Use of lipophilic cation to monitor electrical membrane potential in the intact rat lens

Purpose. Tetraphenylphosphonium (TPP⁺) is a permeant lipophilic cation that accumulates in cultured cells and tissues as a function of the electrical membrane potential across the plasma membrane. This study was undertaken to determine whether TPP⁺ can be used for assessing membrane potential in intact lenses in organ culture. Methods. Rat lenses were cultured in media containing 10 μM TPP⁺ and a tracer level of ³H-TPP⁺ for various times. ³H-TPP⁺ levels in whole lenses or dissected portions of lenses were determined by liquid-scintillation counting. Inophores, transport inhibitors, and neurotransmitters were also added to investigate their effects on TPP⁺ uptake. Results. Incubation of lenses in low-K⁺ balanced salt solution and TC-199 medium, containing physiological concentrations of Na⁺ and K⁺, led to a biphasic accumulation of TPP⁺ in the lens that approached equilibrium by 12 to 16 hours of culture. The TPP⁺ equilibrated within 1 hour in the epithelium but penetrated more slowly into the fiber mass. The steady state level of TPP⁺ accumulation in the lens was depressed by 90% when the lenses were cultured in a medium containing high K⁺. The calculated membrane potential for the normal rat lens in TC-199 was -75 ± 3 mV. Monensin (1 μM) and nigericin (1 μM), Na⁺H⁺ and K⁺H⁺ exchangers respectively, as well as the protonophore carbonylcyanide-m- chlorophenylhydrazone (CCCP, 10 μM) and the calcium ionophore A3187 (10 μM), abolished TP⁺ accumulation and caused cloudiness of the lenses. The neurotransmitter acetylcholine at 50 μM decreased TPP⁺ accumulation in the lens, but this effect could be prevented by simultaneous application of 1 mM atropine. Conclusions. TPP⁺ accumulation can be used as an indicator of changes in membrane potential in intact lenses, but because of the long time required to reach steady state, its utility is limited. The slow accumulation of TPP⁺ and its slow efflux from the lens under conditions known to depolarize membranes are consistent with a diffusion barrier in the deep cortex and nucleus of the lens.