Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.

Acute adaptive cellular base uptake in rat duodenal epithelium.

We studied the role of duodenal cellular ion transport in epithelial defense mechanisms in response to rapid shifts of luminal pH. We used in vivo microscopy to measure duodenal epithelial cell intracellular pH (pH(i)), mucus gel thickness, blood flow, and HCO secretion in anesthetized rats with or without the Na(+)/H(+) exchange inhibitor 5-(N,N-dimethyl)-amiloride (DMA) or the anion transport inhibitor DIDS. During acid perfusion pH(i) decreased, whereas mucus gel thickness and blood flow increased, with pH(i) increasing to over baseline (overshoot) and blood flow and gel thickness returning to basal levels during subsequent neutral solution perfusion. During a second brief acid challenge, pH(i) decrease was lessened (adaptation). These are best explained by augmented cellular HCO uptake in response to perfused acid. DIDS, but not DMA, abolished the overshoot and pH(i) adaptation and decreased acid-enhanced HCO secretion. In perfused duodenum, effluent total CO(2) output was not increased by acid perfusion, despite a massive increase of titratable alkalinity, consistent with substantial acid back diffusion and modest CO(2) back diffusion during acid perfusions. Rapid shifts of luminal pH increased duodenal epithelial buffering power, which protected the cells from perfused acid, presumably by activation of Na(+)-HCO cotransport. This adaptation may be a novel, important, and early duodenal protective mechanism against rapid physiological shifts of luminal acidity.

Sensory pathways and cyclooxygenase regulate mucus gel thickness in rat duodenum.

We previously showed that the duodenal hyperemic response to acid occurs through activation of capsaicin-sensitive afferent nerves with subsequent release of vasodilatory substances such as calcitonin gene-related peptide (CGRP) and nitric oxide. We then tested the hypothesis that similar factors regulate duodenal mucus gel thickness. Gel thickness was optically measured using in vivo microscopy in anesthetized rats. Duodenal mucosae were superfused with pH 7.0 buffer with vanilloid receptor agonist capsaicin, bradykinin, or PGE(2) injection or were challenged with pH 2.2 solution, with or without the vanilloid antagonist capsazepine, human CGRP-(8-37), N(G)-nitro-L-arginine methyl ester, and indomethacin. Other rats underwent sensory ablation with high-dose capsaicin pretreatment. Acid, bradykinin, capsaicin, and PGE(2) all quickly thickened the gel. Antagonism of vanilloid and CGRP receptors, inhibition of nitric oxide synthase, and sensory deafferentation delayed gel thickening, suggesting that the capsaicin pathway mediated the initial burst of mucus secretion that thickened the gel. Indomethacin abolished gel thickening due to acid, bradykinin, and capsaicin. Inhibition of gel thickening by indomethacin in response to multiple agonists suggests that cyclooxygenase activity is essential for duodenal gel thickness regulation. Duodenal afferent neural pathways play an important role in the modulation of cyclooxygenase-mediated physiological control of gel thickness.

Dynamic regulation of mucus gel thickness in rat duodenum.

We examined the dynamic regulation of mucus gel thickness (MGT) in vivo in rat duodenum in response to luminal acid, cyclooxygenase (COX) inhibition, and exogenous PGE(2). An in vivo microscopic technique was used to measure MGT with fluorescent microspheres in urethan-anesthetized rats. Duodenal mucosa was topically superfused with pH 7.0 or pH 2.2 solutions with or without PGE(2) and indomethacin treatments. Glycoprotein concentration of duodenal loop perfusates was measured with periodic acid/Schiff (PAS) or Alcian blue (AB) staining. MGT and perfusate glycoprotein concentration were stable during a 35-min perfusion with pH 7.0 solution. Acid exposure increased MGT and PAS- and AB-positive perfusate glycoprotein concentrations. Indomethacin pretreatment increased both PAS- and AB-positive perfusate glycoprotein at baseline; subsequent acid superfusion decreased perfusate glycoproteins and gel thickness. PGE(2) (1 mg/kg iv) simultaneously increased MGT and PAS-positive perfusate glycoprotein concentrations followed by a transient increase in AB-positive glycoprotein concentration, suggesting contributions from goblet cells and Brunner's glands. Parallel changes in MGT and perfusate glycoprotein concentration in response to luminal acid and PGE(2) suggest that rapid MGT variations reflect alterations in the balance between mucus secretion and exudation, which in turn are regulated by a COX-related pathway. Luminal acid and PGE(2) augment mucus secretion from goblet cells and Brunner's glands.

Acid-sensing pathways of rat duodenum.

We tested the hypothesis that the duodenal hyperemic response to acid occurs through activation of capsaicin-sensitive afferent nerves with subsequent release of vasodilatory substances such as calcitonin gene-related peptide (CGRP) and nitric oxide (NO). Laser-Doppler flowmetry was used to measure duodenal blood flow in urethan-anesthetized rats. Duodenal mucosa was superfused with pH 7. 0 buffer with capsaicin or bradykinin or was acid challenged with pH 2.2 solution, with or without vanilloid receptor antagonists, a CGRP receptor antagonist, an NO synthase (NOS) inhibitor, or a cyclooxygenase inhibitor. The selective vanilloid receptor antagonist capsazepine (CPZ) dose dependently inhibited the hyperemic response to acid and capsaicin but did not affect bradykinin-induced hyperemia. Ruthenium red was less inhibitory than capsazepine. Selective ablation of capsaicin-sensitive nerves, CGRP-(8-37), and N(G)-nitro-L-arginine methyl ester inhibited acid-induced hyperemia, but indomethacin did not. We conclude that luminal acid, but not bradykinin, stimulates CPZ-sensitive receptors on capsaicin-sensitive afferent nerves of rat duodenum. Activation of these receptors produces vasodilation via the CGRP-NO pathway but not via the cyclooxygenase pathway. Acid appears to be the endogenous ligand for duodenal vanilloid receptors.