Primary active transport of peptidic endothelin antagonists by rat hepatic canalicular membrane

The biliary excretion mechanism of three derivatives of BQ-123, an anionic cyclopentapeptide, was examined using isolated canalicular membrane vesicles (CMVs) from Sprague-Dawley rats. The uptake by CMV of BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, was stimulated by ATP. An "overshoot" phenomenon and saturation were observed for the ATP-dependent uptake of these three peptides. The Michaelis-Menten constants (Km) for the uptake of BQ-485 and BQ-518 were comparable to the inhibition constants (Ki) for their inhibitory effects on ATP-dependent [3H]BQ-123 uptake. The uptake of BQ-485 showed the highest value and was inhibited by BQ-123 with a Ki that was comparable to the Km for BQ-123 uptake. The ATP-dependent uptake of BQ-123, BQ-485, and BQ-518 was much lower in CMVs from Eisai hyperbilirubinemic rats, a strain having a hereditary defect of the canalicular multispecific organic anion transporter (cMOAT). These results suggest that both BQ-485 and BQ-518 principally share the cMOAT transporter with BQ-123. Compound A almost completely inhibited BQ-123 uptake, although its ATP-dependent uptake was much lower than that of the other three peptides. The ATP-dependent uptake of compound A was not very different in Sprague-Dawley rats and Eisai hyperbilirubinemic rats and was not inhibited by S-(2, 4-dinitrophenyl)-glutathione, a typical substrate for cMOAT. Thus, although compound A inhibits cMOAT-mediated transport, its own transport by cMOAT is minimal and mediated by another transporter. This low degree of primary active transport by cMOAT may be the principal reason for its relatively longer residence in the circulation.     

High Helicobacter pylori eradication rate with a 1-week regimen containing ranitidine bismuth citrate

HSR-903 is a newly synthesized quinolone antibacterial agent with low toxicity. The biliary and urinary excretion of unchanged HSR-903, its R-isomer, and their glucuronides was determined after iv bolus administration (5 mg/kg) to normal Sprague-Dawley rats (SDR) and Eisai hyperbilirubinemic mutant rats (EHBR). The values for the biliary excretion clearance of HSR-903 and its glucuronide in EHBR were decreased to approximately 40 and 2% of those in SDR, respectively, whereas the values for the urinary excretion clearance of HSR-903 and its glucuronide were comparable in SDR and EHBR. The biliary excretion clearance values for the R-isomer and its glucuronide were approximately 3 times greater than those for HSR-903. These results demonstrated that the enantiomers of HSR-903 and their conjugates were excreted into bile in a stereospecific manner. The hepatic uptake of [14C]HSR-903 in vivo was evaluated by means of integration plot analysis. The results indicated that the hepatic uptake of [14C]HSR-903 was very fast and was blood flow-limited. To clarify the mechanism of excretion of HSR-903 into bile, the uptake and efflux of [14C]HSR-903 were studied using isolated hepatocytes from SDR and EHBR. The initial uptake of HSR-903 by hepatocytes was temperature-dependent, saturable, and stereospecific. Unlabeled HSR-903 (S-isomer), the R-isomer, grepafloxacin, and sparfloxacin significantly inhibited the uptake of [14C]HSR-903. The efflux of [14C]HSR-903 from hepatocytes from EHBR was significantly slower than that from hepatocytes from SDR. The addition of sodium azide or bromosulfophthalein reduced the efflux of [14C]HSR-903. These results demonstrate that HSR-903 is actively excreted into bile via the canalicular multispecific organic anion transporter, which is deficient in EHBR.     

Modulators of the protein kinase C system influence biliary excretion of cationic drugs

To investigate whether hepatobiliary transport of organic cations is under regulatory control, we studied transport of tri-n-butylmethylammonium in the isolated perfused rat liver and in isolated rat hepatocytes. Transport was investigated in the presence of modulators of the protein kinase C and the cyclic AMP second-messenger system. In the isolated perfused rat liver, it was observed that compounds modulating protein kinase C activity clearly affected the biliary excretion process of the cation tri-n-butylmethylammonium. Phorbol 12-myristate 13-acetate, a compound that directly stimulates protein kinase C, elevated the biliary excretion rate of tri-n-butylmethylammonium in a concentration-dependent manner, reaching a twofold increase at 60 nmol/L of the phorbol ester. The inactive derivative 4 alpha-phorbol 12, 13-didecanoate (60 nmol/L) did not show any effect. Vasopressin (48 nmol/L), a receptor-mediated activator of protein kinase C, stimulated the excretion rate of the cation by about 50%. Staurosporin (1 mumol/L), an inhibitor of protein kinase C, clearly decreased the biliary excretion rate of the cation and also blocked its stimulation by phorbol 12-myristate 13-acetate. Neither phorbol 12-myristate 13-acetate nor vasopressin (at concentrations ranging from 10(-9) to 10(-6) mol/L) affected the initial uptake velocity of tri-n-butylmethylammonium in isolated hepatocytes and isolated perfused livers, whereas staurosporin (1 mumol/L) showed only a modest inhibition of the uptake of the cation. It is inferred that the effect of protein kinase C modulators on hepatobiliary transport of organic cations occurs at the level of carrier-mediated transport in the canalicular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carrier-mediated hepatic uptake of quinolone antibiotics in the rat

The systemic clearance of many quinolone antibiotics is mainly via metabolism and urinary excretion; by contrast, biliary excretion is a major route of elimination for a new quinolone grepafloxacin (GPFX). Accordingly, we studied the hepatic uptake of GPFX because it is the first step in the drug's hepatobiliary transport. The hepatic uptake of GPFX in vivo after i.v. administration was found to approach the hepatic blood flow, suggesting the existence of an effective hepatic uptake mechanism. To clarify this transport mechanism, GPFX uptake by isolated rat hepatocytes was examined and found to consist of a saturable component (Km 173 microM, Vmax 6.96 nmol/min/mg) and a nonspecific diffusion component. The inhibition of GPFX uptake by ATP-depletors and a lack of effect after replacing Na+ with choline demonstrated that the uptake was an Na+-independent carrier-mediated active process. This uptake was inhibited by other quinolones and for lomefloxacin this was competitive in nature. Mutual inhibition studies were undertaken to investigate whether the transporter for GPFX might be the same as other transporters so far identified. GPFX inhibited the uptake of taurocholic acid, pravastatin (organic anion), cimetidine (organic cation) and ouabain (neutral steroid). However, GPFX uptake was not inhibited by these compounds. Confirmation that GPFX uptake is blood flow limited was obtained by extrapolation of the in vitro data based on mathematical modeling. In conclusion, the effective hepatic uptake of quinolone antibiotics are via carrier-mediated active transport, which is distinct from that involved in the transport of bile acids, organic anions, organic cations or neutral steroids.     

Endothelin A-receptor blockade worsens endotoxin-induced hepatic microcirculatory changes and necrosis

BACKGROUND & AIMS: Endothelin 1 is considered to be an important regulator of sinusoidal blood flow and increases during endotoxemia. The purpose of this study was to investigate the role of endothelin 1 in hepatic microcirculation, oxygen transport, and liver injury during endotoxemia. METHODS: Male Sprague-Dawley rats were continuously infused with 2.5 mL/h of saline, 0.8 mg . kg-1 . h-1 of lipopolysaccharide (LPS), 3 mg . kg-1 . h-1 of BQ-485, an endothelin A-receptor antagonist, or LPS plus BQ-485 for 7 hours. RESULTS: BQ-485 infusion had no significant effect on hepatic microcirculation and liver injury. LPS increased the plasma levels of aspartate aminotransferase (AST) and total bilirubin and decreased the hepatic adenosine triphosphate (ATP) level and bile flow rate. LPS + BQ-485 infusion further increased the plasma levels of AST and total bilirubin and decreased the bile flow rate and the hepatic ATP level. Dual-spot microspectroscopy revealed mild decreases in sinusoidal erythrocyte velocity and oxygen transport in the LPS group and profound decreases in these parameters in the LPS + BQ-485 group. Histological examinations revealed massive necrotic changes in the pericentral regions of the LPS + BQ-485 group. CONCLUSIONS: These results suggest that blockade of endothelin A receptors disturbs hepatic microcirculation and oxygen transport and aggravates the necrotic injury induced by endotoxin.     

Noninvasive assessment of hepatobiliary and renal elimination of cysteinyl leukotrienes by positron emission tomography

N-Acetyl-leukotriene E4 has been identified as an endogenous, biologically less active cysteinyl leukotriene metabolite in rodents and humans. To evaluate the ratio of hepatobiliary to renal elimination of leukotrienes noninvasively by positron emission tomography (PET), we synthesized N-[11C]acetyl-leukotriene E4 by chemical N-acetylation of leukotriene E4. After the intravenous injection of N-[11C]acetyl-leukotriene E4 in normal rats and monkey, uptake by the liver and subsequent excretion into bile were largely responsible for its rapid elimination from blood. In the Cynomolgus monkey, renal excretion of the leukotriene into urine was of additional quantitative importance. Kinetic modeling indicated a mean transit time through the liver of 17 minutes and 34 minutes in rat and monkey, respectively; the corresponding hepatic excretion half-times amounted to 8.5 minutes and 16 minutes. In a mutant rat strain deficient in the hepatobiliary excretion of cysteinyl leukotrienes across the canalicular membrane, the apparent mean liver transit time was 54 minutes, and the hepatic excretion half-time was 29 minutes, indicating prolonged organ storage and metabolism. After transport from the liver back into the circulating blood of omega-oxidized and beta-oxidized metabolites of N-[11C]acetyl-leukotriene E4, renal excretion compensated for the impairment of hepatobiliary elimination in the transport mutant. Metabolite analyses in urine after intravenous injection of N-[3H]acetyl-leukotriene E4 indicated the extensive inactivation of N-acetyl-leukotriene E4 by beta-oxidation from the omega-end in the mutants. A similar shift from hepatobiliary to renal cysteinyl leukotriene elimination was monitored in rats with cholestasis due to bile duct obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multispecific organic anion transporter is responsible for the biliary excretion of the camptothecin derivative irinotecan and its metabolites in rats

Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11), is a potent anticancer drug that is increasingly used in chemotherapy. A frequent limiting side effect involves gastrointestinal toxicity (diarrhea), which is thought to be related to the biliary excretion of CPT-11 and its metabolites. Accordingly, the biliary excretion mechanisms for both the lactone and carboxylate forms of CPT-11 and its metabolites, SN-38 and its glucuronide (SN38-Glu), were investigated using Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR), with the latter being mutant rats with a genetic deficiency of the canalicular multispecific organic anion transporter. After i.v. administration of CPT-11, the biliary excretion clearance, defined as the biliary excretion rate normalized to the hepatic concentration, of both the lactone and carboxylate forms of SN38-Glu was much lower in EHBR. The biliary excretion clearance for the carboxylate form of both CPT-11 and SN-38 was also substantially smaller in EHBR and showed marked saturation with increasing dose only in SD rats. On the other hand, the biliary excretion clearance for the lactone forms of CPT-11 and SN-38 showed only a minimal difference in EHBR, compared with SD rats. These results suggest that, for the carboxylate form of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu, there exists a specific transport system at the bile canalicular membrane that is deficient in EHBR. To confirm this hypothesis, the uptake of these substrates by isolated hepatic canalicular membrane vesicles (CMV) was examined. ATP-dependence was clearly observed for the uptake of these four compounds by CMV prepared from SD rats but not by CMV from EHBR. In addition, the compounds inhibited the ATP-dependent uptake of S-(2,4-dinitrophenyl) glutathione by CMV from SD rats, in a concentration-dependent manner. These results suggest that the biliary excretion of the carboxylate forms of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu is mediated by the multispecific organic anion transporter, which is deficient in EHBR.     

Functional integrity of hepatocyte canalicular membrane transport of taurocholate and bilirubin diglucuronide in Eisai hyperbilirubinuria rats

Eisai hyperbilirubinuria rats (EHBR) are characterized by conjugated hyperbilirubinemia, and impaired or defective excretion of bilirubin, reduced glutathione and other organic anions from hepatocytes. Hepatocyte canalicular membrane vesicles (CMV) from EHBR and normal SD rats were studied with regard to taurocholate (TC) transport driven by ATP or a membrane potential and bicarbonate-stimulated bilirubin diglucuronide (BDG) transport. ATP-dependent uptake or association of BDG with CMV was also studied in both strains of rats. No significant differences in the uptake of TC and BDG by CMV were observed. This indicates the functional integrity of the canalicular transporters for both organic anions in EHBR. Biliary excretion of taurolithocholic acid sulfate (TLCS) is defective in EHBR. However, TLCS inhibited ATP-dependent TC uptake by SD rat CMV competitively, which may be against the hypothesis that a common organic anion carrier is defective in canalicular membranes of jaundiced rats.     

Systemic and renal effects of an ET(A) receptor subtype-specific antagonist in healthy subjects

1. Endothelins (ETs) might play a pathophysiological role in a variety of vascular diseases. The aim of the present study was to characterize the effects of BQ-123, a specific ET(A) receptor antagonist on systemic and renal haemodynamics in healthy subjects. This was done at baseline and during infusion of exogenous ET-1. 2. The study was performed in a balanced, randomized, placebo-controlled, double blind 4 way cross-over design in 10 healthy male subjects. Subjects received co-infusions of ET-1 (2.5 ng kg(-1) min(-1) for 120 min) or placebo and BQ-123 (15 microg min(-1) for 60 min and subsequently 60 microg min(-1) for 60 min) or placebo. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were assessed by the para-aminohippurate (PAH) and the inulin plasma clearance method, respectively. 3. BQ-123 alone had no renal or systemic haemodynamic effect. ET-1 significantly reduced RPF (-24%, P<0.001) and GFR (-12%, P=0.034). These effects were abolished by co-infusion of either dose of BQ-123 (RPF: P=0.0012; GFR: P=0.020). 4. BQ-123 reversed the renal haemodynamic effects induced by exogenous ET-1 in vivo. This indicates that vasoconstriction in the kidney provoked by ET-1 is predominantly mediated by the ET(A) receptor subtype.     

Selective ETA receptor antagonism with BQ-123 is insufficient to inhibit angioplasty induced neointima formation in the rat

OBJECTIVE: The aim was to assess whether or not the endothelin ETA receptor selective antagonist BQ-123 could inhibit neointima formation in vivo following balloon angioplasty. METHODS: The effect of either acute administration of BQ-123 (0.1 mg.kg-1.min-1 intra-arterial infusion for 1 h before and 1 h after angioplasty) or chronic administration (bolus intraperitoneal injection, 2.5 mg.kg-1 twice daily; continuous intraperitoneal infusion, 0.8 and 8 mg.kg-1.d-1) on neointima formation was examined in rats which had undergone left common carotid artery balloon angioplasty. RESULTS: Neither acute intra-arterial infusion nor either mode of chronic intraperitoneal administration of BQ-123 had a significant effect on the degree of neointima formation observed following balloon angioplasty. CONCLUSIONS: Neither acute nor chronic ETA receptor blockade is sufficient to inhibit angioplasty induced neointima formation in the rat. Since it was previously shown that the ETA/B antagonist SB 209670 was effective in this model, while the ETA selective antagonist BQ-123 is now found to be ineffective, the data implicate the ETB receptor subtype in the pathogenesis of neointima formation.     

Expression of the transcriptional regulator Egr-1 in experimental glomerulonephritis: requirement for mesangial cell proliferation

BACKGROUND/AIMS: The hepatic transport of bile salts can be regulated by changes in bile salt pool size and/or in the flux of bile salts through the liver. Prolonged bile salt pool depletion is associated with down-regulation of maximum taurocholate transport and decreased canalicular membrane specific bile salt binding sites. This study was undertaken to investigate: a) whether adaptive down-regulation of maximum hepatic bile salt transport occurs to the same extent for bile acids of different hydrophobicity; and b) the role of microtubule-dependent vesicular pathway in the adaptive changes of bile salt transport capacity. METHODS: Male rats were subjected to 24-h or 48-h external biliary diversion to induce bile salt pool depletion. Basal bile flow, bile salt secretion and lipid secretion, maximum secretory rate of three bile salts of different hydrophobicity (tauroursodeoxycholate, taurocholate and taurochenodeoxycholate) and changes in the biliary excretion of two markers of the microtubule-dependent vesicular pathway (horseradish peroxidase and polyethyleneglycol molecular weight-900) were measured in control and bile salt-depleted rats. Taurocholate-stimulated horseradish peroxidase biliary excretion was also assessed in order to define whether the restoration of bile salt flux across the hepatocytes increased the excretion of this marker in bile salt-depleted rats. RESULTS: The reduction in the maximum secretory rate of the three bile salts under study observed after prolonged biliary diversion was clearly related to their hydrophobicity, with greater reduction for taurochenodeoxycholate and smaller reduction for tauroursodeoxycholate, compared with taurocholate. The biliary excretion of vesicular transport markers was significantly reduced in bile salt-depleted rats. However, when stimulated by taurocholate, biliary excretion of horseradish peroxidase was similar to controls. CONCLUSIONS: The magnitude of the decrease of the hepatic bile salt maximum transport capacity seen after bile salt pool depletion is directly related to the hydrophobicity of the bile salt infused. A functionally depressed vesicular transport pathway appears to be also a contributing factor to this phenomenon.     

Carrier-mediated active transport of the glucuronide and sulfate of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) into rat liver: quantitative comparison of permeability in isolated hepatocytes, perfused liver and liver in vivo

The hepatic uptake of glucuronic acid and sulfate conjugates of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), a dual inhibitor of 5-lipoxygenase and thromboxane A2 synthetase, was investigated in rats. The biliary excretion clearance values for the glucuronide and the sulfate, obtained after i.v. administration of E3040, were similar and corresponded to approximately 30% of the hepatic blood flow rate. The influx clearance values of E3040 conjugates in the presence of 3% bovine serum albumin, measured by a multiple indicator dilution method in the perfused liver, were 1.20 ml/min/g liver for the glucuronide and 0.74 ml/min/g liver for the sulfate, which were twice and equal to the normal hepatic plasma flow rate, respectively, which suggests the presence of an efficient transport system(s). The uptake of E3040 conjugates into the isolated hepatocytes is mediated by Na(+)-independent active transport system(s), which is inhibited by dibromosulfophthalein and bile acids. The uptake for the sulfate had high-affinity and high-capacity transport activity (Km = 25 microM; Vmax = 7.8 nmol/min/10(6) cells) compared with that for the glucuronide (Km = 59 microM; Vmax = 2.2 nmol/min/10(6) cells). The uptakes of E3040 conjugates (glucuronide, sulfate) exhibited a mutual competitive inhibition. It is suggested that both conjugates share a multispecific organic anion transporter located on the sinusoidal membrane.     

Effect of a bacterial lipopolysaccharide on biliary excretion of a beta-lactam antibiotic, cefoperazone, in rats

Klebsiella pneumoniae O3 lipopolysaccharide (LPS) has been found to dramatically modify the pharmacokinetics of the beta-lactam antibiotic cefazolin in rats. This study investigated the effect of LPS on the biliary excretion of the beta-lactam antibiotic cefoperazone (CPZ) in rats. CPZ is known to be actively secreted into the bile by a carrier-mediated transport system. LPS (250 micrograms/kg of body weight) was infused for 20 to 30 min 2 h before an intravenous administration of CPZ (20 mg/kg). The pharmacokinetic parameters of CPZ were estimated by a noncompartment model. LPS induced a significant decrease in the systemic clearance (by approximately 50%) and an increase in the mean residence time of CPZ. Significant decreases were also seen in the bile flow rate and in the biliary recovery of unchanged CPZ in the LPS-treated rats. LPS tended to increase the proportion of urinary excretion of CPZ. LPS significantly decreased the biliary clearance (by approximately 55%) and renal clearance (by approximately 35%) of CPZ. However, no changes in the volume of distribution at steady state for CPZ were observed between the treatment groups. Our findings suggest that LPS induces changes in the pharmacokinetics of CPZ as a result of changes occurring in the biliary secretory system.     

Biliary excretion mechanism of CPT-11 and its metabolites in humans: involvement of primary active transporters

After administration of CTP-11, a camptothecin derivative exhibiting a wide spectrum of antitumor activity, dose-limiting gastrointestinal toxicity with great interpatient variability is observed. Because the biliary excretion is a major elimination pathway for CPT-11 and its metabolites [an active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), and its glucuronide, SN38-Glu], several hypotheses for the toxicity involve biliary excretion. Here, we investigated whether primary active transport is involved in the biliary excretion of anionic forms of CPT-11 and its metabolites in humans using bile canalicular membrane vesicles (cMVs). Uptake of the carboxylate form of CPT-11 and the carboxylate and lactone forms of SN38-Glu by cMVs prepared from five human liver samples was ATP dependent. The concentration dependence of the ATP-dependent uptake of the carboxylate form of CPT-11 and SN38-Glu suggests the involvement of at least two saturable transport components, both with lower affinity and higher capacity than in rats. The ATP-dependent uptake of the carboxylate form of SN-38 showed a single saturable component but was detectable only in one human cMV sample. Both carboxylate and lactone forms of SN38-Glu uptake also showed a large intersample variability, although the variability was less than that observed for the carboxylate form of SN-38. On the other hand, the carboxylate form of CPT-11 exhibited much less variability. The carboxylate forms of SN38-Glu and SN-38 almost completely inhibited the ATP-dependent uptake of leukotriene C4, a well-known substrate of canalicular multispecific organic anion transporter, whereas the inhibition by the carboxylate form of CPT-11 was not as marked. Thus, multiple primary active transport systems are responsible for the biliary excretion of CPT-11 and its metabolites, and the major transport system for CPT-11 differs from that for the other two compounds. A greater degree of inter-cMV variability in the uptake of SN-38 and SN38-Glu may imply that interindividual variability in biliary excretion of these metabolites might contribute to interpatient variability in the toxicity caused by CPT-11.     

Hepatic uptake and biliary excretion of d-tubocurarine and trimethyltubocurarine in the rat in vivo and in isolated perfused rat livers

The clearance from perfusion medium and the biliary excretion of d-tubocurarine (d-TC) and trimethyltubocurarine (tMeTC) was studied in isolated perfused rat livers. Despite the related structure, d-TC exhibited considerably higher lipophilicity and plasma protein binding than its trimethyl derivative. Significant differences in hepatic disposition of the two agents were found. The clearance constant of elimination from the perfusate for d-TC was 2.00 and 0.41 ml/min for tMeTC. Fifty-one percent of the administered d-TC was excreted in the bile during 2 hours of perfusion. For tMeTC this amounted to only 16%. Bile/plasma concentration ratios of d-TC were 10 times those of tMeTC. There was no evidence for biotransformation of the substances. The unequal biliary output cannot be explained by differences in subcellular distribution. After injection into rats in vivo, the major part of drug in the liver is confined to the particulate fractions. Subfractionation studies indicate binding to lysosomes. The hepatocyte cytosol concentrations of d-TC and tMeTC are in the same order and are lower than the concomitant plasma concentrations. Both bile/liver and liver/plasms concentration ratios were higher for d-TC. The results support the idea that the balance of hydrophilic and hydrophobic properties is an important factor determining hepatic transport of organic compounds.     

Probenecid protects against In vivo acetaminophen-induced nephrotoxicity in male Wistar rats

Renal effects of acetaminophen (APAP) were studied in rats pretreated with probenecid to analyze whether acute APAP-induced nephrotoxicity could be related to a probenecid-sensitive transport system for APAP or its S-derived conjugates. The administration of probenecid (200 mg/kg b.wt. i.p.) 30 min before APAP administration (1000 mg/kg b.wt. i.p.) improved urine flow rate and protected against the alterations on glomerular filtration rate and urea and creatinine plasma levels induced by APAP. Fewer epithelial cells and granular casts and a decrease in the urinary excretion of protein and glucose were observed in rats pretreated with probenecid. Probenecid pretreatment promoted an elevation in the urinary 16-hr excretion of APAP and a diminution in the plasma levels attained by APAP. These results suggest that protection afforded by probenecid in vivo could be a consequence of the inhibition of APAP S-conjugate renal uptake and/or an increase in APAP renal clearance. The effects of APAP in presence of probenecid were studied with the isolated perfused kidney model. Perfusion with probenecid (0.1 mM) before APAP (10 mM) did not change APAP direct renal effects, APAP urinary excretion, or APAP renal clearance relative to glomerular filtration rate. Our results suggest that protection afforded by probenecid in vivo could be the result of the inhibition of the uptake of nephrotoxic APAP metabolites and/or a diuresis-induced enhanced APAP renal excretion.     

Teratocarcinosarcoma of the paranasal sinuses: a clinicopathologic and immunohistochemical study

We observed endothelin (ET)-induced contractile responses on prostatic and epididymal segments, as well as the facilitation of an electrically stimulated tone on prostatic segments of isolated rat vas deferens. In both segments, the selective ET(B)-receptor agonists, IRL 1620 and sarafotoxin S6c, produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of contraction potencies (pD2 value) was ET-1 = ET-2 > ET-3 > sarafotoxin S6c = IRL 1620. The maximum responses of ET-induced contractions in the prostatic segments were larger than those in the epididymal segments. The contractile response to ET-3 was antagonized by pretreatment for 30 min with BQ-123 (10 nM), a selective ET(A) receptor antagonist, and BQ-788 (1 microM), a selective ET(B) receptor antagonist. The contractile responses to ET-1 were antagonized by pretreatment with BQ-123 (10 microM), but not with BQ-788 (1 microM). The ET-3-induced facilitation on the twitch response to electrical stimulation in the prostatic segment of the vas deferens was antagonized by BQ-123 (0.1 microM) and BQ-788 (1 microM). The ET-1-induced facilitation was antagonized by pretreatment with BQ-123 (3 microM), but not with BQ-788 (10 microM). These results suggest that in rat vas deferens the ET(A) receptors are divided into BQ-123-sensitive ET(A1) and BQ-123-insensitive ET(A2) subtypes, and the production of a contractile response of smooth muscle as well as the facilitation of neurotransmission are accomplished through mediation by ET(A1)- and ET(A2)-subtypes.     

Antihypertensive effect of a newly synthesized endothelin antagonist, BQ-123, in a genetic hypertensive model

A newly synthesized ET(A)-selective antagonist, BQ-123, was examined with respect to its anti-endothelin(ET) action in vitro and in vivo and its effect on blood pressure in Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP). In isolated porcine coronary arteries, BQ-123 (0.07 microM to 6.0 microM) shifted the concentration-response curve for ET-1 to the right without affecting the maximal response of ET-1, its pA2 value being 7.35. Intravenous infusion of BQ-123 at a rate of 1.2 and 30 mg/kg/hr produced a significant decrease in blood pressure in 20- to 29-week-old SHRSP, but did not alter blood pressure in 13- to 16-week-old WKY or in 18- to 19-week-old and 40-week-old SHR. The hypotensive effect of BQ-123 depended on the pretreatment blood pressure level. These results suggest that ET-1 is involved in part in the maintenance of high blood pressure in malignant hypertension, as exemplified by SHRSP.     

Endothelial modulation of neural sympathetic vascular tone in canine skeletal muscle

The effect of nitric oxide synthase (NOS) inhibition and endothelin-A (ETA)-receptor blockade on neural sympathetic control of vascular tone in the gastrocnemius muscle was examined in anesthetized dogs under conditions of constant flow. Muscle perfusion pressure (MPP) was measured before and after NOS inhibition (Nomega-nitro-L-arginine methyl ester; L-NAME) and ETA-receptor blockade [cyclo-(D-Trp-d-Asp-Pro-D-Val-Leu); BQ-123]. Zero and maximum sympathetic nerve activities were achieved by sciatic nerve cold block and stimulation, respectively. In group 1 (n = 6), MPP was measured 1) before nerve cold block, 2) during nerve cold block, and 3) during nerve stimulation. Measurements under these conditions were repeated after L-NAME and then BQ-123. The same protocol was followed in group 2 (n = 6) except that the order of L-NAME and BQ-123 was reversed. MPP and muscle vascular resistance (MVR) increased after L-NAME and then decreased to control values after BQ-123. MVR decreased after BQ-123 alone and, with the addition of L-NAME, increased to a level not different from that observed during the control period. MVR fell during nerve cold block. This response was not affected by administration of L-NAME followed by BQ-123, but it was attenuated by administration of BQ-123 before L-NAME. The constrictor response during sympathetic nerve stimulation was enhanced by L-NAME; no further effect was observed with BQ-123, nor was the response affected when BQ-123 was given first. These findings indicate that endothelin contributes to 1) basal vascular tone in skeletal muscle and 2) the increase in skeletal muscle vascular resistance after NOS inhibition. Finally, nitric oxide "buffers" the degree of constriction in skeletal muscle vasculature during maximal sympathetic stimulation.     

Hepatic sequestration and modulation of the canalicular transport of the organic cation, daunorubicin, in the Rat

In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments. Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated. We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF-B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR). Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only approximately 10% of the dose administered (0.2-20 micromol) was excreted in bile after 85 minutes. Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF-B cells. Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A. Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively. The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR. No effect on secretion was noted in TR- mutant rats deficient in mrp2. Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion. While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures. These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated.