The duodenum is the site of mixing of massive amounts of gastric H + with secreted HCO 3 -, generating CO 2 and H 2 O accompanied by the neutralization of H + . We examined the role of membrane-bound and soluble carbonic anhydrases (CA) by which H + is neutralized, CO 2 is absorbed, and HCO 3 -is secreted. Rat duodena were perfused with solutions of different pH and P CO 2 with or without a cell-permeant CA inhibitor methazolamide (MTZ) or impermeant CA inhibitors. Flow-through pH and P CO 2 electrodes simultaneously measured perfusate and effluent pH and P CO 2 . High CO 2 (34.7 kPa) perfusion increased net CO 2 loss from the perfusate compared with controls (pH 6.4 saline, P CO 2 ≈ 0) accompanied by portal venous (PV) acidification and P CO 2 increase. Impermeant CA inhibitors abolished net perfusate CO 2 loss and increased net HCO 3 -gain, whereas all CA inhibitors inhibited PV acidification and P CO 2 increase. The changes in luminal and PV pH and [CO 2 ] were also inhibited by the Na + -H + exchanger-1 (NHE1) inhibitor dimethylamiloride, but not by the NHE3 inhibitor S3226. Luminal acid decreased total CO 2 output and increased H + loss with PV acidification and P CO 2 increase, all inhibited by all CA inhibitors. During perfusion of a 30% CO 2 buffer, loss of CO 2 from the lumen was CA dependent as was transepithelial transport of perfused 13 CO 2 . H + and CO 2 loss from the perfusate were accompanied by increases of PV H + and tracer CO 2 , but unchanged PV total CO 2 , consistent with CA-dependent transmucosal H + and CO 2 movement. Inhibition of membrane-bound CAs augments the apparent rate of net basal HCO 3 -secretion. Luminal H + traverses the apical membrane as CO 2 , is converted back to cytosolic H + , which is extruded via NHE1. Membrane-bound and cytosolic CAs cooperatively facilitate secretion of HCO 3 -into the lumen and CO 2 diffusion into duodenal mucosa, serving as important acid-base regulators.
The proximal duodenum is exposed to extreme elevations of P(CO(2)) because of the continuous mixture of secreted HCO(3)(-) with gastric acid. These elevations (up to 80 kPa) are likely to place the mucosal cells under severe acid stress. Furthermore, we hypothesized that, unlike most other cells, the principal source of CO(2) for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal P(CO(2)) on duodenal HCO(3)(-) secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO(2) challenge, the duodenum was superfused with a high Pco(2) solution. Intracellular pH (pH(i)) of duodenal epithelial cells was measured by ratio microfluorometry. CO(2) challenge, but not isohydric solutions, strongly increased DBS to approximately two times basal for up to 1 h. Preperfusion of the membrane-permeant carbonic anhydrase inhibitor methazolamide, or continuous exposure with indomethacin, fully inhibited CO(2)-augmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO(2)-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral sodium-HCO(3)(-) cotransporter, also inhibited CO(2)-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO(2) decreased epithelial cell pH(i), followed by an overshoot after removal of the CO(2) solution. We conclude that luminal CO(2) diffused in the duodenal epithelial cells and was converted to H(+) and HCO(3)(-) by carbonic anhydrase. H(+) initially exited the cell, followed by secretion of HCO(3)(-). Secretion was dependent on a functioning basolateral sodium/proton exchanger, a functioning basolateral HCO(3)(-) uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO(2) into HCO(3)(-) and H(+).
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