RESUMEN
OBJECTIVE: The objective of this study was to investigate whether gadolinium (Gd(3+))-sensitive stretch-activated ion channels (SAC) are basally active in left ventricular (LV) myocytes in early stages of heart failure (HF) induced by volume overload. METHODS: The aortocaval fistula (ACF) model was employed to induce HF due to volume overload in rat. At specific time-points, LV myocytes were acutely isolated using a modified Langendorff apparatus. Whole-cell currents were measured using the patch-clamp technique and intracellular Ca(2+)(Ca(2+)(i)) was examined using fluorescence imaging and the Ca(2+)-sensitive dye Fura-2. RESULTS: Current-voltage data were obtained from sham and ACF myocytes at 5-d and 2-, 6-, 8- and 10-wk post surgery. Compared to data from matching sham rats, a 10 microM Gd(3+)-sensitive current at -100 mV comprised a larger fraction of total current in myocytes from 5-d, 2-wk, and 6-wk ACF rats. In general, the Gd(3+)-sensitive current contributed to inward currents at mV< or =-80 and outward currents at >+20 mV. The enhanced Gd(3+)-sensitive current was absent in myocytes from 8- and 10-wk ACF rats. 10 or 100 microM Gd(3+) had no appreciable effect on resting Ca(2+)(i) of myocytes from 5-d ACF or corresponding sham rats. The Gd(3+)-sensitive current in 5-d ACF myocytes was i) sensitive to the cation-selective SAC inhibitor, GsMTx-4, ii) non-selective for Na(+)/K(+), and iii) impermeable to Ca(2+). CONCLUSION: A basally-active, Gd(3+)- and GsMTx-4-sensitive SAC current that is non-selective for Na(+) and K(+), but impermeable to Ca(2+) under resting conditions is transiently elevated in LV myocytes from rats in early stages of volume overload-induced HF.
Asunto(s)
Gadolinio/farmacología , Insuficiencia Cardíaca/metabolismo , Canales Iónicos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Potenciales de Acción/efectos de los fármacos , Adaptación Fisiológica , Animales , Calcio/análisis , Calcio/metabolismo , Colorantes Fluorescentes , Fura-2 , Ventrículos Cardíacos , Péptidos y Proteínas de Señalización Intercelular , Microscopía Fluorescente , Modelos Animales , Técnicas de Placa-Clamp , Péptidos/farmacología , Perfusión , Ratas , Venenos de Araña/farmacología , Estimulación Química , Estrés Mecánico , Factores de TiempoRESUMEN
Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO(2)/HCO(3)(-) solution displayed all the hallmarks of an electrogenic Na(+)/HCO(3)(-) cotransporter: (a) a DIDS-sensitive pH(i) recovery following the initial CO(2)-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na(+)-dependent outward current under voltage-clamp conditions (-60 mV). All three variants had similar external HCO(3)(-) dependencies (apparent K(M) of 4-6 mM) and external Na(+) dependencies (apparent K(M) of 21-36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (-60 mV) expressing NBCe1-A exhibited peak HCO(3)(-)-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current-voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (V(m) = -60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO(3)(-)-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Variación Genética/fisiología , Oocitos , Simportadores de Sodio-Bicarbonato/fisiología , Animales , Femenino , Regulación de la Expresión Génica/genética , Variación Genética/genética , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/genética , Ratas , Simportadores de Sodio-Bicarbonato/biosíntesis , Simportadores de Sodio-Bicarbonato/química , Simportadores de Sodio-Bicarbonato/genética , Transfección , Xenopus laevisRESUMEN
We investigated the mechanisms of endogenous nitric oxide (NO) modulation of lung sodium (Na(+)) transport. C57BL/6 mice injected intraperitoneally with the specific inducible NO synthase (iNOS) inhibitor 1400W (10 mg/kg every 8 h for 72 h) exhibited decreased alveolar nitrite levels and Na(+)-dependent amiloride-sensitive alveolar fluid clearance as compared with mice injected with vehicle. Similarly, pretreatment of mouse tracheal epithelial cells with 1400W abolished the inhibitory effects of amiloride on their Na(+) short circuit currents. On the other hand, mouse tracheal epithelial cells pretreated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of guanylate cyclase, had lower levels of cGMP, but normal values of amiloride-sensitive Na(+) currents. Amiloride also inhibited whole-cell Na(+) currents across A549 cells treated with vehicle (K(i) = 249 nM), but had no effect in A549 cells treated with 1400W. Western blotting studies showed significantly lower levels of alpha and gammaENaC in lung tissues and alveolar type II (ATII) cells from iNOS(-/-) as well as iNOS(+/+) mice treated with 1400W, as compared with the corresponding values from vehicle-treated iNOS(+/+) mice. Similar values for ratios of alpha, beta, and gammaenac to gapdh were obtained by real-time polymerase chain reaction for iNOS(+/+) mice and iNOS(-/-) mice. We concluded that NO derived from iNOS under basal conditions is necessary for amiloride-sensitive Na(+) transport across lung epithelial cells and modulates the amount of alpha and gammaENaC via post-transcriptional, cGMP-independent mechanisms.
Asunto(s)
Amilorida/metabolismo , Pulmón/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Sodio/metabolismo , Amidinas/metabolismo , Animales , Bencilaminas/metabolismo , Transporte Biológico/fisiología , Línea Celular , Diuréticos/metabolismo , Inhibidores Enzimáticos/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio , Humanos , Pulmón/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Técnicas de Placa-Clamp , Isoformas de Proteínas/metabolismo , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Canales de Sodio/metabolismoRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999) J. Biol. Chem. 274, 3602-3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr(1424)-X-X-Ile(1427) where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.