Assuntos
Cálcio/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/metabolismo , Fibroblastos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/patologia , Fibroblastos/patologia , Humanos , Pele/patologiaRESUMO
This study examined mechanisms of Cl- transport in rat lymphocytes under a variety of conditions. Basal intracellular Cl- concentration ([Cl-]i) was not different between cells assayed in the presence of HCO3- or its absence (HEPES). Removal of external Cl- resulted in a fall in [Cl-]i and a rapid rise in intracellular pH (pH(i)). Both Cl- efflux and the rise in pH(i) were blocked by DIDS or removal of external Na+ but were unaffected by furosemide. The mechanisms governing Cl- influx were assessed in cells that had been Cl- depleted for 1 h. Reexposure to Cl- resulted in a rapid rise in [Cl-]i that was partially inhibited by pretreatment with DIDS (57%) and partially inhibited by pretreatment with furosemide (45%). Pretreatment with both compounds together completely blocked Cl- influx. Cl- depletion caused a marked increase in pH(i) that rapidly declined toward normal when the cells were reexposed to Cl-. Preincubation with DIDS completely blocked this decrease in pH(i). In contrast, neither removal of Na+ nor preincubation with furosemide affected the decline in pH(i) when the cells were reexposed to Cl-. We conclude that, in thymic lymphocytes, Cl-/HCO3- (or Cl-/base exchange) regulates both Cl- influx and efflux. Cl- efflux is totally inhibited by DIDS and is mediated by a Na+-dependent Cl-/HCO3- exchanger. Cl- influx is partially DIDS sensitive and partially furosemide sensitive and is mediated by both a Na+-independent Cl-/HCO3- exchanger and by a Na+-K+-2Cl- cotransporter.
Assuntos
Antiporters/metabolismo , Cloretos/metabolismo , Linfócitos T/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Antiporters/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Antiportadores de Cloreto-Bicarbonato , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Concentração de Íons de Hidrogênio , Compostos de Quinolínio/farmacologia , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio/farmacologia , Simportadores de Cloreto de Sódio-Potássio , Linfócitos T/efeitos dos fármacosRESUMO
The present study examined the abundance of NHE-1 protein in cultured vascular smooth muscle cells (VSMCs), freshly isolated thymocytes, and fresh aortic tissue from spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. Two sets of affinity-purified antibodies (Ab[765-778] and Ab[698-711]) against different epitopes of the NHE-1 isoform of the Na+-H+ antiporter were used. Each set of antibodies recognized a major protein band at 105 to 110 kD that was more abundant in protein lysates prepared from cultured VSMCs from the SHR than those from WKY rats (Ab[765-778] 0.047 +/- 0.011 vs 0.010 +/- 0.002 O.D. units/10 microg protein, P<.001 for SHR and WKY, respectively; and Ab(698-711) 0.173 +/- 0.026 vs 0.087 +/- 0.028 O.D. units/10 microg protein, P<.05, for SHR and WKY, respectively). The increase in NHE-1 protein abundance in cultured VSMCs from the SHR was associated with a greater Vmax of the Na+-H+ antiporter as compared to those from WKY rats (17.93 +/- 2.07 vs 8.16 +/- 1.05 mmol H+/min, P<.001, respectively). In contrast to cultured VSMCs, there was no difference in the relative abundance of NHE-1 protein in fresh aortic tissue (0.075 +/- 0.018 vs 0.083 +/- 0.017 O.D. units/10 microg protein, from SHR and WKY, respectively) or in freshly isolated thymocytes (0.158 +/- 0.046 vs 0.226 +/- 0.054 O.D. units/10 microg protein, from SHR and WKY, respectively). We conclude that the increase in the Vmax of the Na+-H+ antiporter in cultured VSMCs from the SHR, compared to those from WKY rats, is due, at least in part, to increased levels of NHE-1 protein.
Assuntos
Linfócitos/metabolismo , Músculo Liso Vascular/metabolismo , Ratos Endogâmicos SHR/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Células Cultivadas , Músculo Liso Vascular/citologia , Ratos , Ratos Endogâmicos WKY , Timo/citologia , Timo/metabolismoRESUMO
An increased activity of the Na+/H+ antiporter in cells from patients with insulin-dependent diabetes mellitus (IDDM) has been proposed as a potential marker of nephropathy. We evaluated Na+/H+ antiporter activity and its relationship to DNA and protein synthesis in cultured skin fibroblasts from patients with IDDM classified as having either overt nephropathy or absence of nephropathy on the basis of urinary albumin excretion and kidney biopsy findings. In IDDM patients with overt nephropathy, Na+/H+ antiporter activity in serum stimulated cells was increased as compared to cells from control subjects (9.62 +/- 0.89 vs. 5.67 +/- 0.97 mmol H+/min, P < 0.005, respectively) and cells from IDDM patients without nephropathy (7.22 +/- 0.67 mmol H+/min, P < 0.025). By contrast, in cells made quiescent by serum deprivation Na+/H+ antiporter activity was lower than in serum-stimulated cells and there were no significant differences between the three groups. DNA synthesis assessed by [3H] thymidine incorporation was increased in the IDDM group with nephropathy as compared to the group without nephropathy (138 +/- 14 vs. 105 +/- 13 cpm/1000 cells, respectively, P < 0.05) and as compared to control subjects (65 +/- 11 cpm/1000 cells, P < 0.001). By contrast, protein synthesis assessed by [14C] L-leucine incorporation was not increased in fibroblasts from IDDM patients with nephropathy, suggesting that cellular hypertrophy is not a feature of their altered growth phenotype. After chronic inhibition of the Na+/H+ antiporter using EIPA (25 microM), [3H] thymidine incorporation was reduced by about 20% both in cells from IDDM patients and controls. This parameter therefore remained higher in cells from IDDM patients with nephropathy than in those from controls (81 +/- 16 vs. 40 +/- 6 cpm/1000 cells, P < 0.05), while in cells from IDDM patients without nephropathy [3H] thymidine incorporation after EIPA (56 +/- 7.0 cpm/1000 cells) was intermediate between cells from controls and IDDM patients with nephropathy. We argue that cultured skin fibroblasts from IDDM patients, with nephropathy display an abnormal growth phenotype characterized by cell hyperplasia. This growth phenotype is associated with overactivity of the Na+/H+ antiporter during serum stimulation but not when cells are made quiescent and persists after inhibition of the Na+/H+ antiporter. Our data, therefore, further shows that overactivity of the Na+/H+ antiporter is not required for the expression of the altered growth phenotype of cultured skin fibroblasts from IDDM patients with nephropathy.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Nefropatias Diabéticas/fisiopatologia , Trocadores de Sódio-Hidrogênio/metabolismo , Adulto , Albuminúria/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Antiarrítmicos/farmacologia , Divisão Celular/fisiologia , Células Cultivadas , Feminino , Fibroblastos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Fenótipo , Pele/citologia , Timidina/metabolismoRESUMO
An increase in Na+/H+ antiporter activity may be involved in hyperproliferation of vascular smooth muscle cells (VSMC) and possibly in the vascular hyperplasia characteristic of hypertension. The present study was designed to examine cell proliferation, Na+/H+ exchange activity, and mRNA levels of the NHE-1 isoform of the Na+/H+ antiporter in cultured aortic VSMC derived from the spontaneously hypertensive rat (SHR) and from normotensive controls, the Wistar/Kyoto rat (WKY). VSMC derived from the SHR grown in early (2 to 6), but not in later (7 to 10) sub-passages, exhibited an increase in [3H]-thymidine incorporation and shorter doubling times as compared to those derived from WKY rats. Na+/H+ exchange activity assayed in the nominal absence of HCO3-/CO2, as the rate of intracellular pH (pHi) recovery after cell acidification was significantly higher in cells from SHR than in those from WKY rats when cells were studied in early sub-passages, but not in cells studied in later sub-passages. In cells grown in early sub-passage, Na+/H+ exchange activity assessed as the initial rate of Na+i accumulation following acute cell acidification was also significantly higher in SHR than WKY cells both in the nominal absence (10.22 +/- 1.15 and 6.98 +/- 1.17 mmol Na+i/90 seconds, P < 0.05, respectively) and in the presence of HCO3-/CO2 (9.94 +/- 1.02 and 5.59 +/- 0.86 mmol Na+/90 seconds, P < 0.01, respectively). There were no detectable differences in the level of steady-state Na+/H+ antiporter (NHE-1) mRNA between VSMC from SHR and WKY rats. Our findings indicate that Na+/H+ exchange activity is increased in cultured aortic VSMC derived from SHR as compared to those derived from WKY rats. The higher functional activity of the Na+/H+ antiporter in VSMC from the SHR is due to a post-transcriptional event(s) and may be related to enhanced growth in culture.
Assuntos
Hipertensão/patologia , Músculo Liso Vascular/química , Trocadores de Sódio-Hidrogênio/análise , Animais , Aorta Torácica , Divisão Celular , Células Cultivadas , Meios de Cultura , Concentração de Íons de Hidrogênio , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Sódio/metabolismoRESUMO
Abnormal growth of vascular smooth muscle (VSM) is seen in various pathologic conditions such as hypertension and atherosclerosis. Many classic vasoconstrictors have now been shown to be mitogenic, either by themselves or in conjunction with other cofactors, such as insulin. The mitogenic effects of vasoconstrictors may be due, in part, to activation of similar second messenger pathways, including stimulation of the Na+/H+ antiporter. It has been suggested, therefore, that an enhanced proliferation rate may be, in part, the consequence of elevated Na+/H+ exchange. This hypothesis is supported by several observations of the close association between Na+/H+ exchange activity and DNA synthesis in some cell types including fibroblasts and VSM. Stimulation of Na+/H+ exchange may play a permissive role in optimal growth by preventing H+ accumulation (a fall in intracellular pH [pHi]) due to the increased metabolic activity during cell stimulation. Enhancement of Na+/H+ exchange activity increases Na+ influx into the cell, and secondarily increases K+ entry through activation of Na+/K+ ATPase activity. Although the Na+/H+ antiporter may influence cell proliferation through various ionic mechanisms, it is not clear that enhanced proliferation is the consequence of overactivity of this antiporter. In VSM, there are also differences in the pattern of activation of the Na+/H+ antiporter by hyperplastic and hypertrophic agents. Although pHi is increased in response to both acute and chronic stimulation by hyperplastic factors, such as platelet-derived growth factor, a hypertrophic agonist such as angiotensin II increases pHi acutely but lowers it chronically. Likewise, hyperplastic factors increase the Na+/H+ antiporter (NHE-1) mRNA levels, whereas angiotensin II does not.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Músculo Liso Vascular/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Angiotensina II/farmacologia , Animais , Arginina Vasopressina/farmacologia , Divisão Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Hiperplasia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologiaRESUMO
This study examined the mechanisms whereby alterations of intracellular pH (pHi) impact on free cytosolic calcium (Cai2+) in cultured rat aortic vascular smooth muscle cells (VSMC) assayed in the presence of HCO3/CO2. Rapid cell alkalinization, effected by the exposure to NH4Cl or removal of CO2 from the superfusate, produced a rapid increase in Cai2+. The rise in Cai2+ was markedly diminished when sarcoplasmic reticulum (SR) Ca2+ stores had been depleted by prior exposure to arginine vasopressin (AVP) in Ca(2+)-free media or when SR release and reuptake of Ca2+ were blocked by the addition of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), but was unaffected by the removal of external Ca2+ or inhibition of Ca2+ entry using NiCl2. Cell acidification also resulted in a rapid increase in Cai2+. This Cai2+ increase was most apparent when pHi was very low (< 6.6) and was unaffected by removal of external Ca2+ or NiCl2 addition. Unlike the effect of cell alkalinization, the increase in Cai2+ associated with cell acidification was not prevented by pretreatment with AVP or TMB-8. We conclude that, in cultured VSMC, acute intracellular alkalinization and, to a lesser extent, acidification result in release of Ca2+ from internal stores. Alkalinization increases Cai2+ by promoting its release from a store which is AVP and TMB-8 sensitive, most likely the SR. Cell acidification increases Cai2+ from an intracellular store(s) that is neither AVP nor TMB-8 sensitive. The increase in Cai2+ produced by cell acidification may be explained on the basis of cell buffering such that, as cytosolic H+ increases, it displaces Cai2+ from internal buffers with similar affinities for Ca2+ and H+.
Assuntos
Aorta Torácica/metabolismo , Cálcio/metabolismo , Concentração de Íons de Hidrogênio , Músculo Liso Vascular/metabolismo , Cloreto de Amônio/farmacologia , Animais , Arginina Vasopressina/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Células Cultivadas , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ácido Egtázico/farmacologia , Líquido Intracelular/metabolismo , Cinética , Masculino , Níquel/farmacologia , Ratos , Sódio/antagonistas & inibidores , Fatores de TempoRESUMO
This study examined contribution of Na(+)-dependent processes to the regulation of free cytosolic calcium (Ca2+i) in cultured vascular smooth muscle cells (VSMC) using fura-2. Removal of Na+ from superfusate (replacement with choline) resulted in an increment of Ca2+i that was greatly augmented by pretreatment with ouabain. Under both conditions, Ca2+i increase was followed by partial recovery to a new steady state that was still significantly higher than that seen before removal of external Na+ (Na+o). In ouabain-pretreated cells lowering of Na+o caused progressive increases in Ca2+i. Addition of NiCl2, a Na(+)-Ca2+ exchange inhibitor, completely blocked the increase in Ca2+i produced by removal of Na+o, indicating that the Na(+)-Ca2+ antiporter was responsible for observed Ca2+i changes. Ca2+i increase produced by reduction of Na+o was also seen after depletion of inositol trisphosphate-sensitive Ca2+ stores with repeated pulses of angiotensin II or after blockade of sarcoplasmatic reticulum Ca2+ release with TMB-8 but was not observed in the absence of external Ca2+. These observations indicate that the source of Ca2+i increase in response to changes in the transmembrane Na+ gradient is largely external, and potentiation of the Ca2+i surge by ouabain suggests Ca2+ influx via the Na(+)-Ca2+ exchanger operating in the reverse mode. The relative contribution of a Na(+)-dependent and -independent component of Ca2+i recovery was investigated by superfusing cells with ionomycin in a Na(+)-free medium and later adding Na+ to the medium. This Ca2+ ionophore increased Ca2+i to a peak, and this was followed by a rapid but partial recovery to a new steady state. Readdition of varying amounts of Na+ to the superfusate, in the continued presence of ionomycin, resulted in concentration-related decline in Ca2+i, thereby uncovering a substantial contribution of a Na(+)-dependent mechanism of Ca2+i regulation. Decline of Ca2+i produced by readdition of Na+ was blocked by addition of NiCl2 to the superfusate. Our findings thereby provide evidence for Ca2+i regulation in VSMC via a Na(+)-dependent mechanism, consistent with a Na(+)-Ca2+ exchanger, which acts as a Ca2+ efflux mechanism when Ca2+i is elevated. Na(+)-Ca2+ exchanger acts as a Ca2+ influx mechanism when intracellular Na+ is elevated by prior exposure to ouabain.
Assuntos
Aorta/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Espaço Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Sódio/metabolismo , Animais , Aorta/citologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Concentração de Íons de Hidrogênio , Ionomicina/farmacologia , Músculo Liso Vascular/citologia , Concentração Osmolar , Retículo Sarcoplasmático/metabolismoRESUMO
Although numerous studies have documented the effects of the renal nerves on kidney function, the mechanisms involved in the diuresis have yet to be elucidated. The present study was undertaken to examine the effect of acute unilateral renal denervation (DNX) on proximal tubular absorption of fluid and bicarbonate and to determine if acute DNX was associated with changes in Na-K-ATPase activity. Acute DNX caused significant increases in urine flow and absolute and fractional excretions of Na, HCO3 and K compared to the contralateral control kidney (INN) or sham denervated kidneys in normal rats as well as in rats made alkalotic by the I.V. infusion of 150 mM NaHCO3. These effects were seen without significant changes in GFR. When proximal convoluted tubules (PCT) were perfused with bicarbonate-Ringer's solution DNX resulted in a 67% decrease in fluid reabsorption (INN: 3.0 +/- 0.2 vs DNX: 1.0 +/- 0.1 nl/min/mm; p less than 0.001) and a 40% decline in bicarbonate (total CO2) reabsorption (INN: 151.3 +/- 8.8 vs DNX: 94.5 +/- 10.1 pmol/min/mm; p less than 0.01). Acute DNX caused a significant reduction in Na-K-ATPase activity measured in microsomes derived from the outer cortex of the kidney (INN: 13.2 +/- 1.3 vs DNX: 10.9 +/- 0.7 mumol PO4/mg prot/hr; p less than 0.01) while Mg-ATPase was unaffected. Sham denervation had no effect on any of the above parameters. These results indicate that the renal nerves play an important role in the regulation of bicarbonate and fluid reabsorption in the PCT. The diuresis, natriuresis, and bicarbonaturia associated with acute unilateral renal denervation may be the direct result of inhibition of Na-K-ATPase activity.
Assuntos
Bicarbonatos/metabolismo , Córtex Renal/enzimologia , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Equilíbrio Ácido-Base/fisiologia , Animais , Bicarbonatos/urina , Gasometria , Denervação , Masculino , Membranas/enzimologia , Perfusão , Potássio/urina , Ratos , Ratos Endogâmicos , Sódio/urinaRESUMO
Free intracellular calcium was measured in renal proximal tubules obtained from spontaneously hypertensive rats (SHR) and from age-matched Wistar-Kyoto rats (WKY) ingesting a normal diet. Experiments were performed on renal proximal tubule suspensions using fura-2 to monitor cytosolic calcium. In 4-week-old rats, when systolic blood pressure was not significantly different between the two groups, renal proximal tubule cytosolic calcium was similar (143 +/- 28 and 144 +/- 15 nM, respectively). By the age of 5 weeks, cytosolic calcium increased significantly in both SHR and WKY (214 +/- 24 and 262 +/- 34 nM, respectively, p less than 0.05). Calcium, however, was not significantly different between the two groups, even though at this age blood pressure was higher in SHR than in WKY. As compared with values in 4-week-old rats, cytosolic calcium was also found increased in tubules from both SHR and WKY aged 10 to 12 weeks (261 +/- 42 and 279 +/- 30 nM, respectively) and 20 to 24 weeks (263 +/- 42 and 308 +/- 28 nM, respectively). However, no significant differences in cytosolic calcium were found between SHR and WKY even though at these ages systolic blood pressure increased markedly in the SHR. Moreover, regression analysis failed to reveal a correlation between cytosolic calcium and blood pressure when data from either group of rats of all ages studied were pooled. Exposure to ouabain (10(-3) M) to inhibit Na+,K+-adenosine triphosphatase and increase intracellular sodium had no significant effect on cytosolic calcium in tubules from either SHR or WKY (260 +/- 69 and 250 +/- 45 nM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)