RESUMO
To understand the regulation of Na(+)/H(+) exchanger regulatory factor (NHERF1) in polycystic ovarian syndrome, we studied the expression of NHERF1 in uterus of Wistar rats injected with (6 mg/kg) of dehydroepiandrosterone (DHEA) for 7 and 20 days. Immunohistochemistry analysis of NHERF1 showed a substantial shift in the intracellular localization of NHERF1 in endometrial glands and areas of luminal epithelium as early as 7 days of DHEA administration. The NHERF1 accumulated in the "Golgi apparatus area" virtually in all the glands in the 7-day protocol, and in the majority of the glands of 20-day protocol. In contrast, NHERF1 is expressed in the apical membrane and slightly in the cytoplasm of the control epithelium. The subcellular redistribution of NHERF1 could affect the sorting of proteins to the apical membrane and the organization of the apical compartment.
Assuntos
Desidroepiandrosterona/farmacologia , Endométrio/metabolismo , Ovário/metabolismo , Fosfoproteínas/biossíntese , Trocadores de Sódio-Hidrogênio/biossíntese , Útero/metabolismo , Animais , Endométrio/citologia , Endométrio/efeitos dos fármacos , Feminino , Ovário/citologia , Ovário/efeitos dos fármacos , Ratos , Ratos Wistar , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Útero/citologia , Útero/efeitos dos fármacosRESUMO
The Na(+)/H(+) exchanger (NHE) is involved in the regulation of intracellular pH and volume by mediating the electroneutral transport of H(+) against an influx of Na(+) ions. Since NHE1 regulates pH in neurons and astrocytes and it is expressed in nociceptive nerve fibers, it is likely that NHE may modulate neuronal excitability and pain transmission. The purpose of this study was to assess the participation of peripheral and spinal NHE in the secondary allodynia/hyperalgesia induced by formalin. In addition, we determined whether formalin injection modifies the expression of NHE1 in lumbar dorsal root ganglia (DRG) and dorsal spinal cord. Subcutaneous injection of 0.5% formalin into the dorsal surface of the hind paw produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-lasting bilateral secondary mechanical allodynia/hyperalgesia. Peripheral and intrathecal pre-treatment (-10min) with selective NHE inhibitors 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.3-30µM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 0.3-30µM) and [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine dihydrochloride (zoniporide, 0.03-3µM) significantly increased 0.5% formalin-induced bilateral long-lasting secondary allodynia/hyperalgesia. Contrariwise, local peripheral or intrathecal post-treatment (day 6 postinjection) with these NHE inhibitors did not affect formalin-induced nociceptive behaviors. Formalin injection reduced NHE1 expression in ipsilateral and contralateral spinal dorsal horns from day 1 to 12. In addition, formalin diminished NHE1 protein expression in DRG at day 12. These results suggest that NHE1 plays a role in pain processing at peripheral and spinal levels in formalin-induced long-lasting nociceptive behaviors. Additionally, these results suggest that proteins involved in pH regulation could be targets for the development of new analgesic drugs.
Assuntos
Hiperalgesia/enzimologia , Medição da Dor/métodos , Nervos Periféricos/enzimologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/biossíntese , Medula Espinal/enzimologia , Amilorida/administração & dosagem , Amilorida/análogos & derivados , Animais , Feminino , Hiperalgesia/induzido quimicamente , Injeções Espinhais , Medição da Dor/efeitos dos fármacos , Nervos Periféricos/efeitos dos fármacos , Estimulação Física/efeitos adversos , Ratos , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/fisiologia , Medula Espinal/efeitos dos fármacosRESUMO
Lithium (Li)-treated patients often develop urinary concentrating defect and polyuria, a condition known as nephrogenic diabetes insipidus (NDI). In a rat model of Li-induced NDI, we studied the effect that sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, has on renal expression of aquaporin-2 (AQP2), urea transporter UT-A1, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), epithelial Na channel (ENaC; α-, ß-, and γ-subunits), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase. We also evaluated cGMP levels in medullary collecting duct cells in suspension. For 4 wk, Wistar rats received Li (40 mmol/kg food) or no treatment (control), some receiving, in weeks 2-4, Sil (200 mg/kg food) or Li and Sil (Li+Sil). In Li+Sil rats, urine output and free water clearance were markedly lower, whereas urinary osmolality was higher, than in Li rats. The cGMP levels in the suspensions of medullary collecting duct cells were markedly higher in the Li+Sil and Sil groups than in the control and Li groups. Semiquantitative immunoblotting revealed the following: in Li+Sil rats, AQP2 expression was partially normalized, whereas that of UT-A1, γ-ENaC, and eNOS was completely normalized; and expression of NKCC2 and NHE3 was significantly higher in Li rats than in controls. Inulin clearance was normal in all groups. Mean arterial pressure and plasma arginine vasopressin did not differ among the groups. Sil completely reversed the Li-induced increase in renal vascular resistance. We conclude that, in experimental Li-induced NDI, Sil reduces polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1.
Assuntos
Diabetes Insípido Nefrogênico/fisiopatologia , Compostos de Lítio/efeitos adversos , Piperazinas/uso terapêutico , Poliúria/tratamento farmacológico , Sulfonas/uso terapêutico , Animais , Aquaporina 2/biossíntese , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/biossíntese , Diabetes Insípido Nefrogênico/induzido quimicamente , Ingestão de Líquidos/efeitos dos fármacos , Canais Epiteliais de Sódio/biossíntese , Taxa de Filtração Glomerular/efeitos dos fármacos , Rim/metabolismo , Medula Renal/enzimologia , Masculino , Proteínas de Membrana Transportadoras/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo III/biossíntese , Purinas/uso terapêutico , Ratos , Citrato de Sildenafila , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/biossíntese , Simportadores de Cloreto de Sódio-Potássio/biossíntese , Membro 1 da Família 12 de Carreador de Soluto , Transportadores de UreiaRESUMO
In normal embryonic fibroblasts, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) stabilizes E-cadherin/ß-catenin binding and the lack of NHERF1 expression promotes cell transformation thus acting as a tumor suppressor gene. We here tested the hypothesis that NHERF1 could act as a tumor suppressor gene in colon cancer as a mediator of estrogens' protective actions in colon carcinogenesis. We studied the expression and localization of NHERF1 and ß-catenin by immunohistochemistry in colonic tumors induced by 1,2 dimethylhidrazine (DMH) in Sprague-Dawley rats. One group of the rats treated with the carcinogen was ovariectomized (OVX) in the middle of the tumor induction, simulating a human menopausal condition. We observed a protective role of estrogens in colon cancer, as non-ovariectomized rats (DMH) had a reduced tumor area compared with the ovariectomized group (DMH + OVX; mean ± SE) 28.98 ± 4.65 vs. 67.58 ± 8.69 (p < 0.00380). Despite the lack of plasma estrogen stimulation, we found abundant expression of NHERF1 in colon tumors from ovariectomized rats. NHERF1 was mainly localized in the cytoplasm of the adenocarcinoma cells and lost the apical localization previously reported in normal colon tissue. We also detected expression of NHERF1 by western blot in the SW48, CACO-2, and HT29 colon cancer cell lines. Non-estrogenic factors in plasma or the tumor microenvironment may regulate NHERF1 expression in transformed colon epithelial cells. Further studies are required to understand the regulation of NHERF1 expression in colon cancer tissue.
Assuntos
Neoplasias do Colo/metabolismo , Genes Supressores de Tumor , Hormônios Esteroides Gonadais/sangue , Fosfoproteínas/biossíntese , Trocadores de Sódio-Hidrogênio/biossíntese , 1,2-Dimetilidrazina/toxicidade , Animais , Western Blotting , Células CACO-2 , Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Feminino , Células HCT116 , Humanos , Imuno-Histoquímica , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.
Assuntos
Colo/metabolismo , Estrogênios/fisiologia , Ciclo Estral/fisiologia , Fosfoproteínas/biossíntese , Trocadores de Sódio-Hidrogênio/biossíntese , Útero/metabolismo , Animais , Colo/efeitos dos fármacos , Diestro/metabolismo , Estro/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Proestro/metabolismo , Ratos , Ratos Wistar , Útero/efeitos dos fármacosRESUMO
The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.
Assuntos
Gambás/fisiologia , Hormônio Paratireóideo/farmacologia , Trocadores de Sódio-Hidrogênio/biossíntese , Animais , Biotinilação , Carbazóis/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Luciferases/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Pirróis/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type-3 Na+/H+ exchanger (NHE3) and a thick ascending limb Na+-K+-2Cl(-) cotransporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.
Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/biossíntese , Simportadores de Cloreto de Sódio-Potássio/biossíntese , Doença de Weil/tratamento farmacológico , Acetilcisteína/administração & dosagem , Acetilcisteína/farmacologia , Ampicilina/administração & dosagem , Animais , Antibacterianos/administração & dosagem , Antígenos de Bactérias/análise , Cricetinae , Regulação para Baixo , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/farmacologia , Perfilação da Expressão Gênica , Rim/patologia , Rim/fisiopatologia , Fígado/patologia , Mesocricetus , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/análise , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Simportadores de Cloreto de Sódio-Potássio/análise , Simportadores de Cloreto de Sódio-Potássio/efeitos dos fármacos , Membro 1 da Família 12 de Carreador de Soluto , Tiobarbitúricos/sangue , Doença de Weil/patologia , Doença de Weil/fisiopatologiaRESUMO
Rats exposed to prolonged administration of the NHE-1 inhibitor cariporide showed enhanced activity of the exchanger in cardiac tissue, as assessed by the rise in the steady-state pHi value in the absence of bicarbonate (7.15+/-0.01 in control vs 7.49+/-0.06 and 7.41+/-0.05 in cariporide-treated for 1 or 2 months, respectively, P<0.05). In the presence of bicarbonate, the change in pHi was blunted due to a compensatory activation of acid loading pHi regulatory mechanisms. The enhancement of NHE activity disappeared after 1 week of the inhibitor withdrawal. The kinetic analysis of H+ fluxes after an acid load revealed an increased net H+ efflux (JH+) at any given pHi value and an alkaline shift of the apparent "set-point" of the exchanger (from 7.11+/-0.02 to 7.38+/-0.04,P <0.05) in treated rats. In the presence of the PKC inhibitor chelerythrine, the "set-point" of the exchanger was normalized in the cariporide-treated rats while JH+ at acidic pHi values persisted elevated. Cardiac NHE-1 mRNA levels and protein expression were increased in cariporide-treated rats. In addition to the increased protein expression after the treatment, the normalization of the augmented "set-point" by chelerythrine suggests an increased turnover rate of the units through a PKC dependent pathway. These data demonstrate that long-term treatment with the NHE-1 inhibitor cariporide enhances the antiporter activity in cardiac tissue through an increase of the number and turnover of functional units. This finding deserves further experimental and clinical evaluations to consider whether it would be advisable a gradual withdrawal of prolonged NHE inhibition to avoid an enhanced response when the exchanger is stimulated.