Dual-function radiation sensitizers and bioreductive drugs: factors affecting cellular uptake and sensitizing efficiency in analogues of RSU 1069

Alkyl aziridine analogues of the hypoxic cell radiosensitizer RSU 1069 have been synthesized and one of these, RB 7040, containing the tetramethyl substituted aziridine, is a more efficient sensitizer in vitro than RSU 1069 (Ahmed et al., 1986). The extent to which variation in drug uptake can influence the sensitizing efficiency of RSU 1069 and its analogues has been investigated by determining the cellular uptake of these weakly basic sensitizers as a function of the pH of the extracellular medium (pHe) over the range 5.4-8.4. Following exposure of V79 cells to these agents for 1 h at room temperature, the ratio of intra- to extracellular concentration (Ci/Ce) was near unity at pH 5.4. Increasing pHe to 8.4 resulted in no change in the ratio Ci/Ce for RSU 1069 (pKa = 6.04). In contrast, the values of Ci/Ce increased three-fold for RSU 1165 (pKa = 7.38) and eleven-fold for RB 7040 (pKa = 8.45). Radiosensitization by RSU 1069 showed little dependence on pHe over the range studied, whereas increasing pH caused an apparent increase in sensitizing efficiency of both RSU 1165 and RB 7040. However, when the enhancement ratios for sensitization were normalized to take account of the effect of extracellular pH on drug uptake, efficiency of sensitization was independent of pHe. This study suggests that changes in basicity (pKa) may have wider potential for therapeutic exploitation on the basis of selective tumour uptake for this type of agent.     

Evidence for vesicles that mediate long-chain fatty acid uptake by human microvascular endothelial cells

This study analyzes the mechanisms of long-chain fatty acid (LCFA) uptake by human microvascular endothelial cells (HMEC). The time course revealed the presence of an early, carrier-mediated uptake component and a later component mediated by clathrin-coated vesicles (CCV) and caveolae, as evidenced by three different experimental approaches: 1) significant reduction of [3H]oleate uptake over 5 min by either inhibition of CCV formation by potassium depletion or hypertonic medium, or disruption of caveolae by filipin III or cyclodextrin. 2) Co-localization of intracellular 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]octadecanoic acid with CCV and caveolae using confocal laser scanning microscopy. 3) Enrichment of [3H]oleate in a subcellular fraction containing CCV and caveolae. Within 10 min, more than 75% of intracellular [3H]oleate remained unmetabolized, suggesting that HMEC preferentially shuttle LCFA through the cell using CCV and caveolae as carriers. The uptake of albumin paralleled that of oleate within the first 10 min, suggesting internalization of at least some LCFA bound to albumin. Compared to oleate and albumin, the uptake of sucrose and dextran was low, indicating a potential minor contribution of fluid-phase endocytosis to the total vesicular LCFA uptake. The data indicate a previously unrecognized role of both CCV and caveolae for the uptake of LCFA by HMEC.     

Ammonium uptake in carrot cell structures is influenced by pH-dependent cell aggregation

Semicontinuously grown wild carrot ( Daucus carota L.) cells were used in an investigation of the effect of culture medium pH on ammonium uptake in suspension cultures as a first step in exploring the relationship between pH and anthocyanin biosynthesis. In contrast to published data showing decreasing uptake rates with decreasing culture medium pH, ammonium-limited, semicontinuous carrot cell cultures showed a 25% greater ammonium uptake rate at pH 4.5 than at pH 5.5. When cells that had been grown semicontinuously in medium with a pH of 4.5 or 5.5 were grown in batch cultures at pH 4.5, 5.5 or 6.5 the ammonium uptake rates were those of the semicontinuous cultures, indicating that the pH of the batch culture medium had no effect on ammonium uptake rates over 7 days. The cell culture was composed of very small aggregates when it was grown semicontinuously in medium at pH 4.5, but was composed of large aggregates when it was grown semicontinuously in medium at pH 5.5. The aggregation/disaggregation of the cells was pH dependent, as changing the pH of the semicontinuous culture medium altered the extent of the aggregation. We conclude that the change in culture medium pH caused the cells to aggregate or disaggregate which in turn decreased or increased the rate of ammonium uptake from the medium.     

Structural determinants of cationic amphiphilic amines which induce clear cytoplasmic vacuoles in cultured cells

Disobutamide (D), an antiarrhythmic cationic amphiphilic amine (CAA), was withdrawn from clinical testing when clear cytoplasmic vacuoles (CCV) were found in the rat and dog during toxicity studies. To delineate the structural determinants of amines that induce CCV, we exposed cultured rat urinary bladder carcinoma and rabbit aorta muscle cells to numerous cationic drugs and chemicals and examined cells by phase light microscopy. The cationic moiety of these CAA was responsible for the induction of CCV. The very potent inducers were compounds that had two strongly basic amine (cationic) centers. The bis tertiary amines were particularly potent inducers. Aliphatic diamines of minimal lipophilicity-induced CCV, thus showing that an "amphiphilic" structural feature, though present in many CAA drugs, is not necessary for CCV induction. The distance between the two cationic centers was irrelevant to the induction of CCV. These results support the concept that CCV are a manifestation of intracellular (e.g., intralysosomal) drug storage. These structural delineations will be useful in future drug design and for further understanding of drug-cell interactions. Based on these findings, we were able to synthesize an antiarrhythmic CAA which did not induce CCV.     

Aluminum Uptake by Neuroblastoma Cells

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 x 10(6) cells/ml, pH 6.0, and 25 microM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60-70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the postmitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum-fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.     

Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects.

The pH dependence of proton uptake and electron transfers during the reaction between fully reduced cytochrome c oxidase and oxygen has been studied using the flow-flash method. Proton uptake was monitored using different pH indicators. We have also investigated the effect of D2O on the electron-transfer reactions. Proton uptake was biphasic throughout the pH range studied (6.3-9.3), and the decrease of the observed rate constants at increasing pH could be described by titration curves with pKa values of 8-8.5. Of the four phases resolved in the redox reaction, the rate constants for the first two were independent of pH, whereas that of the third decreased at increasing pH with a pKa of 7.9. All phases except the first were slower in D2O than in H2O. The values obtained for kH/kD were 1.0 for the first phase, 1.4 for the second and third phases, and 2.5 for the fourth phase. We suggest from these results that the fast phase of proton uptake is initiated by the second phase of the redox reaction and that this step includes a partially rate-limiting internal proton transfer. The third and fourth phases of the redox reaction are suggested to be rate limited by proton uptake from the medium. The pH dependencies of the proton uptake reactions are consistent with the participation of a titrable group in the protein in proton transfer from the medium to the oxygen-binding site.     

Physiological properties of Saccharomyces cerevisiae during sine-modulated and stepwise changes in the medium pH

The responses of a chemostat Saccharomyces cerevisiae culture (D = 0.1 h-1) to a stepwise increase or decrease in the pH of the medium were shown to be asymmetric. When the pH was lowered from 6.5 to a value above 0.3, the rates of oxygen uptake and carbon dioxide evolution rose for a sort period of time whereas the optical density of the culture fell down. The detected changes in the properties of the culture were identical with those which had been observed in the course of spontaneous undamped oscillations in the physiological parameters of the continuous C. cerevisiae culture. Apparently, in both cases, the energy status of cells changed when the oxidative type of metabolism was substituted by fermentation. When the pH of the medium was elevated within the same range (4.7-6.5), the response of the culture was three times as low and its properties changed in the opposite direction. When the pH of the medium was changed in a cyclic sinusoidal manner, oscillations in the physiological characteristics of the culture, identical with spontaneous oscillations were induced at certain values of the amplitude and the frequency of pH changes.     

Interaction of hyperthermia and pentamidine in HeLa S-3 cells

Pentamidine is similar to rhodamine-123 in chemical structure and state of electron charge, and rhodamine-123 was previously shown to be a hyperthermic sensitizer under appropriate cell culture conditions. The present experiments were carried out to determine whether pentamidine would potentiate hyperthermic cell killing and, if so, under what cultural conditions. Exposure of HeLa cells to pentamidine (80 microM) up to 4 h was not cytotoxic in culture medium in the presence or absence of glucose at 37 degrees C and pH 7.4. Cells in the glucose-deprived medium became progressively sensitive to killing as temperature and drug concentration were increased. On the other hand, there was a moderate level of enhanced cell killing in the glucose-fed medium at 42 degrees C. The enhanced effects of heat by the drug were most pronounced under alkaline pH of the culture medium. The cell kill under acidic pH was far less than that observed at neutral or alkaline pH; these effects may be a result of decreased cellular uptake of pentamidine. Together with our previous results on rhodamine-123 and glycolytic inhibitors, the present data with pentamidine are consistent with the concept that deprivation of cellular energy increases sensitivity to cell killing by heat.     

Comparison of the membrane transport of anthracycline derivatives in drug-resistant and drug-sensitive K562 cells.

One of the phenotypes of multidrug resistance is characterized by a decrease in the intracellular concentration of drug in resistant cells as compared to sensitive cells. This is correlated with the presence in the membrane of resistant cells of a 150-180-kDa glycoprotein, P-glycoprotein, responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to compare the membrane transport of five anthracycline derivatives: adriamycin, daunorubucin, 4'-o-tetrahydropyranyladriamycin, carminomycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells. The initial rate of uptake of these five drugs has been measured as a function of the extracellular pH, pHe. The data show that the uptake occurs through free permeation of the neutral form of the drug. For each drug an influx coefficient kpHe, characteristic of the drug and of the cell type has been defined and calculated: k+(7.2) = V+/[D]e.n where V+ and [D]e are the initial rate of uptake and the concentration of drug in the medium at pHe = 7.2 respectively and n is the number of cells. This coefficient is characteristic of a passive diffusion of the neutral form of the drug through the lipid bilayer. Efflux coefficients k-(7.2)- at pHi = 7.2 (the intracellular pH value) have also been calculated. In the case of sensitive cells, k+(7.2) and k-(7.2)- are equal. For resistant cells, the efflux coefficient is composed of two terms: (a) (k-)p corresponding to the passive diffusion of the neutral form of the drug and (k-)p = k+; (b) (k-)a corresponding to an active efflux mediated by the P-glycoprotein. Our data suggest that the anthracycline derivatives efflux actively in the neutral form.     

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

pH effect on cellular uptake of Sn(IV) chlorine e6 dichloride trisodium salt by cancer cells in vitro

The effects of pH value and presence of serum in an incubation medium on photosensitizer drug cellular uptake in MCF7 cancer cells have been investigated. The results showed that the presence of serum in an incubation medium reduced the drug cellular uptake at all pH values. It has been found that decreasing on pH values of the incubation medium increased the cellular uptake of the drug, demonstrating selective uptake of the sensitizer. The HepG2 liver cancer cells exhibited more drug cellular uptake than CCD-18CO normal colon cells, which assessed the selectivity uptake of photosensitizer on cancerous cells. The concentration of photosensitizer measured in 10⁶ cells showed a good correlation to the incubation time. Fluorescence and absorption spectroscopy been have used to examine the cells.     

Changes in 45Ca and 109Cd uptake, membrane potential and cell pH in Saccharomyces cerevisiae provoked by Cd2+

The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and [14C]tetraphenylphosphonium (TPP) uptake and cell pH was examined. At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and [14C]TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly. The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells. The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+. Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.     

Hexose uptake by Catharanthus roseus cell suspensions is inhibited by a high medium salt content

Summary The uptake of glucose and fructose from the medium by Catharanthus roseus cell suspensions was strongly inhibited by high medium salt concentration, such as found in LS (Linsmaier and Skoog 1965) medium. After inoculation into standard LS nutrient medium with less than 5 mM hexose no uptake occurred, while in low salt medium hexose was completely depleted. At a hexose concentration of 50 mM the uptake rate was higher in low salt medium than in standard medium. The lower rate of uptake at high salt concentration was not the result of a pH or osmotic effect of the salts. Probably the affinity of the hexose carrier is affected by the ion concentration of the medium. The decrease in medium salt concentration during normal batch culture probably will have a considerable effect on hexose uptake.Abbreviations LS Linsmaier and Skoog - S sucrose - N mineral nitrogen - K K₂SO₄ - F fructose     

Transport and intracellular accumulation of vitamin C in endothelial cells: relevance to collagen synthesis

Endothelial cells preserve vascular integrity in part by synthesizing type IV collagen for the basement membrane of blood vessels. Vitamin C, which at physiologic pH is largely the ascorbate mono-anion, both protects these cells from oxidant stress and is required for collagen synthesis. Therefore, cultured endothelial cells were used to correlate intracellular concentrations of ascorbate with its uptake and ability to stimulate collagen release into the culture medium. The kinetics and inhibitor specificity of ascorbate transport into EA.hy926 endothelial cells were similar to those observed in other cell types, indicative of a specific high affinity transport process. Further, transport of the vitamin generated intracellular ascorbate concentrations that were 80-100-fold higher than concentrations in the medium following overnight culture, and transport inhibition with sulfinpyrazone and phloretin partially prevented such ascorbate accumulation. On the other hand, low millimolar intracellular concentrations of ascorbate impaired its transport measured after overnight culture. Synthesis and release of type IV collagen into the culture medium was markedly stimulated by ascorbate in a time-dependent manner, and was saturable with increasing medium concentrations of the vitamin. Optimal rates of collagen synthesis required intracellular concentrations of the vitamin up to 2 mM. Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium.     

Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells

Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma cells, when incubated with plasminogen at a variety of pH values, generated angiostatic peptides and miniplasminogen. The enzyme(s) responsible for this reaction was purified and identified as procathepsin D. The purified procathepsin D, as well as cathepsin D, generated two angiostatic peptides having the same NH(2)-terminal amino acid sequences and comprising kringles 1-4 of plasminogen in the pH range of 3.0-6.8, most strongly at pH 4.0 in vitro. This reaction required the concomitant conversion of procathepsin D to catalytically active pseudocathepsin D. The conversion of pseudocathepsin D to the mature cathepsin D was not observed by the prolonged incubation. The affinity-purified angiostatic peptides inhibited angiogenesis both in vitro and in vivo. Importantly, procathepsin D secreted by human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that by human prostate carcinoma cells. Since deglycosylated procathepsin D from both prostate and breast carcinoma cells exhibited a similar low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in carbohydrate structures of procathepsin D molecules between the two cell types. The seminal vesicle fluid from patients with prostate carcinoma contained the mature cathepsin D and procathepsin D, but not pseudocathepsin D, suggesting that pseudocathepsin D is not a normal intermediate of procathepsin D processing in vivo. The present study provides evidence for the first time that cathepsin D secreted by human prostate carcinoma cells is responsible for angiostatin generation, thereby causing the prevention of tumor growth and angiogenesis-dependent growth of metastases.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Morphological study on the entry of African swine fever virus into cells

The early interactions between African swine fever virus (ASFV) and monkey kidney cells in culture, and the effect of chloroquine were studied by electron microscopy. Our results indicate that ASFV uptake occurs by endocytosis: after attachment to the cell surface, the virions were seen in coated pits and were internalized by endocytosis in endosomes and finally in lysosomes. Virions in coated vesicles were never seen. All these steps were completed in about 15 min. Direct penetration of viruses through the plasma membrane was never observed. In order to elucidate the participation of an acidic intracellular compartment in the penetration of ASFV, we studied the effect of chloroquine, a lysosomotropic drug known to increase the pH of acidic intracellular vacuoles and to inhibit ASFV infection. In the presence of this drug there were no apparent alterations on binding, endocytosis and intracellular distribution of the viral particles. The main effect of chloroquine was to retain the virions in lysosomes. When the drug was removed from the medium, the viral particles disappeared and images of binding of viral membranes with the membranes of the intracellular vacuoles were obtained, suggesting that the inhibited step is the uncoating of the virus. Viral fusion with the plasma membrane was obtained when the medium was acidified to pH 5-6. These results suggest that ASFV enters the cells by adsorptive endocytosis and that the uncoating process takes place intracellularly in a way similar to that described for Semliki Forest virus and other enveloped viruses.     

Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.     

Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.