Molecular mechanisms of membrane polarity in renal epithelial cells.

Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context.

Pharmacokinetics of fluorouracil in humans.

A high-pressure liquid chromatography method has been used to investigate the pharmacokinetics of fluorouracil after single i.v. doses to patients. The method is simple and is specific for fluorouracil. No interference was observed in fluorouracil determinations due to other administered medication. Plasma levels of fluorouracil declined rapidly after dosing. The mean half-life was 11.4 min, and drug was essentially cleared from plasma in 1 hr. Individual differences in plasma fluorouracil levels, and also in derived pharmacokinetic constants, were considerably less than those reported previously.

A mutation linked with Bartter's syndrome locks Kir 1.1a (ROMK1) channels in a closed state.

Mutations in the inward rectifying renal K(+) channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332-391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP-wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332-351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland.

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K(+) (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K(+) currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (beta(1)gamma(2)) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K(+) currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.

Prediction of drug dosage in patients with renal failure using data derived from normal subjects.

A one-compartment model designed to predict alterations in persistence of drugs in uremic patients was constructed using information obtained from normal subjects. Data obtained from the literature in which the fraction of absorbed drug eliminated unchanged in the urine and the apparent elimination rate constants were compared in both normal control subjects and in severely uremic patients. Twenty-two drugs were examined. Despite changes in apparent volume of distribution and metabolism reported in uremia, the model was able to predict overall elimination rate constants in severe uremia with an error under 10% for 12 and under 20% for 7 additional drugs. The method appears to be most informative for drugs that tend to be retained in the presence of renal failure. Great error was observed with doxycycline and erythromycin. Studies with erythromycin lactobionate and a cupplate assay reduced the error for this drug from 56% for the glucoheptonate to 18%. Doxycycline is known to have a complex enterohepatic circulation. The model is offered as a useful approach to predict dosage adjustment in uremic patients with drugs for which data are not available.

Error dependent on renal function when monoexponential equation assumed. Serum clearances of 125I-iothalamate and 131I-o-iodohippurate.

An example from the literature has been used to demonstrate errors involved in calculating drug clearance by inappropriate use of the apparent drug distribution volume Vdext. The Vdext is always an overestimate of the true volume of distribution in a multicompartment system, and the degree of overestimation in using it to calculate clearance for such a system will increase as renal function increases. Drug dosages calculated on the basis of overestimated clearance values may give rise to overdosage in normal individuals, or therapeutic failure in severely uremic patients. Problems associated with the use of an oversimplified pharmacokinetic model for clearance calculations are discussed, together with the concept of model-independent calculations.

Influence of diet and fluid on bioavailability of theophylline.

The influence of various test meals, and of fluid volumes, on the bioavailability of theophylline from a solid dosage form has been studied in healthy male volunteers. Absorption of a drug was faster after dosing immediately following a high protein meal than after a high fat or a high carbohydrate meal. Absorption from a solution was faster than from a solid dosage form in all treatments; areas under serum level time curves after dosing were also significantly higher up to 12 hr. Areas up to 12 hr after dosing also tended to be higher after the high protein meal and after dosing with 500 ml water on an empty stomach than after other solid dose treatments.

Reduction in oral penicillamine absorption by food, antacid, and ferrous sulfate.

Plasma levels of penicillamine, urinary recovery of penicillamine and its oxidized metabolites, and urinary excretion of copper were examined after single 500-mg oral doses of penicillamine to six healthy men. Penicillamine was given after an overnight fast, a standard breakfast, and after antacid and ferrous sulfate. Following the fasting dose, the mean peak plasma level of 3.05 micrograms/ml developed at 3.8 hr and the drug was cleared from plasma with a t1/2 of 2.1 hr. Penicillamine levels were reduced to 52%, 35%, and 66% of those from the fasting dose after food, ferrous sulfate, and antacid. The rates of penicillamine appearance and disappearance from plasma were essentially treatment independent. There were good correlations between urinary recovery of total penicillamine (r = 0.875), between urinary copper excretion (r = 0.758) and the penicillamine plasma concentration AUCs. The availability of oral penicillamine is very susceptible to interactions with other substances. Further studies may be necessary to assess the full clinical significance of these interactions.

Propoxyphene and norpropoxyphene: influence of diet and fluid on plasma levels.

The influence of various test meals and ingested fluid volumes on the bioavailability and pharmacokinetics of propoxyphene and its major metabolite norporpoxyphene has been studied in healthy human subjects. The absorption of drug was delayed by all test meals, but the overall efficiency of absorption was either not affected or was slightly increased. Increased fluid volume intake decreased propoxyphene bioavailability. Plasma levels of metabolite correlated well with levels of unchanged drug, particularly in the first 2 hr after dosing, but were not markedly influenced by treatments.

The theophylline-enoxacin interaction: I. Effect of enoxacin dose size on theophylline disposition.

Theophylline interacts pharmacokinetically with a variety of other drugs. Recently enoxacin was found to change theophylline's disposition. In a four-subject, four-way crossover study enoxacin was administered every 12 hours at four levels (0, 25, 100, and 400 mg) for 14 doses. With the ninth dose of enoxacin, 200 mg theophylline was coadministered. Blood and urine samples were assayed by sensitive and specific assays for the parent drugs and their metabolites. Significant reduction in the formation of theophylline's three major metabolites occurred on coadministration of enoxacin. At the 400 mg dose level, enoxacin caused a threefold decrease in theophylline's plasma clearance, a fourfold decrease in the urinary recovery of 3-methylxanthine and 1,3-dimethylurate, and a threefold decrease in the recovery of 1-methylurate.

Pharmacokinetic disposition of 14C-glyburide in patients with varying renal function.

The pharmacokinetics of 14C-labeled glyburide were studied in 13 men with varying degrees of renal impairment. Patients received a single, 5 mg oral dose of glyburide as a solution (10 microCi/ml/mg) after a high-carbohydrate breakfast. Serial plasma and breath samples were collected for 48 hours and urine and feces were collected for 5 to 7 days. Patients with normal to moderately impaired renal function (creatinine clearance [CLCR] of 29 to 131 ml/min/1.7 m2) had glyburide plasma t1/2 values of 2.0 to 5.0 hours, with no relationship between CLCR and glyburide clearance. One subject with severe renal impairment (CLCR = 5 ml/min/1.7 m2) had decreased glyburide clearance that resulted in a t1/2 of 11 hours. The elimination of metabolites was more dependent on renal status but was only significantly affected in the patient with severe renal impairment.

The theophylline-enoxacin interaction: II. Changes in the disposition of theophylline and its metabolites during intermittent administration of enoxacin.

The pharmacokinetics of theophylline and its three major metabolites, 3-methylxanthine, 1-methylurate, and 1,3-dimethylurate, were studied during intermittent administration of enoxacin. The addition of enoxacin (400 mg, twice daily) to a theophylline dosing regimen (150 mg, twice daily) resulted in an immediate fall in plasma theophylline metabolite concentrations. Mean steady-state theophylline concentration in plasma during the dosing interval increased from 3.17 to 8.23 micrograms/ml. The mean 12-hour recovery of total theophylline metabolite decrease from 76.3 to 38.6 mg. After the discontinuation of enoxacin, but not theophylline, the plasma theophylline metabolite levels immediately increased to near or above the concentrations observed before enoxacin coadministration. Concurrently, theophylline concentrations decreased to levels equivalent to those observed before enoxacin coadministration. In general, the changes in plasma theophylline concentrations observed after the addition of discontinuation of enoxacin were complete within 3 days.

Interactions affecting drug absorption.

The influence of drug-drug and drug-food interactions affecting the absorption of orally administered medication is reviewed. Drug-drug interactions can be classified in terms of indirect effects by one drug on gastrointestinal tract physiology influencing the absorption of other drugs, or direct interactions involving altered pH, adsorption, absorption, or chelation. Most, but not all, drug-drug interactions result in reduced or delayed systemic drug availability. Drug-food interactions may result in reduced, delayed, or increased systemic drug availability. The absorption of only a small number of drugs is unaffected by concomitant food intake. The degree of interaction and whether it positively or negatively affects drug absorption depends on a number of factors including the physical and chemical nature of the drug, the formulation, the type of meal, and the time interval between eating and dosing. Mechanisms of drug-food interactions are not well characterised. They clearly involve both direct and indirect factors in a similar fashion to drug-drug interactions, but indirect factors probably predominate. Reduced or delayed drug absorption is generally attributed, at least in part, to delayed stomach-emptying due to food. Increased absorption may also result from delayed stomach-emptying facilitating greater drug dissolution before it passes from the stomach into the small intestine. Increased bioavailability of some drugs, e.g. propranolol, metoprolol and labetalol, may be related to reduced presystemic clearance. The potential clinical implications of drug-drug and drug-food interactions must be taken into account with oral medications in order to minimise variations in systemic drug availability and hence in clinical efficacy.

Evidence for isoxicam binding to site I as a primary site and to site II as a secondary site of human serum albumin.

Isoxicam binding to HSA was studied using equilibrium dialysis and fluorescence methods. It was shown that this drug binds to or near site I (warfarin or azapropazone site) and to site II (the diazepam site) as a secondary site, although it is generally considered that their respective drug structural requirements are often exclusive. The binding parameters were calculated with different mathematical models; a site oriented model with or without fixing the number of binding sites as integer values and a stoichiometric model. The relevant results are in good agreement under the selected experimental conditions. The stoichiometric method indicates that no positive cooperativity occurred during the binding process but other interactions between the two sites cannot be excluded.

Graphic methods in pharmacokinetics: the basics.

A considerable amount of information regarding the kinetics of drug absorption, distribution, metabolism, and excretion can be obtained by graphic analysis of plasma concentration and/or urinary excretion data. The simplest pharmacokinetic model is used to describe the analysis of drug concentration profiles in plasma after administration intravenous or nonvascular routes. Methods to calculate pharmacokinetic and pharmacologic parameters include urinary excretion rate, cumulative excretion, and the construction and analysis of Sigma-minus plots. The treatment for this simple pharmacokinetic model provides a basis for graphic analysis of more complex models.

Calculation of serum digoxin levels in patients with normal and impaired renal function.

Serum digoxin levels were examined in 55 patients with varying degrees of renal function impairment who were receiving chronic oral digoxin therapy. Three methods of predicting digoxin serum levels were investigated. Each method may be applied using serum creatinine values and does not require urinary data. Correlations between calculated and actual digoxin levels in combined male and female patients were improved when changes in digoxin distribution volumes in renal impairment were considered. Correlations between calculated and actual digoxin levels were poor in male patients but were again improved by incorporating changes in drug distribution volume. Correlations obtained in female patients were superior to those obtained in male patients and appeared to be independent of the method of calculation employed.

Pharmacokinetics of phenobarbital following single and repeated doses.

Serum levels of phenobarbital, and also urinary excretion of phenobarbital and p-hydroxyphenobarbital, were examined after single and repeated oral doses of phenobarbital to three male subjects. Serum levels of phenobarbital at steady state were approximately ten times as high as those after a single dose. The overall elimination rate constant for loss of phenobarbital from serum, Kel, was significantly reduced after repeated doses, and Cmax infinity values calculated from single-dose data poorly predicted observed Cmax infinity values. Five-day urinary excretion of phenobarbital and p-hydroxyphenobarbital accounted for 16 and 21 per cent, respectively, of the initial dose. Due to extensive drug accumulation, 83 per cent of the final dose was excreted in five-day urine as phenobarbital and 85 per cent, as p-hydroxyphenobarbital. Comparison of plasma and renal clearances indicated that the rate of phenobarbital metabolism was reduced owing to repeated dosing, while the rate of urinary excretion of parent drug was unchanged.

Bioavailability of oral and intramuscular phenobarbital.

The absorption of phenobarbital was compared in healthy adult subjects after oral and intramuscular therapeutic doses. Serum levels of phenobarbital were determined for 21 days after dosing by means of radioimmunoassay. Serum levels were similar from both dosage routes, with peak levels occurring at 1-3 hours after dosing and then declining slowly with an elimination half-life of about 90 hours. The overall efficiency of phenobarbital absorption from intramuscular doses was approximately 80 per cent of that from equivalent oral doses. Except in cases where oral dosing is not appropriate, there is no clinical advantage in giving phenobarbital intramuscularly to adult patients.

Pharmacokinetics of alcohol following single low doses to fasted and nonfasted subjects.

The absorption and elimination characteristics of alcohol have been studied in healthy fasted and nonfasted human volunteers using low single doses. In non-fasted subjects, carbohydrate reduced overall alcohol bioavailability by about 96 percent, compared to 90 per cent for fat and 75 per cent for protein. Inhibition of absorption in nonfasted subjects appeared to be due to less alcohol being available for absorption rather than a reduced absorption rate. Serum alcohol levels in fasted subjects were interpreted in terms of both first-order and zero-order absorption followed by first-order elimination. Of the two proposed models, that utilizing zero-order absorption provided a marginally better fit to observed data.

Pharmacokinetics of 125I-iothalamate and 131I-o-iodohippurate in man.

The pharmacokinetics of 125I-iothalamate (IOT125I) and 131I-o-iodohippurate (OIH131I) have been studied in patients with varying degrees of renal insufficiency. Both compounds have been shown to obey two-compartment model kinetics after intravenous administration. The clearances are closely related to renal function, but poor correlations were obtained between renal function and compartmental distribution characteristics. This study provides further evidence that IOT125I and OIH131I may be used as indicators of renal function in patients after a single intravenous injection.