RESUMO
Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.
Assuntos
Cardiotônicos/metabolismo , Isoenzimas/metabolismo , Miocárdio/enzimologia , Ouabaína/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Cardiotônicos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Humanos , Técnicas In Vitro , Ouabaína/farmacologia , TrítioRESUMO
Human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-subunit isoforms have a similar high affinity for cardiac glycosides. K(0.5) values for antagonism of ouabain binding by K(+) were ranked in order as follows: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (0.9 +/- 0.6 mM), and K(0.5) values for Na(+) antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha(1)beta(1) (6,652 min(-1)) was about twice as high as that by alpha(3)beta(1) (3,145 min(-1)). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na(+)-K(+)-ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na(+) pumps expressed in Xenopus oocytes, the alpha(2)beta(1) complex in yeast membranes was significantly less stable than alpha(1)beta(1) or alpha(3)beta(1), resulting in a lower functional expression level. The alpha(2)beta(1) complex was also more easily denatured by SDS than was the alpha(1)beta(1) or the alpha(3)beta(1) complex.
Assuntos
Inibidores Enzimáticos/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Humanos , Microssomos/enzimologia , Saccharomyces cerevisiae , Especificidade por SubstratoRESUMO
Urea transporters have been cloned from kidney medulla (UT-A) and erythrocytes (UT-B). We determined whether UT-A proteins could be detected in heart and whether their abundance was altered by uremia or hypertension or in human heart failure. In normal rat heart, bands were detected at 56, 51, and 39 kDa. In uremic rats, the abundance of the 56-kDa protein increased 1.9-fold compared with pair-fed, sham-operated rats, whereas the 51- and 39-kDa proteins were unchanged. We also detected UT-A2 mRNA in hearts from control and uremic rats. Because uremia is accompanied by hypertension, the effects of hypertension per se were studied in uninephrectomized deoxycorticosterone acetate salt-treated rats, where the abundance of the 56-kDa protein increased 2-fold versus controls, and in angiotensin II-infused rats, where the abundance of the 56 kDa protein increased 1.8-fold versus controls. The 51- and 39-kDa proteins were unchanged in both hypertensive models. In human left ventricle myocardium, UT-A proteins were detected at 97, 56, and 51 kDa. In failing left ventricle (taken at transplant, New York Heart Association class IV), the abundance of the 56-kDa protein increased 1.4-fold, and the 51-kDa protein increased 4.3-fold versus nonfailing left ventricle (donor hearts). We conclude that (1) multiple UT-A proteins are detected in rat and human heart; (2) the 56-kDa protein is upregulated in rat heart in uremia or models of hypertension; and (3) the rat results can be extended to human heart, where 56- and 51-kDa proteins are increased during heart failure.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Miocárdio/metabolismo , Adulto , Animais , Western Blotting , Proteínas de Transporte/genética , Feminino , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Uremia/genética , Uremia/metabolismo , Transportadores de UreiaRESUMO
Na,K-ATPase (NKA, Na-pump), an alphabeta heteromer, is the receptor for cardiac glycosides (CG) which exert a positive inotropic effect by inhibiting enzyme activity, decreasing the driving force for Na,Ca-exchange (NCX) and increasing cellular content and release of Ca2+ during depolarization. Our previous study of regional distribution of NKA in non-failing human hearts demonstrated that Na-pump alpha2-, alpha3- and beta1-isoforms were 30-50% lower in right atrium (RA) compared with left ventricle (LV), resulting in overall lower NKA activity and CG binding site number and increased sensitivity to inotropic stimulation. In failing human heart LV Na-pump alpha1, alpha3 and beta1 proteins were reduced 30-40%, with no change in alpha2 or NCX; NKA activity and CG binding sites decreased 40%, and sensitivity to inotropic stimulation increased, all compared to LV from non-failing hearts. In this study we investigated the influence of region specific factors (e.g. hemodynamics) on the regulation of NKA isoform and NCX expression in heart failure by comparing the pattern of change in right atrial myocardium during heart failure with that previously determined for LV. In RA samples from failing hearts, alpha1-, alpha2- and beta1-isoform protein expression were decreased by 40, 50 and 25%, respectively, with no significant change in alpha3 or NCX levels relative to non-failing hearts (both n= 12). Thus, alphabeta1 decreases in both RA and LV during heart failure, while alpha2beta1 is reduced only in RA and alpha3beta1 only in LV. This indicates that there are not only regional differences in normal cardiac Na-pump isoform expression but also regional differences in the pattern of isoform expression as a function of failure that may have distinct functional consequences in the adaptive process of heart failure. The mechanisms underlying Na,K-ATPase regulation and effect of hemodynamics remain to be investigated.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Adulto , Calibragem , Cardiomiopatia Dilatada/enzimologia , Feminino , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Humanos , Immunoblotting , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Extratos de TecidosRESUMO
Fourteen-day adrenal steroid treatment increases [(3)H]ouabain binding sites 22-48% in muscle biopsies from patients treated with adrenal steroids for chronic obstructive lung disease and in rats treated with dexamethasone (Dex). Ouabain binding measures plasma membrane sodium pumps (Na(+)-K(+)-ATPase) with isoform-dependent affinity. In this study we have established the specific pattern of Dex regulation of sodium pump isoform protein and mRNA levels in muscle. Rats were infused with Dex (0.1 mg/kg per day) or vehicle for 14 days. Abundance of sodium pump catalytic alpha(1)- and alpha(2)-subunits and glycoprotein beta(1)- and beta(2)-subunits was determined by immunoblot in soleus, extensor digitorum longus, whole gastrocnemius, and diaphragm and was normalized to the mean vehicle control value. Dex increased alpha(2) and beta(1) protein in all muscle types by 53-78% and ~50%, respectively. Dex increased alpha(1) protein only in diaphragm (65 +/- 7%). At the mRNA level in whole hindlimb muscle, Dex increased alpha(2) (6.4 +/- 0.5-fold) and beta(1) (1.54 +/- 0.15-fold) and decreased beta(2) (to 0.36 +/- 0.6 of control). In summary, alpha(2)beta(1) is the Dex-responsive pump in all skeletal muscles, and changes in alpha(2) and beta(1) mRNA levels can drive the 50% change in alpha(2)beta(1)-subunits, which can account for the reported increase in [(3)H]ouabain binding.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Feminino , Músculo Esquelético/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
We aimed to test the feasibility of quantifying insulin action on cellular K(+) uptake in vivo in the conscious rat by measuring the exogenous K(+) infusion rate needed to maintain constant plasma K(+) concentration ([K(+)]) during insulin infusion. In this "K(+) clamp" the K(+) infusion rate required to clamp plasma [K(+)] is a measure of insulin action to increase net plasma K(+) disappearance. K(+) infusion rate required to clamp plasma [K(+)] was insulin dose dependent. Renal K(+) excretion was not significantly affected by insulin at a physiological concentration ( approximately 90 microU/ml, P > 0.05), indicating that most of insulin-mediated plasma K(+) disappearance was due to K(+) uptake by extrarenal tissues. In rats deprived of K(+) for 2 days, plasma [K(+)] fell from 4.2 to 3.8 mM, insulin-mediated plasma glucose clearance was normal, but insulin-mediated plasma K(+) disappearance decreased to 20% of control, even though there was no change in muscle Na-K-ATPase activity or expression, which is believed to be the main K(+) uptake route. After 10 days K(+) deprivation, plasma [K(+)] fell to 2.9 mM, insulin-mediated K(+) disappearance decreased to 6% of control (glucose clearance normal), and there were 50% decreases in Na-K-ATPase activity and alpha2-subunit levels. In conclusion, the present study proves the feasibility of the K(+) clamp technique and demonstrates that short-term K(+) deprivation leads to a near complete insulin resistance of cellular K(+) uptake that precedes changes in muscle sodium pump expression.
Assuntos
Resistência à Insulina/fisiologia , Insulina/farmacologia , Rim/metabolismo , Deficiência de Potássio/fisiopatologia , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico , Glicemia/metabolismo , Técnica Clamp de Glucose , Hiperinsulinismo , Infusões Intravenosas , Insulina/administração & dosagem , Rim/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Acute hypertension in Sprague-Dawley rats (SD) provokes a decrease in renal proximal tubule (PT) salt and fluid reabsorption, redistribution of apical Na/H exchanger isoform 3 (NHE3) and Na-P(i) cotransporter type 2 (NaPi2) out of the brush border into higher density membranes, and inhibition of renal cortical Na-K-ATPase (NKA) activity (41). The aims of this study were to determine 1) whether an increase in arterial pressure affects distribution or activity of Na transporters in the spontaneously hypertensive rat (SHR) and 2) whether development of chronic hypertension in SHR leads to persistent adaptive changes in NHE3 and NaPi2 distribution and/or NKA activity. Renal cortex Na transporter protein density distributions and activities were compared by subcellular fractionation in 1) adult SHR with an acute increase or decrease in arterial pressure and 2) young SD (YSD) and young SHR (YSHR) vs. adult SD and SHR. In adult hypertensive SHR NHE3 was shifted to membranes of higher densities, analogous to SD with acute hypertension, and there were no further changes with a further increase or decrease in arterial pressure. There was no change in total pool size of NHE3 in cortex in YSHR vs. SHR. NHE3, NaPi2, megalin, NKA alpha-/beta-subunit, dipeptidyl peptidase IV (DPPIV), and villin distributions were the same in YSHR vs. YSD. NHE3, NaPi2, and megalin shifted to higher densities in adult SHR, but not SD, with age. Basolateral NKA and apical alkaline phosphatase activities were 40% greater in YSHR than YSD and decreased to SD levels in adults. We conclude that there are persistent changes in Na(+) transporter distributions and activity in response to chronic hypertension in SHR that mimic the responses to acute hypertension seen in SD rats and that elevated sodium pump activity per transporter in YSHR may contribute to the generation of hypertension.
Assuntos
Proteínas de Transporte/metabolismo , Hipertensão/metabolismo , Túbulos Renais Proximais/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores , Doença Aguda , Envelhecimento/fisiologia , Animais , Pressão Sanguínea , Doença Crônica , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Túbulos Renais Proximais/enzimologia , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Trocador 3 de Sódio-Hidrogênio , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II , ATPase Trocadora de Sódio-Potássio/metabolismo , Especificidade da Espécie , Distribuição TecidualRESUMO
Renal Na,K-ATPase drives active reabsorption of sodium and cotransported solutes along the nephron. There is a large increase in net Na(+) reabsorption in the postnatal rat kidney. It has previously been established that the postnatal increase in expression of sodium pump isoforms in the brain, but not the heart, is blunted in the hypothyroid neonate. The aims of this study were to establish whether the developmental increase in renal sodium transport is associated with coordinate increases in the abundance of the sodium pump alpha(1) catalytic and beta(1) glycoprotein subunits and Na,K-ATPase activity, and to determine whether thyroid status influences the postnatal increase in renal Na,K-ATPase expression. Pregnant rats were made hypothyroid with low iodine diet, propylthiouracil and perchlorate. Offspring were hypothyroid assessed by triiodothyronine/thyroxine RIA. Renal cortical membranes were prepared from euthyroid and hypothyroid rats from 6 to 24 days of age. There was no change in Na,K-ATPase activity or expression between 6 and 15 days. Between 15 and 24 days, Na,K-ATPase activity increased 1.35-fold while sodium pump alpha(1) and beta(1) subunit abundance increased coordinately to 1.7- and 2-fold over the previous period, respectively. In hypothyroid neonates, kidney weight was less than in euthyroids, and Na,K-ATPase activity, alpha(1) and beta(1) subunit pool sizes did not significantly increase as a function of age between 6 and 24 days. We conclude that the postnatal increase in sodium pump activity can be accounted for by coordinate increases in the pool sizes of alpha(1) and beta(1) subunits and that, like in brain, this increased Na,K-ATPase expression is dependent on normal thyroid status.
Assuntos
Hipotireoidismo/enzimologia , Córtex Renal/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Feminino , Córtex Renal/crescimento & desenvolvimento , Gravidez , Radioimunoensaio , Ratos , Ratos Sprague-DawleyRESUMO
Acute hypertension provokes a rapid decrease in proximal tubule salt and water reabsorption that increases the levels of sodium chloride at the macula densa, the error signal to increase arteriolar resistance to autoregulate renal blood flow and glomerular filtration rate, and contributes to pressure natriuresis. The molecular mechanisms responsible for this critical homeostatic adjustment are beginning to be dissected: apical sodium transporters in the proximal tubule are redistributed out of the brush border to intermicrovillar and endosomal stores and sodium pump activity is inhibited. These responses are strikingly similar to the cellular responses to parathyroid hormone, and are mediated by similar signalling pathways.
Assuntos
Hipertensão Renal/metabolismo , Túbulos Renais Proximais/fisiopatologia , Sódio/metabolismo , Simportadores , Doença Aguda , Membrana Basal/metabolismo , Proteínas de Transporte/metabolismo , Doença Crônica , Humanos , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
When K+ output exceeds input, skeletal muscle releases intracellular fluid K+ to buffer the fall in extracellular fluid (ECF) K+. To investigate the mechanisms and muscle specificity of the K+ shift, rats were fed K+-deficient chow for 2-10 days, and two muscles at phenotypic extremes were studied: slow-twitch oxidative soleus and fast-twitch glycolytic white gastrocnemius (WG). After 2 days of low-K+ chow, plasma K+ concentration ([K+]) fell from 4.6 to 3.7 mM, and Na+-K+-ATPase alpha2 (not alpha1) protein levels in both muscles, measured by immunoblotting, decreased 36%. Cell [K+] decreased from 116 to 106 mM in soleus and insignificantly in WG, indicating that alpha2 can decrease before cell [K+]. After 5 days, there were further decreases in alpha2 (70%) and beta2 (22%) in WG, not in soleus, whereas cell [K+] decreased and cell [Na+] increased by 10 mM in both muscles. By 10 days, plasma [K+] fell to 2.9 mM, with further decreases in WG alpha2 (94%) and beta2 (70%); cell [K+] fell 19 mM in soleus and 24 mM in WG compared with the control, and cell [Na+] increased 9 mM in soleus and 15 mM in WG; total homogenate Na+-K+-ATPase activity decreased 19% in WG and insignificantly in soleus. Levels of alpha2, beta1, and beta2 mRNA were unchanged over 10 days. The ratios of alpha2 to alpha1 protein levels in both control muscles were found to be nearly 1 by using the relative changes in alpha-isoforms vs. beta1- (soleus) or beta2-isoforms (WG). We conclude that the patterns of regulation of Na+ pump isoforms in oxidative and glycolytic muscles during K+ deprivation mediated by posttranscriptional regulation of alpha2beta1 and alpha2beta2 are distinct and that decreases in alpha2-isoform pools can occur early enough in both muscles to account for the shift of K+ to the ECF.
Assuntos
Cátions/metabolismo , Hipopotassemia/metabolismo , Músculo Esquelético/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Espaço Extracelular/metabolismo , Glicólise , Hipopotassemia/enzimologia , Masculino , Músculo Esquelético/enzimologia , Concentração Osmolar , Oxirredução , Potássio/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/genética , Fatores de TempoRESUMO
The aim of this study was to test the hypothesis that in vivo administration of parathyroid hormone (PTH) provokes diuresis/natriuresis through redistribution of proximal tubule apical sodium cotransporters (NHE3 and NaPi2) to internal stores and inhibition of basolateral Na-K-ATPase activity and to determine whether the same cellular signals drive the changes in apical and basolateral transporters. PTH-(1-34) (20 U), which couples to adenylate cyclase (AC), phospholipase C (PLC), and phospholipase A2 (PLA2), or [Nle8,18,Tyr34]PTH-(3-34) (10 U), which couples to PLC and PLA2 but not AC, were given to anesthetized rats as an intravenous bolus followed by low-dose infusion (1 U. kg-1. min-1 for 1 h). Renal cortex membranes were fractionated on sorbitol density gradients. PTH-(1-34) increased urinary cAMP excretion 3-fold, urine output (V) 2.0 +/- 0.1-fold, and lithium clearance (CLi) 2.8 +/- 0.3-fold. With this diuresis/natriuresis, 25% of NHE3 and 18% of NaPi2 immunoreactivity redistributed from apical membranes to higher density fractions containing intracellular membrane markers, and basolateral Na-K-ATPase activity decreased 25%. [Nle8,18,Tyr34]PTH-(3-34) failed to increase V or CLi or to provoke redistribution of NHE3 or NaPi2, but it did inhibit Na-K-ATPase activity 25%. We conclude that in vivo PTH stimulates natriuresis/diuresis associated with internalization of apical NHE3 and NaPi2 and inhibition of Na-K-ATPase activity, that cAMP-protein kinase A stimulation is necessary for the natriuresis/diuresis and NHE3 and NaPi2 internalization, and that Na-K-ATPase inhibition is not secondary to depressed apical Na+ transport.
Assuntos
Proteínas de Transporte/metabolismo , Fragmentos de Peptídeos/farmacologia , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Simportadores , Teriparatida/análogos & derivados , Animais , Transporte Biológico/efeitos dos fármacos , AMP Cíclico/urina , Ativação Enzimática/efeitos dos fármacos , Rim/química , Rim/efeitos dos fármacos , Rim/enzimologia , Lítio/urina , Masculino , Hormônio Paratireóideo/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio , Proteínas Cotransportadoras de Sódio-Fosfato , Teriparatida/farmacologia , UrinaRESUMO
BACKGROUND: Cardiac glycosides initiate an increase in force of contraction by inhibiting the sarcolemmal sodium pump (Na+, K+-ATPase), thereby decreasing Ca2+ extrusion by the Na+-Ca2+ exchanger, which increases the cellular content of Ca2+. In patients with heart failure the sensitivity toward cardiac glycosides is enhanced. METHODS AND RESULTS: Because the inotropic effect of cardiac glycosides may be a function of the sodium pump and Na+-Ca2+ exchanger (NCE) expression levels, the present study aimed to investigate protein expression of both transporters (immunoblot with specific antibodies against the sodium pump catalytic alpha1-, alpha2-, alpha3-, and glycoprotein beta1-isoforms and against NCE) in left ventricle from failing (heart transplantations, New York Heart Association class IV, n=21) compared with nonfailing (donor hearts, NF, n=22) human myocardium. The density of 3H-ouabain-binding sites (Bmax) and the Na+,K+-ATPase activity were also measured. In NYHA class IV, protein levels of Na+,K+-ATPase alpha1- (0.62+/-0.06 of control), alpha3- (0.70+/-0.09), and beta1- (0.61+/-0.04) but not alpha2-isoforms were significantly reduced (P<0.01), whereas levels of NCE (0.92+/-0.13 of control) and calsequestrin (0.98+/-0.06) remained unchanged. Both Na+,K+-ATPase activity (NF: 1.9+/-0.29; NYHA class IV: 1.1+/-0.17 micromol ATP/min per milligram of protein) and the 3H-ouabain binding sites (Bmax NF: 15.9+/-1.9 pmol/mg protein; NYHA class IV: 9.7+/-1.5) were reduced in NYHA class IV and correlated significantly to each other (r2=0. 73; P<0.0001), as did beta1-subunit expression. In left ventricular papillary muscle strips from NYHA class IV compared with nonfailing tissue the Na+-channel modulator BDF 9198 exerted an increase in force of contraction with unchanged effectiveness but enhanced potency. CONCLUSIONS: The enhanced sensitivity of failing human myocardium toward cardiac glycosides may be, at least in part, attributed to a reduced protein expression and activity of the sarcolemmal Na+,K+-ATPase without a change in Na+-Ca2+ exchanger protein expression.
Assuntos
Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Adolescente , Adulto , Sítios de Ligação , Calsequestrina/metabolismo , Cardiomiopatia Dilatada/metabolismo , Feminino , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Ventrículos do Coração , Humanos , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Ouabaína/metabolismo , Músculos Papilares/fisiologia , Músculos Papilares/fisiopatologia , Valores de ReferênciaAssuntos
Espaço Extracelular/fisiologia , Líquido Intracelular/fisiologia , Rim/fisiologia , Canais de Sódio/fisiologia , Animais , Canais Epiteliais de Sódio , Homeostase , Humanos , Modelos Biológicos , Oócitos/fisiologia , Pressão Osmótica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Canais de Sódio/biossíntese , XenopusRESUMO
Redistribution of apical Na+/H+ exchangers (NHE) in the proximal tubules as a plausible mechanism of pressure natriuresis was investigated with confocal immunofluorescence microscopy in Sprague-Dawley rats (SD), spontaneously hypertensive rats (SHR), and two-kidney, one-clip Goldblatt hypertensive rats (GH). NHE isoform NHE3 was localized in the brush border of proximal tubules in SD. Twenty minutes of induced acute hypertension (20-40 mmHg) resulted in a pronounced redistribution of isoform NHE3 from the brush border into the base of microvilli, where clathrin-coated pits were localized. Prehypertensive young SHR (5 wk old, mean blood pressure 105 +/- 3 mmHg, n = 11) produced similar findings. However, NHE3 was found to concentrate in the base of microvilli in adult SHR (12 wk old, mean blood pressure 134 +/- 6 mmHg, n = 12) and nonclipped kidneys of GH (mean blood pressure 131 +/- 6 mmHg, n = 6). In clipped kidneys of GH, which were not exposed to the hypertension because of the arterial clips, NHE3 was localized on the brush border as in normal SD. No further redistribution of NHE3 was detected in adult SHR or GH when acute hypertension was induced. Since both acute and chronic increase of arterial pressure can provoke the redistribution of apical NHE in proximal tubules, the pressure-induced NHE redistribution could be a physiological response and an integral part of pressure natriuresis.
Assuntos
Hipertensão Renovascular/metabolismo , Hipertensão/metabolismo , Túbulos Renais Proximais/metabolismo , Microvilosidades/metabolismo , Trocadores de Sódio-Hidrogênio/biossíntese , Doença Aguda , Animais , Pressão Sanguínea , Doença Crônica , Invaginações Revestidas da Membrana Celular/metabolismo , Dipeptidil Peptidase 4/metabolismo , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertensão Renovascular/fisiopatologia , Imuno-Histoquímica , Túbulos Renais Proximais/fisiologia , Túbulos Renais Proximais/fisiopatologia , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Trocador 3 de Sódio-HidrogênioRESUMO
Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F1004-F1014, 1996]. To determine the reversibility and specificity of these responses, rats were subjected to 1) elevation of blood pressure (BP) of 50 mmHg for 5 min, 2) restoration of normotension after the first protocol, or 3) sham operation. Systolic hypertension increased urine output and endogenous lithium clearance three- to fivefold within 5 min, but these returned to basal levels only 15 min after BP was restored. Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions of the apical cytoskeleton protein villin were unaltered. After 20 min of normalized BP, all the NHE3 and smaller fractions of the other apical membrane proteins returned to their original distributions. These findings suggest that the dynamic regulation of proximal tubule sodium transport by acute changes in BP may be mediated by rapid reversible regulation of sodium pump activity and relocation of apical sodium transporters.
Assuntos
Hipertensão/metabolismo , Túbulos Renais Proximais/metabolismo , Doença Aguda , Animais , Pressão Sanguínea/fisiologia , Córtex Renal/enzimologia , Túbulos Renais Proximais/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Acute systolic arterial hypertension provokes a rapid decrease in proximal tubule sodium reabsorption and diuresis associated with inhibition of renal cortex Na,K-ATPase activity and redistribution of apical membrane Na/H exchanger (NHE-3) to heavier density membranes containing markers of intermicrovillar cleft and endosomes. Because cytochrome P-450-dependent arachidonate metabolites participate in the regulation of renal sodium transport and BP, this study tested the hypothesis that these renal responses to acute hypertension would be prevented if cytochrome P-450 metabolism were inhibited by cobalt chloride (CoCl2). Four groups of rats (n = 4 to 5) were studied: (1) sham-operated; (2) 50 mg of CoCl2/kg subcutaneously for 2 d; (3) acute hypertension by constricting arteries for 5 min; and (4) acute hypertension after CoCl2 treatment as in group 3. Renal cortex was analyzed after sorbitol density gradient fractionation. CoCl2 treatment alone did not significantly affect the rate of urine output, endogenous lithium clearance (an inverse measure of proximal tubule sodium reabsorption), maximal activity of Na,K-ATPase, or subcellular distribution of NHE-3-containing membranes. In non-CoCl2-treated animals, acute hypertension provoked a three- to fourfold increase in urine output and endogenous lithium clearance, 33% inhibition of renal cortex Na,K-ATPase activity, and redistribution of NHE-3 out of the apical membrane peak. In CoCl2-treated animals, acute urine output and endogenous lithium clearance increased only twofold during acute hypertension, there was no inhibition of Na,K-ATPase activity, and there was no redistribution of NHE-3 immunoreactivity to higher density membranes. These findings demonstrate that CoCl2 treatment both attenuates the inhibition of proximal tubule sodium reabsorption and diuresis and abolishes Na,K-ATPase inhibition and NHE-3 redistribution during acute hypertension, evidence that these responses may be mediated by cytochrome P-450 arachidonate metabolites.
Assuntos
Cobalto/administração & dosagem , Inibidores das Enzimas do Citocromo P-450 , Hipertensão/fisiopatologia , Córtex Renal/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Doença Aguda , Análise de Variância , Animais , Técnicas de Cultura , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Immunoblotting , Córtex Renal/efeitos dos fármacos , Lítio/urina , Masculino , Ratos , Ratos Sprague-Dawley , Valores de Referência , Circulação Renal/efeitos dos fármacos , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , UrinaRESUMO
We measured Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity and subunit abundance in renal cortical homogenates and basolateral membranes (BLM) from fetal, newborn, and adult guinea pigs. Pump specific activity increased four- to fivefold in cortical homogenates and BLM during the transition from fetus to newborn. Immunoblots of BLM showed that alpha- and beta-subunit abundance increased four- to seven- and fourfold, respectively, during the transition from fetus to newborn. Immunoblots of cortical homogenates revealed similar developmental patterns, with newborns having 3.5-fold (alpha) and 2.3-fold (beta) greater subunit abundances than fetuses. Therefore, the bulk of the postnatal increase in BLM-Na(+)-K(+)-ATPase abundance resulted from increased pump production or decreased pump degradation, rather than from redistribution of pumps from intracellular pools. Despite the developmental increase in alpha- and beta-subunit protein levels, newborn guinea pig kidneys had only 1.4- to 2.1-fold greater alpha 1-subunit mRNA abundance and only a 1.5-fold greater beta 1-subunit mRNA abundance than fetal kidneys. These results demonstrate large increases in renal cortical Na(+)-K(+)-ATPase specific activity and protein abundance immediately after birth. These increases, which appear to result largely from posttranscriptional upregulation, may play an important role in mediating the rapid postnatal increase in tubular NaCl reabsorption.
Assuntos
Animais Recém-Nascidos/metabolismo , Córtex Renal/enzimologia , Processamento de Proteína Pós-Traducional , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Northern Blotting , Cobaias , Immunoblotting , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
During potassium deprivation, skeletal muscle loses K+ to buffer the fall in extracellular K+. Decreased active K+ uptake via the sodium pump, Na,K-ATPase, contributes to the adjustment. Skeletal muscle expresses alpha1, alpha2, beta1, and beta2 isoforms of the Na, K-ATPase alphabeta heterodimer. This study was directed at testing the hypothesis that K+ loss from muscle during K+ deprivation is a function of decreased expression of specific isoforms expressed in a muscle type-specific pattern. Isoform abundance was measured in soleus, red and white gastrocnemius, extensor digitorum longus, and diaphragm by immunoblot. alpha2 expression was uniform across control muscles, whereas alpha1 and beta1 were twice as high in oxidative (soleus and diaphragm) as in fast glycolytic (white gastrocnemius) muscles, and beta2 expression was reciprocal: highest in white gastrocnemius and barely detectable in soleus and diaphragm. Following 10 days of potassium deprivation plasma K+ fell from 4.0 to 2.3 mM, and there were distinct responses in glycolytic versus oxidative muscles. In glycolytic white gastrocnemius alpha2 and beta2 fell 94 and 70%, respectively; in mixed red gastrocnemius and extensor digitorum longus both fell 60%, and beta1 fell 25%. In oxidative soleus and diaphragm alpha2 fell 55 and 30%, respectively, with only minor changes in beta1. Although decreases in alpha2 and beta2 expression are much greater in glycolytic than oxidative muscles during K+ deprivation, both types of muscle lose tissue K+ to the same extent, a 20% decrease, suggesting that multiple mechanisms are in place to regulate the release of skeletal muscle cell K+.
Assuntos
Hipopotassemia/enzimologia , Isoenzimas/metabolismo , Músculo Esquelético/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Masculino , Testes de Precipitina , Ratos , Ratos Sprague-DawleyRESUMO
Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 microM). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.
Assuntos
Proteínas de Transporte/isolamento & purificação , Isoenzimas/isolamento & purificação , Miocárdio/enzimologia , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , Adulto , Azetidinas/metabolismo , Transporte Biológico , Cálcio/metabolismo , Proteínas de Transporte/genética , Feminino , Átrios do Coração/enzimologia , Septos Cardíacos/enzimologia , Ventrículos do Coração/enzimologia , Humanos , Immunoblotting , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Ouabaína/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Trocador de Sódio e Cálcio , ATPase Trocadora de Sódio-Potássio/genética , Distribuição Tecidual , Doadores de TecidosRESUMO
Acute arterial hypertension provokes a rapid decrease in proximal tubule (PT) Na+ reabsorption, increasing flow to the macula densa, the signal for tubuloglomerular feedback. We tested the hypothesis, in rats, that Na+ transport is decreased due to rapid redistribution of apical Na+/H+ exchangers and basolateral Na+ pumps to internal membranes. Arterial pressure was increased 50 mmHg by constricting various arteries. We also tested whether transporter internalization occurred when PT Na+ reabsorption was inhibited with the carbonic anhydrase inhibitor benzolamide. Five minutes after initiating either natriuretic stimuli, cortex was removed, and membranes were fractionated by density gradient centrifugation. Urine output and endogenous lithium clearance increased threefold in response to either stimuli. Acute hypertension provoked a redistribution of apical Na+/H+ exchanger NHE3, alkaline phosphatase, and dipeptidyl peptidase IV to higher density membranes enriched in the intracellular membrane markers. Basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity decreased 50%, 25-30% of the alpha 1-and beta 1-subunits redistributed to higher density membranes, and the remainder is attributed to decreased activity of the transporters. Benzolamide did not alter Na+ transporter activity or distribution, implying that decreasing apical Na+ uptake does not initiate redistribution or inhibition of basolateral Na(+)-K(+)-ATPase. We conclude that PT natriuresis provoked by acute arterial pressure is mediated by both endocytic removal of apical Na+/H+ exchangers and basolateral Na+ pumps as well as decreased total Na+ pump activity.