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Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (kH+) was determined by addition of vesicles (pHi = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 x 10(-3) (gastric parietal cell stimulation-associated vesicles; SAV) and 5 x 10(-3) (duodenal brush-border membrane vesicles; dBBMV) to 11 x 10(-3) sec-1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mM, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in kH+ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na+/H+ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pHi less than pHo) 22Na+ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K(+)-diffusion potential ([K+]i much much greater than [K+]o). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K(+)-ionophore valinomycin, indicating that the native K+ permeability is rate limiting. In the presence of valinomycin, the K(+)-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K+ permeability. Valinomycin was without effect, but the K+/H+ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The rabbit gastric gland model was used to study the nature of the muscarinic cholinergic and gastrin responses of parietal cells. Carbachol (100 microM) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist pirenzepine with an IC50 of 13 microM; by the M2 antagonist 11,2-(diethylamino)methyl-1-piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4-benzodiazepin-6-one (AF-DX 116) with an IC50 of 110 microM; and by the M3 antagonist diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP) with an IC50 of 35nM. The three antagonists displayed similar IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H(+)-H(+)-ATPase. Intracellular calcium levels wer measured in gastric glands loaded with FURA2. Carbachol was shown both to release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or metabolism. However, the rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM. Image analysis confirmed that the effect of carbachol and of the antagonists on intracellular calcium was occurring in the partial cell. In particular, the high-affinity inhibition of calcium release by 4-DAMP occurs in the parietal cell. Accordingly, it appears that the secretory receptor of the parietal cell is of the M3 type, and acid secretion depends on the entry of calcium rather than on calcium release from intracellular stores. In parallel experiments gastrin (G-17-sulfated) produced a dose-dependent increase in intracellular calcium (EC50, 0.14 +/- 0.013 microM). No stimulation of acid secretion was observed, but pepsinogen secretion was stimulated dose-dependently (EC50 = 1.17 +/- 0.21 microM).

The passive proton permeability (Pnet) of apical membrane vesicles from rabbit parietal cells (gastric) and duodenal and renal cortical brush-border membranes (BBM) was determined by acridine orange fluorescence quenching. Values of Pnet were found to be gastric (4 x 10(-4) cm/s) less than duodenal (10(-3) cm/s) much less than renal (10(-2) cm/s). Arrhenius plots of the temperature profile of proton permeation of gastric vesicles was linear, whereas that of duodenal BBM displayed a discontinuity at 30-33 degrees C. Alcohols (octyl, benzyl, ethyl) increased Pnet in a concentration-dependent manner, with efficacy related to their oil-water partition coefficients. In a parallel series of experiments, structural parameters of the vesicle membrane lipids (fluidity) were monitored from both the steady-state and time-resolved fluorescence anisotropy of diphenylhexatriene. Fluidity of the membranes was unrelated to Pnet (renal congruent to duodenal less than gastric). Gastric vesicles demonstrated a linear Arrhenius plot of temperature dependence for fluidity, whereas duodenal BBM demonstrated a discontinuity. Membrane fluidity of gastric and duodenal vesicles was increased by alcohols, with the same potency as for Pnet, and these two variables were significantly correlated after perturbation with alcohols. Thus the fluidity of the lipid bilayer is not the major determinant of Pnet, but alteration of its structural parameters, as reflected by fluidity, produces parallel changes in Pnet.

This paper investigates acid disposal by gastric and duodenal mucosa with particular reference to the mechanisms of acid-stimulated luminal alkalinization in the duodenum. The bulk of solute flux across duodenal epithelium occurs by paracellular permeation, and passive diffusion of HCO3 via shunt pathways contributes substantially to luminal alkalinization by this tissue in vitro. Effects on epithelial permeability of two treatments which stimulate mucosal alkaline secretion (PGE2 and low luminal pH) were studied in vivo by measuring transmucosal fluxes of radiolabeled urea (mol wt 76) and inulin (mol wt approximately 5000). Rats were anesthetized and rates of alkalinization in segments of distal duodenum perfused with saline were monitored by continuous titration. PGE2 (10 microM, topically) increased alkaline secretion from 1.63 +/- 0.34 to 3.07 +/- 0.54 microeq/cm/hr (mean +/- SEM, N = 5). Luminal acid exposure (10 mM HCl for 10 min) increased alkalinization rate from 1.54 +/- 0.43 to 2.69 +/- 0.76; subsequent addition of PGE2 induced a further rise to 3.27 +/- 0.54. Recovery of inulin in the luminal perfusate following intravenous injection was less than 10% of that of urea. Topical PGE2 had little or no effect on recovery of either marker. Luminal acidification increased the rate of appearance of urea by 130 +/- 30% (P less than 0.01, N = 6); recovery of inulin rose slightly but did not achieve statistical significance compared with control. Thus stimulation of mucosal alkaline secretion by luminal PGE2 or acid are associated with differential effects on mucosal permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Duodenal and jejunal brush border membrane vesicle integrity was studied after in vitro treatment of rabbit tissue with ethyl, benzyl or octyl alcohol. The effects of the alcohols on gastric parietal cell apical and microsomal membrane vesicle integrity was also studied. Membrane vesicle integrity was determined from the enclosed volume of the vesicle preparations, measured as [14C]glucose space at equilibrium. Exposure of vesicles to the three alcohols caused concentration dependent decreases in enclosed volume. The rank order of potency of the alcohol was octyl greater than benzyl greater than ethyl. Concentrations greater than or equal to 10 mM benzyl alcohol significantly reduced the enclosed volume of duodenal or jejunal vesicles; jejunal vesicles were disrupted by 625 mM ethanol, whereas 2 M ethanol was required to disrupt the duodenal vesicles. Gastric apical membrane integrity was reduced with 0.25 M ethanol, the vesicles being approximately an order of magnitude more sensitive to ethanol than gross estimates of gastric mucosal damage, but 1 M ethanol was required to significantly damage gastric microsomes. All concentrations of benzyl or octyl alcohol tested (greater than or equal to 5 mM) reduced the enclosed volume of both gastric apical membrane vesicles and gastric microsomes. As determined by shrink-swell techniques, benzyl alcohol permeated duodenal vesicles at a faster rate than NH4Cl (apparent rate constant of 9.89 (0.71) X 10(-3)s-1 compared with 4.48 (0.23) X 10(-3)s-1). Therefore, reductions in enclosed volume in response to alcohol treatment could not be explained by alcohol induced osmotic shrinkage. The enclosed volume of the vesicles after alcohol treatment was negatively correlated with membrane fluidity suggesting a common causal effect, the increased fluidity increasing membrane fragility. Duodenal vesicles were more resistant to disruption by the alcohols compared with gastric and jejunal vesicles.