RESUMO
Primary cultures of immunodissected rabbit connecting tubule and cortical collecting duct cells were used to investigate the effect of apical Na+ entry rate on aldosterone-induced transepithelial Na+ transport, which was measured as benzamil-sensitive short-circuit current (I(sc)). Stimulation of the apical Na+ entry, by long-term short-circuiting of the monolayers, suppressed the aldosterone-stimulated benzamil-sensitive I(sc) from 320 +/- 49 to 117 +/- 14%, whereas in the presence of benzamil this inhibitory effect was not observed (335 +/- 74%). Immunoprecipitation of [(35)S]methionine-labeled beta-rabbit epithelial Na+ channel (rbENaC) revealed that the effects of modulation of apical Na+ entry on transepithelial Na+ transport are exactly mirrored by beta-rbENaC protein levels, because short-circuiting the monolayers decreased aldosterone-induced beta-rbENaC protein synthesis from 310 +/- 51 to 56 +/- 17%. Exposure to benzamil doubled the beta-rbENaC protein level to 281 +/- 68% in control cells but had no significant effect on aldosterone-stimulated beta-rbENaC levels (282 +/- 68%). In conclusion, stimulation of apical Na+ entry suppresses the aldosterone-induced increase in transepithelial Na+ transport. This negative-feedback inhibition is reflected in a decrease in beta-rbENaC synthesis or in an increase in beta-rbENaC degradation.
Assuntos
Aldosterona/farmacologia , Túbulos Renais Coletores/metabolismo , Canais de Sódio/biossíntese , Sódio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Polaridade Celular/fisiologia , Células Cultivadas , Canais Epiteliais de Sódio , Espaço Extracelular/metabolismo , Retroalimentação/fisiologia , Túbulos Renais Coletores/citologia , Coelhos , Radioisótopos de EnxofreRESUMO
The epithelial Na+ channel (ENaC) functions as the rate-limiting factor in aldosterone-regulated transcellular Na+ transport. In the study described here, the effect of aldosterone on ENaC mRNA levels, protein synthesis and benzamil-sensitive Na+ transport was investigated using primary cultures of immunodissected rabbit kidney connecting tubule and cortical collecting duct cells (CNT and CCD, respectively). After a lag time of 3 h, aldosterone caused transepithelial Na+ transport to increase, reaching maximal level of 260+/-44% after 16 h of incubation. The alpha, beta and gamma rabbit ENaC (rbENaC) mRNA levels, measured by semi-quantitative reverse transcriptase-polymerase chain reaction, were not changed by aldosterone during the first 3 h, but a twofold increase was apparent after 6 h; levels remained elevated for up to 16 h of incubation. Immunoprecipitation of [35S]methionine-labeled rbENaC revealed a rise in protein levels of the alpha and beta subunits, but the protein level of the gamma subunit remained constant. In conclusion, our data suggest that in rabbit CNT and CCD the early increase in Na+ transport caused by aldosterone is due to the activation or insertion of existing Na+ channels into the apical membrane, and that the late response is mediated by increased synthesis of the alpha and beta rbENaC subunits.
Assuntos
Aldosterona/farmacologia , Amilorida/farmacologia , Córtex Renal/metabolismo , Canais de Sódio/fisiologia , Amilorida/análogos & derivados , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Condutividade Elétrica , Epitélio/metabolismo , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Córtex Renal/química , Cinética , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , Canais de Sódio/análise , Canais de Sódio/genética , Distribuição TecidualRESUMO
Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Ibeta or a membrane-bound cGK II/Ibeta chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.