Acid/base transporters in human duodenal enterocytes.

BACKGROUND: Duodenal mucosal bicarbonate secretion serves as a key defensive factor against mucosal injury. The purpose of the present study was to isolate human proximal duodenal enterocytes and identify their inherent acid/base transporters that participate in duodenal alkaline secretion. METHODS: Biopsy specimens were obtained from the duodenal bulb in 18 healthy volunteers. Individual duodenal epithelial cells were isolated by means of a combination of calcium chelation and collagenase. Intracellular pH (pHi) was measured by the pH-sensitive dye BCECF and dynamic fluorescence ratio imaging. RESULTS: Cytologic and histologic examination confirmed that isolated cells were of epithelial origin. In HCO3--free media, pHi recovery after acidification with NH4Cl was amiloride-sensitive and Na+-dependent, indicating the presence of an Na+/H+ exchanger. pHi recovery after acidification was significantly enhanced by the presence of HCO3-, showing the presence of an HCO3--dependent recovery mechanism (that is, a base loader/acid extruder). HCO3--dependent recovery required external Na+ yet was Cl-- and amiloride-insensitive, characteristic of an NaHCO3 cotransporter. In the presence of HCO3-, a Cl--dependent anion exchanger serving as a base extruder was shown, indicative of a Cl-/HCO3- exchanger. CONCLUSIONS: Human duodenal enterocytes contain at least three acid/base transporters: an Na+/H+ exchanger that serves as to extrude acid, an NaHCO3 cotransporter that functions as base loader, and a Cl-/HCO3- exchanger that operates as a base extruder.

Acid-base transport in isolated rabbit duodenal villus and crypt cells.

BACKGROUND: Duodenal mucosal bicarbonate secretion is an important first line of defense against gastric acid. Studies in the ileum indicate that the secretion originates from the crypt cells, whereas villus cells are mainly absorptive. Data on acid/base transporters along the crypt-villus axis in duodenal epithelia are not available. It was our purpose to identify and compare acid/base transporters in isolated mammalian duodenal villus and crypt cells. METHODS: The proximal duodenum of rabbits was excised, and duodenal epithelial cells were isolated in five fractions by a modified calcium chelation technique. Intracellular pH (pHi) was measured with a pH-sensitive dye and dynamic fluorescence ratio imaging. RESULTS: In both villus and crypt cells incubated in Hepes buffer, removal of Na+ or addition of amiloride decreased basal pHi and pHi recovery after intracellular acidification, indicating an Na+/H+ exchanger in both cell types. In both cell types acid extrusion rates in bicarbonate-buffered Ringer's solution were significantly higher than in Hepes buffer. The bicarbonate-dependent acid extruder was unaffected by removal of Cl- or addition of amiloride but was blocked by removal of Na+, indicating the presence of a NaHCO3 cotransporter in both villus and crypt cells. Removal of external Cl induced a reversible increase in pHi (inhibited by H2DIDS) in both villus and crypt cells, indicating a Cl-/HCO3- exchanger in both. CONCLUSIONS: Mammalian duodenal villus and crypt cells have identical acid-base transporters. These findings tend to negate the theory of a functional difference in acid-base transporters between duodenal villus and crypt cells and instead imply alkaline secretion by both cell fractions. However, as these experiments were performed in unpolarized, single cells, additional studies with either membrane vesicles or polarized cells are needed.

Cyclic adenosine-3',5'-monophosphate production is greater in rabbit duodenal crypt than in villus cells.

BACKGROUND: Duodenal surface epithelial cells secrete bicarbonate. Agonists of duodenal alkaline secretion (such as vasoactive intestinal polypeptide (VIP), prostaglandin E2 (PGE(2)), and forskolin) increase intracellular cyclic adenosine-3', 5-monophosphate (cAMP), and cAMP stimulates Cl-HCO(3)- exchange in duodenal brush border membrane vesicles. As intestinal villus and crypt cells differ in function, our aims were to contrast cAMP generation in duodenal villus versus crypt cells in response to VIP, PGE(2), and forskolin. METHODS: Villus and crypt rabbit duodenal enterocytes were isolated by calcium chelation. To prevent the degradation of cAMP in vitro, phosphodiesterase activity was inhibited. cAMP production was quantitated in response to VIP (10(-10)-10(-5)M), PGE(2) (10(-10)-10(-4)M), and forskolin (10(-8)-10(-3)M). RESULTS: In crypt cells cAMP generation was approximately 10-fold greater (P < 0.001) in response to VIP, PGE(2), and forskolin than to villus cells. The relative orders of potency (that is, D(50), VIP > PGE(2) > forskolin) and efficacy (that is, V max, forskolin > VIP and PGE(2)) were similar in villus and crypt cells. CONCLUSION: cAMP production is greater in duodenal crypt than in villus enterocytes at rest and in response to forskolin, VIP, and PGE(2), suggesting that alkaline secretion may differ along the villus-to-crypt axis.