RESUMO
Transepithelial Cl(-) secretion mediated by apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels plays a key role in cyst fluid accumulation in autosomal dominant polycystic kidney disease (ADPKD). The molecular identity of the basolateral transporter(s) responsible for Cl(-) entry in ADPKD cells is unknown, although pharmacological studies suggest that a bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC/BSC) is involved. We investigated the expression of NKCC1, CFTR and anion exchanger type I (AE1) in ADPKD kidneys and cultured ADPKD cells. Immunoblotting and immunoprecipitation detected NKCC1 at ~170 kDa in ADPKD cells and kidney extracts. Immunostaining located NKCC1 in one-third of ADPKD cysts, with a pattern of basolateral reactivity. Staining of serial sections showed that cysts positive for NKCC1 also stained for CFTR. Additional studies demonstrated that AE1 is expressed in ADPKD kidneys, and is located at the basolateral pole of CFTR-positive ADPKD cysts that do not express NKCC1. RT-PCR and sequence analyses confirmed the selective expression of NKCC1 or AEI in cultured ADPKD cells that also express CFTR. The fact that most CFTR-positive ADPKD cysts also express NKCC1 suggests that transepithelial Cl(-) secretion in ADPKD involves molecular mechanisms similar to secretory epithelia. AE1 might be an alternative basolateral pathway for Cl(-) in a minority of cysts.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Neoplasias do Colo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Humanos , Immunoblotting , Fenótipo , Rim Policístico Autossômico Dominante/fisiopatologia , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 2 da Família 12 de Carreador de Soluto , Células Tumorais CultivadasRESUMO
We studied the phosphorylation of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to beta-adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by beta-adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.
