cAMP-mediated regulation of murine intestinal/pancreatic Na⁺/HCO 3 − cotransporter subtype pNBC1

Abstract

Seidler. cAMP-mediated regulation of murine intestinal/ pancreatic Na ϩ /HCO 3 Ϫ cotransporter subtype pNBC1. Am J Physiol Gastrointest Liver Physiol 284: G37-G45, 2003. First published September 18, 2002 10.1152/ajpgi.00209.2002Basolateral Na ϩ -HCO 3 Ϫ 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 ϩ -HCO 3 Ϫ cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na ϩ -HCO 3 Ϫ transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na ϩ -HCO 3 Ϫ cotransport rates were 5.07 Ϯ 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 ϩ -HCO 3 Ϫ cotransport activation was secondary to secretion-associated changes in HCO 3 Ϫ or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na ϩ -HCO 3 Ϫ transport activity in CFTR Ϫ/Ϫ crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na ϩ -HCO 3 Ϫ transport rates in CFTR Ϫ/Ϫ compared with CFTR ϩ/ϩ crypts but similar Na ϩ -HCO 3 Ϫ cotransport activation by forskolin. These studies establish the existence of an intracellular HCO 3 Ϫ concentration-and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP. bicarbonate secretion; colon; ion transport; chloride secretion; 2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein ALL SEGMENTS OF THE INTESTINAL tract secrete HCO 3 Ϫ , and in all segments, its secretory rate is inhibited both by azetazolamide, which inhibits the hydration of CO 2 , and by stilbene derivatives, which inhibit several classes of anion transporters, among them all currently known isoforms of the recently discovered gene family of the Na ϩ -HCO 3 Ϫ cotransporters. Similar to the pancreas, functional data suggest that HCO 3 Ϫ is imported by Na ϩ -HCO 3 Ϫ cotransport during cAMP-induced HCO 3 Ϫ secretion and that the basolateral uptake and/or intracellular generation of HCO 3 Ϫ is the rate-limiting step for intestinal and pancreatic HCO 3 Ϫ secretion (11,17,18,42). Thus more information about the agonistdependent regulation of Na ϩ -HCO 3 Ϫ contransport rates in the gastrointestinal (GI) tract is desirable to design pharmacological strategies to influence it during both excess intestinal HCO 3 Ϫ loss, as in diarrheal states, and insufficient secretion, as in cystic fibrosis.After the cloning of the renal Na ϩ -HCO 3 Ϫ cotransporter NBC1 in 1997 (29), other isoforms of this gene family were rapidly discovered. These were named either according to the sequence of their cloning [NBC1 (2, 28), NBC2 (15), NBC3 (4), and NBC4 (25)] or named NBC1 for the first electrogen...

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3− secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

“…PTH might also be capable of controlling cellular volume because it could directly stimulate Cl Ϫ efflux through the outward rectifying apical Cl Ϫ channels in cultured rabbit renal proximal tubular cells (53). Since intestinal epithelial cells are equipped with a number of cAMP-dependent transporters, such as cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 in the apical membrane, and NHE1 and electrogenic Na ϩ -HCO 3 Ϫ cotransporter-1 [NBCe1; also known as solute carrier (SLC) 4A4] (4,62) in the basolateral membrane, as well as PTH signaling machinery, including PTH receptor (PTHR) isoform-1 (PTHR1) (37), the direct effect of PTH on intestinal ion transport was anticipated.…”

Differential Cl-and HCO3-mediated anion secretion by different colonic cell types in response to tetromethylpyrazine

“…This suggests that the secretory role of villus/ surface epithelium is distinct from that of crypts which are mainly responsible for Cl -secretion on which colonic fluid secretion depends. The differential expression of NBC, the major basolateral transport mechanism for HCO 3 -entry [23] , in the two human colonic cells lines also supports this contention.…”

“…This was supported by the observation that the TMPinduced response in T84 was sensitive to extracellular Cl -replacement or an inhibitor of basolateral NKCC, bumetanide, while the response in Caco-2 cells was relatively insensitive to these treatments, at least to a much less extent. Instead, the response in Caco-2 cells was sensitive to the removal of extracellular HCO 3 -and an inhibitor of basolateral NBC, which has been shown to mediate HCO 3 -entry into the cells in a number of tissues including the pancreas [21,22] , intestine [23] , kidney [24] and endometrium [25] . Although DIDS is also known to inhibit Cl -/HCO 3 -exchanger (AE), the involvement of AE in this case was unlikely since it was not electrogenic.…”

Localized Na+/H+ Exchanger 1 Expression Protects Ca2+-regulated Adenylyl Cyclases from Changes in Intracellular pH

“…It is expected that other channels or exchangers mediating cellular pH i changes could also give rise to relatively large pH i shifts beneath the plasma membrane. There is also potential for the feedback stimulation of pH regulatory mechanisms as a consequence of raised local cAMP levels (6,43,44) or the functional coupling of G protein-linked receptors to AC and acid extruders via divergent signaling mechanisms (45 Very significantly, however, we were able to prove that NHE1 was localized to the same subcellular compartments (caveolae) that expressed high levels of functional AC8. A similar distribution of NHE1 was also observed in membrane fractions from C6-2B glioma cells.…”