Pharmacokinetic interactions between nelfinavir and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors atorvastatin and simvastatin.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are effective agents in lowering cholesterol and triglycerides and are being used by human immunodeficiency virus-positive patients to treat the lipid elevation that may be associated with antiretroviral therapy. Many HMG-CoA reductase inhibitors and protease inhibitors are metabolized by the same cytochrome P450 enzyme 3A4 (CYP3A4). In addition, many protease inhibitors are potent inhibitors of CYP3A4. Therefore, coadministration of these two classes of drugs may cause significant drug interactions. This open-label, multiple-dose study was performed to determine the interactions between nelfinavir, a protease inhibitor, and two HMG-CoA reductase inhibitors, atorvastatin and simvastatin, in healthy volunteers. Thirty-two healthy subjects received either atorvastatin calcium (10 mg once a day) or simvastatin (20 mg once a day) for the first 14 days of the study. Nelfinavir (1,250 mg twice a day) was added on days 15 to 28. Pharmacokinetic assessment was performed on days 14 and 28. The study drugs were well tolerated. Nelfinavir increased the steady-state area under the plasma concentration-time curve during one dosing period (AUC(tau)) of atorvastatin 74% and the maximum concentration (C(max)) of atorvastatin 122% and increased the AUC(tau) of simvastatin 505% and the C(max) of simvastatin 517%. Neither atorvastatin nor simvastatin appeared to alter the pharmacokinetics of nelfinavir. It is recommended that coadministration of simvastatin with nelfinavir should be avoided, whereas atorvastatin should be used with nelfinavir with caution.

Lack of effect of cimetidine on the pharmacokinetics of sustained-release bupropion.

The objective of this study was to assess whether cimetidine affects the pharmacokinetics of sustained-release (SR) bupropion hydrochloride and the active metabolite, hydroxybupropion. This randomized, open-label, two-period crossover study was conducted in 24 healthy volunteers 18 to 45 years of age. ANOVA showed that administration of two 150 mg bupropion SR tablets with one 800 mg cimetidine tablet following an overnight fast did not change values for AUC infinity, Cmax, tmax, t1/2, and CL/F (CL/F calculated for bupropion only) for either bupropion or hydroxybupropion as compared with two 150 mg bupropion SR tablets alone. In this study, it appears that there is no effect of cimetidine on the pharmacokinetics of bupropion SR.

Effect of cationic drugs on the renal secretion of ranitidine in the rat isolated perfused kidney.

1. The rat isolated perfused kidney (IPK) was used to determine whether the renal tubular secretion of ranitidine is influenced by clinically relevant concentrations of other organic cationic drugs (amantadine, pseudoephedrine, triamterene and trimethoprim) that also undergo tubular secretion. 2. Ranitidine and [3H]-ranitidine were administered to the recirculating perfusion medium as a loading dose followed by a constant infusion to maintain clinically relevant perfusate ranitidine concentrations in the range 400-700 ng/mL. The renal clearance of ranitidine (CL[R]) was calculated, as was glomerular filtration rate (GFR), from the renal clearance of [14C]-inulin. 3. A total of 20 perfusions were performed and, in each case, ranitidine was administered for 80 min. In four control IPK, no drug other than ranitidine was administered. In the remaining IPK, amantadine, pseudoephedrine, triamterene or trimethoprim (n = 4 in each case) were administered to achieve low, medium and high concentrations during the 20-40, 40-60 and 60-80 min periods, respectively. 4. The mean (+/- SD) unbound fraction of ranitidine in the perfusion medium was 0.889 +/- 0.046 and was not altered (P>0.05) by the presence of the other drugs. 5. The CL(R)/GFR ratio for ranitidine in all kidneys was substantially greater than unity and had a mean value of 10.65 or greater in control kidneys, indicating extensive net tubular secretion. 6. The CL(R)/GFR was not affected (P>0.05) by amantadine, pseudoephedrine or triamterene at any concentration or by trimethoprim at the low concentration. However, medium (2000 ng/mL) and high (5000 ng/mL) concentrations of trimethoprim caused significant reductions in CL(R)/GFR of 20 and 28%, respectively (P<0.05). 7. The results indicate that at clinically relevant concentrations the renal tubular secretion of ranitidine is inhibited by trimethoprim, but not by amantadine, pseudoephedrine or triamterene.

Pharmacokinetics of bupropion and its metabolites in cigarette smokers versus nonsmokers.

Bupropion is an antidepressant agent that is also effective as an aid to quit cigarette smoking. A single 150-mg tablet of sustained-release bupropion hydrochloride was administered to two groups of volunteers, cigarette smokers and nonsmokers, who were matched for race, gender, body frame, age, and weight. Pharmacokinetic parameters were calculated for bupropion, and three major metabolites (hydroxybupropion and the aminoalcohol isomers, threohydrobupropion and erythrohydrobupropion, expressed as a composite total). Mean (+/-SD) values of area under the concentration-time curve from time 0 extrapolated to infinity (AUC0-infinity), maximum concentration (Cmax), time to reach Cmax (tmax), and half-life (t1/2) of bupropion in smokers and nonsmokers, respectively, were 1,164 +/- 220 ng.hr/mL and 1,161 +/- 292 ng.hr/mL; 144 +/- 28 ng/mL and 143 +/- 39 ng/mL; 3.00 +/- 0.50 hours and 2.88 +/- 0.49 hours; and 19 +/- 5 hours and 18 +/- 3 hours. No clinically significant differences between smokers and nonsmokers or between male and female volunteers were observed for the pharmacokinetics of bupropion or its metabolites. The absence of pharmacokinetic differences indicates that dosage adjustments are not necessary when bupropion is prescribed to male and female cigarette smokers.

Pharmacokinetics of amiloride after inhalation and oral administration in adolescents and adults with cystic fibrosis.

STUDY OBJECTIVE: To compare the pharmacokinetics and systemic exposure of nebulized and oral amiloride in adolescents and adults with mild to moderate cystic fibrosis (CF). DESIGN: Open-label, randomized, two-way crossover, single-dose pharmacokinetic study. SETTING: University hospital clinical research unit. PATIENTS: Nine adolescents and 10 adults with mild to moderate CF (forced expiratory volume in 1 sec > or = 50% predicted, Brasfield score > or = 15). INTERVENTIONS: Patients received amiloride solution orally (10 mg of amiloride 1-mg/ml solution) and by inhalation [4.5 ml amiloride of 1-mg/ml solution in 12% saline (approximately 3.8 mmol/L) by DeVilbiss 646 nebulizer] during two study phases separated by a 7- to 28-day washout period. Serial blood and urine samples were collected for 48 and 72 hours, respectively. MEASUREMENTS AND MAIN RESULTS: After oral dosing, the mean +/- SD maximum peak concentration (Cmax) was 20.6 +/- 10.0 ng/ml at 3.2 +/- 1.2 hours in adults and 21.7 +/- 4.88 at 2.9 +/- 0.6 hours in the adolescents. Mean area under the concentration-time curve (AUC) from time zero to infinity hours was 275 +/- 115 and 254 +/- 60 ng.hr/ml in the adult and adolescent groups; half-life was 16.0 +/- 0.7 and 13.4 +/- 1.4 hours, respectively. After nebulization, 14 of 19 subjects exhibited two concentration peaks (Cmax1 and Cmax2) with mean values of 1.57 +/- 1.67 ng/ml at 0.5 +/- 0.2 hours and 1.37 +/- 1.21 ng/ml at 4.0 +/- 1.0 hours for adults, and 1.49 +/- 0.99 ng/ml at 0.5 +/- 0.1 hours and 1.52 +/- 0.81 ng/ml at 3.3 +/- 0.5 hours for adolescents. Estimated mean +/- SD dose nebulized was 1.91 +/- 0.66 and 2.28 +/- 0.30 mg in the adult and adolescent groups, respectively. Mean +/- SD AUC from time zero to the last measurable plasma amiloride concentration after inhalation was 14.4 +/- 17.6 and 15.4 +/- 10.1 ng.hr/ml in the adults and adolescents. No significant adverse events occurred during the study. Pharmacokinetic parameters were not statistically different between the adolescent and adult groups by route of administration. However significant differences in peak amiloride concentration, AUC, and urinary amiloride excretion were evident when comparing oral versus inhalation administration within each group. CONCLUSIONS: Mean amiloride plasma concentration peaks and AUC after inhalation were significantly lower than after oral dosing. In addition, the second amiloride plasma concentration peak may be due to oral ingestion of the nebulized amiloride, whereas the earlier Cmax1 after inhalation may be due to pulmonary absorption of amiloride. These results suggest that single-dose amiloride inhalation in patients with mild to moderate CF results in minimal systemic exposure compared with oral dosing, and that drug disposition is similar in adolescents and adults with CF.

Pharmacokinetics of lamivudine in human immunodeficiency virus-infected patients with renal dysfunction.

The purpose of this study was to determine the safety and pharmacokinetics of lamivudine (3TC), a nucleoside analog that has shown potent in vitro and recent in vivo activity against human immunodeficiency virus. Sixteen human immunodeficiency virus-infected patients, six with normal renal function (creatinine clearance [CLCR], > or = 60 ml/min), four with moderate renal impairment (CLCR, 10 to 40 ml/min), and six with severe renal impairment (CLCR, < 10 ml/min), were enrolled in the study. After an overnight fast, patients were administered 300 mg of 3TC orally. Blood was obtained before 3TC administration and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 32, 40, and 48 h afterward. Timed urine collections were performed for patients able to produce urine. Serum and urine were assayed for 3TC by reverse-phase high-performance liquid chromatography with UV detection. Pharmacokinetic parameters were calculated by using standard noncompartmental techniques. The peak concentration of 3TC increased with decreasing renal function; geometric means were 2,524, 3,538, and 5,684 ng/ml for patients with normal renal function, moderate renal impairment, and severe renal impairment, respectively. The terminal half-life also increased with decreasing renal function; geometric means were 11.5, 14.1, and 20.7 h for patients with normal renal function, moderate renal impairment, and severe renal impairment, respectively. Both oral and renal clearances were linearly correlated with CLCR. A 300-mg dose of 3TC was well tolerated by all three patient groups. The pharmacokinetics of 3TC is profoundly affected by impaired renal function. Dosage adjustment, by either dose reduction or lengthening of the dosing interval, is warranted.

Renal disposition and drug interaction screening of (-)-2'-deoxy-3'-thiacytidine (3TC) in the isolated perfused rat kidney.

PURPOSE: Dideoxynucleoside bases are used for the treatment of acquired immune deficiency syndrome (AIDS), acting by inhibiting reverse transcriptase and preventing human immunodeficiency virus (HIV) replication. Currently, AZT (zidovudine), ddC (zalcitibine), and ddI (didanosine) are available to the medical community to prevent the onset of AIDS in HIV-infected individuals. 3TC (-)-2'-deoxy-3'-thiacytidine, lamivudine), a new dideoxynucleoside base, is currently undergoing Phase II/III trials, and has exhibited anti-HIV replication activity, a favorable adverse event safety profile, and is eliminated via renal mechanisms. Concomitantly administered drugs could potentiate the effects of 3TC due to interaction in the kidney. METHODS: An isolated perfused rat kidney (IPK) technique was used to screen several clinically relevant drugs for potential interaction with 3TC. The following perfusions were performed: baseline 3TC; and 500 ng/mL 3TC with clinically relevant concentrations of AZT, ddC, ddI, probenecid, trimethoprim, sulfamethoxazole, ranitidine, and cimetidine. RESULTS: Renal clearance of 3TC was nonlinear between 500 and 5000 ng/mL, decreasing from 3.06 to 1.74 mL/min. Excretion ratio also decreased, from 3.67 (500 ng/mL) to 2.49 (5000 ng/mL), consistent with a decrease in 3TC secretion. AZT, ddI, and ddC elicited no or minimal effects on 3TC elimination at the concentrations studied. However, trimethoprim caused significant reductions in 3TC elimination parameters: clearance and excretion ratio decreased to 1.25 mL/min and 1.43, respectively. CONCLUSIONS: These results indicate that caution should be exercised when the combination of 3TC and trimethoprim are administered to AIDS patients.

Pharmacokinetics and cyclooxygenase inhibition of itazigrel in normal volunteers after single oral doses.

The pharmacokinetics and cyclooxygenase inhibition of itazigrel were studied in normal male volunteers. In a low-dose study, subjects received a single oral dose of 5-100 mg of itazigrel. Serum concentration and the production rate of thromboxane B2, an indicator of cyclooxygenase activity, were monitored for 48 h. In a high-dose study, single oral doses of 100-600 mg of itazigrel were administered. Serum concentrations were monitored for 72 h. Production rates of thromboxane B2 and leukotriene B4, an indicator of lipoxygenase activity, were monitored for the first 2 h after drug administration. Pharmacokinetics of itazigrel appeared to follow biexponential elimination with an alpha half-life between 1.2 and 2 h and a beta half-life between 23 and 28 h. The relationship between dose and area under the serum concentration curve was nonlinear, probably due to saturable systemic metabolism or saturable first-pass metabolism. Cyclooxygenase inhibition by itazigrel was related to the serum concentration by the Hill's equation with a mean IC50 value of 2.1 ng/mL. Itazigrel did not appear to affect the lipoxygenase activity in the study.

Pharmacokinetics of a novel retinoid AGN 190168 and its metabolite AGN 190299 after intravenous administration of AGN 190168 to rats.

The pharmacokinetics of AGN 190168, a novel synthetic retinoid, and its major metabolite, AGN 190299, in rat blood after intravenous administration was investigated. Approximately 4.4 mg kg-1 (high dose) or 0.49 mg kg-1 (low dose) of AGN 190168 was administered to rats via the femoral vein. Blood was collected from the femoral artery at various time points during an 8 h period. Blood concentrations of AGN 190168 and AGN 190299 were determined by a specific and sensitive high-pressure liquid chromatographic (HPLC) method. AGN 190168 was rapidly metabolized in rats. The only detectable drug-related species in the blood was AGN 190299. Therefore, only pharmacokinetics of AGN 190299 were calculated. Elimination of AGN 190299 appeared to be non-linear after administration of the high dose, and linear after administration of the low dose. The maximum elimination rate (Vmax) and the concentration at half of the Vmax (km), as estimated by a Michaelis-Menten one-compartment model, were 7.58 +/- 2.42 micrograms min-1 (mean +/- SD) and 6.10 +/- 1.58 micrograms mL-1, respectively. The value of the area under the blood concentration time curve (AUC) was 9.54 +/- 1.68 micrograms h mL-1 after administration of the high dose and 0.594 +/- 0.095 micrograms h mL-1 after administration of the low dose. The clearance value was 7.79 +/- 1.20 mL min-1 kg-1 after the high dose, statistically significantly different from that after the low dose (p < 0.05), 14.0 +/- 2.2 mL min-1 kg-1. The terminal half-life (t1/2) was 1.25 +/- 0.74 h for the high-dose group and 0.95 +/- 0.16 h for the low-dose group. Study results demonstrate rapid systemic metabolism of AGN 190168 to AGN 190299, non-linear pharmacokinetics of AGN 190299 after the 4.4 mg kg-1 dose, and the lack of difference in disposition profiles between sexes after intravenous administration of AGN 190168 to rats.

Oral ondansetron pharmacokinetics: the effect of chemotherapy.

The effect of a typical 5-day chemotherapy treatment with cisplatin (20-40 mg/m2 per day) and 5-fluorouracil (5-FU, 1 gm/m2 per day) on the pharmacokinetics of ondansetron was investigated. Twenty cancer patients received 8 mg of ondansetron in three periods, including an oral tablet on day 1, an intravenous infusion on day 4, and an oral tablet on day 5. Absolute bioavailability after the oral dosing on day 1 was 87.5 +/- 31.3%, and on day 5 was 85.2 +/- 22.1% (P > .05). Mean values of AUC, Cmax, Tmax, and half life on days 1 and 5 were 399 +/- 275 and 381 +/- 222 ng.hour/mL, 53.3 +/- 26.8 and 43.6 +/- 21.7 ng/mL, 1.9 +/- 1.4 and 2 +/- 1.4 hours, and 5.21 +/- 1.78 and 6.19 +/- 1.99 hours, respectively. These values were not significantly different (P > .05). In summary, this study showed that cisplatin and 5-FU did not significantly alter the pharmacokinetics of oral ondansetron in cancer patients during the 5 days of chemotherapy. Oral bioavailability of ondansetron appeared to be greater in cancer patients than in healthy subjects.

Automated high-performance liquid chromatographic analysis of (-)-2'-deoxy-3'-thiacytidine in biological fluids using the automated sequential trace enrichment of dialysate systems.

A fully automated HPLC procedure was developed for the analysis of a small volume of perfusion solutions from an isolated perfused rat kidney study. The method involved separation of (-)-2'-deoxy-3'-thiacytidine (3TC) from the matrix by dialysis with 10 mM potassium phosphate buffer pH 3.0. 3TC was subsequently separated from the dialysate as it flowed through a SCX cation-exchange cartridge. The trapped 3TC was then eluted with a mobile phase of 50 mM ammonium acetate buffer (pH 5.5)-methanol (90.5:9.5, v/v) at a flow-rate of 1.0 ml/min for 2 min. The eluent was directed to the HPLC system and chromatographed with a BDS C18 analytical column at a temperature of 45 degrees C. Detection of 3TC was carried out by UV absorption at 274 nm. The procedure was validated from 25 to 10,000 ng/ml. Coefficients of variance (C.V.) of 3TC quality control samples were less than 9%. C.V.s of the standard curve samples were also less than 10% except for the 25 ng/ml samples (11.5%). The mean interpolated concentrations were within 8% of the nominal concentrations for all samples. No interference from concurrent drugs was observed. Preliminary results suggested that this procedure may also be used for human serum and urine samples.

Validation of a liquid chromatography post-column derivatization assay for the determination of cisplatin in plasma.

Method validation results are described for a cisplatin LC post-column derivatization assay. Cisplatin plasma samples were treated with acetonitrile and a citrate buffer solution to enhance cisplatin stability. Processed samples were analysed on a chemically generated anion exchange column using a customized post-column derivatization platform and refrigerated autosampler. The UV response was monitored at 290 nm. The retention time of cisplatin was 9 min. The assay was linear from 0.06 to 30.0 micrograms ml-1 (r > 0.998) with inter-run precisions (RSD) of 8.2% (n = 8), 5.9% (n = 8) and 4.0% (n = 8) for low (0.18 microgram ml-1), medium (1.5 microgram ml-1) and high (24.0 micrograms ml-1) quality control samples, respectively. The validated assay was used to monitor cisplatin levels in cisplatin drug interaction studies.

Comparison of the pharmacokinetics of an ondansetron solution (8 mg) when administered intravenously, orally, to the colon, and to the rectum.

Ondansetron, an antagonist of the serotonin type 3 (5-HT3) receptor, is indicated for the treatment of chemotherapy-induced emesis. This study compares the pharmacokinetics, especially the bioavailability, of an ondansetron 8-mg solution when administered intravenously, orally, to the colon via nasogastric intubation, and to the rectum using a retention enema. Six healthy, male volunteers received ondansetron infused into the colon during the first treatment period. These subjects then received the remaining three treatments in random order, with a minimum 1-week washout period between treatments. Serial plasma samples were obtained for up to 24 hr after dosing in each treatment period. Absolute bioavailability after the oral dosing, colonic infusion, and rectal administration averaged 71 +/- 14, 74 +/- 26, and 58 +/- 18%, respectively. These values were not significantly different (P > 0.05). Values of Tmax and Cmax were also not significantly different among the nonparenteral routes. Mean absorption half-lives were 0.66, 1.1, and 0.75 hr after the oral, colonic, and rectal administrations, respectively. These results indicate that ondansetron is well absorbed in the intestinal segments studied including the upper small intestine, the colon, and the rectum and that sustained-release and suppository formulations of ondansetron are feasible.

Mechanism of intestinal absorption of ranitidine and ondansetron: transport across Caco-2 cell monolayers.

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability co-efficients (Papp) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. Papp values for ranitidine and ondansetron (bioavailability of 50 and approximately 100% in humans, respectively) were 1.03 +/- 0.17 x 10(-7) and 1.83 +/- 0.055 x 10(-5) cm/sec, respectively. The Papp value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.

Pharmacokinetic interactions between arbaprostil and aspirin in humans.

Arbaprostil is an orally active prostaglandin E2 analogue. It has been developed as a drug to treat ulcers induced by non-steroidal anti-inflammatory drugs. In this study, pharmacokinetic interactions between arbaprostil and aspirin were examined in humans after chronic doses of both drugs. Subjects received either arbaprostil (50 micrograms), aspirin (975 mg) or arbaprostil (50 micrograms) and aspirin (975 mg) four times a day for 6 days and one dose on 7th day. Blood and urine samples were collected after the last dose for 6 h. Pharmacokinetic parameters of arbaprostil, aspirin, and salicylate were determined. Coadministration of arbaprostil significantly lowered the area under curve (5.09 +/- 0.32 micrograms hml-1 vs 5.78 +/- 0.29 micrograms hml-1, mean +/- SE, p less than 0.05) and time (0.45 +/- 0.07 h vs 0.70 +/- 0.12 h, p less than 0.05) to reach maximal plasma concentration of aspirin (acetylsalicylate). The pharmacokinetics of salicylate were not changed by arbaprostil, nor were the pharmacokinetics of arbaprostil affected by aspirin. Coadministration of these two drugs did not appear to potentiate the side-effects of either drug. The results suggest that arbaprostil and aspirin may be administered together without clinically significant changes in pharmacokinetics or adverse side-effects.

Renal transport of drugs: an overview of methodology with application to cimetidine.

The development of new methods to study transport processes in renal epithelia has greatly enhanced our knowledge of the mechanisms involved in the transport of a number of endogenous compounds. More recently, these methods have been applied to study mechanisms of specific drug transport. This article is intended to provide an overview of the various methods used to study renal elimination of compounds. References to more detailed reviews of the individual methods are provided. Studies of the renal transport of cimetidine, a histamine H2-receptor antagonist, are presented to illustrate the application of these methods to the study of specific drugs. Methods such as clearance techniques and the Sperber chicken preparation used to study renal elimination of compounds in whole animals are briefly described. Techniques to identify the site of renal transport including stop flow, isolated perfused tubules, and micropuncture methods are discussed and references to more technical reviews are cited. The more recently developed methods of isolated membrane vesicles for studying transport across the individual polar membranes of the proximal tubule are discussed along with the relevant studies of the use of these membranes in elucidating the mechanisms involved in the renal transport of cimetidine. Finally, the use of cultured renal epithelial cell lines in studying renal transport is described. Knowledge of drug transport mechanisms in the kidney is important both in drug targeting to the kidney and in understanding the pharmacokinetics of renally eliminated drugs. As exemplified by the studies with cimetidine, only by combining the data from experiments using diverse methodology can the mechanisms involved in the renal excretion of compounds be delineated.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of organic anions with the organic cation transporter in renal BBMV.

It is generally assumed that the organic cation transport system in the renal proximal tubule is specific for organic cations and the transport of organic cations is not affected by organic anions. However, there are also data in the literature demonstrating that probenecid, a classical inhibitor of organic anion transport systems, inhibits the transport of an organic cation, cimetidine, in the renal proximal tubule. In this study we investigated the effects of probenecid and furosemide on the transport of N'-methylnicotinamide (NMN) the classical substrate of the organic cation transporter, in brush-border membrane vesicles prepared from rabbit renal cortex. In the presence of a pH gradient, both probenecid (10 mM) and furosemide reduced the initial uptake of NMN. Probenecid reduced the initial uptake of NMN to 12.1% of the control values (1.19 +/- 0.26 pmol/mg) and furosemide reduced the initial uptake of NMN to 39.2%. Probenecid (10 mM) also decreased the initial transport of NMN in the absence of a pH gradient. Inhibition of the transport of NMN by probenecid was concentration dependent, with the concentration of probenecid resulting in 50% inhibition of the transport of NMN equal to 2.31 +/- 1.18 mM in the presence of a pH gradient. Probenecid appeared to be a competitive inhibitor of NMN transport. The apparent Km (mean +/- SE) of NMN transport (2.01 +/- 0.78 mM) was increased to 18.7 +/- 10 mM (P less than 0.05) by probenecid (10 mM), whereas the Vmax was not changed (125 +/- 19.2 pmol.s-1.mg-1 vs. 186 +/- 94 pmol.s-1.mg-1, P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Essential tyrosine residues in transport of organic cations in renal BBMV.

The effects of tyrosine modifying agents on organic cation transport in brush-border membrane vesicles prepared from rabbit renal cortex were investigated. Treatment of membranes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and N-acetylimidazole reduced the initial rate of transport of N'-methylnicotinamide (NMN) significantly. The inactivation of NMN transport by NBD-Cl was concentration and time dependent. The maximal transport rate (Tmax) of NMN transport (18.4 +/- 4.3 pmol X s-1 X mg protein-1) in vesicles treated with NBD-Cl (0.15 mM) was reduced to 56% of the Tmax in the control vesicles (32.6 +/- 8.4 pmol X s-1 X mg protein-1, P less than 0.05); whereas the Km was not changed. Treatment with 2-mercaptoethanol reversed the reaction of NBD-Cl with sulfhydryl groups but did not significantly change the transport of NMN in the control or the treated membranes, suggesting that tyrosine but not sulfhydryl residues are involved. The overshoot of NMN uptake in the presence of a proton gradient was 3.38 +/- 0.67 pmol/mg protein in the untreated membranes and was reduced to 2.05 +/- 0.71 pmol/mg protein in the NBD-Cl-treated membranes (P less than 0.05). Studies with the pH-sensitive dye, acridine orange, demonstrated that NBD-Cl-treated vesicles were not leakier with respect to protons, suggesting that NBD-Cl may have specifically affected the organic cation-proton transporter.(ABSTRACT TRUNCATED AT 250 WORDS)