Faculty Bibliography

To determine the role of carbonic anhydrase (CA) in colonic electrolyte transport, we studied Car-2(0) mice, mutants deficient in cytosolic CA II. Ion fluxes were measured under short-circuit conditions in an Ussing chamber. CA was analyzed by assay and Western blots. In Car-2(0) mouse colonic mucosa, total CA activity was reduced 80% and cytosolic CA I and membrane-bound CA IV activities were not increased. Western blots confirmed the absence of CA II in Car-2(0) mice. Normal mouse distal colon exhibited net Na(+) and Cl(-) absorption, a serosa-positive PD, and was specifically sensitive to pH. Decrease in pH stimulated active Na(+) and Cl(-) absorption whether it was caused by increasing solution PCO(2), reducing HCO(-)(3) concentration, or reducing pH in CO(2)/HCO(-)(3)-free HEPES-Ringer solution. Membrane-permeant methazolamide, but not impermeant benzolamide, at 0.1 mM prevented the effects of pH. Car-2(0) mice exhibited similar basal transport rates and responses to pH and CA inhibitors. We conclude that basal and pH-stimulated colonic electrolyte absorption in mice requires CA I. CA II and IV may have accessory roles

Short chain fatty acids (SCFA) prevent and reverse cyclic 3',5'-adenosine monophosphate (cAMP) but not Ca(2+)-mediated Cl- secretion. Mucosal [HCO3-]i has an opposite effect on these secretagogues. We examined whether SCFA and [HCO3-]i affect cyclic 3',5'-guanosine monophosphate (cGMP)-induced secretion. Stripped segments of male Sprague-Dawley rat (Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Using chambers, and pHi and [HCO3-]i were determined. Mucosal [cGMP] was measured in proximal colon. In T84 cells, the increase in Cl- secretion (measured as Isc) induced by mucosal 0.25 microM Escherichia coli heat-stable enterotoxin (STa) was prevented/reversed by bilateral 50 mM Na+ butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%). In proximal colon in HCO3- Ringer, basal Cl- secretion was not affected by [HCO3-]i or 25 mM butyrate. Mucosal 0.25 microM STa decreased net Na+ and Cl- absorption. Bilateral but not mucosal 25 mM SCFA reversed STa-induced effects on Na+ absorption and Cl- secretion. Bilateral and mucosal 25 mM SCFA but not [HCO3-]i prevented STa-induced Cl- secretion and increases in mucosal [cGMP]. STa did not produce Cl- secretion in distal colon. It was concluded that SCFA but not [HCO3-]i can prevent and reverse cGMP-induced colonic Cl- secretion

Short-chain fatty acid (SCFA) transport across the colon may occur by nonionic diffusion and/or via apical membrane SCFA-/HCO3- exchange. To examine the relative importance of these processes, stripped segments of rat (Ratus ratus) proximal and distal colon were studied in Ussing chambers, and the unidirectional fluxes of radiolabeled SCFA butyrate, propionate, or weakly metabolized isobutyrate were measured. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or 1 or 5 mM HCO3- Ringer, decreases in mucosal pH stimulated mucosal-to-serosal flux (Jm-->s) of all SCFA, decreases in serosal pH stimulated serosal-to-mucosal flux (Js-->m), and bilateral pH decreases stimulated both fluxes equally. These effects were observed whether the SCFA was present on one or both sides of the tissue, in both proximal and distal colon, in the absence of luminal Na+, and in the presence of either luminal or serosal ouabain. Changes in intracellular pH or intracellular [HCO3-] did not account for the effects of extracellular pH. Luminal Cl- removal, to evaluate the role of apical membrane Cl-/SCFA- exchange, had no effect on Jm-->s but decreased Js-->m 32% at pH 6.5 and 22% at 7.2. Increasing SCFA concentration from 1 to 100 mM, at pH 6.4 or 7.4, caused a linear increase in Jm-->s. We conclude that SCFA are mainly transported across the rat colon by nonionic diffusion

Increasing intracellular bicarbonate concentration ([HCO3-]i) inhibits calcium-mediated Cl- secretion in rat distal colon and T84 cells. We investigated the effect of [HCO3-]i on Cl- secretion in rat ileum. Segments of intact ileum from Sprague-Dawley rats were studied in Ussing chambers and villus and crypt intracellular pH and [HCO3-]i were determined using BCECF. A range of crypt and villus [HCO3-]i from 0 to 31 mM was obtained by varying Ringer's composition. Basal serosal-to-mucosal Cl- flux (JsmCl) averaged 8.5 +/- 0.2 mu eq.h-1.cm-2 and was unaffected by changing [HCO3-]i or serosal bumetanide. Carbachol increased JsmCl by 3.9 +/- 0.5 mu eq.h-1.cm-2 at [HCO3-]i = 0 mM but only by 1.0 +/- 0.3 mu eq.h-1.cm-2 at high crypt and villus [HCO3-]i. Dibutyryl-cAMP increased JsmCl by 2.5 +/- 0.2 mu eq.h-1.cm-2 at all [HCO3-]i. Carbachol and db-cAMP showed mutual antagonism at low [HCO3-]i and near-additivity at high [HCO3-]i. We conclude that like rat colon and T84 cells, calcium-mediated but not cAMP-mediated Cl- secretion in the ileum is inhibited by increasing [HCO3-]i. Mutual antagonism between carbachol and db-cAMP at low [HCO3-]i was present in ileum and distal colon but not in T84 cells

Butyrate stimulates salt absorption in mammalian colon. We examined whether butyrate also affects Cl- secretion. Mucosal segments of distal colon of male Sprague-Dawley rats and T84 cells were studied in Ussing chambers. In control colon, 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) increased short-circuit current (Isc) and serosal-to-mucosal Cl- flux (JsmCl) by 3.2 +/- 0.8 and 2.9 +/- 0.8 mueq.cm-2.h-1, respectively. Mucosal or serosal 25 mM butyrate prevented DBcAMP-induced increases in Isc and JsmCl. Four and eight millimolar butyrate caused half-maximal inhibition of the increases in JsmCl and Isc, respectively. Butyrate also inhibited basal JsmCl (by 2.0 +/- 0.4 mueq.cm-2.h-1) but not carbachol-mediated Cl- secretion. The relative inhibitory potency at 25 mM of other short-chain fatty acids (SCFA) paralleled their degree of cellular metabolism: butyrate > acetate = propionate > isobutyrate. At 25 mM, all SCFA reduced mucosal intracellular pH (pHi) transiently by 0.1 pH unit. In intact T84 cells, 50 mM butyrate inhibited the DBcAMP-induced rise in Isc by 55%. In T84 cells with nystatin-permeabilized basolateral membranes, butyrate inhibited the increase in Isc by 82%. We conclude that butyrate inhibits basal and cAMP-mediated Cl- secretion by a mechanism independent of pHi, possibly located at the apical membrane

Only minimal quantities of ingested and normally secreted solutes and water are excreted in the stool. This near 100% bioavailability means that the diet and kidneys are relatively more important determinants of solute, water and acid-base balance than the intestine. Intestinal bioavailability is based on excess transport capacity under normal conditions and the ability to adapt to altered or abnormal conditions. Indeed, the regulatory system of the intestine is as complex, segmented and multi factorial as in the kidney. Alterations in the rate and intestinal site of absorption reflect this regulation, and the diagnosis and treatment of various clinical abnormalities depend on the integrity of intestinal absorptive processes. However, the basis for this regulation an bioavailability are uncertain. Perhaps they had survival value for mammals, a phylogenic class that faced the twin threats of intestinal pathogens and shortages of solutes and water

Intracellular acidification by stimuli rather than CO2 fails to stimulate colonic apical Na/H ex-change and Na absorption. We examined whether Na absorption could be stimulated in the absence of changes in cytoplasmic pH (pHi). Distal colon of male Sprague-Dawley rats was used for pHi measurements with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and for flux measurements in Ussing chambers. In 21 mM HCO3-Ringer, increasing PCO2 from 20 to 70 mmHg decreased pHi from 7.51 to 7.03 and increased net Na flux (JnetNa) from 4.2 +/- 0.4 to 6.8 +/- 0.6 mu eq.cm-2.h-1. Similar increases in JnetNa occurred in the absence of mucosal CI and in the presence of phalloidin to inhibit microfilaments or penzolamide to inhibit membrane-bound carbonic anhydrase. sohydric increases in Pco2 did not alter pHi but stimulated JnetNa from 5.1 +/- 0.6 to 7.2 +/- 0.8 mu eq.cm-2.h-1. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased pHi from 7.45 to 7.35 but did not stimulate JnetNa. Butyrate (25 mM) decreased pHi from 7.15 to 7.02 with recovery to baseline within 6 min; however, JnetNa increased by 2.2 mu eq.cm-2.h-1 for 60 min. We conclude that apical Na/H exchange activity is unresponsive to changes in bulk pHi and is independent of Cl/HCO3 exchange, microfilaments, and membrane-bound carbonic anhydrase. The presence of an H-tight, CO2, and butyrate-permeable subapical domain is postulated