Carbachol increases Na+-HCO3− cotransport activity in murine colonic crypts in a M3−, Ca2+/calmodulin-, and PKC-dependent manner

Abstract

The Na+-HCO3− cotransporter (NBC) mediates HCO3− import into the colonocyte via its pNBC1 isoform. Whereas renal kNBC1 is inhibited by increased cAMP levels, pNBC1 is stimulated. Cholinergic stimulation activates renal NBC, but the effect on intestinal NBC is unknown. Therefore, crypts were isolated from the murine proximal colon by Ca2+ chelation and loaded with the pH-sensitive dye 2′,7′-bis-carboxyethyl-5,6-carboxyfluorescein. Na+-HCO3− cotransport activity was calculated from the dimethylamiloride-insensitive (500 μM) intracellular pH recovery from an acid load in the presence of CO2-HCO3− and the intracellular buffering capacity. Carbachol strongly increased Na+-HCO3− cotransport activity compared with control rates. Ca2+ chelation with BAPTA-AM, blockade of the M3 subtype of muscarinergic receptors with 4-diphenylacetoxy- N-methylpiperidine methiodide, and inhibition of Ca2+/calmodulin kinase II with KN-62 all caused significant inhibition of the carbachol-induced NBC activity increase. Furthermore, PKC inhibition with Gö-6976 and Gö-6850 significantly reduced the carbachol effect, which may be related to the unique NH2-terminal consensus site for PKC-dependent phosphorylation of pNBC1. We conclude that NBC in the murine colon is thus activated by carbachol, consistent with its presumed function as an anion uptake pathway during intestinal anion secretion, but that the signal transductions pathways are distinct from those involved in the cholinergic activation of renal NBC1.