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Characterization of the ileal Na+/bile salt co-transporter in brush border membrane vesicles and functional expression in Xenopus laevis oocytes.

ABSTRACT: The Na+/bile salt co-transporter of the pig ileal brush border membrane has been expressed in Xenopus laevis oocytes. Injection of pig ileal poly (A)+ RNA into oocytes resulted in the functional expression of an Na(+)-gradient-stimulated taurocholate uptake within 2-5 days. The expressed Na(+)-dependent taurocholate uptake exhibited saturation kinetics (apparent Km of 48 microM), and displayed similar competitive substrate inhibition by taurodeoxycholate as the native brush border Na+/bile salt co-transporter studied in pig ileal brush border membrane vesicles. Interestingly, injection of pig proximal and mid intestinal poly (A)+ RNA into oocytes also resulted in the expression of the Na+/bile salt co-transporter, though the Na(+)-dependent transport of bile salts does not occur in brush border membrane vesicles (BBMV) isolated from pig proximal and mid intestine. This suggests that the mRNA coding for the co-transporter is present in the enterocytes lining the whole length of the small intestine, but that the function is only expressed in the brush border of the distal small intestine. The transport of D-glucose into BBMV, and the transport of methyl-alpha-D-glucopyranoside (a non-metabolizable hexose derivative) into oocytes were used throughout the study as methods of confirming the integrity of vesicles and oocytes.

SUBMITTER: Mullins JG

PROVIDER: S-EPMC1132864 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

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Project description:In ileal Na+ absorptive cells, carbachol inhibits NaCl absorption and its component brush-border Na+/H+ exchanger, acting via basolateral membrane (BLM) receptors. This carbachol effect involves brush-border but not BLM protein kinase C. In the present work we describe another asymmetric aspect of signal transduction in these epithelial cells, this time involving phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC). Thirty seconds and 1 min after carbachol treatment, brush-border PIP2-specific PLC activity increased, returning to control levels by 2.5 min. Involvement of brush-border tyrosine kinase(s) in this effect was suggested by inhibition of the carbachol effect on NaCl absorption by the tyrosine kinase inhibitor genistein, added to the mucosal but not the serosal surface. Luminal genistein pretreatment also prevented the carbachol-induced increase in brush-border PLC activity. In contrast, carbachol exposure did not change the BLM PIP2-specific PLC activity. Western analysis and immunoprecipitation demonstrated that PLC-gamma 1 is present in the brush border and that carbachol increases the PLC-gamma 1 amount in the brush border. Both the brush border and BLM contain PLC-beta 3 and a small amount of PLC-delta 1 but no PLC-beta 1, whereas BLM lacks detectable PLC-gamma 1. No change in PLC-beta 3 or PLC-delta 1 amount in the brush border occurred with carbachol exposure. No change in tyrosine phosphorylation of brush-border PLC-gamma 1 occurred with carbachol treatment. The Ca2+ ionophore A23187 did not alter PIP2-specific PLC activity in either the brush border or the BLM. These studies demonstrate that carbachol but not Ca2+ ionophore effects on brush-border NaCl absorption are associated with increases in brush-border but not BLM PIP2-specific PLC activity and in the amount of brush-border PLC-gamma 1, and involve tyrosine phosphorylation. This asymmetric aspect of epithelial signal transduction, together with the previous demonstration of localization of high-sensitivity IP3 stores to the apical membrane area in intestinal epithelial cells, shows that different aspects of signal transduction occur at the apical and basolateral membranes in epithelial and requires studies in both domains to define mechanisms of intracellular signalling.

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Characterization of the ileal Na+/bile salt co-transporter in brush border membrane vesicles and functional expression in Xenopus laevis oocytes.

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