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Qiufang Cheng, David Lichtstein, Paul Russell, J. Samuel Zigler; Use of a Lipophilic Cation to Monitor Electrical Membrane Potential in the Intact Rat Lens. Invest. Ophthalmol. Vis. Sci. 2000;41(2):482-487.
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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. Ionophores, 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
(CCCP, 10 μM) and the calcium ionophore A23187 (10 μM), abolished
TPP+ 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
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