The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment.

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure  to  PEF indicate a strong coupling between these parts of the cell.

The evaluations of 99mTc cyclopentadienyl tricarbonyl triphenyl phosphonium cation for multidrug resistance.

A triphenylphosphonium cation, [99mTc]Technetium cyclopentadienyltricarbonyl-6-hexanoyl-triphenylphosphonium cation ([99mTc]3) was prepared to target multidrug resistance (MDR). The radiotracer was evaluated in the MDR-negative MCF-7 and MDR-positive MCF-7/ADR cell lines in vitro, as well as animal models in vivo. [99mTc]3 was proofed to be a substrate of P-glycoprotein and multidrug resistant protein 1, and showed a higher accumulation in the MDR-negative MCF-7 cells compared to 99mTc-sestamibi in vitro. The MCF-7 tumor-to-MCF-7/ADR tumor ratio of [99mTc]3 was ∼3 at 1hp.i. in the biodistribution study. These results demonstrated the capability of the radiotracer to detect multidrug resistance in tumor cells.

Influence of pH on transungual passive and iontophoretic transport.

The present study investigated the effects of pH on nail permeability and the transport of ions such as sodium (Na) and chloride (Cl) ions endogenous to nail and hydronium and hydroxide ions present at low and high pH, which might compete with drug transport across hydrated nail plate during iontophoresis. Nail hydration and passive transport of water across the nail at pH 1-13 were assessed. Subsequently, passive and iontophoretic transport experiments were conducted using (22)Na and (36)Cl ions under various pH conditions. Nail hydration was independent of pH under moderate pH conditions and increased significantly under extreme pH conditions (pH >11). Likewise, nail permeability for water was pH independent at pH 1-10 and an order of magnitude higher at pH 13. The results of passive and iontophoretic transport of Na and Cl ions are consistent with the permselective property of nail. Interestingly, extremely acidic conditions (e.g., pH 1) altered nail permselectivity with the effect lasting several days at the higher pH conditions. Hydronium and hydroxide ion competition in iontophoretic transport was generally negligible at pH 3-11 was significant at the extreme pH conditions studied.

Effects of targeting moiety, linker, bifunctional chelator, and molecular charge on biological properties of 64Cu-labeled triphenylphosphonium cations.

In this report, we present the synthesis and evaluation of six new 64Cu-labeled triphenylphosphonium (TPP) cations. Biodistribution studies were performed using the athymic nude mice bearing U87MG human glioma xenografts to explore the impact of TPP moieties, linkers, bifunctional chelators (BFCs), and molecular charge on biological properties of 64Cu radiotracers. On the basis of the results from this study, it is concluded that (1) mTPP (tris(4-methoxyphenyl)phosphonium) is a better mitochondrion-targeting molecule than TPP and 3mTPP (tris(2,4,6-trimethoxyphenyl)phosphonium); (2) DO3A (1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid) and DO2A (1,4,7,10-tetraazacyclododecane-4,7-diacetic acid) are suitable BFCs for the 64Cu-labeling of TPP cations; (3) NOTA-Bn ( S-2-(4-thioureidobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid) has a significant adverse effect on the radiotracer tumor uptake and tumor-to-background ratios; and (4) monoanionic BFCs should be avoided to ensure that 64Cu chelate has a neutral or negative charge. Considering the tumor uptake and tumor/liver ratios, 64Cu(DO2A-xy-TPP)+ is the best candidate for more extensive evaluations in different tumor-bearing animal models.

Degradation of tetraphenylphosphonium bromide at high pH and its effect on radionuclide solubility.

Recently, tetraphenylphosphonium bromide (TPPB) has been used to remove technetium from some radioactive waste streams. However, before TPPB could be approved for use it was necessary to show that TPPB and its degradation products would not have a significant detrimental effect on post-closure performance of a radioactive waste repository. TPPB is known to be stable at neutral pH, however, under alkaline conditions it degrades by an alkaline hydrolysis mechanism to triphenylphosphonium oxide (TPPO). Degradation can also occur by radiolysis to produce triphenylphosphine (TPP). The kinetics of the alkaline hydrolysis degradation of TPPB is described and the solubility of europium, iodine, nickel, technetium(VII) and uranium(VI) in aqueous solutions of TPPB and its degradation products is reported. These results were used to support the use of TPPB in removing technetium from some waste streams.

A new strategy to screen molecular imaging probe uptake in cell culture without radiolabeling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

UNLABELLED: Numerous new molecular targets for diseases are rapidly being identified and validated in the postgenomic era, urging scientists to explore novel techniques for accelerating molecular probe development. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated as a potential tool for high-throughput screening and characterization of molecular imaging probes. Specifically, MALDI-TOF-MS was used to screen a small library of phosphonium cations for their ability to accumulate in cells. METHODS: C6 cells incubated with phosphonium cations at room temperature were collected and lysed for experiments. Calibration curves for the internal standard, methyltriphenyl phosphonium, and for tetraphenylphosphonium bromide (TPP) and other phosphonium cations were first established. The time course of TPP uptake by C6 cells was then quantified using both MALDI-TOF-MS and liquid scintillation counting with (3)H-TPP. In addition, MALDI-TOF-MS was used to screen a library of 8 phosphonium cations and subsequently rank their ability to penetrate membranes and accumulate in cells. Finally, the accumulation of 4-fluorophenyltriphenyl phosphonium (FTPP) in the membrane potential-modulated cells was also measured by MALDI-TOF-MS. RESULTS: MALDI-TOF-MS spectra clearly revealed that TPP was easily identified from cell lysates even as early as 10 min after incubation and that levels as low as 0.11 fmol of TPP per cell could be detected, suggesting the high sensitivity of this technique. The time course of TPP influx determined by both MALDI-TOF-MS and radioactivity counting showed no statistically significant difference (P > 0.05 for all time points). These data validated MALDI-TOF-MS as an alternative approach for accurately measuring uptake of phosphonium cations by cells. TPP and FTPP demonstrated greater accumulation in cells than did the other cations evaluated in this study. Furthermore, uptake profiles suggested that FTPP preserves the membrane potential-dependent uptake property of TPP in cell cultures. Taken together, these data justify further synthesis and evaluation of (18)F-FTPP as a molecular probe for imaging mitochondrial dysfunction. CONCLUSION: These results demonstrate that MALDI-TOF-MS is a powerful analytic tool for rapid screening and characterization of phosphonium cations as molecular probes. This technique can potentially be applied to the evaluation of other imaging probes or drugs and thus may facilitate their rational design and development.

Bacterial cytoplasmic membrane permeability assay using ion-selective electrodes.

We used K(+) and tetraphenylphosphonium (TPP(+)) electrodes simultaneously to evaluate the ability of antimicrobial peptides to form channels (or more generally to increase permeability) and to abolish membrane potential in bacterial cytoplasmic membranes in situ. Such evaluations are usually made independently by colorimetric monitoring of the hydrolysis of a chromogenic substrate by a cytoplasmic enzyme or by fluorimetric determination of membrane depolarization using a membrane potential-sensitive dye. In the present study, the K(+) electrode was used to evaluate channel-forming ability by monitoring the efflux of K(+) originally present in the cytoplasm of bacteria, while the TPP(+) electrode was used to examine membrane depolarization causing the efflux of TPP(+) accumulated in the cytoplasm of bacteria dependent on membrane potential. Thus, the combination of these two electrodes enabled us to clarify how the peptide-induced formation of ion channels is involved in disrupting the energy-generating system in situ.

Tetraphenylphosphonium as a novel molecular probe for imaging tumors.

UNLABELLED: Mitochondrial membrane potential (DeltaPsim)-dependent enhanced uptake of phosphonium salts, including (3)H-tetraphenylphosphonium ((3)H-TPP), in tumor cells, suggests the potential use of phosphonium salts as tracers for tumor imaging. In this study, we characterize the tumor accumulation of (3)H-TPP and compare it with (18)F-FDG in cell culture and in xenograft, metastatic, and inflammation models in living animals. METHODS: (3)H-TPP and (3)H-FDG accumulation was compared in cell culture with a variety of cell lines in different glucose concentrations. Normal biodistribution and tumor uptake were assessed using nude mice with or without subcutaneous xenograft tumors (C6). To compare the accumulation of (3)H-TPP and (18)F-FDG in a metastatic tumor, severe combined immunodeficiency mice were tail-vein injected with human melanoma cell lines (A375-FL). To characterize the accumulation of (3)H-TPP and (18)F-FDG in inflammation, an inflammatory reaction was induced by subcutaneous injection of Complete Freund's Adjuvant in the left hind paw of Sprague-Dawley rat. RESULTS: The DeltaPsim data from a separate study and the current (3)H-TPP uptake data showed good correlation (r(2) = 0.82, P < 0.05). (3)H-TPP accumulation was significantly greater than that of (3)H-FDG for glucose >/=100 mg/dL. The biodistribution study of (3)H-TPP showed low uptake in most tissues but high accumulation in the heart and kidneys. (3)H-TPP accumulation in xenograft or metastatic tumors was comparable with that of (18)F-FDG, whereas (3)H-TPP accumulation in inflammatory tissues was markedly lower than that of (18)F-FDG. CONCLUSION: The sensitive tumor accumulation of (3)H-TPP with less propensity for inflammatory regions warrants further investigation of radiolabeled phosphonium analogs for tumor imaging in living subjects.

Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and -2 and membrane hyperpolarization in human umbilical vein endothelial cells.

Insulin induces vasodilatation in human subjects and increases L-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. L-arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca2+, L-arginine and L-citrulline levels, L-[3H]citrulline formation from L-[(3)H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased L-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca2+, L-[3H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the L-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.

Supertargeted chemistry: identifying relationships between molecular structures and their sub-cellular distribution.

Supertargeted chemistry is the study of how chemical structures localize or direct molecules to specific sub-cellular compartments in living cells. Supertargeting can be used to increase the activity or specificity of an inhibitor against its target, by concentrating the inhibitor in the particular organelle where the target is active. But, unlike structure-activity relationships, structure-localization relationships are not a simple function of compound concentration. Various aspects of mitochondrial physiology, proteomics and pharmacology have made this the organelle of choice for supertargeting studies. While exploration of supertargeting strategies to this and the other organelles has been limited, combinatorial chemical libraries of fluorescent molecules are beginning to illuminate new supertargeting mechanisms at the sub-cellular level. Moreover, predictive approaches that determine the relationship between a molecule's features and sub-cellular localization are being developed in the related field of functional genomics. Applied to the small molecules, such strategies could prove useful for predicting structure-localization relationships amongst large libraries of compounds.

Rapid stimulation of L-arginine transport by D-glucose involves p42/44(mapk) and nitric oxide in human umbilical vein endothelium.

D-glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells. High D-glucose (25 mmol/L, 2 minutes) induced membrane hyperpolarization and an increase of L-arginine transport (V(max) 6.1+/-0.7 versus 4.4+/-0.1 pmol/ microg protein per minute) with no change in transport affinity (K(m) 105+/-9 versus 111+/-16 micromol/L). L-[3H]citrulline formation and intracellular cGMP, but not intracellular Ca2+, were increased by high D-glucose. The effects of D-glucose were mimicked by levcromakalim (ATP-sensitive K+ channel blocker), paralleled by p42/p44(mapk) and Ser(1177)-endothelial NO synthase phosphorylation, inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; NO synthesis inhibitor), glibenclamide (ATP-sensitive K+ channel blocker), KT-5823 (protein kinase G inhibitor), PD-98059 (mitogen-activated protein kinase kinase 1/2 inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor), but they were unaffected by calphostin C (protein kinase C inhibitor). Elevated D-glucose did not alter superoxide dismutase activity. Our findings demonstrate that the human fetal endothelial L-arginine/NO signaling pathway is rapidly activated by elevated D-glucose via NO and p42/44(mapk). This could be determinant in pathologies in which rapid fluctuations of plasma D-glucose may occur and may underlie the reported vasodilatation in early stages of diabetes mellitus.

Preferential accumulation of (3)H-tetraphenylphosphonium in non-small cell lung carcinoma in mice: comparison with (99m)Tc-MIBI.

UNLABELLED: We have previously shown enhanced accumulation of the delocalized lipophilic cation (11)C-triphenylmethylphosphonium in canine brain glioma, suggesting its potential use for tumor staging in humans using PET. Here, we extend our studies of phosphonium cations to nonbrain tumors and characterize the biodistribution and tumor specificity of (3)H-tetraphenylphosphonium ((3)H-TPP) in non-small cell lung carcinoma in mice. METHODS: (3)H-TPP accumulation in isolated malignant lung nodules of the Lewis lung carcinoma (LLC) cell line, in LLC-bearing lung, and in control lung was measured at various intervals after inoculation. Tumor uptake and biodistribution of (3)H-TPP were compared with those of (99m)Tc-methoxyisobutylisonitrile (MIBI). RESULTS: (3)H-TPP accumulation in LLC nodules at 14 d was significantly greater than that in controls, peaked at 21 d, and declined to lower values at 28 d after injection. At 21 d after injection, (3)H-TPP uptake in LLC nodules was greater than that in control lung tissue and in LLC-bearing lung tissue-by 549% and 230%, respectively-whereas (99m)Tc-MIBI nodule uptake was greater by 90% and 30%, respectively. CONCLUSION: The high tumor accumulation and sensitivity to the phase of tumor development suggest the potential use of radiolabeled phosphonium analogs for in vivo tumor staging and as a tool for investigating tumor evolution.

Primary causes of decreased mitochondrial oxygen consumption during metabolic depression in snail cells.

Cells isolated from the hepatopancreas of estivating snails (Helix aspersa) have strongly depressed mitochondrial respiration compared with controls. Mitochondrial respiration was divided into substrate oxidation (which produces the mitochondrial membrane potential) and ATP turnover and proton leak (which consume it). The activity of substrate oxidation (and probably ATP turnover) decreased, whereas the activity of proton leak remained constant in estivation. These primary changes resulted in a lower mitochondrial membrane potential in hepatopancreas cells from estivating compared with active snails, leading to secondary decreases in respiration to drive ATP turnover and proton leak. The respiration to drive ATP turnover and proton leak decreased in proportion to the overall decrease in mitochondrial respiration, so that the amount of ATP turned over per O2 consumed remained relatively constant and aerobic efficiency was maintained in this hypometabolic state. At least 75% of the total response of mitochondrial respiration to estivation was caused by primary changes in the kinetics of substrate oxidation, with only 25% or less of the response occurring through primary effects on ATP turnover.

The ostensible paradox of multidrug recognition.

The ability of multidrug-efflux transporters to recognize scores of dissimilar organic compounds has always been considered paradoxical because of its apparent contradiction to some of the basic dogmas of biochemistry. In order to understand, at least in principle, how a protein can recognize multiple compounds, we analysed the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr. This regulator, BmrR, binds multiple dissimilar hydrophobic cations and, by activating expression of the Bmr transporter, causes their expulsion from the cell. Crystallographic analysis of the complexes of the inducer-binding domain of BmrR with some of its inducers revealed that ligands penetrate the hydrophobic core of the protein, where they form multiple van der Waals and stacking interactions with hydrophobic amino acids and an electrostatic bond with the buried glutamate. Mutational analysis of the binding site suggests that each ligand forms a unique set of atomic contacts with the protein: each tested mutation exerted disparate effects on the binding of different ligands. The example of BmrR demonstrates that a protein can bind multiple hydrophobic compounds with micromolar affinities by using only electrostatic and hydrophobic interactions. Its ligand specificity can be further broadened by the flexibility of the binding site. It appears, therefore, that the commonly expressed fascination with the relaxed substrate specificity of multidrug transporters is misdirected and originates from an almost exclusive familiarity with the more sophisticated processes of specific molecular recognition that predominate among proteins analyzed to date.

Comparative in vitro evaluation of dequalinium B, a new boron carrier for neutron capture therapy (NCT).

A boronated derivative of dequalinium, a delocalized lipophilic cation (DLC), was synthesized as a potential boron carrier for the selective targeting of mitochondria in malignant versus benign cells for boron neutron capture therapy (BNCT), a binary modality for the treatment of cancer. This agent, designated DEQ-B, was taken up and retained in vitro in the KB, F98, and C6 tumor cell lines but not in the normal epithelial cell line CV1. DEQ-B was also less toxic in the latter cell line at lower exposure concentrations The uptake, retention, and toxicity profiles of DEQ-B are comparable to those of the non-boronated DLCs, dequalinium, MKT 077, RH 123, and tetraphenylphosphonium chloride. Our results suggest that the synthesis and further evaluation of boronated DLCs as potential delivery agents for BNCT is warranted.

Limitations of cyclosporin A inhibition of the permeability transition in CNS mitochondria.

Activation of the mitochondrial permeability transition may contribute to excitotoxic neuronal death (Ankarcrona et al., 1996; Dubinsky and Levi, 1998). However, cyclosporin A (CsA), a potent inhibitor of the permeability transition in liver mitochondria, only protects against neuronal injury by limited doses of glutamate and selected ischemic paradigms. The lack of consistent CsA inhibition of the mitochondrial permeability transition was analyzed with the use of isolated brain mitochondria. Changes in the permeability of the inner mitochondrial membrane were evaluated by monitoring mitochondrial membrane potential (Deltapsi), using the distribution of tetraphenylphosphonium, and by monitoring mitochondrial swelling, using light absorbance measurements. Metabolic impairments, large Ca(2+) loads, omission of external Mg(2+), or low doses of palmitic acid or the protonophore FCCP exacerbated Ca(2+)-induced sustained depolarizations and swelling and eliminated CsA inhibition. BSA restored CsA inhibition in mitochondria challenged with 50 microm Ca(2+), but not with 100 microm Ca(2+). CsA failed to prevent Ca(2+)-induced depolarization or to repolarize mitochondria when mitochondria were depolarized excessively. Similarly, CsA failed to prevent mitochondrial swelling or PEG-induced shrinkage after swelling when the Ca(2+) challenge produced a strong, sustained depolarization. Thus in brain mitochondria CsA may be effective only as an inhibitor of the permeability transition and the Ca(2+)-activated low permeability state under conditions of partial depolarization. In contrast, ADP plus oligomycin inhibited both permeabilities under all of the conditions that were tested. In situ, the neuroprotective action of CsA may be limited to glutamate challenges sufficiently toxic to induce the permeability transition but not so severe that mitochondrial depolarization exceeds threshold.

Cadmium hepatotoxicity and alterations of the mitochondrial function.

OBJECTIVE: To examine the effect of low concentrations of cadmium on isolated liver mitochondrial function as related to hepatotoxicity. METHODS: Tetraphenyl phosphonium ion uptake and retention, estimated with a tetraphenyl phosphonium-sensitive electrode, was used to monitor changes in liver inner mitochondrial membrane potential. Ca2+ efflux was measured spectrophotometrically with the Ca2+ indicator Arsenazo III. Mitochondrial swelling was measured spectrophotometrically at 540 nm. Oxygen consumption was measured with a Clark-type oxygen microelectrode. RESULTS: Incubation of isolated liver mitochondria with cadmium (5-30 microM) altered mitochondrial function as indicated by swelling, inhibition of respiration, loss of inner mitochondrial membrane potential, and loss of preaccumulated Ca2+. The presence of dithiothreitol (2 mM) in the incubation medium restored mitochondrial function to almost the control level. Cyclosporin A (1 microM), however, did not provide any protection against cadmium toxicity. CONCLUSIONS: The findings point to a direct effect of cadmium on liver mitochondrial function. Cadmium toxicity may be due to loss of reduced glutathione rather than to increased mitochondrial inner membrane permeability. The effect of cadmium on liver mitochondria seems to be an early event in cadmium-induced hepatotoxicity.

Factors that determine the plasma-membrane potential in bloodstream forms of Trypanosoma brucei.

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.

Properties of a polyamine transporter regulated by antizyme.

The regulation of polyamine transport by antizyme, a protein that is involved in the rapid degradation of ornithine decarboxylase (ODC), was studied in FM3A mouse cells overproducing ODC. Both artificial (Z1) and natural antizymes not only inhibited polyamine uptake but also stimulated polyamine excretion. The properties of the polyamine transporter regulated by antizyme were characterized. The uptake of radiolabelled polyamines was inhibited by excess acetylpolyamines and a protonophore, CCCP (carbonyl cyanide m-chlorophenylhydrazone), whereas the excretion of radiolabelled polyamines was stimulated by unlabelled polyamines, acetylpolyamines and CCCP in the medium. Furthermore, it is shown that polyamines and acetylpolyamines are excreted from cells. On the basis of the results, it is discussed how antizyme regulates polyamine transport negatively.

Use of a 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 microM 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 microM) and nigericin (1 microM), Na+H+ and K+H+ exchangers respectively, as well as the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP, 10 microM) and the calcium ionophore A23187 (10 microM), abolished TPP+ accumulation and caused cloudiness of the lenses. The neurotransmitter acetylcholine at 50 microM 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.