membranas/metabolismo , Ratones , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Ouabaína/farmacología , Ratas , Receptores Nicotínicos/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Torpedo , XenopusRESUMEN
The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.
Asunto(s)
Encéfalo/enzimología , Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fluoruro de Sodio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Calcio/farmacología , Bovinos , Membrana Celular/enzimología , Citratos/farmacología , Digitonina/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Ligandos , Magnesio/farmacología , Masculino , Citrato de SodioRESUMEN
In experiments on isolated rat diaphragm muscle, acetylcholine (100 nmol/l) hyperpolarized muscle fibres due to activation of the alpha 2 isoform of Na,K-ATPase. This hyperpolarization was blocked in a dose-dependent manner by ouabain (K0.5 = 8 +/- 4 nmol/l) as well as by a solution of porcine kidney extract (10 kDa cut-off filtration), with the K0.5 approximately equal to a 1:20,000-fold dilution. The inhibitory activity of the developed slowly over a period of 3 hours and, in contrast to ouabain, was still present after 1 hour of washing. Ouabain, but not the extract, inhibits Rb+ uptake in human erythrocytes that only express the alpha = 1 isoform of Na, K-ATPase. Our data suggest that in rat skeletal muscle the alpha 1 isoform of Na,K-ATPase is primarily responsible for ionic homeostasis, while the alpha 2 isoform provides a "regulatable" function and may be controlled by cholinergic stimulation and/or endogenous digitalis-like factors (EDLFs). Porcine kidney extract contains a factor (M. W. < 10 kDa) that selectively inhibits the rat alpha 2 isoform and differs from ouabain. Our experimental protocol can be used as a highly sensitive physiological assay for factors that selectively inhibit the alpha 2 isoform of Na,K-ATPase.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Riñón/química , Músculo Esquelético/enzimología , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Extractos de Tejidos/farmacología , Animales , Diafragma/efectos de los fármacos , Diafragma/enzimología , Diafragma/fisiología , Inhibidores Enzimáticos/aislamiento & purificación , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Ratas , Porcinos , Extractos de Tejidos/aislamiento & purificaciónRESUMEN
The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.