Biomedical subjects

A J Dudley

Publications and source records attributed to A J Dudley.

3 recordsLinked to original sources

PubMed1996

Chloride channel blockers decrease intracellular pH in cultured renal epithelial LLC-PK1 cells.

The effects of chloride channel blockers upon intracellular pH (pHi) were examined in renal epithelial monolayers of LLC-PK1 cells. A significant intracellular acidification was found with addition of 100 microM 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), niflumic acid, flufenamate and diphenylamine-2-carboxylate (DPC) but not with 4,4'-diisothiocyanatostilbene-2-2'disulphonic acid (DIDS). The effects of these agents upon pHi was dose-dependent with apparent K0.5 values of: 16.7 +/- 0.3 microM, 34.2 +/- 0.9 microM and 740 +/- 13 microM for niflumic acid, flufenamate and DPC respectively. The results indicate that at concentrations commonly used to block channel activity these chloride channel blockers have profound effects upon pHi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

Mediation of cimetidine secretion by P-glycoprotein and a novel H(+)-coupled mechanism in cultured renal epithelial monolayers of LLC-PK1 cells.

1. Previous studies have shown that the weak base, cimetidine, is actively secreted by the renal proximal tubule. In this study we have examined the transport of cimetidine by renal LLC-PK1 epithelial cell monolayers. 2. In LLC-PK1 cell monolayers the basal-to-apical flux of cimetidine was significantly greater than the apical-to basal flux, consistent with net secretion of cimetidine in a basal-to-apical direction. 3. Net secretion of cimetidine was significantly (70%) reduced by the addition of either 100 microM verapamil or 100 microM nifedipine to the apical membrane. The reduction in net secretion was the result of an inhibition of basal-to-apical flux; these agents had no effect upon flux in the apical-to-basal direction. These results suggest that cimetidine secretion is mediated primarily by P-glycoprotein located in the apical membrane. In addition we found no evidence of a role for organic cation antiport in the secretion of cimetidine. 4. In the presence of an inwardly directed proton gradient across the apical membrane (pH 6.0), cimetidine secretion was significantly reduced compared to that measured at an apical pH of 7.4. The reduction in net secretion at pH 6.0 was the result of a stimulation of cimetidine uptake across the apical membrane. This pH-dependent uptake mechanism was sensitive to inhibition by DIDS (100 microM). 5. Experiments with BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) loaded monolayers demonstrated that cimetidine influx across the apical membrane was associated with proton flow into the cell and was sensitive to inhibition by DIDS. 6. These results suggest that net secretion of cimetidine across the apical membrane is a function of the relative magnitudes of cimetidine secretion mediated by P-glycoprotein and cimetidine absorption mediated by a novel proton-coupled, DIDS-sensitive transport mechanism.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

pH-dependent transport of procainamide in cultured renal epithelial monolayers of OK cells: consistent with nonionic diffusion.

1. Previous studies suggest that procainamide is a substrate for organic cation/proton antiport. In order to study the coupling between procainamide flux and proton flux in greater detail we investigated the effects of extracellular procainamide addition upon intracellular pH in cultured monolayers of renal OK cells. Intracellular pH was monitored by use of BCECF as a probe. 2. Apical addition of procainamide (10 mM) caused a significant alkalinisation of intracellular pH. Basolateral addition of procainamide was equally effective in raising intracellular pH. A similar alkalinisation was found in two other renal cell lines: MDCK strain 1 and LLCPK1. 3. In contrast, both tetraethylammonium and N-methylnicotinamide, archetypal substrates for organic cation/proton antiport were without effect upon intracellular pH. 4. At physiological pH values, procainamide exists as a neutral weak base (B) and its conjugate weak acid (BH+). To test which species of procainamide was responsible for the alkalinisation, experiments in which [B] was kept constant whilst [BH+] was varied from 1.15 mM to 7.25 mM were performed. The results suggested that the neutral weak base (B) was the permeant species. 5. Procainamide efflux from procainamide-loaded cell monolayers resulted in a significant acidification of intracellular pH. As with procainamide uptake, this result could be ascribed to the movement of neutral weak base. 6. These effects of procainamide upon intracellular pH are consistent with nonionic diffusion of procainamide rather than an interaction of procainamide with the organic cation/proton antiporter. In addition, the results suggest that organic cation/proton antiport is not highly expressed in OK cells.

Animals↗