Effect of thiamine on H-MPP uptake by Caco-2 cells

Recent studies on the intestinal uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP⁺) showed that transport of this compound occurs through human extraneuronal monoamine transporter (hEMT). Moreover, it was recently described that alkaline phosphatase (ALP), an ecto-phosphatase anchored to the plasma membrane and able to dephosphorylate extracellular substrates or cell-surface proteins, is directly or indirectly involved in the modulation of MPP⁺ uptake by Caco-2 cells. The present study investigated a putative modulation of MPP⁺ intestinal apical uptake and ecto-ALP activity by thiamine (T⁺) and thiamine pyrophosphate (TPP, a T⁺ dietary precursor). For this purpose, we used Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, as an intestinal model. Ecto-ALP activity and N-[methyl-³H]-4-phenylpyridinium acetate (³H-MPP⁺) uptake were evaluated in intact Caco-2 cells. T⁺ and TPP were able to increase ecto-ALP activity, with an equal potency, and to decrease ³H-MPP⁺ apical uptake, with a similar potency. The effects of both compounds on ecto-ALP activity and ³H-MPP⁺ uptake were concentration-dependent. The results suggest that the effect of T⁺ and TPP on ecto-ALP activity may lead to inhibition of the intestinal absorption of other organic cations present in the diet. Another important conclusion is that the intestinal absorption of T⁺ may occur through hEMT, in Caco-2 cells.     

Interaction of green tea catechins with renal organic cation transporter 2

1.?Green tea extract (GTE) and EGCG have previously shown to increase the uptake of MPP⁺ into Caco-2 cells. However, whether GTE and its derivatives interact with renal basolateral organic cation transporter 2 (Oct2) which plays a crucial role for cationic clearance remains unknown. Thus, this study assessed the potential of drug-green tea (GT) catechins and its derivatives interactions with rat Oct2 using renal cortical slices and S2 stably expressing rat Oct2 (S2rOct2).

2.?Both GTE and ECG inhibited MPP⁺ uptake in renal slices in a concentration-dependent manner (IC₅₀?=?2.71?±?0.360?mg/ml and 0.87?±?0.151?mM), and this inhibitory effect was reversible. Inhibition of [³H]MPP⁺ transport in S2rOct2 by either GTE or ECG (IC₅₀?=?1.90?±?0.087?mg/ml and 1.67?±?0.088?mM) was also observed.

3.?The weak and reversible interactions of GTE and ECG with rOct2 indicate that consumption of GT beverages could not interfere with cationic drugs secreted via renal OCT2 in humans. However, the rise of therapeutic use of GTE and ECG might have to take into account the significant possibility of adverse drug–green tea catechins interactions which could alter renal organic cation drug clearance.     

Transport of small organic cations in the rat liver

The kidneys and the liver are the principal organs for the inactivation of circulating organic cations. Recently, an organic cation transporter (OCT1) has been cloned from rat kidney. In order to answer the question whether OCT1 is involved also in hepatic uptake of organic cations, the pharmacological characteristics of organic cation transport in hepatocytes were compared to the characteristics of transiently expressed OCT1.Primary cultures of rat hepatocytes avidly accumulated the small organic cation ³H-1-methyl-4-phenylpyridinium (³H-MPP⁺). At equilibrium, the hepatocytes accumulated ³H-MPP⁺ 56-fold. Initial rates of specific ³H-MPP⁺ transport in hepatocytes were saturable. The half-saturating concentration was 13 mol/l. ³H-MPP⁺ transport was sensitive to quinine (K_i = 0.79 mol/l) and cyanine863 (K_i = 0.097 µmol/l). Quinine and cyanine863 are known inhibitors of type I hepatic transport of cationic drugs and of renal excretion of organic cations, respectively. To compare the functional characteristics of ³H-MPP⁺ transport in hepatocytes with those of OCT1, OCT1 has been heterologously expressed and characterized in a mammalian cell line (293 cells). Initial rates of ³H-MPP⁺ transport were saturable, the K_m being 13 mol/l. The rank order of inhibitory potencies of various inhibitors was almost identical in hepatocytes and 293 cells transiently transfected with OCT1. There was a positive correlation between the K_i's for the inhibition of ³H-MPP⁺ transport in isolated hepatocytes and transfected 293 cells (r = 0.85; P<0.01; n = 8).The results indicate that OCT1 is functionally expressed not only in the kidney but also in hepatocytes where it is responsible for the transport of small organic cations which, in the past, have been classified as type I substrates.     

Enhancement effect of methylxanthines on the intestinal absorption of poorly absorbable dyes from the rat small intestine.

The effect of theophylline or caffeine on the absorption of phenol red (PR) or bromphenol blue (BPB) (poorly absorbable dyes) from the rat small intestine was investigated by using the in situ recirculation technique. The disappearance of PR and BPB from the recirculated solution was increased by pretreatment of the intestinal lumen with 5 mM theophylline and 5 mM caffeine. With BPB, increases in tissue accumulation and net absorption were observed in the presence of 5 mM theophylline or 5 mM caffeine. However, the administration of 15 μmoles theophylline or 25 μmoles caffeine into a femoral vein by a single injection failed to produce an increasing effect on the disappearance of PR or BPB from the recirculated solution. A possible mechanism of the enhancement effect of the methylxanthines on dye absorption was discussed.     

Epigallocatechin-3-gallate increases intracellular [Ca<Superscript>2+</Superscript>] in U87 cells mainly by influx of extracellular Ca<Superscript>2+</Superscript> and partly by release of intracellular stores

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [³H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca²⁺] ([Ca²⁺]_i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca²⁺]_i. The EGCG-induced [Ca²⁺]_i increases were reduced to 20.9% of control by removal of extracellular Ca²⁺. The increases were also inhibited markedly by treatment with the non-specific Ca²⁺ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca²⁺ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca²⁺-ATPase thapsigargin (1 µM) also significantly inhibited the EGCG-induced [Ca²⁺]_i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 µM) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 µM) had no effect. EGCG increased [³H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 µM) and flufenamic acid (100 µM), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca²⁺]_i increase in non-treated and thapsigargin-treated cells but indomethacin (100 µM) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca²⁺]_i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca²⁺ and partly by mobilizing intracellular Ca²⁺ stores by PLC activation. The EGCG-induced [Ca²⁺]_i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.     

L-type Ca<Superscript>2+</Superscript> channels activation and contraction elicited by myricetin on vascular smooth muscles

The effects of myricetin (3,3,4,5,5,7-hesahydroxyflavone), a natural flavonoid found in edible plants, were studied on vascular smooth muscle L-type Ca²⁺ channels by comparing its mechanical, radioligand binding, and electrophysiological properties to those of the Ca²⁺ channel agonist (S)-(-)-Bay K 8644.In rat aorta rings, both myricetin and (S)-(-)-Bay K 8644 induced contractile responses, which were dependent upon prior exposure to K⁺. At 15 mM K⁺ (K15) the pEC₅₀ values for myricetin and (S)-(-)-Bay K 8644 were 4.43±0.03 and 7.92±0.13, respectively. Furthermore, the maximum tension response to myricetin was not significantly different from that elicited by either (S)-(-)-Bay K 8644 or K60. The Ca²⁺ channel blockers nifedipine, verapamil and diltiazem antagonised and fully reverted myricetin-, (S)-(-)-Bay K 8644- as well as K60-induced contractions. Both myricetin and (S)-(-)-Bay K 8644 potentiated rat aorta ring responses to K⁺, shifting the K⁺ concentration-response curve to the left. (S)-(-)-Bay K 8644, but not myricetin, inhibited in a concentration-dependent manner (+)-[³H]PN200–110 binding in porcine aortic membranes. Electrophysiological recordings from single rat tail artery myocytes, under amphotericin B-perforated as well as conventional methods, showed that both myricetin and (S)-(-)-Bay K 8644 increased L-type Ba²⁺ current (I_Ba(L)) and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarising direction, without, however, modifying the threshold potential. Furthermore, (S)-(-)-Bay K 8644 accelerated both activation and inactivation kinetics of I_Ba(L) while myricetin slowed down the activation kinetics. Finally, both (S)-(-)-Bay K 8644 and myricetin slowed down deactivation kinetics of I_Ba(L).These results suggest that myricetin induces vasoconstriction by activating L-type Ca²⁺ channel with similar efficacy but a site of action different to that of (S)-(-)-Bay K 8644.Abbreviations I_Ba(L) L-type Ba²⁺ current - PSS Physiological salt solution - V_h Holding potential     

Radiolabeling of EGCG with <Superscript>131</Superscript>I and biodistribution in rats

Epigallocatechin gallate (EGCG), is the most abundant and widely studied catechin in green tea (Camellia sinensis Theaceae). The inhibitory effects of EGCG and green tea extract on carcinogenesis in various organs in rodents have now been demonstrated over the past decade. The aim of study was to label EGCG with I-131, to determinate its structure and to evaluate its biodistribution in Wistar rats. Radiolabeling was carried out by direct electrophilic iodination method (iodogen) and yield was determined by radio thin layer chromatography (RTLC). Radiolabelling yield is determined as 89 ± 1.0%. Besides, determination of structure of iodinated molecule, serum stability, and partition coefficient experiments was performed. The structure analysis of synthesized cold ¹²⁷I-EGCG complex was assessed with LC–MS–MS and ¹H-NMR. ¹H-NMR and LC–MS–MS results of iodinated EGCG (¹²⁷I-EGCG) show that oxidize iodine reacts electrophilic with aromatic ring. Serum stability results showed that in vitro stability of ¹³¹I-EGCG was quite high. It is observed that labeling percentage decreased 83 ± 2% at 24th, Partition coefficient results show that the partition coefficient of EGCG was calculated as theoretical partition coefficient = 2.04 ± 0.42 and the experimental partition coefficient of ¹³¹I-EGCG was found as 1.46 ± 0.2. The biodistribution data shown that the maximum uptake of the radioiodinated EGCG was seen in lung and pancreas at 30 min. The blocking assay results indicated that the uptake of ¹³¹I-EGCG in lung was not significantly change (0.25, 0.23, and 0.22%ID/g at 30, 60, and 150 min, respectively). Biodistribution data showed no significant uptake in a specific organ of the rat. Hence radiolabeled EGCG is seen in some organs (lung, liver, pancreas, kidney, etc.).     

Inward transport of 3H-MPP+ in freshly isolated rat hepatocytes: evidence for interaction with catecholamines

1-Methyl-4-phenylpyridinium (MPP⁺), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is efficiently taken up and accumulated by rat hepatocytes. However, the nature of the mechanism(s) involved in the hepatic uptake of MPP⁺ remains partially unknown. The aim of the present study was to further characterize the hepatic uptake of ³H-MPP⁺, namely by investigating the interactions of catecholamines (which are also efficiently taken up by rat hepatocytes) with MPP¹ transport.The accumulation of ³H-MPP⁺ in isolated rat hepatocytes occurred through saturable and non-saturable mechanisms. The kinetics of the saturable component of ³H-MPP⁺ uptake was as follows: V_max = 181.3 ± 11.1 pmol mg protein^–1 min^–1 and K_m = 47.1 M (27.9, 66.3) (n = 5). The diffusion constant (in ml mg protein^–1 min^–1) for the non-saturable uptake of ³H-MPP⁺ was 0.00068 (0.00052, 0.00083) (n = 5). From the analysis of the time course of ³H-MPP⁺ accumulation at a substrate concentration of 100 nM ³H-MPP⁺, it was found that the rate constant of inward transport of ³H-MPP⁺ into hepatocytes (k_in) was 15.7 ± 3.8 l mg protein^–1 min^–1, the rate constant of outward transport of ³H-MPP⁺ from hepatocytes (k_out) was 0.077 ± 0.023 min^–1 and the equilibrium accumulation (A_max) of ³H-MPP⁺ was 20.2 ± 2.0 pmol mg protein^–1 (n = 36). Decynium22 (1,1-diethyl-2,2-cyanide; 1 M) significantly reduced k_in to 6.1 ± 1.8 l mg protein^–1 min^–1 (P < 0.05) and the equilibrium accumulation (A_max) of ³H-MPP⁺ to 9.6 ± 1.3 pmol mg protein^–1 (P < 0.005) (n = 36). ³H-MPP⁺ accumulation (in cells incubated with 200 nM ³H-MPP⁺) was sensitive to (–)-adrenaline, (–)-isoprenaline, (–)-dopamine, (±)-adrenaline and (–)-noradrenaline. The most potent catecholamine in inhibiting ³H-MPP⁺ uptake was (–)-adrenaline, with an IC₅₀ of 99 (22, 449) M (n = 6). (–)-Adrenaline competitively inhibited ³H-MPP⁺ uptake, as it significantly increased the K_m value of ³H-MPP⁺ uptake (to 125.4 M (63.6; 187.1); P < 0.02; n = 3) but did not change the V_max value. The cyanide-derivatives decynium22 and cyanine863 (1-ethyl-2-([1,4-dimethyl-2-phenyl-6-pyrimidinylidene]methyl)quinolinium), which inhibit uptake₂ as well as the apical type of the renal transporter for organic cations, potently inhibited ³H-MPP⁺ uptake with IC₅₀'s of 1.4 (0.4–5.3) (n = 6) and 6.5 (2.6–16) (n = 4) M, respectively. Under conditions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) inhibition with either pargyline (500 M + Ro01-2812) (3,5-dinitropyrocatechol; 2 M) or pargyline (500 M) + U-0521(3,4-dihidroxy-2-methyl-propiophenone; l2 M)), (–)-adrenaline (up to 1 mM) had no inhibitory effect on the uptake of ³H-MPP⁺. Moreover, the uptake of ³H-MPP⁺ in the presence of pargyline + Ro 01-2812 was significantly lower (66.9 ± 30.4%; P < 0.05; n = 4) than in the absence of these compounds. Therefore, the effect of these MAO and COMT inhibitors on ³H-MPP⁺ uptake was examined. Interestingly enough, pargyline, Ro 01-2812 and U-0521 were found to inhibit the uptake of ³H-MPP⁺ (in cells incubated with 200 nM ³H-MPP⁺): 500 M pargyline, 2 M Ro 012812 and 100 M U-0521 decreased the accumulation of ³H-MPP⁺ to 38.1 ± 6.8 (n = 5), 60.5 ± 10.1(n = 7) and 71.3 ± 14.5 (n = 7) % of control, respectively.It is concluded that ³H-MPP⁺ is efficiently taken up by rat hepatocytes by a carrier-mediated mechanism sensitive to catecholamines, decynium22 and cy anine863, and to the enzyme inhibitors pargyline, Ro 01-2812 and U-0521.     

Saturable Absorptive Transport of the Hydrophilic Organic Cation Ranitidine in Caco-2 Cells: Role of pH-Dependent Organic Cation Uptake System and P-Glycoprotein

Purpose The purpose of this work was to investigate the involvement of carrier-mediated apical (AP) uptake and efflux mechanisms in the absorptive intestinal transport of the hydrophilic cationic drug ranitidine in Caco-2 cells. Methods Absorptive transport and AP uptake of ranitidine were determined in Caco-2 cells as a function of concentration. Permeability of ranitidine in the absorptive and secretory directions was assessed in the absence or presence of the P-glycoprotein (P-gp) inhibitor, GW918. Characterization of the uptake mechanism was performed with respect to inhibitor specificity, pH, energy, membrane potential, and Na⁺ dependence. Efflux from preloaded monolayers was evaluated over a range of concentrations and in the absence or presence of high extracellular ranitidine concentrations. Results Saturable absorptive transport and AP uptake of ranitidine were observed with K_m values of 0.27 and 0.45 mM, respectively. The ranitidine absorptive permeability increased and secretory permeability decreased upon inhibition of P-gp. AP ranitidine uptake was inhibited in a concentration-dependent fashion by a diverse set of organic cations including tetraethylammonium, 1-methyl-4-phenylpyridinium, famotidine, and quinidine. AP ranitidine uptake was pH and membrane potential dependent and reduced under conditions that deplete metabolic energy. Efflux of [³H]ranitidine across the basolateral membrane was neither saturable as a function of concentration nor trans stimulated by unlabeled ranitidine. Conclusions Saturable absorptive transport of ranitidine in Caco-2 cells is partially mediated via a pH-dependent uptake transporter for organic cations and is subject to attenuation by P-gp. Inhibition and driving force studies suggest the uptake carrier exhibits similar properties to cloned human organic cation transporters. The results also imply ranitidine transport is not solely restricted to the paracellular space.     

Influences of Immobilization and Footshock Stress on Pharmacokinetics of Theophylline and Caffeine in Rats

The influences of immobilization and footshock stress on pharmacokinetics of theophylline (20 mg kg^?1) and caffeine (30 mg kg^?1) administered orally were examined in rats. The immobilization stress for 30 min or 1 h immediately after oral administration caused marked immobilization period-related decreases in plasma theophylline concentrations during the absorption phase, but did not affect plasma caffeine concentrations. The k_a and C_max values for theophylline were significantly decreased, and the t_max was significantly increased. On the other hand, when the immobilization stress was loaded for 1 or 3 h before the oral administration, the plasma theophylline or caffeine concentrations were not affected. The footshock stress for 30 min immediately after oral administration did not significantly decrease plasma theophylline concentrations during the absorption phase. These results suggest that the pharmacokinetics of theophylline are influenced by strong stress, possibly due to the inhibition of its absorption from the gastrointestinal tract, but the pharmacokinetics of caffeine are not influenced by stress, probably due to its central action.     

R 56865 exerts cardioprotective properties independent of the intracellular Na<Superscript>+</Superscript>-overload in the guinea pig heart

Reducing intracellular Na⁺-accumulation during ischemia exerts cardioprotective effects in reperfusion in a variety of models. Since slowly-inactivating Na⁺-channels may contribute to Na⁺-influx in ischemia, we investigated whether the ischemia-protective properties of R 56865, an inhibitor of slowly inactivating Na⁺-channels, are mediated by inhibition of the ischemic Na⁺-overload. Monitoring intracellular Na⁺ (Na⁺_i) by ²³Na-NMR-spectroscopy revealed a continuous rise of Na⁺_i during ischemia in the isolated perfused guinea pig heart. Within 30 and 60 min of ischemia, respectively, Na⁺_i had risen 2.6±0.2- and 4.4±0.2-fold compared to baseline (n=6). R 56865 (1 M) did not influence the time course of the Na⁺_i-accumulation at any point of the ischemic period. R 56865, however, showed marked cardioprotective properties: in the reperfusion period the agent markedly improved the restoration of left ventricular developed pressure (29.1±6.8 mm Hg vs. 2.4±2.0 mm Hg), ATP (2.8±0.3 mM vs. 1.7±0.6 mM) and phosphocreatine (10.9±2.2 mM vs. 6.8±1.1 mM), furthermore contracture development was reduced. The present study strongly suggests slowly-inactivating Na⁺-channels being at best a minor port of Na⁺-entry in the ischemic guinea pig heart. It clearly demonstrates that the potent cardioprotective properties of R 56865 are unrelated to intracellular sodium homeostasis.     

The extraneuronal transport mechanism for noradrenaline (uptake2) avidly transports 1-methyl-4-phenylpyridinium (MPP+)

Summary The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake₂) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake₂ has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake₂ in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP⁺).Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake₂. The initial rate of ³H-noradrenaline transport [k_in = 0.58 l/(mg protein · min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (k_{O-methylation} = 0.54 min^–1). The neurotoxin MPP⁺ was found to be a good substrate of uptake₂. Initial rates of specific ³H-MPP⁺ transport into Caki-1 cells were saturable, the K_m being 24 mol/l and the V_max being 420 pmol/(mg protein · min). The rate constant of specific inward transport was 34 times higher [19.6 mol/l (mg protein · min)] than that of ³H-noradrenaline. The ratio specific over non-specific transport was considerably higher for ³H-MPP⁺ (12.6) than for ³H-noradrenaline (3.0). ³H-MPP⁺ transport into Caki-1 cells was inhibited by various inhibitors of uptake₂. The highly significant positive correlation (p < 0.001, r = 0.986, n = 7) between the IC₅₀'s for the inhibition of the transport of ³H-noradrenaline and ³H-MPP⁺, respectively, proves the hypothesis that MPP⁺ enters Caki-1 cells via uptake₂. ³H-MPP⁺ is taken up via uptake₂ not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake₂.Tritiated MPP⁺ is a new and convenient tool for the investigation of uptake₂. The rate constant for inward transport, the factor of accumulation and the ratio specific over non-specific transport are considerably higher for ³H-MPP⁺ than for ³H-noradrenaline. In uptake studies with ³H-MPP⁺ inhibition of intracellular noradrenaline-metabolizing enzymes is not necessary. In tissues and tissue cultures which possess fewer uptake₂ carriers than Caki-1 cells or the rat heart, the identification and characterization of uptake₂ can be expected to be greatly facilitated by the use of ³H-MPP⁺.Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to H. Russ at the above address     

Influence of Osmolality of Solutions Used for K+ Contraction on Relaxant Responses to Pinacidil,Verapamil, Theophylline and Terbutaline in Isolated Airway Smooth Muscle

Abstract: Concentration-relaxation profiles for pinacidil, verapamil, terbutaline and theophylline were studied in guinea-pig trachealis contracted by two commonly applied techniques for K⁺ depolarization. All drugs were much less effective on contractions induced by hyperosmolar 124 mM K⁺ solution (added KCl) than on contractions elicited by an isoosmolar 124 mM K⁺ Krebs solution (substituted KCl). The maximal relaxant responses were (isoosmolar K⁺/hyperosmolar K⁺): pinacidil 100%/40%, verapamil 100%/60%, theophylline 100%/0%, terbutaline 50%/0%. Addition of mannitol to establish the same hyperosmolarity as with 124 mM KCl also produced contraction of guinea-pig trachealis. Concentration-relaxation curves for the drugs on mannitol-induced contractions had close resemblance to those obtained in hyperosmolar 124 mM K⁺ solution. When contraction was elicited by 30 mM K⁺, pinacidil showed seven times higher relaxant potency in hyperosmolar compared to isoosmolar solution, whereas the relaxant responses to verapamil, theophylline and terbutaline were not influenced by osmolality. When K⁺ depolarization is used as a tool for evaluation of drug action in airway smooth muscle, the two different techniques produce dissimilar results. The influence of hyperosmolarity pec se appears to be an important and unwanted feature when K⁺ depolarization is produced by addition of KCl.     

Carrier-mediated intestinal absorption of valacyclovir,the L-valyl ester prodrug of acyclovir. 1. Interactions with peptides,organic anions and organic cations in rats

The mechanism of intestinal transport of valacyclovir (VACV), the L-valyl ester prodrug of acyclovir, was investigated in rats using an in situ intestinal perfusion technique. VACV demonstrates an oral bioavailability that is three to five time greater than acyclovir, concentration dependent, and saturable in humans. Homogenate and perfused buffer stability results demonstrated that VACV was increasingly unstable with increasing pH. VACV was converted to ACV in a concentration dependent manner during a single pass through the intestinal segment. Perfusions were performed at 37°C, pH 6.5, and under iso-osmotic conditions (290±10 mOsm L⁻¹). Intestinal outlet concentrations were corrected for VACV that was converted to ACV during the perfusion. The effective dimensionless intestinal permeability (P_e*) of VACV was concentration dependent, saturable (intrinsic K_m = 1.2±0.7 mM), and significantly reduced (p <0.05) in the presence of peptide analogues (amoxicillin, ampicillin, cefadroxil, and cephradine), by the organic anion, p -amino hippuric acid and by the organic cation quinine. VACV transport was not inhibited by classical nucleoside competitive substrates or inhibitors or by valine. These results suggest that H⁺–oligopeptide, H⁺–organic cation, and organic anion transporters are involved in the small-intestinal uptake of VACV. The permeability of VACV in the colon was very low, indicating that VACV is predominantly absorbed from the small intestine. VACV P_e* was not altered in the presence of glucose-induced convective fluid flow, suggesting that carrier-mediated, transcellular uptake is the predominant absorption pathway of VACV in rat small intestine. Based on these results, the oral bioavailability of VACV appears to be significantly influenced by the preabsorptive conversion of VACV to the poorly absorbed ACV, by the involvement of multiple transporters in VACV small-intestinal uptake, and by the low permeability of VACV in the colon. © 1998 John Wiley & Sons, Ltd.     

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.     

Construction of a Functional Transporter Analysis System Using <Emphasis Type="BoldItalic">MDR1</Emphasis> Knockdown Caco-2 Cells

Purpose The efflux transporter, P-glycoprotein (P-gp), located in the brush-border membrane of intestinal absorptive cells, reduces the bioavailability of a wide range of orally administered drugs. Using P-gp inhibitors in transport experiments in Caco-2 cell monolayers is widely accepted as an efficient way to estimate the contribution of P-gp to the intestinal absorption of drugs. However, there still remain some arguments that the inhibitors might affect the function of other proteins. Multidrug resistance 1 gene (MDR1) specifically inhibited Caco-2 cells were constructed, therefore, as a better in vitro evaluation system of intestinal drug absorption.Methods The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNA^val-shRNA expression vector.Results In siRNA stably expressed Caco-2 cells, the expression level of MDR1 was reduced at mRNA and protein levels. Transcellular transport studies using digoxin revealed that the P-gp function was suppressed completely, similar to that in verapamil-treated cells.Conclusions MDR1 stable knockdown Caco-2 cells were successfully constructed by RNAi technology. This will consequently allow the development of a selection system for candidate drugs with improved absorption properties.     

Voltage Dependence of Transepithelial Guanidine Permeation Across Caco-2 Epithelia Allows Determination of the Paracellular Flux Component

Purpose The aim of this study was to investigate transepithelial ionic permeation via the paracellular pathway of human Caco-2 epithelial monolayers and its contribution to absorption of the base guanidine. Methods Confluent monolayers of Caco-2 epithelial cells were mounted in Ussing chambers and the transepithelial conductance and electrical potential difference (p.d.) determined after NaCl dilution or medium Na substitution (bi-ionic conditions). Guanidine absorption (J_a–b) was measured ± transepithelial potential gradients using bi-ionic p.d.'s. Results Basal NaCl replacement with mannitol gives a transepithelial dilution p.d. of 28.0 ± 3.1 mV basal solution electropositive (P_Cl/P_Na = 0.34). Bi-ionic p.d.'s (basal replacements) indicate a cation selectivity of NH₄⁺ > K⁺∼Cs⁺ > Na⁺ > Li⁺ > tetraethylammonium⁺ > N-methyl-d-glucamine⁺∼choline⁺. Transepithelial conductances show good correspondence with bi-ionic potential data. Guanidine J_a–b was markedly sensitive to imposed transepithelial potential difference. The ratio of guanidine to mannitol permeability (measured simultaneously) increased from 3.6 in the absence of an imposed p.d. to 13.8 (basolateral negative p.d.). Conclusions Hydrated monovalent ions preferentially permeate the paracellular pathway (Eisenman sequence 2 or 3). Guanidine may access the paracellular pathway because absorptive flux is sensitive to the transepithelial potential difference. An alternative method to assess paracellular-mediated flux of charged organic molecules is suggested.     

Possible involvement of Ca2+-induced Ca2+ release mechanism in Ag+-induced contracture in frog skeletal muscle

To determine if an Ag⁺-induced contracture is associated with the Ca²⁺-induced Ca²⁺ release mechanism in the sarcoplasmic reticulum, effects of Ca²⁺-induced Ca²⁺ release modulators on the Ag⁺-induced contracture were studied with single fibers of frog toe skeletal muscle. The fiber treated with 1 mM caffeine contracted significantly much more than controls without caffeine at Ag⁺ concentrations below 1 μM. Procaine shifted the Ag⁺ concentration-tension curve to the right, dose-dependently. When 10 mM procaine was applied to contracting fibers not treated with caffeine, the duration of 5 μM Ag⁺-induced contracture was shortened with a little decrease in tension amplitude, that was different from the effect of procaine on caffeine contracture. In caffeine solution, 0.5 μM Ag⁺ caused a long-lasting contracture with sometimes two peaks. 2 mM procaine led to disappearance of such two peaks, resulting in shortening of the contracture. K⁺ contracture was potentiated by 1 mM caffeine only at lower concentrations of K⁺, and inhibited by 10 mM procaine. These results suggest that the Ag⁺-induced contracture is composed of two components: Ca²⁺-induced Ca²⁺ release-dependent and -independent. 5 μM Ag⁺-induced contracture slowly relaxed with a wavy tension pattern to the resting level when 0.05 mM dithiothreitol was applied around peak of the tension. This relaxation was accelerated by procaine application. These findings may be explained by attributing a portion of Ag⁺-induced contracture to the effect of Ca²⁺ released through the Ca²⁺-induced Ca²⁺ release mechanism in the sarcoplasmic reticulum.     

Mechanism and Kinetics of Transcellular Transport of a New β-Lactam Antibiotic Loracarbef Across an Intestinal Epithelial Membrane Model System (Caco-2)

Various processes involved in the transcellular transport (TT) of loracarbef (LOR) were studied in the Caco-2 cell monolayer, a cell culture model of the small intestinal epithelium. The results provide support for presence of two AP to BL peptide TT pathways in the intestinal epithelial cell monolayer (Caco-2). The H⁺ gradient-dependent pathway (K_m = 0.789 mM, and J_max = 163 pmol/min per cm²) is relatively high affinity and low capacity compared to H⁺ gradient-independent pathway (K_m = 8.28 mM, and J_max = 316 pmol/min per cm²). In addition, TT of LOR in the presence of a H⁺ gradient was inhibited 77% to 88% (p < 0.05) by 10 mM of cephalexin, enalapril, Gly-Pro and Phe-Pro, while TT of LOR in the absence of a H⁺ gradient was only inhibited 42% to 48% (p < 0.05) by 10 mM of Gly-Pro and Phe-Pro. Since AP uptake is H⁺ gradient-dependent and saturable while the BL efflux is mostly nonsaturable and not driven by a H⁺ gradient, these two transmembrane transport processes must be different, which could be the result of two different peptide carriers. In vivo, these two transport processes must have worked in concert to produce transcellular flux of loracarbef. To explain the differences between kinetic characteristics of AP uptake and TT transport, a cellular pharmacokinetic (PK) model was developed and the results indicate that the PK model appropriately described the kinetics of LOR TT. The use of this PK model may provide an additional advantage to the use of the cell culture model because kinetic parameters at both sides of the intestinal epithelial membrane may be obtained using the same preparation. Taken together, the Caco-2 model system represents an excellent model system for the study of carrier-mediated processes involved in the TT of peptides and peptide-like drugs.