Intestinal transport of beta-thioglycosides by Na+/glucose cotransporter

Intestinal metabolism and transport of p-nitrophenyl beta-D-thioglucoside (p-NPbetaSglc) and p-nitrophenyl beta-D-thiogalactoside (p-NPbetaSgal) by the Na+/glucose cotransporter were studied in excised small intestine of the rat. p-NPbetaSglc and p-NPbetaSgal were stable enough on the mucosal side to be transported to the serosal side. Transport of p-NPbetaSglc was inhibited in the presence of phloridzin (a Na+/glucose cotransporter inhibitor), glucose, or 3-O-methylglucose (3-OMG). p-NPbetaSglc transport was dependent on Na+ concentration in a sigmoidal manner. The activation energy for transport was 19.4 kcal mol(-1). The distribution of transport activity of p-NPbetaSglc in each region of the small intestine correlated well with that of 3-OMG. These results indicate that p-NPbetaSglc is transported by the Na+/glucose cotransporter in small intestine. The order of turnover rate for glycoside transport by Na+/glucose cotransporter was 3-OMG > p-nitrophenyl beta-O-glucoside > p-NPbetaSglc > p-NPbetaSgal, indicating that the presence of a galactose moiety and a sulphur between the monosaccharide moiety and aglycone decreases the turnover rate of the Na+/glucose cotransporter in the transport of glycosides. In an inhibition study using stable p-NPbetaSglc as a Na+/glucose cotransporter-transportable marker glucoside, it was also shown that the Na+/glucose cotransporter recognized several types of glycosides. In conclusion, displacement of the oxygen at carbon C-1 of glucose by sulphur in thioglycosides decreases the turnover rate of the Na+/glucose cotransporter, but thioglycosides are stable in the small intestine and are transported by the Na+/glucose cotransporter.     

Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity. I. An improved method for preparation of proximal tubular cells and their functional characterization by alpha-methylglucose uptake

Rat renal proximal tubular cells were isolated by successive EGTA and collagenase perfusions and purified by filtration and isopycnic centrifugation. The method is rapid and provides a much higher fraction of proximal tubular cells (90-95%) than comparable methods. The yield of viable (97 +/- 3%) cells is high (30 X 10(6) cells/g kidney). The intracellular ATP was 16 +/- 2 nmol/mg protein and remained essentially constant for at least 3 hr. The cells were characterized for transport of organic ions and glucose. Glucose transport was studied by alpha-[14C]methylglucose uptake; apparent Km and Vmax values were 3.4 +/- 0.5 mM and 4.1 +/- 0.9 nmol/min.mg protein, respectively. This transport could almost be completely inhibited by phloridzin, indicating that the uptake is mediated by the brush border glucose carrier.     

Regional difference in P-glycoprotein function in rat intestine

It has been reported that inhibition of the P-glycoprotein (P-gp) results in the improved absorption of P-gp substrate in the intestinal tract. In fact, the increased permeability of P-gp substrate across the intestinal epithelium was observed following inhibition of P-gp in in vitro experiments. To develop the formulation containing P-gp inhibitor and P-gp substrate for practical use, it is necessary to know whether the results obtained in the in vitro experiments are reproducible at whole body level. It is also important to find out the regional difference of the P-gp activity in the intestinal tract. In this study, we examined whether verapamil, a specific inhibitor of P-gp, improves the absorption of rhodamine123 (Rho123), a substrate of P-gp, from the jejunum, ileum, and colon of rats using the in situ loop method. The water content in the loop decreased during the experiment, resulting in a significant change of the Rho123 concentration in the loop. Thus, to accurately determine the absorption rate of Rho123, it was necessary to measure the water movement. It was found that there was a regional difference in the water movement, i.e., greatest in colon, followed by ileum. Verapamil did not change the water movement in any intestinal regions. When the concentration of Rho123 in the loop was corrected by water movement, the Rho123 clearance was in the order of ileum (1.15 microL/min/cm), colon (0.83 microL/min/cm) and jejunum (0.47 microL/min/cm). In the presence of verapamil, the Rho123 clearance was significantly increased at jejunum and ileum but not in colon (ileum: 2.08 microL/min/cm, colon: 1.14 microL/min/cm, jejunum: 1.28 microL/min/cm). These results suggest that P-gp inhibits the drug absorption in jejunum and ileum. From these results, it is possible to evaluate the role of P-gp and its regional difference in the in situ experiments. In particular, the inhibition of P-gp results in an increase in absorption of the P-gp substrate limited to jejunum and ileum.     

In situ studies of regional absorption of lobucavir and ganciclovir from rabbit intestine and predictions of dose-limited absorption and associated variability in humans

The regional absorption of lobucavir (LBV), an experimental antiviral agent, and ganciclovir (DHPG) was investigated in rabbit intestine using an in situ single-pass perfusion technique. Duodenal, jejunal, and colonic segments in anesthetized rabbits were perfused with drug solutions in a hypotonic buffer at 0.2 mL/min. Effluent perfusate samples for drug analysis were collected every 10 min for 180 min. To account for water absorption during perfusion, an intestinal absorption model was developed to estimate the absorptive clearance (PeA): PeA=Qavexln((QinxCin)/(QoutxCout)), where Qave is a logarithmic average of the inflow (Qin) and outflow perfusion rate (Qout); Cin and C(out) are drug inflow and outflow concentrations. The PeA of LBV in the duodenum and jejunum was 2.1+/-0.77 and 1.7+/-0.46 microL/min/cm (n=3), respectively, 4.8- and 3.0-fold higher than that of DHPG in the same animals. However, LBV PeA decreased significantly in the colon (0.47+/-0.11 microL/min/cm) and was similar to that of DHPG which exhibited no regional differences in absorption. The interplay between PeA and solubility was studied using a compartmental absorption and transit model, and simulations were performed to investigate dose-limited absorption and the sources of variability in absorption where two compounds differ significantly. The dose range where absorption started to decrease was predicted using the model, with LBV exhibiting the phenomenon at a lower dose than DHPG (450 vs. 750 mg). Furthermore, the intersubject variability in human absorption of both compounds was reproduced when the variability in both PeA and the small intestinal transit time was considered in the model. The variability in the ascending colonic transit time also contributed to the intersubject variability observed for DHPG. The results demonstrate value of integrating in situ studies and modeling in predicting these absorption characteristics.     

Kinetic analysis about the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) by isolated rat hepatocytes

The purpose of the present study was to investigate the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was saturable, with a Km of 29.1 +/- 3.2 microM and Vmax of 2.9 +/- 0.1 mmol/min/mg protein. Subsequently, the initial efflux rate of ANS from isolated hepatocytes was determined by resuspending preloaded cells to 3.0% (w/v) BSA buffer. The efflux process for total ANS revealed a little saturability. The mean value of the efflux clearance was 2.2 +/- 0.1 microL/min/mg protein. The efflux rate of ANS from hepatocytes was markedly decreased at 4 degrees C, indicating that the apparent efflux of ANS might not be attributed to the release of ANS bound to the cell surface, but to the efflux of ANS from intracellular space. The efflux clearance was furthermore corrected for the unbound intracellular ANS concentration on the basis of its binding parameters to cytosol. The relation between efflux rate and unbound ANS concentration was fitted well to the Michaelis-Menten equation with a saturable and a nonsaturable components. The Vmax and Km values were 0.54 mmol/min/mg protein, and 10.0 microM, respectively. Based on the comparison of the ratios of Vmax to Km (Vmax/Km) corresponding to the transport clearance, the influx clearance was two times higher than the efflux clearance. Together with our preliminary studies that ATP suppression in hepatocytes substantially inhibited ANS influx rate, we concluded that the hepatic uptake of ANS is actively taken up into hepatocytes via the carrier mediated transport system.     

Carrier-mediated uptake of grepafloxacin, a fluoroquinolone antibiotic, by the isolated rat lung cells

Grepafloxacin (GPFX) is a new quinolone antibiotic (NQ) which is highly distributed to the lung and other tissues. In the present study, to characterize the distribution mechanism of GPFX to the lung, the uptake of GPFX by isolated rat lung cells was examined in vitro. GPFX was rapidly taken up by the cells, and the uptake reached a steady-state within 5 min. The cell-to-medium concentration ratio at equilibrium was 56.8+/-1.9 microL/mg protein, which was much higher than the cellular volume. GPFX uptake consisted of a saturable component (Km: 264+/-181 microM, Vmax: 2.94+/-2.33 nmol/min/mg protein) and a nonsaturable component (Pdif: 7.04+/-2.17 microL/min/mg protein). The uptake of GPFX was reduced in the presence of ATP-depletors (FCCP and Rotenone) and by the replacement of sodium with choline in the medium, suggesting that GPFX uptake is at least partially mediated by an Na+- and energy-dependent process. GPFX uptake tended to be reduced in the presence of other NQs such as levofloxacin, lomefloxacin and sparfloxacin, but was only minimally affected by the substrates of several uptake mechanisms already identified in the liver and kidney such as taurocholate, p-aminohippurate, L-carnitine and tetraethylammonium. These results suggested that GPFX is taken up by the lung partially via carrier-mediated transport system(s), distinct from the identified transporters, and such active transport systems may at least partially account for the efficient distribution of GPFX to the lung.     

Intestinal first-pass metabolism via carboxylesterase in rat jejunum and ileum.

To determine the activity of a major intestinal esterase in the first-pass hydrolysis of O-isovaleryl-propranolol (isovaleryl-PL), a model ester compound, rat intestinal jejunum and blood vessels were perfused simultaneously after inhibition of a carboxylesterase (CES) by bis-p-nitrophenyl phosphate (BNPP). BNPP specifically inhibits approximately 90% of CES activity without influencing aminopeptidase activity or the transport of L-leucyl-p-nitroanilide and p-nitroaniline, nonester compounds. When isovaleryl-PL was perfused into the jejunal lumen after BNPP treatment, its absorption clearance (7.60 +/- 0.74 microl/min) increased approximately 3-fold compared with control, whereas its degradation clearance (32.5 +/- 5.40 microl/min) decreased to 23% of control. Therefore, CES seems to be mainly responsible for the intestinal first-pass hydrolysis of isovaleryl-PL. This finding is consistent with the results from studies of in vitro BNPP inhibition in the mucosal S9 fraction. V(max) values for valeryl-PL, isovaleryl-PL, and p-nitrophenyl acetate in the jejunal S9 fraction were 1.7- to 2.5-fold higher than that in the ileal S9 fraction, which agreed with the jejunum/ileum ratio (approximately 1.5-fold) of mRNA expression levels for the CES2 isozymes, AB010635 and AY034877. These findings indicated that CESs expressed in the intestine markedly contribute to first-pass hydrolysis in both jejunum and ileum.     

Transport of quercetin di-sodium salt in the human intestinal epithelial Caco-2 cell monolayer 139.

Quercetin di-sodium salt (QDS), a water-soluble derivative of quercetin (Q), is a potent free radical scavenger. The aim of this study was to examine the in vitro intestinal transport of QDS compared to that of Q using the Caco-2 human intestinal epithelial cell line. The apical (A) to basolateral (B) transport of QDS was found to be higher than the B to A transport of this compound. This polarized transport involved the presence of a carrier protein system. The involvement of the sodium/glucose transporter-1 (SGLT-1) was shown by using phloridzin, a selective inhibitor of this conveyor system. However, the transport of Q was not affected by this inhibitor. Moreover, the influx of QDS was pH-sensitive and decreased at pH 5.5 compared with that observed at pH 7.4 and 6.5. The permeability of QDS was 10-fold higher than that of Q. This could be explained by the involvement of SLGT-1 and the absence of an active efflux pump in the absorption of QDS in comparison with Q. This finding was supported by comparing the solubility of Q with that of QDS. This study indicates that both the higher solubility of QDS and its dependence on the SGLT-1 transport system resulted in more efficient permeability compared to Q.     

Carrier-mediated transport of glycerol in the perfused rat small intestine

Studies using the closed loop and everted sacs of the rat small intestine recently prompted us to suggest that carrier-mediated transport is involved in the intestinal absorption of glycerol. Although it could be mediated by a novel carrier system, little information is available. The aim of the present study was to kinetically characterize carrier-mediated glycerol transport in the perfused rat small intestine to help in identifying the carrier involved and to explore the possibility that the carrier might be used as a pathway for oral drug delivery and a target for drug development. In situ single-pass perfusion was conducted using a 10-cm midgut segment of the male Wistar rat, and the absorption of [3H]glycerol was evaluated by its disappearance from the intestinal lumen. The absorption of glycerol was saturable and significantly reduced by removing Na+ from the perfusion solution, suggesting the involvement of a Na+-dependent carrier-mediated transport system. The concentration-dependent absorption profile was successfully analyzed by assuming Michaelis-Menten type carrier-mediated transport and simultaneous passive (diffusive) transport. The maximum transport rate (J(max)) was 77.0 pmol/s/cm2 and the Michaelis constant (K(m)) was 1.04 mM, giving a J(max)/K(m) of 7.39 x 10(-5) cm/s. The membrane permeability coefficient for passive transport (P(m,d)) was 6.89 x 10(-5) cm/s, slightly smaller than J(max)/K(m). Therefore, it could be the major mechanism of intestinal glycerol absorption in the low concentration range where carrier-mediated transport conforms to linear kinetics represented by J(max)/K(m). Furthermore, carrier-mediated glycerol transport was found to be inhibited by glycerol 3-phosphate, monoacetin and diglycerol, indicating that the carrier system may be shared by these structural analogues. Thus, the present study has successfully demonstrated and characterized carrier-mediated glycerol transport in the perfused rat small intestine which is a physiologically relevant model.     

Effects of capsaicin on intestinal cephalexin absorption in rats

The effects of capsaicin on intestinal cephalexin absorption were investigated by means of in situ single pass perfusion in rats to clarify whether this pungent compound present in spice is a potential factor altering the intestinal drug absorption processes. Under the control condition, cephalexin was absorbed at a rate of 1.16+/-0.08 and 0.90+/-0.06 nmol/min/cm in the jejunum and ileum, respectively. The intestinal cephalexin absorption rate was decreased when capsaicin was dissolved in the perfusate at a concentration of 400 microM, being 0.54+/-0.07 and 0.46+/-0.10 nmol/min/cm in the jejunum and ileum, respectively. The inhibitive effect of capsaicin on intestinal cephalexin absorption was diminished when ruthenium red, a non-selective inhibitor of the transient receptor potential (TRP) cation channels, was intravenously infused into the rat during the experiment. Moreover, when we evaluated the paracellular permeability of cephalexin by utilizing a competitive inhibitor, glycylsarcosine, it was demonstrated that glycylsarcosine-insensitive intestinal cephalexin absorption in the jejunum was increased by 4.5 times in the presence of 400 microM capsaicin. These findings indicate that capsaicin affects both transcellular and paracellular pathways of intestinal cephalexin absorption by interacting with the TRP cation channels in intestinal tissues, in which capsaicin seems to change the transport activity of H+/peptide co-transporter 1 (PEPT1), and to a lesser degree, it seems to alter the paracellular permeability of the intestinal epithelia.     

Single dose pharmacokinetics of tiaprofenic acid. Effects of food and severe renal insufficiency

The single oral dose pharmacokinetics of tiaprofenic acid (Surgam) has been investigated in fasting and non-fasting healthy volunteers (200 and 300 mg) and in fasting patients with severe renal insufficiency (200 mg). A dose independent pharmacokinetics of tiaprofenic acid was shown in fasting healthy volunteers and the following parameters were calculated after administration of 200 mg: tl = 0.53 +/- 0.15 h, tm = 1.28 +/- 0.19 h, cm = 27.1 micrograms/ml, ka = 2.79 +/- 0.93 h-1, lambda 2 = 1.06 +/- 0.14 h-1, t1/2 = 3.0 +/- 0.2 h, AUCl-infinity = 80 +/- 7 mg X h/l, Clt = 43.8 +/- 3.7 ml/min and V beta = 11.1 +/- 0.8 l. A small, but significant positive deviation from linearity was observed with increasing dose for cm and AUCl-infinity in non-fasting healthy volunteers, probably due to a slightly higher bioavailability of the 300 mg formulation in the non-fasting state as compared with the 200 mg formulation. Intake of food decreased cm significantly at both dosage levels from 27.1 to 19.1 micrograms/ml and from 47.9 to 39.1 micrograms/ml for 200 and 300 mg, respectively. The absorption kinetics of tiaprofenic acid was not significantly different in fasting healthy volunteers and in fasting patients with severe renal insufficiency. However, a significant increase in t1/2 and AUCl-infinity to 5.8 +/- 0.9 h and 173 +/- 34 mg X h/l, respectively, and a significant decrease in total body clearance to 25.5 +/- 5.3 ml/min were observed in this category of patients. No correlation was found between creatinine clearance and tiaprofenic acid clearance.     

Involvement of active transport systems in the mobilization of cadmium by dithiocarbamates in vivo.

Previously, we reported that the action of cadmium (Cd) complexing dithiocarbamates, such as N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD), in removing Cd from the kidney involves a probenecid-sensitive organic anion transport system. However, other mechanisms responsible for Cd mobilizing effects of BGD and HBGD are still unclear. Therefore, in the present study we examined the effects of phloretin (an inhibitor of plasma membrane glucose carrier), phloridzin (an inhibitor of Na(+)-dependent active hexose transport) and alpha-aminoisobutyric acid (AIB, an inhibitor of amino acid transport) on the excretion and distribution of the chelating agents and Cd in mice. Phloretin pretreatment markedly decreased the biliary and urinary excretions of BGD and HBGD. Phloridzin pretreatment also decreased the biliary and urinary excretions of HBGD, but had no effect on the BGD. AIB pretreatment had no effect on the excretions of either BGD or HBGD. Phloretin pretreatment increased the hepatic and renal contents of BGD and HBGD. Contrary to this, phloridzin pretreatment decreased the hepatic content of BGD and hepatic and renal contents of HBGD, while AIB pretreatment decreased the renal contents of BGD and HBGD. The mobilizing effects of BGD and HBGD on the hepatic and renal Cd was also investigated using Cd-exposed mice. Phloretin or phloridzin pretreatment decreased the mobilizing effect of BGD and HBGD on the hepatic Cd, but had no effect on the renal Cd. These results suggest that BGD and HBGD are taken up into the liver and kidney by phloridzin- and phloretin-sensitive transport system, respectively; that Cd-BGD and Cd-HBGD complexes formed in the hepatic cells are secreted to the bile by phloridzin- and phloretin-sensitive transport systems; and that free BGD and HBGD secreted from these organ to the bile and urine might have occurred, at least in part, by different mechanisms.     

Transport and recognition of aminopeptidase-resistant cellobiose-coupled tyrosylglycylglycine by intestinal Na+/glucose cotransporter (SGLT1): recognition of sugar conjugates by SGLT1 is much less restricted than transport.

Transport and recognition of aminopeptidase-resistant cellobiose-coupled tyrosylglycylglycine (CcpTGG) by intestinal Na+/glucose cotransporter (SGLT1) was examined in rat small intestine. Inhibitory effect of phloridzin (SGLT1 inhibitor) on the CcpTGG transport was extremely low. Concentration dependence of the CcpTGG transport was observed, but the primary component of transport was passive diffusion. However, CcpTGG significantly inhibited SGLT1-mediated transport, indicating its recognition by SGLT1. Other glucose-conjugates also inhibited SGLT1-mediated transport. These results indicate that recognition of sugar conjugates by SGLT1 is much less restricted than transport, and that it should be relatively easier to design SGLT1 inhibitors than SGLT1-transportable sugar conjugates.     

Inhibition of esterolysis of enalapril by paraoxon increases the urinary clearance in isolated perfused rat kidney.

The effect of competing elimination pathways on the metabolic and excretory clearance estimates was examined with tracer concentrations of [(3)H]enalapril, which was both metabolized and excreted by the rat kidney. Perturbation was achieved with use of the carboxylesterase inhibitor paraoxon, which inhibited [(3)H]enalapril metabolism to [(3)H]enalaprilat in rat renal S9 fraction. At 0.1, 0.5, 1, and 10 microM paraoxon, esterolysis of enalapril was inhibited by 76 +/- 7, 93 +/- 5, 96 +/- 5, and 93 +/- 6%, respectively. The lowest concentration (0.1 microM) of paraoxon was chosen for single-pass isolated perfused kidney (IPK) studies because viability was least compromised, and the sodium and glucose reabsorptive functions of the IPK remained constant. After an equilibration period (15-20 min at constant pressure, 90-100 mm Hg), perfusion of the rat kidney with [(3)H]enalapril was carried out under constant flow (8 ml/min) for 30 min in the absence and presence of paraoxon (0.1 microM). The metabolic (from 1.83 +/- 0.52 to 1.48 +/- 0.47 ml/min/g) and total renal (from 1.87 +/- 0.46 to 1. 57 +/- 0.41 ml/min/g) clearances of [(3)H]enalapril in the IPKs were decreased significantly (p <.05) in the presence of paraoxon when compared with controls. Concomitantly, the urinary clearance (from 0. 04 +/- 0.07 to 0.09 +/- 0.09 ml/min/g) and the fractional excretion (from 0.23 +/- 0.18 to 0.52 +/- 0.25) of [(3)H]enalapril doubled (p <.05). The study illustrates that a reduction in cellular metabolism of the kidney brings forth a rise in the estimate of clearance of its complimentary pathway, estimate of the excretory (urinary) clearance.     

Transport of the organic cations gonyautoxin 2/3 epimers, a paralytic shellfish poison toxin, through the human and rat intestinal epitheliums.

The aim of this work is to study the mechanisms involved in gonyautoxins (GTXs) intestinal absorption. For this purpose, we studied the transport of GTX 2/3 epimers by intestinal epithelial cell lines (IEC-6 and Caco-2) cultured on polycarbonate filters. Specific transport was calculated by subtracting from the flux of GTX 2/3 measured at 37 degrees C that occurring at 4 degrees C, this being an indication of transcellular transport. The transcellular apical-to-basolateral (A-B) flux in Caco-2 cell monolayers, was greater than that in the opposite direction, suggesting the involvement of an active transport system favoring the absorption of the toxin. However, in IEC-6 cells the transcellular basolateral-to-apical (B-A) specific transport of the toxin was greater than that in the opposite direction. The A-B and B-A fluxes were, respectively, 127 +/- 26 and 205 +/- 23 nmol/min, suggesting the presence of a prevalent secretive process of the toxin in IEC-6 cells. The A-B transport of GTX 2/3 epimers in Caco-2 cells, but not in IEC-6 cells, was partially Na(+)-dependent and significantly inhibited by adenosine. TEA and verapamil in both Caco-2 and IEC-6 cells failed to affect the A-B and B-A transport of GTX 2/3 epimers. Cyanine in IEC-6 cells, but not in Caco-2 cells, increased the A-B flux of the toxin, suggesting the involvement of the organic cation transporter in the absorption of GTX 2/3 epimers. The mitochondrial energetic uncoupler 2,4-dinitrophenol significantly inhibited the A-B and the B-A transport in both cell lines. In conclusion, IEC-6 cells secrete actively the toxins, whereas Caco-2 cells were found to absorb the toxins in a process that was inhibited in the presence of adenosine and the absorption was dependent of Na(+).     

The transport of a reversible proton pump antagonist, 5, 6-dimethyl-2-(4-Fluorophenylamino)-4-(1-methyl-1,2,3, 4-tetrahydroisoquinoline-2-yl) pyrimidine hydrochloride (YH1885), across caco-2 cell monolayers.

5,6-Dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3, 4-tetrahydroisoquinoline-2-yl) pyrimidine hydrochloride (YH1885) is under development as a novel acid pump antagonist by Yuhan Research Center. Previous studies have suggested that the AUC and C(max) of orally dosed YH1885 are dose-dependent in the range of 2 to 500 mg/kg. The objective of the present study was to investigate the absorption mechanism of YH1885 using a human colon carcinoma cell line, Caco-2. The cells were grown to confluency on a permeable polycarbonate membrane insert to permit loading of YH1885 on either the apical or basolateral side of the cell monolayer. The flux across the monolayer from the apical to basolateral side was 3 to 5 times greater than that from the basolateral to apical side. The uptake of YH1885 into the Caco-2 cell monolayer was saturable and appeared to be mediated by a high-affinity transporter, with an apparent K(m) of 1.47+/-0.21 microM and a V(max) of 25.14+/-1.16 pmol/cm(2)/40 s. The apical to basolateral transport across the monolayer was Na(+)-independent, H(+)-sensitive, and energy-dependent. The transport was inhibited significantly by the presence of structural analogs of YH1885 (e.g., YH957, YH1070, and YH1041), some pyrimidine nucleobases (uracil and 5-methyluracil), and nucleobase transport inhibitors (e.g., papaverine, dipyridamole, and phloridzin). These results demonstrate that the apical to basolateral transport of YH1885 across the Caco-2 cell monolayer is partially mediated by a nucleobase transport system, which exhibits high-affinity and energy-dependent properties for YH1885. Saturation of this transport system, in addition to the limited solubility of YH1885 (i.e., approximately 5.3 microM), appears to contribute to the dose-dependent bioavailability of the drug.     

Nucleoside transport in primary cultured rabbit tracheal epithelial cells

The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or analogous molecules.     

Inhibition of renal brush border phosphate transport and stimulation of renal gluconeogenesis by cyclic amp and parathyroid hormone

The aims of the study were to determine whether 8-bromo-cyclic AMP (8BcAMP) in vivo mimics the inhibitory action of parathyroid hormone (PTH) on phosphate transport across the brush border membrane (BBM) of the renal proximal tubule, and to examine whether changes in BBM transport are accompanied by changes in the rate of renal gluconeogenesis. Thyroparathyroidectomized dogs were anesthetized and equilibrated, and control urine collections were obtained prior to removing the left kidney. Subsequent intravenous infusion of 8BcAMP at 50 mg/hr for 2 hr increased fractional excretion of phosphate from 4 +/- 1 (controls) to 29 +/- 4% (P less than 0.001) without changing glomerular filtration. In BBM vesicles isolated from the renal cortex, the initial Na+-dependent transport of phosphate was decreased from 747 +/- 135 (controls) to 564 +/- 126 pmoles per mg per 0.25 min after 8BcAMP (P less than 0.025), but Na+-independent phosphate uptake and Na+-dependent L-proline uptake were not changed significantly. Renal gluconeogenesis in the same animals was increased from 2.5 +/- 0.3 (controls) to 5.3 +/- 0.5 mumoles glucose per g tissue per hr after infusion of 8BcAMP (P less than 0.001). Infusion of PTH, like 8BcAMP, inhibited BBM phosphate transport and stimulated renal gluconeogenesis. We conclude that the inhibitory action of cyclic AMP and PTH on BBM phosphate transport is accompanied by stimulation of gluconeogenesis which suggests, indirectly, that changes in gluconeogenesis may be part of the intracellular mechanism for regulating BBM phosphate uptake in response to certain stimuli.     

In-vivo and in-vitro evidence of a carrier-mediated efflux transport system for oestrone-3-sulphate across the blood-cerebrospinal fluid barrier

The efflux transport of oestrone-3-sulphate, a steroid hormone sulphate, across the blood-cerebrospinal fluid barrier has been examined following its intracerebroventricular administration. [3H]Oestrone-3-sulphate was eliminated from cerebrospinal fluid (CSF) with an apparent efflux clearance of 205 microL min(-1) per rat. There was 25% of unmetabolized [3H]oestrone-3-sulphate in the plasma 5 min after intracerebroventricular administration, indicating that at least a part of [3H]oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier. This efflux transport was inhibited by co-administration of excess oestrone-3-sulphate (25 mM 10 microL = 0.25 micromol) into rat cerebral ventricle. To characterize the oestrone-3-sulphate transport process, an in-vitro uptake experiment was performed using isolated rat choroid plexus. Oestrone-3-sulphate uptake by isolated rat choroid plexus was found to be a saturable process with a Michaelis-Menten constant (Km) of 18.1 +/- 6.3 microM, and a maximum uptake rate (Vmax) of 48.0 +/- 15.1 pmol min(-1) microL(-1) of tissue. The oestrone-3-sulphate transport process was temperature dependent and was inhibited by metabolic inhibitors such as 2,4-dinitrophenol and rotenone, suggesting an energy dependence. This uptake process was also inhibited by steroid hormone sulphates (1 mM dehydroepiandrosterone sulphate and 1 mM oestrone sulphate), bile acids (1 mM taurocholic acid and 1 mM cholic acid) and organic anions (1 mM sulphobromophthalein and 1 mM phenolsulphonphthalein), whereas 1 mM p-aminohippuric acid, 1 mM p-nitrophenol sulphate, 0.1 mM methotrexate and the cardiac glycoside, 2.5 microM digoxin, had little effect. In conclusion, these results provide evidence that oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier via a carrier-mediated efflux transport system.