Reduction of intracellular pH by tenidap: Involvement of cellular anion transporters in the pH change

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Tenidap [5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1 H-indole-1-carboxamide], a novel antirheumatic agent, produces a rapid and sustained intracellular acidification when applied to cells in culture. To investigate the mechanism by which this change in ionic homeostasis is achieved, the acidification activities of structural analogs of tenidap were determined, and the movements of [ 14C]tenidap into and out of cells were explored. The acidification activity of tenidap was enhanced by lowering extracellular pH, suggesting that the free acid species was required for this process. Consistent with this requirement, a non-acidic analog of tenidap did not produce a change in intracellular pH (pHi). In contrast, multihalogenated derivatives of tenidap produced greater changes in pHi than did tenidap, and one analog produced a transient acidification from which the cell recovered; this recovery, however, was blocked by an inhibitor of the Na + H + antiporter. Fibroblasts incubated with [ 14C]tenidap achieved within 5 min a level of cell-associated drug that remained constant during longer incubations. Simultaneous addition of the electrogenic ionophore valinomycin or the P-glycoprotein inhibitor 4-(3,4-dihydro-6,7-dimethoxy-2(1 H)-isoquinolinyl)- N-[2-(3,4-dimethoxyphenyl)ethyl]-6,7-dimethoxy-2-quinazolinamine (CP-100,356) caused a time- and concentration-dependent increase in the level of cell-associated [ 14C]tenidap; other agents tested did not promote this enhanced cellular accumulation. [ 14C]Tenidap accumulated by fibroblasts in the presence of CP-100,356 subsequently was released when these cells were placed in a tenidap- and CP-100,356-free medium. Importantly, several agents that are known to inhibit anion transport processes, including a-cyano-β-(1-phenylindol-3-yl) acrylate, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and meclofenamic acid, inhibited efflux of [ 14C]tenidap. In contrast, ethacrynic acid and 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid did not impair the efflux process. Likewise, tenidap analogs that produced a sustained intracellular acidification blocked the efflux of [ 14C]tenidap, but non-acidifying species did not. These data suggest that movements of tenidap into and/or out of cells is a facilitated process subject to pharmacological intervention. Together, the structural selectivity of the acidification response and the evidence of facilitated transport suggest that the pHi modulating activity of tenidap is dependent on its unique physicochemical properties. Due to the dependence of these physicochemical properties on environmental and cellular conditions, in vivo expression of the acidification activity is likely to occur only within restricted environments that favor this tenidap-induced process.