Expression, activity and regulation of CYP3A in human and rodent brain.

The importance of cytochrome P450 (CYP) 3A enzymes in human drug metabolism is well established. The function of these enzymes has been characterized extensively in liver and intestinal tissues but much less is known about their expression, regulation and functional activity in the brain. Several lines of evidence point to the presence and function of multiple forms of CYP enzymes, including CYP3A, in both human and rodent brain. Expression studies suggest that CYP3A enzymes show regional differences in their distribution in the brain, where they may play a role in steroid metabolism. They also metabolize many psychoactive drugs and may have a profound effect on their efficacy and safety. This review explores the tissue, cellular, and subcellular expression of CYP3A isoforms in human and rodent brain and provides insight into their functional roles and regulation.

Decline in Memory, Visuospatial Ability, and Crystalized Cognitive Abilities in Older Adults: Normative Aging or Terminal Decline?

The aim of this study is to explore the pattern of change in multiple measures of cognitive abilities in a sample of oldest-old adults, comparing two different time metrics (chronological age and time to death) and therefore examining both underlying conceptual assumptions (age-related change and terminal decline). Moreover, the association with individual characteristics as sex, education, and dementia diagnosis was also examined. Measures of cognitive status (Mini-Mental State Examination and the Swedish Clock Test) and tests of crystallized (knowledge and synonyms), memory (verbal memory, nonverbal long-term memory, recognition and correspondence, and short-term memory), and visuospatial ability were included. The sample consisted of 671 older Swedish adult participants of the OCTO Twin Study. Linear mixed models with random coefficients were used to analyse change patterns and BIC indexes were used to compare models. Results showed that the time to death model was the best option in analyses of change in all the cognitive measures considered (except for the Information Test). A significant cognitive decline over time was found for all variables. Individuals diagnosed with dementia had lower scores at the study entrance and a faster decline. More educated individuals performed better in all the measures of cognition at study entry than those with poorer education, but no differences were found in the rate of change. Differences were found in age, sex, or time to death at baseline across the different measures. These results support the terminal decline hypothesis when compared to models assuming that cognitive changes are driven by normative aging processes.

Clinical pharmacokinetics of non-opiate abused drugs.

The present review discusses the available data on the kinetic properties of non-opiate abused drugs including psychomotor stimulants, hallucinogens and CNS-depressants. Some of the drugs of abuse reviewed here are illicit drugs (e.g. cannabis, cocaine), while others are effective pharmacological agents but have the potential to be abused (e.g. benzodiazepines). Although some of the drugs mentioned in this review have been in use for centuries (e.g. caffeine, nicotine, cocaine, cannabis), knowledge of their kinetics and metabolism is very recent and in some cases still incomplete. This is partially due to the difficulties inherent in studying drugs of abuse in humans, and to the complex metabolism of some of these drugs (e.g. cannabis, caffeine) which has made it difficult to develop sensitive assays to determine biological pathways. Although drugs of abuse may have entirely different intrinsic pharmacological effects, the kinetic properties of such drugs are factors contributing to abuse and dependence. The pharmacokinetic properties that presumably contribute to self-administration and drug abuse include rapid delivery of the drug into the central nervous system and high free drug clearance. Kinetic characteristics also play an important role in the development of physical dependence and on the appearance of a withdrawal syndrome: the longer the half-life, the greater the likelihood of the development of physical dependence; the shorter the half-life, the earlier and more severe the withdrawal. The balance between these 2 factors, which has not yet been carefully studied, will also influence abuse patterns. The clinical significance of kinetic characteristics with respect to abuse is discussed where possible.

A study of doxorubicin loading onto and release from sulfopropyl dextran ion-exchange microspheres.

The objective of this study was to investigate various factors that influence doxorubicin (Dox) loading onto and release from sulfopropyl dextran ion-exchange microspheres (MS), and to evaluate the anticancer activity of the released drug in vitro. Dox was incorporated into the MS by incubating the MS with aqueous solutions of Dox at room temperature. The drug release was carried out at 37 degrees C in aqueous solutions containing NaCl with or without CaCl2. The kinetics of drug absorption and release, the amount of Dox released, and the stability of Dox after loading, freeze-drying, and release were determined by spectrophotometry. The cytotoxicity of Dox (the original drug or that released from MS) against murine EMT6 breast cancer cells was assessed using a clonogenic assay. An increase in the MS to drug ratio resulted in a higher absorption rate and a higher fraction of the drug extracted from the solution. The release rate and the equilibrium fraction of Dox released increased with a decrease in the initial amount of Dox loaded or an increase in the salt concentration. The addition of divalent ions (Ca2+) promoted drug release compared to NaCl alone. The percent loss of colony forming ability of the cells, a measure of cytotoxicity of the released Dox, was the same as parent Dox solutions, indicating that the drug bioactivity was fully preserved after the drug loading and release cycle. This work demonstrated that various drug release rates were achieved by varying the drug loading and that the MS-delivered Dox was effective against the cancer cells in vitro.

Renal drug transport: a review.

Renal drug elimination involves three major processes: glomerular filtration, tubular secretion, and tubular reabsorption. Drug filtration is a simple unidirectional diffusion process. Renal tubular secretion and reabsorption are bidirectional processes that often involve both passive diffusion and carrier-mediated membrane processes. Various in vivo and in vitro techniques are available to study renal drug elimination and renal drug transport. The complete renal handling of a drug is best understood from data obtained from a combination of in vivo and in vitro methodologies. At the membranes of the renal proximal tubule, a number of carrier systems are involved in the tubular secretion and/or reabsorption of various drugs. Organic acid and base transporters are two major carrier systems important in the tubular transport of a number of organic acid and base drugs, respectively. Nucleoside and P-glycoprotein transporters appear to play an important role in renal tubular transport of dideoxynucleosides (e.g., zidovudine, dideoxyinosine) and digoxin, respectively. Clinically, these transporters are not only necessary for the renal tubular secretion and reabsorption of various drugs, but are also responsible in part for the drug's pharmacologic response (e.g., furosemide), drug-drug interactions of therapeutic or toxic importance, and drug nephrotoxicity.

Interaction of 3'-azido-3'-deoxythymidine with the organic base transporter in a cultured renal epithelium.

In humans and various animal species, 3'-azido-3'-deoxythymidine (AZT) is in part eliminated by the kidneys, where it undergoes significant tubular secretion. The goal of this project was to develop, in a continuous renal epithelial cell line (LLCPK1), a model of AZT transport in which mechanisms of drug interactions could be investigated. Transport properties of H3-AZT were studied in LLCPK1 cells grown as monolayers on permeable filters. This system provides access to the basolateral and apical surfaces of the epithelium and allows the determination of substrate transepithelial flux from the basolateral side to the apical side (B-->A/secretory direction) and apical to basolateral side (A-->B/reabsorptive direction). The B-->A flux of AZT was significantly greater than B-->A flux of mannitol (a nontransported substrate) and was temperature dependent (37 degrees C >> 4 degrees C). The AZT A-->B flux was significantly smaller than the B-->A flux, indicating that the drug is predominantly secreted in this renal epithelium. The B-->A flux was significantly inhibited by the organic bases cimetidine, quinine, quinidine, and trimethoprim. Log concentration dose studies indicate that quinine is a weak inhibitor (IC50 = 9.61 mM) of AZT B-->A flux, and that AZT is a moderate inhibitor (IC50 = 0.69 mM) of the organic base cimetidine. These results suggest that AZT may share the organic base transporter in the renal epithelium, and that this model can be used successfully to study transport properties and renal drug-drug interactions of AZT.

New drug interactions with zidovudine.

Polypharmacy is commonly encountered in human immunodeficiency virus (HIV)-positive patients, and the risk and frequency of drug-drug interactions are significant in this patient population. Most HIV-positive patients receive the antiretroviral drug zidovudine (3'-azido-3'-deoxythymidine, ZDV), the first drug to be approved for the treatment of HIV. Many drug interactions with ZDV have already been reported. As HIV pharmacotherapy becomes more complex, the potential for drug-drug interactions is likely to increase significantly.

In vitro investigation of ionic polysaccharide microspheres for simultaneous delivery of chemosensitizer and antineoplastic agent to multidrug-resistant cells.

Insufficient intratumoral concentration of therapeutic agents and multidrug resistance are major factors responsible for failure of treatment of solid tumors. Simultaneous delivery of chemosensitizing and antineoplastic agents by microspheres could lead to enhanced chemotherapy of multidrug-resistant (MDR) tumors. Ionic polysaccharide microspheres derived from dextran were used to load chemosensitizers (e.g., verapamil) and anticancer drugs such as vinblastine. High drug loading was achieved for both a single agent and dual agents. The equilibrium drug loading was dependent on the ratio of the microspheres (MS) to the drug, as well as the relative affinity of the agents to the MS in the case of dual agents. The drug release from drug-MS involved hydration and swelling of the MS in addition to ion exchange. The effectiveness of MS-delivered chemosensitizers in the reversal of drug resistance was evaluated by measuring the uptake of [3H]vinblastine by MDR cells (CHRC5). The concomitant delivery of verapamil with vinblastine by the MS led to a 6-7-fold increase in the uptake of vinblastine, a level similar to the uptake obtained with free drug solutions. The results suggest that the antineoplastic and chemosensitizing agents were released effectively from the MS and the bioactivity of the chemosensitizer was preserved during the process.

Synthesis and characterization of surface-hydrophobic ion-exchange microspheres and the effect of coating on drug release rate.

Biodegradable, dextran-based ion-exchange microspheres (IE-MS) have been used for localized delivery of anticancer drugs and chemosensitizers. Because of their hydrophilic nature, the IE-MS release their payload quickly. The purpose of this work was to develop an IE-MS system that could provide a broad range of release rates for in vitro and in vivo applications. Sulfopropylated dextran microspheres (SP C25 MS) were surface-modified by acylation. These hydrophobically modified sulfopropylated dextran microspheres (HM-MS) were further coated with the cationic acrylic polymer Eudragit RL100 (EU-MS). The changes in chemical composition after the surface modification and coating were characterized by X-ray photoelectron spectroscopy. The effects of the modification and coating on the surface hydrophobicity, equilibrium swelling, surface morphology, and drug loading capacity were investigated. The HM-MS showed little change in swelling and functionality, despite significantly increased affinity to oil and carbon content on the surface. The coated microspheres (EU-MS) exhibited a profoundly decreased swelling ratio, an even higher affinity to oil, a higher loading capacity, and a lower drug release rate. Through further coating of the EU-MS with different amounts of corn oil, the rate of drug release could be tailored to cover a relatively wide range. These results suggest that a versatile delivery system with various release profiles can be prepared by a combination of hydrophobic modification, polymer coating, and oil coating.

Triton-X-100-modified polymer and microspheres for reversal of multidrug resistance.

Triton X-100 is a non-ionic detergent capable of reversing multidrug resistance (MDR) due to its interaction with cell membranes. However, it interacts with cells in a non-specific way, causing cytotoxicity. This work aimed to develop polymeric chemosensitizers that possess the ability to reverse MDR and lower toxic side effects. When being delivered to tumours, the polymeric chemosensitizers may also have longer retention times in tumours than the free detergent. Triton-X-100-immobilized dextran microspheres (T-MS) and inulin (T-IN) were prepared and characterized. Their cytotoxicity against multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was compared with that of free Triton X-100 solutions. The in-vitro effect of the products on 3H-vinblastine accumulation by CH(R)C5 cells was determined. Both T-MS and T-IN showed a marked decrease in the cytotoxicity, as compared with free Triton solutions at equivalent concentrations. Drug accumulation by CH(R)C5 cells was increased over two fold in the presence of T-MS or T-IN. These results suggest that polymeric drug carriers with MDR-reversing capability and lower cytotoxicity may be prepared by immobilization of chemosensitizers.

Medication use patterns in HIV-positive patients.

Patients with HIV infection or AIDS often receive several medications for treatment or prevention of their primary disease and/or associated complications. The objective of this study was to document patterns of drug use in an HIV-positive, outpatient population. Data were collected via one-on-one interviews with 26 HIV-positive patients; prescription and non-prescription drug use, adverse drug reactions and drug allergies were recorded. Anti-retroviral therapy was received by over 90 % of the patients. Over 90% of patients were on anti-infective agents, commonly cotrimoxazole or dapsone, while 27% received acyclovir. At least 70% of patients used three or more prescription medications concurrently. Every patient reported self-medicating with at least one over-the-counter (OTC) product and over half used three or more OTC products concurrently. Adverse reactions, mainly attributable to zidovudine, were reported by over 80% of patients. Non-compliance was a common drug-related issue; over 70% of patients omitted drug doses. In conclusion, the use of multiple medications in the ambulatory HIV-positive patients presents the health care team with potential drug-related problems that may ultimately affect the efficacy and toxicity of therapy. Thus pharmacists may play an active role in the provision of direct care to these patients.

Pharmacokinetic interaction between zidovudine and trimethoprim/sulphamethoxazole in HIV-1 infected children.

OBJECTIVE: To evaluate the effect of the antimicrobial agent trimethoprim/sulphamethoxazole (TMP/SMX) on the pharmacokinetic properties of the antiretroviral drug zidovudine (ZDV). DESIGN: This single dose, open label, crossover study involved the oral administration of ZDV (150 mg/m²) alone and in combination with oral TMP/SMX (2.5 mg/kg) on two separate occasions. Serial blood samples (0 to 8 h) were collected, and concentrations of ZDV and its glucuronide metabolite were quantified using a radioimmunoassay. ZDV pharmacokinetics were determined by noncompartmental analysis. PATIENTS AND SETTING: Six HIV-1 infected children aged four months to five years were recruited from the HIV clinic at The Hospital for Sick Children, Toronto, Ontario. Only three patients completed both study phases and were included in the pharmacokinetic analysis. MAIN RESULTS: With TMP/SMX therapy, no statistically significant changes were observed in ZDV pharmacokinetic parameters. However, there was a trend towards increased ZDV half-life and area under the concentration versus time curve, as well as decreased apparent oral clearance. Similarly, a trend towards an increased half-life of the ZDV-glucuronide metabolite was also observed. CONCLUSION: The changes in ZDV pharmacokinetics in the presence of TMP/SMX did not reach statistical significance, most likely due to the limited number of patients involved. Despite the limited data, a possible interaction between ZDV and TMP/SMX in young HIV-1 infected children should be considered, and patients may require close clinical monitoring.

Interaction of dipyridamole, a nucleoside transport inhibitor, with the renal transport of organic cations by LLCPK1 cells.

Dipyridamole is a well-known inhibitor of nucleoside transport by various cell membranes and is frequently used in in vitro studies that characterize nucleoside transport properties. Because interactions between the renal transport of organic cations and nucleosides have previously been suggested, we studied the effect of dipyridamole on the renal transport of the typical organic cations cimetidine and N1-methylnicotinamide by LLCPK1 monolayer cells grown on a permeable support. [14C]Mannitol was used to correct for extracellular flux. Basolateral to apical transcellular flux (transepithelial flux-extracellular flux) of [3H]cimetidine was significantly reduced by the monolayer cells (90%) in the presence of 50 microM dipyridamole. In addition, the effect of dipyridamole on cimetidine renal transport was dose dependent (IC50 = 7.7 microM). The dipyridamole inhibitory effect was nearly comparable with the effect of 1 mM quinine (a typical organic cation transport inhibitor), which led to 95% inhibition of cimetidine renal transport over time. The dipyridamole effect on N1-methylnicotinamide renal transport was less potent. The effect of 1 mM of typical probes of the nucleoside transporters (i.e., thymidine, adenosine, uridine) and the effect of 100 nM of another nucleoside transport inhibitor, dilazep, were also studied on cimetidine transport by LLCPK1 monolayer cells. These compounds did not exert any significant effect. These results suggest that dipyridamole, a widely used nucleoside transport inhibitor, is also an inhibitor of organic cation renal transport and they alert us to possible interactions between the renal transport of nucleosides and organic cations. This finding also has relevance to the interpretation of in vitro studies using this agent as a nucleoside membrane transport inhibitor.

Uptake properties of lamivudine (3TC) by a continuous renal epithelial cell line.

The purpose of this study was to characterize the renal uptake properties of the cytidine analog and antiretroviral agent 3TC. The uptake of radiolabelled 3TC was measured at 37 degrees C in a continuous porcine renal epithelial cell line (i.e., LLC-PK1 cells) grown as a monolayer on an impermeable support. 3TC (5 microM) uptake (37 degrees C) by the monolayer cells was saturable (Km = 1.2 +/- 0.2 mM) but not significantly altered by various dideoxynucleoside analog drugs, nucleosides, and nucleoside transport inhibitors, suggesting that a nucleoside transporter is not involved in 3TC uptake. A number of endogenous organic cation probes and inhibitors significantly reduced 3TC uptake by the monolayer cells. Quinine, trimethoprim (TMP), and tetraethylammonium (TEA) inhibited 3TC uptake in a dose dependent manner with IC50 values of 0.6 mM, 0.63 mM, and 1.9 mM, respectively. In turn, the uptake of the typical organic cation substrate TEA was inhibited by high concentrations of 3TC. An outwardly directed proton gradient significantly increased the uptake of 3TC by the monolayer cells, suggesting the involvement of a proton exchange process. Conversely, in the presence of monensin, a Na+/H+ ionophore, the uptake of 3TC was significantly reduced. These results suggest that the uptake of 3TC by a cultured renal epithelium may be mediated by an organic cation-proton exchanger. The observed clinical interaction between 3TC and trimethoprim may be explained by competition for a common renal organic cation tubular transporter.

Role of P-glycoprotein in the renal transport of dideoxynucleoside analog drugs.

P-glycoprotein (P-gp), the MDR1 multidrug transporter, is known to be expressed in several human organs and tissues, including the apical membrane of the renal proximal tubular cells. It has been reported that human immunodeficiency virus 1 (HIV-1) can trigger the expression of P-gp in cultured cells (i.e., H9, a T-lymphocyte cell line, and U937, a monocyte cell line), which may render the cells resistant to antiretrovirals. Since multiple membrane transport systems (i.e., organic cation, organic anion, and nucleoside systems) can be involved in the renal tubular transport of dideoxynucleoside analog drugs (DADs) (i.e., zidovudine and zalcitabine), we have questioned if P-gp is involved in the renal transport of DADs. Chinese hamster ovary colchicine-resistant cells (CH(R)C5), a cell line that is well known to highly express P-gp, and continuous renal epithelial cell lines (LLC-PK1 and OK), which have also been shown to express P-gp, were used. The accumulation of [3H]vinblastine (20 nM), an established P-gp substrate, by the monolayer cells was significantly enhanced in the presence of two P-gp inhibitors (i.e., verapamil and cyclosporin A) and nucleoside transport inhibitors (i.e., dipyridamole and dilazep). In contrast, DADs (i.e., zidovudine, lamivudine, didanosine, and zalcitabine) did not significantly affect vinblastine accumulation by these cell lines. These data suggest that P-gp does not play a significant role in the renal tubular transport of DADs. Dipyridamole and dilazep, two nucleoside membrane transport inhibitors, appear to be P-gp inhibitors.

Characterization of cimetidine transport in LLCPK1 cells.

In this study, cimetidine uptake and its regulation by LLCPK1 monolayers were investigated. Uptake was temperature dependent with kinetic and specificity characteristics typical of a carrier-mediated mechanism. With cimetidine uptake in the presence of an excess concentration of the potent inhibitor quinidine as a measure of nonspecific transport, the estimated kinetic parameters for cimetidine uptake at 37 degrees C under steady-state conditions are Km = 32.3 +/- 6.4 microM and Vmax = 20.2 +/- 2.1 pmol/mg per minute. Amiloride, quinidine, and quinine inhibited cimetidine uptake, whereas N1-methylnicotinamide, tetraethylammonium, and guanidine did not. The uptake of cimetidine was increased in the presence of a cell-->lumen H+ gradient, consistent with the behavior of a cimetidine-H+ antiport system. Furthermore, the activity of both the Na(+)-H+ exchanger and H(+)-ATPase acted to dissipate the cell-->lumen H+ gradient, thereby decreasing net cimetidine transport. These results suggest that there is a cimetidine-H+ exchange system in LLCPK1 cells and that the net secretion of organic base in vivo may be regulated by luminal acidification mechanisms.

A novel zidovudine uptake system in microglia.

In the central nervous system (CNS), brain macrophages and microglia are the primary targets of productive human immunodeficiency virus 1 (HIV-1) infection. Zidovudine (ZDV), a thymidine derivative, has been reported to reduce the progression of the disease and prolong survival in patients with acquired immunodeficiency syndrome (AIDS) and AIDS dementia complex. Although a restricted ZDV distribution has been observed in the CNS, its accumulation in brain parenchyma has not been examined. We have investigated the uptake properties of radiolabeled ZDV by a continuous rat microglia cell line (MLS-9) grown as a monolayer on an impermeable surface. Although the organic cations verapamil, mepiperphenidol, quinidine, cimetidine, and N(1)-methylnicotinamide moderately inhibited ZDV uptake, the organic cation probes tetraethylammonium and 1-methyl-4-phenylpyridinium were weak inhibitors. ZDV uptake was significantly increased when the proton gradient was outward (pH(i) 6.3 < pH(o) 7.4; pH(i) approximately 7.1 < pH 8.0), whereas uptake decreased with extracellular acidification (pH(i) approximately 7.1 > pH(o) 6.0) or in the presence of the Na(+)/H(+) ionophore monensin. ZDV uptake was increased under depolarized membrane conditions (i.e., 138 mM K(+) in external medium) and decreased under hyperpolarized conditions (i.e., 2 mM K(+) in external medium), implying a membrane potential dependence. These results suggest that although ZDV transport system in microglia has some specificity features of an organic cation transporter, it involves a carrier, distinct from other cloned organic cation transporters, that is novel in its sensitivity to pH and membrane potential. This system may play a significant role in the transport of other weak organic cation substrates and/or metabolites in brain parenchyma.

A Na(+)-dependent nucleoside transporter in microglia.

In the central nervous system, HIV-1 has a defined tropism for brain macrophages and microglia. Nucleoside analog drugs such as zidovudine improve the clinical and neuropsychological functions in HIV-demented patients. Multiple carrier-mediated transport systems can play an important role in the membrane permeation of nucleosides and nucleoside analog drugs in a number of cells. The purpose of this project was to characterize the uptake properties of the pyrimidine nucleoside probe thymidine by a continuous rat microglia cell line (MLS-9) grown as a monolayer on an impermeable substratum. Approximately 50% of thymidine (10 microM) uptake by the monolayer cells was found to be Na(+) dependent. Kinetics of specific thymidine uptake showed a single saturation system (K(m) = 44 microM at 37 degrees C) and a Na(+)/thymidine stoichiometry of 2:1. Pyrimidine and purine nucleoside probes (50 microM) exerted a competitive inhibitory effect on specific thymidine uptake with K(i) values of 40, 38, 45, and 39 microM for adenosine, uridine, guanosine, and cytidine, respectively. In addition, nucleoside analog drugs significantly decreased specific thymidine uptake, with IC(50) values of 135.1 microM for abacavir and 0.6 microM for zidovudine, which inhibited in a noncompetitive manner. These results suggest that a Na(+)-dependent nucleoside transport system is present in rat microglia and that long-range interactions between antiretroviral nucleoside analog drugs and the nucleoside substrates may occur at the transporter sites.

Digoxin pharmacokinetics and dosage requirements in pediatric patients.

The pharmacokinetic properties and dosage guidelines for digoxin in pediatric patients with congestive heart failure are reviewed. Interindividual variability in the pharmacokinetics of digoxin in pediatric patients has been reported. The bioavailability of digoxin elixir in newborns and infants is similar to adults; however, the apparent volume of distribution has been reported to be greater in infants than in adults. The total body clearance of digoxin is lowest in premature and full-term neonates and highest in infants aged one month to one year. The elimination half-life of digoxin has been reported to vary significantly among the different age groups of pediatric patients. The usefulness of monitoring digoxin serum concentrations in pediatric patients remains a controversial issue. Serum samples should be drawn under steady-state conditions to evaluate predicted daily maintenance doses. Although infants have been reported to be more tolerant than adults to elevated serum digoxin concentrations, infants experience a higher rate of digoxin toxicity than previously realized. Recent studies have shown appropriate therapeutic response in neonates and infants when low dosages of digoxin are administered. Low digoxin dosage regimens should be used initially for infants with congestive heart failure. If the clinical response is unsatisfactory or if toxicity is suspected, steady-state serum concentrations should be determined and the dosage adjusted.

Gentamicin uptake by LLCPK1 cells: effect of intracellular and extracellular pH changes.

The mechanisms by which aminoglycosides are transported across the luminal membrane of renal proximal tubular cells remain unclear. A luminal organic cation/H+ exchange as well as an adsorptive endocytosis membrane process has been proposed to be involved in gentamicin renal accumulation. The objectives of this work were to explore further the effects of intracellular and extracellular pH changes on gentamicin uptake. [3H]Gentamicin uptake by a continuous renal epithelial cell line, LLCPK1, grown as a monolayer on an impermeable surface was measured at different temperatures and pH conditions and in the presence of various inhibitors. Uptake of gentamicin was found to be carrier mediated (K(m) = 1.26 +/- 0.22 mM, Vmax = 289 +/- 27 pmol.mg-1.min-1), energy dependent (inhibited in part by sodium azide), and temperature dependent (37 degrees C > 4 degrees C). Fifteen-minute gentamicin (10 microM) uptake was inhibited by 1 mM of the organic cations cimetidine (61.0%), quinidine (73.5%), quinine (68.6%), and verapamil (61.5%). More importantly, while an outwardly directed proton gradient did not have a significant effect on gentamicin uptake, extracellular acidification (pH 6.5), which leads to a higher degree of gentamicin ionization, significantly enhanced gentamicin uptake by LLCPK1 monolayer cells. These results suggest that the luminal organic cation/H+ exchanger is not involved in gentamicin uptake by renal cultured epithelial cells. Rather, the cationic charge of gentamicin appears to be one of the primary determinants for renal luminal uptake.