Thyrotropin-Releasing Hormone (TRH) Uptake in Intestinal Brush-Border Membrane Vesicles: Comparison with Proton-Coupled Dipeptide and Na+-Coupled Glucose Transport

The mechanism of thyrotropin-releasing hormone (pGlu-His-Pro-NH₂; TRH) uptake across the luminal membrane of intestinal enterocytes was investigated using brush-border membrane vesicles (BBMV) from rabbit duodenum and jejunum and rat upper small intestine. [¹⁴C]Glucose accumulated within the intestinal vesicles (at 10 sec), in the presence of an inwardly directed Na⁺ gradient, 7- to 14-fold higher than equilibrium values (65 min). The vesicles also accumulated the dipeptide [¹⁴C]Gly-Sar. Dipeptide uptake was greatest in the presence of both an inwardly directed proton gradient and an inside negative membrane potential. The H⁺-dependent Gly-Sar transport was not affected by the presence of an excess (46-fold) of cold TRH. In contrast to the observations with glucose and Gly-Sar, the uptake of [³H]TRH after 10 or 60 sec (in each of the vesicle preparations) was not enhanced by either Na⁺ or H⁺ gradient conditions. The absence of vesicular accumulation of TRH was not due to peptide hydrolysis. For example, after a 60-sec incubation with rabbit jejunal BBMV no degradation of the tripeptide was evident. After 65 min, 6% of [³H]TRH had undergone degradation, by deamidation, to form TRH-OH. These studies provide no evidence for the oral absorption of TRH by a Na⁺- or H⁺-dependent carrier system in the brush-border membrane. Previous observations of TRH absorption in vivo may be accounted for by passive absorption of the peptide combined with its relative resistance to luminal hydrolysis.     

The Intestinal Transport Mechanism of Fluoroquinolones: Inhibitory Effect of Ciprofloxacin,an Enoxacin Derivative,on the Membrane Potential-Dependent Uptake of Enoxacin

Purpose. To clarify the absorption-structure relationship for the fluoroquinolones from the point of view of inhibitory behavior. Methods. The inhibitory effects of ciprofloxacin on the transport process of enoxacin across the rat intestinal brush-border membrane was examined. Results. Ciprofloxacin, which has a similar structure to enoxacin, exhibited a pH-dependent interference with enoxacin absorption from rat jejunal loops. The uptake experiments using BBM vesicles showed that ciprofloxacin significantly reduced not only the initial binding of enoxacin to the membrane surface, but also the K ⁺ - or H⁺-diffusion potential-dependent transport across the membrane. Furthermore, an H ⁺-diffusion potential (interior negative) also exhibited a stimulative uptake of ciprofloxacin. Conclusions. These results suggest that the inhibition behavior of ciprofloxacin from the jejunal loop was closely related to the ionic diffusion potential-dependent uptake of enoxacin across the brush-border membrane.     

Modulation of Organic Cation Transport and Lipid Fluidity by Benzyl Alcohol in Rat Renal Brush-border Membranes

Purpose. Organic cations are actively transported in renal brush-border membranes (BBM) by the H⁺/organic cation antiport system. In the present study, we investigated the relationship between membrane fluidity and organic cation transport in the BBM. Methods. The effects of benzyl alcohol, a membrane fluidizing agent, on the organic cation tetraethylammonium (TEA) uptake were studied using renal BBM vesicles isolated from rat kidney. BBM fluidity was assessed by fluorescence polarization technique. Results. H⁺ gradient-dependent uptake of TEA in BBM vesicles was inhibited by benzyl alcohol in a dose-dependent manner, with an apparent half inhibitory concentration of 18mM. The decrease in fluorescence anisotropy of l,6-diphenyl-l,3,5-hexatriene in BBM, which represents the increase in membrane fluidity, was correlated with the decrease in TEA transport activity. The dissipation rate of H⁺ gradient, a driving force for organic cation transport in BBM, was increased by benzyl alcohol. In addition, H⁺ gradient-independent TEA-TEA exchange was also inhibited by benzyl alcohol. These findings indicate that benzyl alcohol inhibits the uptake of TEA by affecting the intrinsic activity of the organic cation transporter and the H⁺ gradient dissipation rate. Conclusions. The membrane fluidity should be an important determinant for organic cation transport in renal BBM.     

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na⁺-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na⁺-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na⁺-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na⁺ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na⁺-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A₂ (cPLA₂). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA₂ specific inhibitor AACOCF₃. ATP depletion-induced alterations in Na⁺-dependent phosphate uptake and cell viability were prevented by AACOCF₃. Inhibition of Na⁺-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA₂ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Proton-Cotransport of Pravastatin Across Intestinal Brush-Border Membrane

Purpose. The purpose of the present study is to clarify the intestinal brush-border transport mechanism of a weak organic acid, pravastatin, an HMG-CoA reductase inhibitor. Methods. The transport of pravastatin was studied by using intestinal brush-border membrane vesicles prepared from rabbit jejunum, and uptake by the membrane vesicles was measured using rapid filtration technique. Results. The initial uptake of [¹⁴C]pravastatin was markedly increased with decreases in extravesicular pH and showed a clear overshoot phenomenon in the presence of a proton gradient (pH_in/out = 7.5/5.5). A protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the uptake of [¹⁴C]pravastatin. In addition, an ionophore for sodium, potassium and proton, nigericin, stimulated the uptake of [¹⁴C]pravastatin in the presence of a potassium gradient ([K ⁺ ]_in/[K⁺ ]_out = 0/145 mM). On the other hand, neither the imposition of an inwardly directed sodium gradient nor an outwardly directed bicarbonate gradient stimulated the uptake of [¹⁴C]pravastatin. In the presence of a proton gradient (pH_in/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent K_t of 15.2 ± 3.2 mM and J_max of 10.6 ± 1.21 nmol/mg protein/10 sec. The uptake of pravastatin was significantly inhibited by monocarboxylic acid compounds such as acetic acid and nicotinic acid in a competitive manner but not by di- or tri-carboxylic acids, or acidic amino acid. Conclusions. It was concluded that a pH-dependent transport of pravastatin across the brush-border membrane occurs by a proton-gradient dependent carrier-mediated mechanism rather than by simple diffusion of its unionized form.     

Characterization of the transport of uracil across Caco-2 and LLC-PK1 cell monolayers

Purpose. The purpose of this study was to characterize the transport of uracil, a pyrimidine nucleobase, in Caco-2 and LLC-PK₁ cells. Methods. Caco-2 and LLC-PK₁ cells were grown to confluency on a permeable polycarbonate membrane insert to permit transport and uptake experiments after the loading of uracil on either the apical or basolateral side. Results. The vectorial transport of uracil in both directions was saturable with comparable K_m and V_max in Caco-2 cell monolayers, probably because of a Na⁺-independent transport system located on the basolateral membrane. In LLC-PK₁ cell monolayers, two distinct transport systems, namely a Na⁺-dependent and a Na⁺-independent, were functional in the apical to basolateral (A-B) transport of uracil. The first system was saturable with a K_m value of 3.67 ± 0.40 M, a V_max of 11.31 ± 0.91 pmol/cm²/min, and a Na⁺:uracil coupling stoichiometry of 1.28 ± 0.20. The second system was Na⁺ independent and satuable with a low affinity (K_m, 50.37 ± 9.61 M) and V_max (16.01 ± 4.48 pmol/cm²/min). The two transport systems appeared to be located on the apical membrane. Conclusion. The mechanism of uracil transport differs depending on cell lines; a Na⁺-independent system on the basolateral membrane in Caco-2 cells and both Na⁺-dependent and Na⁺-independent systems on the apical membrane in LLC-PK₁ cells seem to be responsible for the difference.     

Transport Characteristics of S-1090, A New Oral Cephem,in Rat Intestinal Brush-Border Membrane Vesicles

Purpose. Elucidating the transport characteristics of S-1090, a new orally active cephalosporin in rat small intestinal brush-border membranes. Methods. A rapid filtration technique. Results. The uptake of S-1090 was stimulated by an inwardly directed H⁺-gradient, but did not show overshooting uptake. To investigate the transport system, the inhibitory and countertransport effects of various compounds on S-1090 uptake were examined. Although the dipeptides and tripeptides composed of amino acids with aliphatic side chains did not inhibit the uptake of S-1090, those having histidine, proline or tryptophan as the N-terminal amino acid showed an inhibitory effect. Among the oral cephems tested, ceftibuten showed marked inhibition, while cefaclor and cephalexin had no inhibitory effect. Countertransport effects on S-1090 uptake were observed only when the vesicles were preloaded with histidyl peptides such as His-Gly or His-Ala, while other compounds which exhibited inhibition had no countertransport effect. Conclusions. Based on the above results, there seems to be heterogeneity (multiplicity) in the oligopeptide transport system which may depend on the structure of the N-terminal amino acid. S-1090 may be dominantly transported via a system that recognizes peptides having histidine as the N-terminal amino acid.     

Taurine Transport in Cultured Choroid Plexus

Purpose. Taurine, a -amino acid, is a neuromodulator which interacts functionally with the glycinergic, GABAergic, cholinergic and adrenergic systems. Although a continuous cell culture model is not available for the choroid plexus epithelia, we recently described a primary cell culture of rabbit choroid plexus epithelia. The goal of the current study was to determine the suitability of this primary cell culture for the study of the Na⁺-taurine transporter in the rabbit choroid plexus. Methods. A primary cell culture of rabbit choroid plexus epithelial cells was grown on semi-permeable filters and kinetics of ³H-taurine uptake were ascertained. Results. Taurine transport in the cultured choroid plexus cells was Na⁺-dependent and saturable (K_m = 156 M). The -amino acids, -alanine and taurine, significantly inhibited Na⁺-driven taurine transport whereas L-alanine partially inhibited taurine transport in the cultured cells. In addition, we observed that the activity of the Na⁺-taurine transporter is affected by exposure to taurine in the media. Conclusions. These results demonstrate that a Na⁺-taurine transporter with characteristics similar to those in the intact tissue is expressed in cultured choroid plexus epithelial cells. The transporter may undergo adaptive regulation and play a role in taurine homeostasis in the central nervous system.     

Stereoselective and Carrier-Mediated Transport of Monocarboxylic Acids Across Caco-2 Cells

Purpose. To characterize the transport mechanism of monocarboxylic acids across intestinal epithelial cells by examining the stereoselectivity of the transcellular transport of several chiral monocarboxylic acids. Methods. The transport of monocarboxylic acids was examined using monolayers of human adenocarcinoma cell line, Caco-2 cells. Results. The permeability of L-[¹⁴C]lactic acid at a tracer concentration (1 µM) exhibited pH- and concentration-dependencies and was significantly greater than that of the D-isomer. The permeabilities of both L-/ D-[¹⁴C]lactic acids involve saturable and nonsaturable processes; the saturable process showed a higher affinity and a lower capacity for L-lactic acid compared with the D-isomer, while no difference between the isomers was seen for the nonsaturable process. The transport of L-lactic acid was inhibited by chiral monocarboxylic acids such as (R)/(S)-mandelic acids and (R)/(S)-ibuprofen in a stereoselective manner. Mutually competitive inhibition was observed between L-lactic acid and (S)-mandelic acid. Conclusions. Some chiral monocarboxylic acids are transported across the intestinal epithelial cells in a stereoselective manner by the specific carrier-mediated transport mechanism.     

Possible Role of Anion Exchanger AE2 as the Intestinal Monocarboxylic Acid/Anion Antiporter

Purpose. The purpose of the present study was to investigate the transport of organic monocarboxylic acids mediated by the anion exchanger AE2, which has been already reported to be present at several tissue cell membranes, including intestinal brush border membrane in rabbit. Methods. Membrane transport of organic monocarboxylic acids by AE2 was investigated by transient AE2-gene expression in HEK 293 cells and subsequent uptake studies by the cells. Results. Functional transfection of AE2 was confirmed by the enhanced ³⁶C1^– efflux from the cells. When preloaded with chloride anion, AE2-transfected cells demonstrated a significantly enhanced [¹⁴C]benzoic acid transport activity compared with mock-transfected cells. The AE2-mediated uptake was saturable with kinetic parameters of Km = 0.26 ± 0.08 mM and Vmax = 6.14 ± 0.52 nmol/mg protein/ 3 min, and the uptake of [¹⁴C]benzoic acid was pH-dependent with a maximal uptake at pH 6.5. AE2-mediated [¹⁴C]benzoic acid uptake was inhibited by Cl^–, HCO₃ ^–, and DIDS. AE2-transfected cells demonstrated significantly enhanced transport activity for nicotinic acid, propionic acid, butyric acid, and valproic acid as well as benzoic acid compared with mock-transfected cells. Conclusions. AE2 is functionally involved in the anion antiport for organic monocarboxylic acids as well as inorganic anions and is supposed to play a partial role in the intestinal transport of organic acids.     

Dipeptide Uptake and Transport Characteristics in Rabbit Tracheal Epithelial Cell Layers Cultured at an Air Interface

Purpose. To determine the functional presence of a H⁺/peptide cotransport process in rabbit tracheal epithelial cell layers cultured at an air-interface and its contribution to transepithelial dipeptide transport. Methods. Rabbit tracheocytes were isolated, plated on Transwells, and cultured at an air-interface. After 5 or 6 days in culture, uptake and transepithelial transport of carnosine were examined. Results. Carnosine uptake by tracheocytes was pH-dependent and was saturable with a Michaelis-Menten constant of 170 M. Moreover, carnosine uptake was inhibited 94% by Gly-L-Phe, 28% by (-Ala-Gly, but not at all by Gly-D-Phe or by the amino acids -Ala and L-His. Unexpectedly, transepithelial carnosine transport at pH 7.4 (i.e., in the absence of a transepithelial pH gradient) was similar in both the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. Lowering the apical fluid pH to 6.5 reduced abtransport 1.6 times without affecting ba transport, consistent with predominantly paracellular diffusion of carnosine under an electrochemical potential gradient. Conclusions. The kinetic behavior of carnosine uptake into cultured tracheal epithelial cell layers is characteristic of a H⁺-coupled dipeptide transport process known to exist in the small intestine and the kidney. Such a process does not appear to be rate-limiting in the transport of carnosine across the tracheal epithelial barrier.     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Transepithelial transport of prodrugs of the HIV protease inhibitors saquinavir,indinavir, and nelfinavir across Caco-2 cell monolayers

Purpose. This study is dedicated to the permeation of various amino acid-, D-glucose-, and PEG-conjugates of indinavir, saquinavir, and nelfinavir across monolayers of Caco-2 cells as models of the intestinal barrier. This screening is aimed at detecting the most promising prodrugs for improving the intestinal absorption of these protease inhibitors. Methods. The bidirectional transport of the prodrugs was investigated using P-gp-expressing Caco-2 monolayers grown on membrane inserts using high-performance liquid chromatography for quantitation. Results. The L-valyl, L-leucyl, and L-phenylalanyl ester conjugates led to an enhancement of the absorptive flux of indinavir or saquinavir. These results are likely attributable to an active transport mechanism and/or to a decrease of their efflux by carriers such as P-gp. Connection of tyrosine through its hydroxyl, of D-glucose, or of polyethylene glycol decreased their absorptive and secretory diffusion. Conclusions. Conjugation of the protease inhibitors to amino acids constitutes a most appealing alternative that could improve their intestinal absorption and oral bioavailability. Whether it could improve their delivery into the central nervous system remains to be explored. D-Glucose conjugation will most probably not improve their intestinal absorption or their crossing of the blood-brain barrier. If some pharmacologic benefits are to be expected from PEG-protease inhibitor conjugates, they must then be administered intravenously.     

The interaction of canrenone with the Na+,K+ pump in human red blood cells

Summary Canrenone inhibits 30–40% of ouabain-sensitive Na⁺ efflux in human red cells. Half-maximal inhibition was obtained with a canrenone concentration=86±37 mol/l (mean±SD of 13 experiments). The partial inhibition of the Na⁺,K⁺ pump appears to be mediated at the digitalis receptor site with an apparent dissociation constant (K _C)=200±130 mol/l (mean±SD). Further evidence suggesting that canrenone is a partial agonist at the digitalis receptor site was obtained by the observation that it decreases the apparent affinity of the Na⁺,K⁺ pump for external K⁺. However, in contrast to ouabain, canrenone decreases the apparent pump affinity for internal Na⁺.Our results show that, at physiological cell Na⁺ levels canrenone is able to enhance the inhibition of the Na⁺,K⁺ pump by low doses of ouabain. Conversely, in cells treated with high concentrations of cardiac glycosides (in which cell Na⁺ content increases), canrenone is able to restimulate the blocked pumps.     

Inhibitory effects of scorpion venom on the uptake of amino acids by synaptosomes and synaptosomal membrane vesicles

Scorpion (Tityus serrulatus) venom strongly inhibited the Na⁺-dependent uptake of [¹⁴C]proline by rat brain synaptosomal preparations. In addition, the efflux of proline was enhanced markedly by scorpion venom. The inhibitory effects of the venom were also demonstrated in synaptosomal vesicle preparations where proline uptake was energized by an artificially imposed Na⁺ gradient. In both preparations, the effect of scorpion venom was additive with the inhibitory effect of veratridine on Na⁺-dependent amino acid uptake. The inhibitory effects of both compounds were abolished by tetrodotoxin. The Na⁺-dependent uptakes of amino acids (e.g. proline, glutamic acid, and γ-aminobutyric acid) were much more sensitive to inhibition by the toxin than the Na⁺⁶-independent uptakes (e.g. leucine and phenylalanine). The results of the present study indicate that the scorpion venom mav exert its inhibitory effect on Na⁺-dependent transport by decreasing the transmembrane Na⁺ gradient. Efflux of accumulated proline, which is presumably controlled by maintenance of this Na⁺ gradient, was stimulated 3- to 4-fold by the scorpion venom.     

Demonstration of a Na+: Mg2+ exchange in human red cells by its sensitivity to tricyclic antidepressant drugs

Summary Iminodibenzyl-, iminostilbene-, dibenzocycloheptadiene-, dibenzooxepine- and dibenzothiepine-derivatives of tricyclic antidepressant drugs were able to inhibit Na⁺-stimulated Mg²⁺ efflux in human erythrocytes at concentrations of 10^–5–10^–3 mol/l. Tricyclic antidepressant drugs belonging to other chemical groups, non-tricyclic antidepressant drugs and phenothiazines were less potent inhibitors (IC₅₀ of 10^–4 mol/l or higher).Imipramine and dothiepine, the most potent compounds, inhibited the Mg" carrier with IC₅₀ of 2.5 and 4 × 10^–5 mol/1 respectively. These IC₅₀ are of similar order of magnitude to those of some classical transport inhibitors (such as furosemide for the [Na⁺K⁺,Cl^–]-cotransport system). In addition, these concentrations of imipramine and dothiepine were free of: i) side effects on other erythrocyte Na and K⁺ transport pathways (with the exception of a slight inhibition of Ca²⁺-sensitive K⁺-channels and [Na⁺,K⁺,Cl^–]- and [K⁺,Cl^–]-cotransport systems) and ii) toxic effects on the membrane leak for divalent or monovalent cations. Therefore, we selected imipramine as an useful tool for investigating fluxes catalyzed by the Na⁺-stimulated Mg²⁺ carrier.Imipramine was tested on the initial rate of ouabain and bumetanide-resistant net Na⁺ influx in Na⁺-depleted, Mg²⁺-loaded erythrocytes. The compound was able to inhibit a Na⁺ influx of about 300–500 mol (l · cells × h)^–1 with an IC₅₀ of about 3 x 10^–5 mol/1. This imipramine-sensitive Na⁺ influx was coupled with an imipramine-sensitive Mg²⁺ efflux in a stoichiometry of 3.03±0.34 (mean±SEM of 7 experiments).Abbreviations MOPS 4-morpholinopropanesulfonic acid - PCMBS p-chloromercuribenzenesulfonate - EGTA ethylene glycol bis-(beta-aminoethyl ether)N,NNN-tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Send offprint requests to R. Garay at the above address     

Transport of Pregabalin in Rat Intestine and Caco-2 Monolayers

Purpose. The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl --aminobutyric acid, isobutyl GAB A), a new anticonvulsant drug, was mediated by amino acid carriers with affinity for large neutral amino acids (LNAA). Methods. Pregabalin transport was studied in rat intestine and Caco-2 monolayers. An in vitro Ussing/diffusion chamber model and an in situ single-pass perfusion model were used to study rat intestinal transport. An in vitro diffusion chamber model was used to evaluate Caco-2 transport. Results. In rat ileum, pregabalin transport was saturable and inhibited by substrates of intestinal LNAA carriers including neurontin (gabapentin), phenylalanine, and proline. Weak substrates of intestinal LNAA carriers (-alanine, --aminobutyric acid, and methyl aminoisobutyric acid) did not significantly change pregabalin transport. In Caco-2 mono-layers that showed a high capacity for phenylalanine transport, pregabalin transport was concentration- and direction-independent and equivalent in magnitude to the paracellular marker, mannitol. The in vitro and in situ rat ileal permeabilities of the LNAA carrier-mediated compounds neurontin, pregabalin, and phenylalanine correlated well with the corresponding in vivo human oral absorption. Conclusions. The transport of pregabalin was mediated by LNAA carriers in rat ileum but not in Caco-2 monolayers. Caco-2 was not an appropriate model for evaluating the in vivo human oral absorption of pregabalin and neurontin.     

Mucosal Uptake of Gabapentin (Neurontin) vs. Pregabalin in the Small Intestine

Purpose. To compare the mucosal membrane transport of gabapentin and pregabalin in animal small intestine. Methods. Uptake of the two drugs by brush-border membrane vesicles (BBMV) from rat and rabbit small intestine was studied as a function of temperature, uptake-medium sodium content, and intestinal region. Amino acid inhibition studies were conducted with pregabalin. Results. Gabapentin uptake by rat and rabbit jejunal BBMV was sodium independent, whereas pregabalin uptake was sodium dependent. Uptake of both drugs in rabbit small intestinal vesicles was greater at 25°C than at 4°C in the absence of sodium and an additional increase in uptake was observed for pregabalin at 25°C in the presence of sodium. Pregabalin uptake in rabbit duodenal, jejunal, and ileal BBMV was equivalent, whereas gabapentin uptake was greater in duodenal and ileal BBMV, compared with jejunal BBMV. Although inhibition is weak, a decrease in BBMV uptake of pregabalin is observed with coincubation of high concentrations of both neutral and basic amino acids. Conclusions. Amino acid carriers mediate the apical uptake of both drugs in the small intestine. Although gabapentin and pregabalin are structurally similar, their small intestinal mucosal uptake differs in sodium dependence and region dependence. Gabapentin uptake is likely mediated by system b^0,+, whereas pregabalin uptake is also mediated by B⁰ and/or B^0,+.