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1.
J Exp Biol ; 215(Pt 7): 1218-30, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22399668

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) is central to anion secretion in both the possum and eutherian small intestine. Here, we investigated its role in the possum proximal colon, which has novel transport properties compared with the eutherian proximal colon. Despite considerable CFTR expression, high doses of the CFTR activator forskolin (EC(50)≈10 µmol l(-1)) were required for a modest, CFTR-dependent increase in short-circuit current (I(sc)) in the proximal colon. Presumably, this is because CFTR is restricted to the apical membrane of a small population of CFTR high expresser (CHE) cells in the surface and upper crypt epithelium. Furthermore, although the forskolin-stimulated I(sc) was dependent on serosal Na(+), Cl(-) and HCO(3)(-), consistent with anion secretion, inhibition of the basolateral Na-K-2Cl(-) (NKCC1) or Na-HCO(3) (pNBCe1) cotransporters did not prevent it. Therefore, although NKCC1 and pNBCe1 are expressed in the colonic epithelium they do not appear to be expressed in CHE cells. At low doses (IC(50)≈1 µmol l(-1)), forskolin also decreased the transepithelial conductance (G(T)) of the colon through inhibition of a 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid-sensitive anion conductance in the basolateral membrane of the CHE cells. This conductance is arranged in series with CFTR in the CHE cells and, therefore, the CHE cells provide a transepithelial Cl(-) conductance for passive Cl(-) absorption across the epithelium. Inhibition of the basolateral Cl(-) conductance of the CHE cells by forskolin will inhibit Na(+) absorption by restricting the movement of its counter-ion Cl(-), assisting in the conversion of the tissue from an absorptive to a secretory state.


Assuntos
Cloretos/metabolismo , Colforsina/farmacologia , Colo/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Trichosurus/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Amilorida/farmacologia , Animais , Western Blotting , Colo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Modelos Biológicos , Nitrobenzoatos/farmacologia , Transporte Proteico/efeitos dos fármacos , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Soluções
2.
J Exp Biol ; 214(Pt 11): 1943-54, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21562182

RESUMO

In eutherian mammals, fluid secretion is essential for intestinal function. This is driven by electrogenic Cl(-) secretion, which involves a NaK2Cl cotransporter (NKCC1) in the enterocyte basolateral membrane and the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, in the possum ileum, NKCC1 expression is low and secretagogues stimulate electrogenic HCO(3)(-) secretion driven by a basolateral NaHCO(3) cotransporter (pNBCe1). Here we investigated whether electrogenic anion secretion occurs in possum duodenum and jejunum and determined the role of CFTR in possum intestinal anion secretion. Prostaglandin E(2) (PGE(2)) and forskolin stimulated a large increase in ileal short-circuit current (I(sc)), consistent with electrogenic HCO(3)(-) secretion, but had little effect on the duodenal and jejunal I(sc). Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH101) inhibited cloned possum CFTR in cultured cells and the PGE(2)-stimulated ileal I(sc), implicating CFTR in ileal HCO(3)(-) secretion. Consistent with this, CFTR is expressed in the apical membrane of ileal crypt and lower villous cells, which also express pNBCe1 in the basolateral membrane. In contrast, duodenal and jejunal CFTR expression is low relative to the ileum. Jejunal pNBCe1 expression is also low, whereas duodenal and ileal pNBCe1 expression are comparable. All regions have low NKCC1 expression. These results indicate that cAMP-dependent electrogenic Cl(-) secretion does not occur in the possum small intestine because of the absence of CFTR and NKCC1. Furthermore, CFTR functions as the apical anion conductance associated with HCO(3)(-) secretion and its distribution limits electrogenic HCO(3)(-) secretion to the ileum.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Íleo/metabolismo , Trichosurus/metabolismo , Animais , Ânions/metabolismo , Duodeno/metabolismo , Jejuno/metabolismo
3.
J Exp Biol ; 212(Pt 16): 2645-55, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19648410

RESUMO

Fluid secretion is essential for intestinal function and, in eutherian mammals, is driven by electrogenic Cl(-) transport, which is dependent upon a bumetanide-sensitive, basolateral Na(+)/K(+)/2 Cl(-) cotransporter, NKCC1. However, ileal secretion in the brushtail possum, a marsupial, involves a fundamentally different process, since NKCC1 expression is low in this tissue and the secretagogue-induced short circuit current (I(sc)) is insensitive to bumetanide. In view of these differences we have investigated the basis of the secretory response of the possum ileum. In the Ussing chamber the secretory I(sc) is independent of Cl(-) but dependent upon Na(+) and serosal HCO(3)(-)/CO(2), suggesting that secretagogues stimulate electrogenic HCO(3)(-) secretion. In agreement with this, serosal DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonate; 1 mmol l(-1)) inhibited the secretory response. However, acetazolamide (1 mmol l(-1)) and serosal amiloride (1 mmol l(-1)) had little effect, indicating that HCO(3)(-) secretion is driven by HCO(3)(-) transport from the serosal solution into the cell, rather than hydration of CO(2) by carbonic anhydrase. Consistent with this the pancreatic variant of the electrogenic Na(+)/HCO(3)(-) cotransporter (pNBC) is highly expressed in the ileal epithelium and is located in the basolateral membrane of the epithelial cells, predominantly in the mid region of the villi, with lower levels of expression in the crypts and no expression in the villous tips. We conclude that the secretory response of the possum ileum involves electrogenic HCO(3)(-) secretion driven by a basolateral pNBC and that the ileal HCO(3)(-) secretion is associated with a specialised function of the possum ileum, most probably related to hindgut fermentation.


Assuntos
Bicarbonatos/metabolismo , Íleo/fisiologia , Simportadores de Sódio-Bicarbonato/fisiologia , Sódio/metabolismo , Trichosurus/fisiologia , Animais , Membrana Celular/fisiologia , DNA Complementar/genética , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Rim/fisiologia , Camundongos , Camundongos Endogâmicos CFTR/genética , Camundongos Knockout , Pâncreas/fisiologia , Isoformas de Proteínas/genética , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 2 da Família 12 de Carreador de Soluto
4.
J Comp Physiol B ; 179(8): 997-1010, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19565248

RESUMO

The colon of the brushtail possum does not have an electrogenic secretory response. Given the functional significance of electrogenic Cl(-) secretion in the intestine of eutherian mammals, we have investigated the secretory response in the small intestine of this marsupial. In the Ussing chamber cAMP-dependent secretagogues stimulated a sustained increase in ileal short-circuit current (Isc), whereas Ca(2+)-dependent secretagogues induced a transient increase. Both the responses were inhibited by mucosal addition of the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 mciromol l(-1)), consistent with an anion secretory response. However, the responses were not inhibited by serosal bumetanide (10 mciromol l(-1)) and were independent of bath Cl(-), indicating that the stimulated ileal Isc does not involve electrogenic Cl(-) secretion driven by the NaK2Cl cotransporter, NKCC1. Consistent with this, there were low levels of NKCC1 expression in the ileal epithelium. In particular, NKCC1 expression in the ileal crypt cells was comparable to that of the villous cells. This differs from eutherian mammals where high levels of NKCC1 expression in the ileal crypt cells are associated with their role in Cl(-) secretion. The cAMP- and Ca(2+)-dependent secretory responses were inhibited by the removal of HCO(3) (-) suggesting that these responses were due to electrogenic HCO(3) (-) secretion. We conclude that the ileum of the possum does not secrete Cl(-) due to low levels of NKCC1 expression. It does however appear to secrete HCO(3) (-). These results are further significant examples of differences in the transport function of the possum intestinal epithelium compared with eutherian mammals.


Assuntos
Bicarbonatos/metabolismo , Cloretos/metabolismo , Íleo/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Trichosurus/metabolismo , Animais , Feminino , Íleo/citologia , Hibridização In Situ , Técnicas In Vitro , Mucosa Intestinal/citologia , Masculino , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Via Secretória/efeitos dos fármacos , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 2 da Família 12 de Carreador de Soluto , Especificidade da Espécie
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