Previous studies have shown that, immediately after the addition of galactose or alanine to the solution bathing the mucosal surface of Necturus small intestine, there is a rapid depolarization of the electrical potential difference across the mucosal membrane (qmC). This is followed by a repolarization of ims that is paralleled by an increase in the ratio of the effective resistance of the mucosal membrane to that of the basolateral membrane (rm/rs); the latter was shown to be, at least in part, due to a marked increase in the conductance of the basolateral membrane. We now report the follow- results in a depolarization of qmc but no subsequent repolarization of And or increase in rm/rs; however, qAmc repolarizes and rm/rs increases when Ba21 is subsequently removed from the serosal bathing solution. We conclude that (i) the basolateral membrane normally possesses a Ba2+-inhibitable K conductance, which appears to be reduced in the presence of metabolic inhibitors; (it) after exposure of the tissue to a hypotonic solution or the addition of galactose to the mucosal solution, this conductance increases; and (iii) these responses can be blocked by metabolic inhibitors. These findings suggest that the delayed response of this tissue to the addition of sugars or amino acids to the mucosal solution may be the result of cell swelling resulting from the intracellular accumulation of these solutes in osmotically active forms.Previous studies from this laboratory have disclosed that the addition of galactose or alanine to the solution bathing the mucosal surface of Necturus small intestine, in vitro, brings about a rapid depolarization of the electrical potential difference across the mucosal (or apical) membrane and an increase in the conductance of that barrier (1). This initial response, which can be attributed to the activation of rheogenic Na'-coupled cotransport processes for sugar and/or amino acid entry across the mucosal membrane, is followed by a slower repolarization of the electrical potential difference across that barrier that is blocked by the presence of metabolic inhibitors in the bathing solutions and appears to be the result of an increase in the K+ conductance of the basolateral membrane (1, 2); however, the "intracellular signal" that elicits this delayed increase in basolateral membrane conductance was not definitively resolved in these studies.It has long been known that sugars and amino acids are accumulated within small intestinal cells in osmotically active forms and, thus, are accompanied by an increase in cell water content and volume (3-5). Recently, studies on several epithelia have indicated that the initial increase in cell volume resulting from exposure to hypotonic bathing solutions is followed by a partial or full restoration of the original cell volume (6-8). It appears that this "volume regulatory response" is due, at least in part, to an increase in the permeability of the basolateral membrane to K+, which results in a loss of K+ from the cells (presumably accompanied by Cl-...

Intracellular pH (pHi) was measured in acini isolated from rabbit mandibular salivary glands using the fluorescent pH-sensitive probe 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pHi was estimated to be 7.13 +/- 0.01 (mean +/- SE of 29 experiments). Stimulation with acetylcholine (ACh) caused an intracellular acidosis followed by a return of pHi toward the control value with a half time of approximately 3 min. The intracellular acidosis was dose dependent and could be abolished by pretreatment of the acini with atropine (10 microM), suggesting that it was due to a receptor-mediated event. Incubation of the acini in HCO3- -free solutions or treatment of the acini with the carbonic anhydrase inhibitor acetazolamide (1 mM) abolished the acidosis, suggesting that the acidosis might be caused by loss of HCO3- from the cell. The acidosis was not affected by either 1) pretreatment of the acini with the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), or 2) equilibration of the acini in Cl- -free solution (Cl- substituted with glucuronate). These results suggest that the postulated HCO3- efflux does not occur by Cl- -HCO3- exchange. However, Cl- -HCO3- exchange did appear to be present because replacement of Cl- caused a large DIDS-sensitive alkalinization of pHi, presumably caused by HCO3- uptake in exchange for Cl-. The recovery of pHi after the initial acidosis on stimulation with ACh could be blocked by 1 mM amiloride, suggesting that the recovery phase was mediated by Na+-H+ exchange.

SUMMARY1. Intracellular pH (pHi) was measured using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in acini isolated from the rabbit mandibular salivary gland.2. Stimulation of the acinar cells with acetylcholine (ACh) evoked an intracellular acidosis, the size of which was dependent on the HCO3-concentration in the bathing medium. A half-maximal acidosis was observed at approximately 10 mm-HCO3-. ACh also evoked an acidosis in HC03--free solutions containing acetate; a halfmaximal acidosis was observed at about 10 mM-acetate.3. Propionate, lactate and butyrate were also able to support the ACh-evoked acidosis to varying extents. In contrast, formate, pyruvate and salicylate did not support the ACh-induced acidosis to any great extent.4. Acetazolamide greatly reduced the size of the acidosis in HCO3--buffered medium, but had no effect in acetate-buffered medium, suggesting that the inhibitory effect of acetazolamide was due to a specific inhibition of carbonic anhydrase activity.5. The Cl-channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (0O5 mM) abolished the ACh-evoked acidosis in both HCO3--and acetate-buffered media. 6. The data are consistent with the presence in the acinar cell of relatively nonspecific anion channels sensitive to DPC and its derivatives. Such channels, activated on stimulation with ACh, would allow HCO3-and other weak acid ions to leave the cell, leading to the observed acidosis. The existence of such channels, located in the apical membrane, could explain why HC03-or acetate can sustain fluid secretion in the intact perfused rabbit mandibular gland in the absence of Cl-.

Intralobular striated ducts have been isolated from rabbit mandibular salivary glands and maintained in primary culture for up to 2 days. Such ducts were loaded with the Cl(-)-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-(6-methoxyquinolinium bromide) (MQAE) and intracellular Cl- concentration ([Cl-]i) monitored using a fluorescence microscope. Intracellular Cl- could be rapidly and reversibly emptied from striated duct cells by replacing Cl- in the superfusing solution with NO(3)-. [Cl-]i could be lowered by removal of external Na+, exposure to 10 microM amiloride or to 10 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Both amiloride and DIDS were able to inhibit the recovery of [Cl-]i after an initial exposure to Na(+)- or Cl(-)-free solution. The amiloride derivatives, benzamil (2 microM) and N-isobutyl-N-methylamiloride (MIBA), (10 microM) also lowered [Cl-]i by similar amounts as 10 microM amiloride. Varying external K+ concentration ([K+]o) also affected [Cl-]i. Increasing [K+]o increased [Cl-]i, but decreasing [K+]o did not decrease [Cl-]i. Instead, [Cl-]i was also increased when [K+]o was lowered below the control value. Bumetanide (0.1 mM) lowered [Cl-]i by only a small amount, while ouabain (1 mM) had no significant effect on [Cl-]i. These data are consistent with current models of electrolyte transport in salivary ducts which include Cl- channels, Na+ channels, and Na+/H+ exchangers in the apical membrane. The effects of low [K+]o can be interpreted in terms of a K(+)-dependent exit mechanism for Cl-.