Hepatic glutathione concentrations and the release of pyrrolic metabolites of the pyrrolizidine alkaloid, monocrotaline, from the isolated perfused liver.

We have examined the relationship between the metabolism of the pyrrolizidine alkaloid, monocrotaline, and glutathione concentration in the isolated, perfused rat liver. On perfusion of monocrotaline (300 microM) through the isolated liver, high concentrations (1.1 mM) of its metabolite glutathionyldehydroretronecine are released into bile, while much lower amounts (4.86 microM; 0.05 mumol/g liver) accumulate in the perfusate over a 1 hr perfusion period. Metabolite concentration in both the bile and perfusate increase when the level of monocrotaline perfused is increased to 900 microM. Metabolite release is also elevated in livers pretreated with phenobarbital. Monocrotaline perfusion lowered glutathione concentrations in the liver from 30 min onwards. Livers from animals treated with buthionine sulfoximine or chloroethanol showed much lower glutathione levels after 60 min perfusion. Livers from chloroethanol-treated (but not buthionine sulfoximine-treated) animals showed significantly lower release of pyrroles into the bile on perfusion with monocrotaline, but there is no effect on the rate of build-up of pyrrolic metabolites in the perfusate. We conclude that hepatic glutathione concentrations and the release of pyrrolic metabolites of monocrotaline mutually interact. Exposure of the liver to monocrotaline reduces glutathione concentrations, while marked depletion of liver glutathione concentration leads to a decrease in the release of monocrotaline metabolites.     

Role of adenosine in catecholamine-induced global coronary functional hyperemia in isolated guinea pig hearts.

This study was designed to test the hypothesis that endogenous adenosine participates in the global coronary functional hyperemia accompanying intracoronary infusions of norepinephrine (NE) and isoproterenol (ISO). Intracoronary adenosine deaminase (ADA) was employed to test the hypothesis in isolated, perfused guinea pig hearts. We measured coronary perfusate flow (CPF) at a constant coronary perfusion pressure. Heart rate (HR), left ventricular pressure, and its rate of development were also measured. Global myocardial oxygen consumption (MVO2) and oxygen extraction were calculated, and blood gases and pH were measured routinely in inflow and outflow perfusate samples. In the absence of ADA, NE and ISO increased HR 67 +/- 6 and 106 +/- 11 beats.min-1, left ventricular pressure development 519 +/- 46 and 375 +/- 35 mm Hg.s-1, MVO2 22 +/- 2 and 28 +/- 3 microliters.min-1.g-1, and CPF 1.6 +/- 0.2 and 2.2 +/- 0.2 ml.min-1.g-1, respectively. With constant infusion of ADA (4.5 U.min-1.g-1, a dose which produces no direct effects on cardiac function) for 4 min, similar increments in HR and left ventricular pressure development were achieved with both catecholamines. Corresponding changes in MVO2 (9 +/- 2 and 6 +/- 3 microliters.min-1.g-1) were significantly less than those seen in the absence of ADA. Moreover, CPF did not increase in response to NE and ISO in the presence of ADA. These findings support an important role for adenosine in catecholamine-induced global coronary functional hyperemia in isolated, perfused guinea pig hearts.     

Propranolol uptake with high capacity by rat perfused lung

Lung isolated from 7-week-old rats was perfused with pH 7.4 Krebs-Ringer bicarbonate buffer solution (35 mL) containing 1 to 100 micrograms mL-1 of propranolol and 3% BSA at the recirculation rate of 8 mL min-1. Almost parallel bi-exponential drug concentration-time curves were obtained at the initial load lower than 10 micrograms mL-1, whereas relatively slow, mono-exponential decline was found after perfusion at 100 micrograms mL-1. Pharmacokinetic analysis for the perfusate propranolol concentration-time curves when loaded at 1 to 10 micrograms mL-1 yielded almost comparable values for the pulmonary perfusion clearance (0.387 +/- 0.092 to 0.486 +/- 0.095 mL min-1 g-1). In contrast, this parameter was significantly reduced at 100 micrograms mL-1 (0.113 +/- 0.042 mL min-1 g-1). The present findings suggest a trend towards saturation kinetics in the in-vitro pulmonary clearance of propranolol.     

Polarity of hepatic glutathione and glutathione S-conjugate efflux, and intraorgan mercapturic acid formation in the skate

Mechanisms of hepatic glutathione and glutathione S-conjugate efflux were investigated in isolated hepatocytes and perfused liver of the little skate (Raja erinacea). Glutathione was released by isolated skate hepatocytes at a rate of 0.12 +/- 0.03 nmol.hr-1.(mg protein)-1. In the perfused liver, glutathione concentrations in bile were high (approximately 0.7 mM) compared to hepatic tissue levels (0.61 +/- 0.11 mumol.g-1). During the first hour of perfusion, the biliary glutathione excretion rate was 3 nmol.hr-1.(g liver)-1, whereas glutathione accumulated in the recirculating perfusate at a rate of only 1.5 nmol.hr-1.(g liver)-1. Release of glutathione by isolated hepatocytes and perfused liver was not affected by the addition of acivicin, an inhibitor of gamma-glutamyltransferase (EC 2.3.2.2), to cell suspension medium or liver perfusate. 1-Chloro-2,4-dinitrobenzene (CDNB) was taken up by isolated hepatocytes, conjugated to glutathione, and released as S-(2,4-dinitrophenyl) (DNP)-glutathione. After infusion of 0.5 mumol CDNB in perfused liver, S-DNP-glutathione was concentrated in bile (0.5 mM) and was associated with choleresis. S-DNP-Conjugates of cysteinylglycine, cysteine and N-acetylcysteine, were also found in bile, suggesting intrahepatic breakdown of S-DNP-glutathione and subsequent acetylation of the resulting cysteine conjugate to form the mercapturic acid, S-DNP-N-acetylcysteine. This mercapturic acid accounted for 31% of the total S-DNP-conjugates collected in bile. In contrast, neither S-DNP-glutathione nor other S-DNP-conjugates were detected in the perfusate (less than 0.5 microM). These findings demonstrate that biliary excretion is the predominant route for efflux of glutathione and a glutathione S-conjugate from skate liver. The results also identify an intrahepatic pathway for mercapturic acid biosynthesis facilitated by biliary glutathione S-conjugate excretion.     

Effects of bovine serum albumin and recirculation rate on the uptake of propranolol by rat perfused lung

Lungs isolated from 7-week-old rats were perfused with pH 7.4, oxygenated Krebs-Ringer bicarbonate buffer solution containing 2.5 micrograms mL-1 of propranolol and 3 to 5% BSA at the recirculation rate of 4 to 16 mL min-1, at 37 degrees C for 60 min. The extent of propranolol metabolism after 60 min was less than 2.3% of the initial load under any in-vitro perfusion condition. Therefore, the amount disappearing from the perfusion medium was considered as being predominantly that taken up by tissue. Under all experimental conditions, perfusate drug level declined bi-exponentially with time. Apparent in-vitro pulmonary clearance of propranolol was not affected by the increase of BSA level from 3 to 5%. When the perfusate BSA level was fixed at 3%, the lowest recirculation rate (4 mL min-1) yielded the smallest clearance (about 0.15 mL min-1 g-1) but almost constant clearance value (about 0.40 mL min-1 g-1) was obtained at the rate ranging from 8 to 16 mL min-1. The tissue to medium concentration ratio of propranolol, after the perfusion with 3 to 5% BSA at the rate of 8 to 16 mL min-1, was approximately 35, whereas that with 3% BSA at 4 mL min-1 was reduced to about 20. The findings suggest evidence for flow-dependent in-vitro pulmonary clearance of propranolol.     

Calcium-dependent effects of cadmium on energy metabolism and function of perfused rat heart

Postequilibrated isolated rat hearts were perfused for 60 min with a standard supporting electrolyte buffer containing one of the following calcium concentrations: 0.9, 1.8, 3.5, or 5.0 mM, either with or without added cadmium. Doses of cadmium which proved to be minimally (0.03 microM Cd)--and maximally (3.0 microM Cd)--effective at 0.9 mM Ca were studied at all other calcium concentrations. A dose-dependent positive inotropy that persisted throughout the 60-min perfusion period was induced by the graded increases in the perfusate calcium concentration throughout the range from 0.9 to 5.0 mM. Atrioventricular node conductivity was prolonged significantly in hearts perfused with 0.9 mM Ca as compared to hearts perfused with higher calcium concentrations. Increasing the perfusate calcium concentration caused a dose-dependent increase in heart glycerol 3-phosphorylcholine (GPC) content. The other measured phosphatic metabolites of the heart were not altered significantly by varying the perfusate calcium level. In contrast, cadmium (3.0 microM Cd) induced extensive functional and metabolic aberrations which varied in magnitude as an inverse function of the perfusate calcium concentration. Contractile tension, rate of tension development (dT/dt), heart rate, coronary flow rate, and atrioventricular node conductivity were decreased significantly in response to cadmium perfusion. Moreover, these hearts characteristically had significantly elevated low energy phosphate (inosine monophosphate and inorganic phosphate) and decreased high energy phosphate (ATP, PCr) levels relative to their respective calcium controls. Furthermore, various phosphorylated intermediates of glycolysis (glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate), as well as glycerol 3-phosphate, and uridine diphosphoglucose accumulated significantly in hearts perfused with cadmium at certain calcium concentrations below 5.0 mM. The calcium-activated increase in heart GPC was inhibited completely by 3 microM cadmium. At the minimally effective dose of cadmium (0.03 microM), demonstrable changes were apparent only at the lowest perfusate calcium concentration examined (0.9 mM). These findings are consistent with the hypothesis that cadmium interferes with calcium-activated and calcium-mediated physiologic and biochemical processes of the mammalian heart. The primary mechanistic basis for the action of cadmium appears to be linked to a competition with calcium for membrane and possibly intracellular binding and activation sites.     

Mechanism of 5-hydroxytryptamine-induced coronary vasodilation assessed by direct detection of nitric oxide production in guinea-pig isolated heart.

1. We assessed whether a submaximal concentration (1 microM) of 5-hydroxytryptamine (5-HT) releases nitric oxide (NO) from the coronary endothelium in guinea-pig perfused heart (n = 5 or 6/group) by direct detection of NO in coronary effluent, and determined whether this accounts for the associated coronary dilation. We also tested whether saponin is a selective and specific tool for examining the role of this mechanism in mediating agonist-induced coronary dilatation. 2. Continuous 5 min perfusion with 5-HT, or acetylcholine (ACh; 1 microM), substance P (1 nM) or sodium nitroprusside (SNP; 1 microM) increased coronary flow from baseline by 3.6 +/- 0.2, 3.4 +/- 0.2, 1.8 +/- 0.1 and 4.1 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Coronary effluent NO content, detected by chemiluminescence, was correspondingly increased from baseline by 715 +/- 85, 920 +/- 136, 1019 +/- 58 and 2333 +/- 114 pmol min-1 g-1, respectively (all P < 0.05). 3. Continuous perfusion for 30 min with NG-nitro-L-arginine methyl ester (L-NAME) 100 microM reduced basal coronary effluent NO content by 370 +/- 32 pmol min-1 g-1 and coronary flow by 7.5 +/- 0.5 ml min-1 g-1 (both P < 0.05). Saponin (three cycles of 2 min of 30 micrograms ml-1 saponin perfusion interrupted by 2 min control perfusion) reduced basal coronary NO content by a similar amount (307 +/- 22 pmol min-1 g-1) but reduced basal coronary flow by only 0.6 +/- 0.2 ml min-1 g-1 (P < 0.05 versus the effect of L-NAME). 4. The increases in coronary flow in response to (5-HT), ACh and substance P were reduced (all P < 0.05) by 100 microM L-NAME to 1.2 +/- 0.3, 1.2 +/- 0.4 and 0.3 +/- 0.3 ml min-1 g-1, respectively. However, the flow increase in response to SNP was not reduced; it was in fact increased slightly to 4.8 +/- 0.4 ml min-1 g-1 (P < 0.05). 5. Similarly, after treatment with saponin, the increases in coronary flow in response to 5-HT, ACh and substance P were reduced to 2.1 +/- 0.3, 1.3 +/- 0.3 and 0.4 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Again, the response to SNP was increased slightly to 4.6 +/- 0.5 ml min-1 g-1 (P < 0.05). 6. L-NAME and saponin also inhibited 5-HT, ACh and substance P-induced NO release (P < 0.05), without affecting equivalent responses to SNP. 7. For substance P, the change in coronary flow (delta CF) correlated with log10 delta NO in the presence and absence of saponin and L-NAME; delta CF = 1.2(log delta NO) 1.9; r = 0.92; P < 0.05. For 5-HT the relationship was delta CF = 2.2(log delta NO-2.7; r = 0.79; P < 0.05, indicating that 5-HT causes a disproportionately greater increase in coronary flow per release of NO. This was taken to indicate that 5-HT relaxes coronary vasculature in part by releasing NO, but in part by additional mechanisms. ACh resembled 5-HT in this respect. 8. Saponin had no effect on cardiac systolic or diastolic contractile function assessed by the construction of Starling curves with an isochoric intraventricular balloon. 9. In conclusion, despite its minimal effect on basal coronary flow, saponin is an effective tool for revealing endothelium-dependent actions of coronary vasodilator substances and has selectivity in that it does not impair endothelium-independent vasodilatation or cardiac contractile function. 5-HT dilates guinea-pig coronary arteries largely by the release of NO from the coronary endothelium.     

Short inhalation exposures of the isolated and perfused rat lung to respirable dry particle aerosols; the detailed pharmacokinetics of budesonide, formoterol, and terbutaline

There is an increasing interest in using the lung as a route of entry for both local and systemic administration of drugs. However, because adequate technologies have been missing in the preclinical setting, few investigators have addressed the detailed disposition of drugs in the lung following short inhalation exposures to highly concentrated dry powder aerosols. New methods are needed to explore the disposition of drugs after short inhalation exposures, thus mimicking a future clinical use. Our aim was to study the pulmonary disposition of budesonide, formoterol, and terbutaline, which are clinically used for the treatment of bronchial asthma. Using the recently developed DustGun aerosol technology, we exposed by inhalation for approximately 1 min the isolated and perfused rat lung (IPL) to respirable dry particle aerosols of the three drugs at high concentrations. The typical aerosol concentration was 1 mug/mL, and the particle size distribution of the tested substances varied with a MMAD ranging from 2.3 to 5.3 mum. The IPL was perfused in single pass mode and repeated samples of the perfusate were taken for up to 80 min postexposure. The concentration of drug in perfusate and in lung extracts was measured using LC-MS/MS. The deposited dose was determined by adding the amounts of drug collected in perfusate to the amount extracted from the tissues at 80 min. Deposited amounts of budesonide, formoterol fumarate, and terbutaline sulphate were 23 +/- 17, 36 +/- 8, and 60 +/- 3.2 mug (mean +/- SD, n = 3), respectively. Retention in lung tissues at the end of the perfusion period expressed as fraction of deposited dose was 0.19 +/- 0.05, 0.19 +/- 0.06, and 0.04 +/- 0.01 (mean +/- SD, n = 3) for budesonide, formoterol, and terbutaline, respectively. Each short inhalation exposure to the highly concentrated aerosols consumed 1-3 mg powder. Hence, this system can be particularly useful for obtaining a detailed pharmacokinetic characterization of inhaled compounds in drug discovery/development.     

Phenacetin and acetaminophen metabolism in the isolated perfused rat liver. Precursor concentration influences the selection of kinetic parameters to assess hypoxic impairment.

Subnormal oxygen concentrations affect a host of cellular functions and can exist under normal or pathological conditions. The present study examined the effects of hypoxia on phenacetin O-deethylation by the mixed-function oxidase system and the subsequent sulfation and glucuronidation of the generated metabolite acetaminophen, compared to hypoxic alterations in conjugation of administered acetaminophen, in isolated perfused rat livers. A recirculating perfusion system containing either 20% (normoxic conditions) or 2.5% (hypoxic conditions) donor rat blood delivered 4.46 and 1.47 mumol/min/g liver oxygen, respectively, resulting in a 44% reduction in oxygen consumption during hypoxia. The total hepatic clearance of phenacetin was diminished significantly during hypoxia, due to a 60% decrease in the formation clearance of acetaminophen. Hypoxia did not influence significantly the total hepatic clearance of either administered or generated acetaminophen. Although the formation clearance for acetaminophen sulfate (AS) remained unchanged, the Vmax for sulfate formation was diminished 35%. The formation clearance (mean +/- SD; N = 4/group) of acetaminophen glucuronide (AG) was greater from administered compared to generated acetaminophen during normoxia (0.47 +/- 0.15 vs. 0.22 +/- 0.06 ml/min, p less than 0.05), and was decreased 2- to 3-fold during hypoxia (0.14 +/- 0.08 vs. 0.11 +/- 0.07 ml/min). Hypoxic conditions did not affect differentially the time lag for the appearance of AG in perfusate, and did not appear to alter the diffusional barrier for AS and AG from perfusate into the hepatocyte.     

Metabolic requirements for release of endogenous noradrenaline during myocardial ischaemia and anoxia.

The metabolic conditions required for noradrenaline (NA) release from ischaemic and anoxic perfused hearts of the rat were studied. Forty minutes of flow reduction to approximately 0.25 ml g-1 min-1 did not elicit enhanced noradrenaline overflow from the isolated heart perfused with normoxic perfusate even in the absence of added substrate. Enhanced overflow did occur when substrate-free ischaemia was induced after a 60 min period of substrate-free perfusion. Noradrenaline overflow was enhanced by perfusion at normal flow rates with an anoxic (Po2 less than or equal to 1 mmHg) perfusate containing no substrate. Such enhanced overflow occurred in the absence of calcium in the perfusate and was almost completely abolished by the addition of 11 mM glucose. Enhanced noradrenaline overflow occurring either during low flow ischaemia after substrate deprivation or during anoxic substrate-free perfusion at normal flow rates was markedly suppressed by desipramine. Exocytotic noradrenaline overflow induced by electrical stimulation of the left cervico-thoracic ganglion continued unchanged during 60 min of anoxia if the perfusate contained 11 mM glucose. In the absence of added substrate there was a decline in the overflow induced by such stimulation which was more rapid with anoxic than normoxic perfusate. Re-introduction of calcium, oxygen and substrate after 10, 20 or 30 min of calcium-free, substrate-free, anoxic perfusion was associated with a massive overflow of the intracellular enzyme lactate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of fexofenadine disposition in the rat isolated perfused liver following injection of bacterial lipopolysaccharide

1. The aim of the present study was to examine the effect of bacterial lipopolysaccharide (LPS) on the disposition of an organic anion transporting polypeptide and P-glycoprotein substrate in the rat isolated perfused liver. 2. Male Sprague-Dawley rats were divided into four groups. Three of the groups received 1, 2.5 or 5 mg/kg, i.p., Escherichia coli LPS in sterile saline. The fourth group received an equivalent volume of sterile saline i.p. Twenty-four hours after treatment, rats were anaesthetized and the liver isolated and perfused with fexofenadine at an initial concentration of 2000 ng/mL in a recirculating system. Perfusate and bile samples were collected for 60 min and the liver was collected at the end of the perfusion. Fexofenadine concentrations were determined by HPLC. Fexofenadine pharmacokinetic parameters, the final liver : perfusate (L : P) and bile : liver (B : L) concentration ratios were determined. 3. Injection of LPS changed the hepatic disposition of fexofenadine. The changes were most marked in the 5 mg/kg LPS group. Notably, clearance from the perfusate (CL) and into the bile (CLB; 5.9 +/- 0.6 and 1.24 +/- 0.20 mL/min, respectively), L : P (44 +/- 11) and B : L (17 +/- 2) were all reduced (P < 0.05) in this group compared with control (CL 10.0 +/- 1.1 mL/min; CLB 2.7 +/- 0.5 mL/min; L : P 87 +/- 14; and B : L 30 +/- 4). 4. In conclusion CL and CLB were reduced following treatment with LPS in a manner consistent with downregulation of both canalicular and sinusoidal transport.     

Cardiac contractile function and electrolyte regulation during hyperosmolal stress: an experimental study in the isolated rabbit heart

Perturbations of the extracellular osmotic environment leads to cell volume changes. The aim of the present study was to evaluate the effects of hyperosmolality on cardiac contractile function and in particular the role of ionic mechanisms anticipated to be operative during hyperosmolal exposure. Paced rabbit hearts were perfused in the Langendorff mode and were exposed to 330, 370, 410, 450 and 600 mOsm kg-1 in 10 min. intervals intervened by 15 min. isosmolal buffer perfusion (by adding mannitol). Thereafter, 370 and 600 mOsm kg-1 perfusates were chosen for investigation of the effects of inhibition of the Na-K-2Cl co-transporter (bumetanide 1 microM and 10 microM), the Na+/H+ exchanger (5-(N-ethyl-N-isopropyl amiloride (EIPA) 100 nM) and the Na+/K(+)-ATPase (ouabain 50 nM). After a rapid and transient decrease in left ventricular developed pressure, all perfusates up to 450 mOsm kg-1 increased LVDP. The 600 mOsm kg-1 perfusate initially reduced LVDP by 50%, but LVDP increased to 85% of initial value at the end of the 10 min. perfusion. EIPA attenuated the recovery of LVDP during perfusion with 600 mOsm kg-1, whereas bumetanide did not affect cardiac contractile function. A net uptake of potassium was observed during hyperosmolal perfusion. Inhibition of the Na+/H+ exchanger resulted in a continued release of cardiac water throughout hyperosmolal perfusion. Isolated perfused rabbit hearts tolerate considerable elevations in perfusate osmolality. Our results suggest that the Na+/H+ antiporter is activated on hyperosmolal exposure with a secondary activation of the Na+/K(+)-ATPase. Since inhibition with bumetanide did not affect contractility or electrolyte movements, the Na-K-2Cl co-transporter does not seem to play an important role in cardiac response to hyperosmolality in rabbits.     

Studies on the mechanism of carbon monoxide-induced vasodilation in the isolated perfused rat heart

We investigated the effects of dissolved CO on isolated potassium-arrested (K+) perfused rat hearts. Hearts from male Sprague-Dawley rats were perfused via the aorta with oxygenated Krebs-Henseleit solution containing 20 mM K+. Coronary flow (Qt) averaged 48.8 +/- 1.6 (SE), 48.1 +/- 1.7, and 55.6 +/- 1.7 ml/min/g dry wt when the perfusate was equilibrated with 95% O2-5% CO2, 5% N2-90% O2-5% CO2, and 5% CO-90% O2-5% CO2, respectively. The change in Qt was statistically significant when CO was present in the perfusion medium, but was not significant when N2 was present. Furthermore, the effect was reversible because coronary flow returned to control levels when CO was removed. Myocardial oxygen consumption (MVO2) did not change significantly when hearts were perfused with either N2 or CO. The magnitude of CO-induced vasodilation was not affected significantly by the addition of either 5 microM propranolol, 2 microM phentolamine, 1 unit of adenosine deaminase, or 0.1 mM indomethacin to the perfusate. In addition, CO reversed the vasoconstrictive effects of the alpha-agonist methoxamine. These results indicate that CO exerts a vasodilatory effect on coronary vasculature that is not the result of decreased O2 content in the perfusate and is not mediated by adrenergic influences, adenosine, or prostaglandins.     

Disposition of amphotericin B in the isolated perfused rat liver

The hepatic disposition and biliary excretion of amphotericin B were investigated in the isolated perfused rat liver (IPRL). Bolus dose of 50 microg, 99 microg and 198 microg amphotericin B in lipoprotein-free perfusate and 198 microg amphotericin B in perfusate with 1 microM high-density lipoprotein (HDL) or 1 microM low-density lipoprotein (LDL) were examined in the IPRL. Amphotericin B concentration in perfusate was measured using a validated HPLC assay. Amphotericin B was eliminated from the perfusate in a biexponential manner. The hepatic clearance (CL(H)) increased in proportion to the dose administered (0.27 +/- 0.05 mL min(-1) at low dose, 0.54 +/- 0.23 mL min(-1) at medium dose and 1.06 +/- 0.24 mL min(-1) at high dose), indicating non-linear hepatic disposition of amphotericin B. The hepatic extraction ratio of amphotericin B was very low (0.066 +/- 0.015). Tissue-to-perfusion partition coefficient, calculated at 120 min, increased 1.5 fold from 9.8 +/- 1.7 at low dose to 15.9 +/- 6.4 at high dose, suggesting the significant uptake and extensive retention of amphotericin B in the liver. Biliary excretion made only minor contribution to amphotericin B elimination in the IPRL, representing around 1-3% of the dose administered. No metabolites were detected in perfusate, bile and liver samples. The hepatic disposition of amphotericin B was not affected by the presence of HDL and LDL in the perfusate. In conclusion, the hepatic disposition of amphotericin B demonstrates restrictive elimination and is concentration-dependent, consistent with carrier-mediated uptake, and lipoproteins do not influence amphotericin B hepatobiliary disposition.     

The mechanism of cardioprotection by S-nitrosoglutathione monoethyl ester in rat isolated heart during cardioplegic ischaemic arrest.

1. This study was designed (i) to assess the effect of S-nitrosoglutathione monoethyl ester (GSNO-MEE), a membrane-permeable analogue of S-nitrosoglutathione (GSNO), on rat isolated heart during cardioplegic ischaemia, and (ii) to monitor the release of nitric oxide (.NO) from GSNO-MEE in intact hearts using endogenous myoglobin as an intracellular .NO trap and the hydrophilic N-methyl glucamine dithiocarbamate-iron (MGD-Fe2+) complex as an extracellular .NO trap. 2. During aerobic perfusion of rat isolated heart with GSNO-MEE (20 mumol 1(-1), there was an increase in cyclic GMP from 105 +/- 11 to 955 +/- 193 pmol g-1 dry wt. (P < 0.05), and a decrease in glycogen content from 119 +/- 3 to 96 +/- 2 mumol g-1 dry wt. (P < 0.05), and glucose-6-phosphate concentration from 258 +/- 22 in control to 185 +/- 17 nmol g-1 dry wt. (P < 0.05). During induction of cardioplegia, GSNO-MEE caused the accumulation of cyclic GMP (100 +/- 6 in control vs. 929 +/- 168 pmol g-1 dry wt. in GSNO-MEE-treated group, P < 0.05), and depletion of glycogen from 117 +/- 3 to 103 +/- 2 mumol g-1 dry wt. (P < 0.05) in myocardial tissue. 3. Inclusion of GSNO-MEE (20 mumol l-1) in the cardioplegic solution improved the recovery of developed pressure (46 +/- 8 vs. 71 +/- 3% of baseline, P < 0.05), and rate-pressure product from 34 +/- 6 to 63 +/- 5% of baseline (P < 0.05), and reduced the diastolic pressure during reperfusion from 61 +/- 7 in control to 35 +/- 5 mmHg (P < 0.05) after 35 min ischaemic arrest. GSH-MEE (20 mumol l-1) in the cardioplegic solution did not elicit the protective effect. 4. During cardioplegic ischaemia, GSNO-MEE (20-200 mumol l-1) induced the formation of nitrosylmyoglobin (MbNO), which was detected by electron spin resonance (ESR) spectroscopy. Inclusion of MGD-Fe2+ (50 mumol l-1 Fe2+ and 500 mumol l-1 MGD) in the cardioplegic solution along with GSNO-MEE yielded an ESR signal characteristic of the MGD-Fe2+ -NO adduct. However, the MGD-Fe2+ trap did not prevent the formation of the intracellular MbNO complex in myocardial tissue. During aerobic reperfusion, denitrosylation of the MbNO complex slowly occurred as shown by the decrease in ESR spectral intensity. GSNO-MEE treatment did not affect ubisemiquinone radical formation during reperfusion. 5. GSNO-MEE (20 microliters l-1) treatment elevated the myocardial cyclic GMP during ischaemia (47 +/- 3 in control vs. 153 +/- 34 pmol g-1 dry wt. after 35 min ischaemia, P < 0.05). The cyclic GMP levels decreased in the control group during ischaemia from 100 +/- 6 after induction of cardioplegia to 47 +/- 3 pmol g-1 dry wt. at the end of ischaemic duration. 6. Glycogen levels were lower in GSNO-MEE (20 mumol l-1)-treated hearts throughout the ischaemic duration (26.7 +/- 3.1 in control vs. 19.7 +/- 2.4 mumol g dry-t wt. in GSNO-MEE-treated group at the end of ischaemic duration), because of rapid depletion of glycogen during induction of cardioplegia. During ischaemia, the amounts of glycogen consumed in both groups were similar. Equivalent amounts of lactate were produced in both groups (148 +/- 4 in control vs. 141 +/- 4 mumol g-1 dry wt. in GSNO-MEE-treated group after 35 min in ischaemia). 7. The mechanism(s) of myocardial protection by GSNO-MEE against ischaemic injury may involve preischaemic glycogen reduction and/or elevated cyclic GMP levels in myocardial tissue during ischaemia.     

Dose effects of adenine on myocardial ATP content in the post-anoxic nonworking rat heart

1. Tissue ATP levels were measured in Langendorff perfused nonworking rat hearts subjected to 50 min anoxia prior to reperfusion with Krebs-Ringer bicarbonate (KRB) buffer alone or supplemented with 50 microM or 1 mM adenine for 60 min. 2. ATP content was restored to the normoxic range in hearts reperfused with 50 microM adenine in KRB (20.82 +/- SEM 1.90 mumol/g dry weight vs 24.95 +/- 0.83 in normoxic hearts, P = NS). 3. Reperfusion with oxygenated KRB alone or buffer with 1 mM adenine failed to improve ATP levels (17.23 +/- 0.91 mumol/g dry weight for buffer alone, 15.60 +/- 0.46 with 1 mM adenine and 13.45 +/- 0.93 for anoxic hearts not reperfused). 4. These findings indicate that adenine at 50 microM dosage can restore ATP concentrations to the normoxic range after 60 in of anoxia in the nonworking rat heart while raising the adenine dose to 1 mM inhibited the tissue ATP content.     

Hepatobiliary disposition of rhodamine 123 in isolated perfused rat livers

The disposition of rhodamine 123 (RH-123), a known marker of P-glycoprotein, and its liver-generated glucuronide metabolite (RH-Glu), a marker of Mrp2, was studied in an isolated perfused rat liver model. Livers were perfused with a buffer containing 0.1 microg ml(-1) RH-123 for 30 or 60 min or for 30 min followed by 90 min of drug-free perfusion, and the concentrations of the drug and its metabolites were determined in the perfusate, bile, and the liver tissue. The outlet perfusate concentrations of RH-123 and RH-Glu reached an apparent plateau during the continuous infusion of the drug, with a very extensive extraction ratio of approximately 96% for the parent drug. However, the biliary excretion rates of both RH-123 and generated RH-Glu continued to rise almost linearly during the entire 60 min of drug infusion. This was associated with a linear increase in the amount of RH-123 recovered in the liver between 30 and 60 min of drug infusion, resulting in a significant (>50% of the administered dose) recovery of the marker in the liver both after 30 and 60 min of perfusion. Additionally, the washout experiments showed that the declines in the biliary excretion rates of RH-123 and RH-Glu were parallel to that of RH-123 concentration in the liver in the absence of drug input. The hepatobiliary disposition of RH-123 in rats is unique because of its substantial and time-dependent accumulation in the liver, resulting in a lack of steady-state in its biliary excretion despite apparent steady-state in the perfusate.     

Ramiprilat increases bradykinin outflow from isolated hearts of rat.

To establish that bradykinin is formed in the heart we measured bradykinin in the venous effluent from rat isolated hearts perfused with Krebs-Henseleit buffer. In addition, we examined the effect on bradykinin outflow of the angiotensin converting enzyme (ACE) inhibitor, ramiprilat. From rat isolated normoxic hearts a bradykinin outflow of 0.85 +/- 0.1 ng ml-1 perfusate g-1 wet weight was measured. Perfusion with ramiprilat increased the bradykinin concentration to 2.8 +/- 0.3 ng ml-1 perfusate g-1 wet weight. During ischaemia bradykinin outflow maximally increased 8.2 fold to 7.0 +/- 0.5 ng ml-1 perfusate g-1, and in ramiprilat-perfused hearts 5.8 fold to 16.0 +/- 1.8 ng ml-1 perfusate g-1. In the reperfusion period bradykinin outflow normalized to values measured in the respective pre-ischaemic period. The presents data show that bradykinin is continuously formed in the rat isolated heart. Ischaemia increases bradykinin outflow from the heart. Presumably by inhibiting degradation of kinins, ACE inhibition significantly increased the bradykinin concentration during normoxia, ischaemia and reperfusion.     

Disposition of tacrolimus in isolated perfused rat liver: influence of troleandomycin, cyclosporine, and gg918.

The disposition of tacrolimus and the influence of cyclosporine, troleandomycin, and GF120918 (GG918, or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine) on its hepatic disposition were examined in the isolated perfused rat liver. Livers from groups of rats were perfused in a recirculatory manner following a bolus dose of tacrolimus (100 microg), a substrate for P-glycoprotein (P-gp) and CYP3A, or with felodipine (200 microg), a substrate only for CYP3A. Perfusions of each substrate were also examined in groups of rats in the presence of the inhibitors: troleandomycin (20 microM, CYP3A inhibitor), GG918 (1 microM, P-gp inhibitor), or cyclosporine (10 microM, CYP3A and P-gp inhibitor). In all experiments, perfusate and bile were collected for 60 min. Tacrolimus, felodipine, and their primary metabolites were determined in perfusate and bile by liquid chromatography/tandem mass spectrometry. The area under the curve (AUC) from 0 to 30 min was determined. For the dual CYP3A and P-gp substrate, tacrolimus, AUC +/- S.D. was decreased from control (2,260 +/- 430 ng. min/ml) by GG918 (1,730 +/- 270 ng. min/ml, P < 0.05) and was increased by troleandomycin (5,200 +/- 2,470 ng. min/ml, P < 0.05) and cyclosporine (4,390 +/- 2,080 ng. min/ml, P < 0.05). For the exclusive CYP3A substrate, felodipine, AUC was unchanged from control by GG918 but increased by troleandomycin and cyclosporine. It is concluded that GG918 increased the hepatic exposure of tacrolimus by inhibiting the canalicular P-gp transport, whereas GG918 has no effect on hepatic disposition of felodipine. These results support our hypothesis that the hepatic metabolic clearance of a dual substrate will be increased by inhibiting the efflux transporter.     

Pinacidil uptake and effects in the isolated rabbit heart

The myocardial accumulation of pinacidil showed one-compartment characteristics with a half-time of 1.11 min., whereas the disposition followed three-compartment kinetics with half-times for the relevant two redistributory and the terminal phases of 0.39, 1.51 and 5.44 min., respectively. At a steady-state drug concentration in the perfusate of 6.12 nmol ml-1, the average concentration of pinacidil in the myocardium was 20.6 nmol g-1. The accumulated amount could predictically be referred with 57% to a central and 31 and 12% to two peripheral (deeper) drug pools. The pharmacodynamic effects of pinacidil in the isolated perfused rabbit heart were studied at stepwise increasing concentrations from 0.15 to 100 microM. Coronary flowrate increased initially up to 24.5% at 1.5 microM pinacidil and then gradually decreased. Amplitude and velocity of contraction were both inhibited in a biphasic way up to 92.7 and 94.1%, respectively. Apparent dynamic steady states developed within 13-15 min. The computer-derived inhibitory Em-values related to the first phase were 49.2 and 52.4% and those related to the second phase were 111.7 and 108.3%, respectively. Heart frequency decreased monophasically and exhibited an inhibitory Em-value of 19.6%. Oxygen consumption decreased at pinacidil concentrations higher than 15 microM and the Em-value was 69.7%. The frequency-corrected QT-interval decreased biphasically and the related inhibitory Em-values were 8.6 and 58.7%. The QRS-interval did not change and the PQ-interval only showed a minor increase at the highest pinacidil concentration. Our findings are compatible with the concept of pinacidil being a potassium channel opener.