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Articles in PresS, published online ahead of print September 18, 2002
Am J Physiol Gastrointest Liver Physiol, 10.1152/ajpgi.00209.2002
Submitted on June 3, 2002
Accepted on September 10, 2002
1 Department of Internal Medicine, University of Tubingen, Tubingen, Germany
2 In Situ Hybridization Core Facility, Beth Israel Deaconess Medical Center, Boston, MA, USA
3 Department of Physiology, University of Cambridge, Cambridge, United Kingdom
4 Cardiff School of Biosciences, Cardiff, United Kingdom
* To whom correspondence should be addressed. E-mail: ursula.seidler{at}uni-tuebingen.de.
Basolateral Na+HCO3- cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity and regulation by cAMP of the Na+HCO3- cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na+HCO3- transport rates were measured fluorometrically in 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF)-loaded crypts, and mRNA expression levels and localization determined by semiquantitative PCR and in situ hybridization. Acid-activated Na+HCO3- cotransport rates were 5.07±0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na+HCO3- cotransport activation was secondary to secretion-associated changes in HCO3- or cell volume, we measured potential forskolin-induced changes in pHi, and assessed Na+HCO3- transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na+HCO3- transport rates in CFTR -/- compared to CFTR +/+ crypts, but similar Na+HCO3- cotransport activation by forskolin. These studies establish the existence of a [HCO3-]i- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.
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