Resonance Raman study of the cytochrome P-450 LM2-halothane intermediate complex.

Resonance Raman (RR) and absorption spectroscopic studies of purified rabbit liver cytochromes P-450 show that the form 2 isomer (LM2) but not the form 4 isomer (LM4) forms a long-lived complex with halothane after dithionite reduction, absorbing light at 470 nm, in which ferric 6-coordinated heme iron in the low-spin configuration is liganded to 2-chloro-1,1-difluoroethylene. The RR data exclude the possibility that the CF3CHCl- carbanion is a ligand and are consistent with the involvement of an active-site pocket in the cytochrome P-450 polypeptide.

Foreign compound metabolism studies with human liver obtained as surgical waste. Relation to donor characteristics and effects of tissue storage.

An investigation has been conducted on the foreign compound-metabolizing activity of human liver collected fresh from surgery or at autopsy from cadavers 3 to 18 hr old, and the effects of low temperature storage on the foreign compound-metabolizing activity of fresh human liver. The enzyme activities studied were microsomal cytochrome P-450 content, biphenyl 4-hydroxylation, benzo-(a)pyrene metabolism, halothane reduction, and 4-hydroxybiphenyl UDP-glucuronosyl transferase, as well as cytosolic thiopurine methyltransferase, thermostable (TS) phenolsulfotransferase, thermolabile (TL) phenolsulfotransferase, and 5-fluorouracil dehydrogenase. Cadaver liver was a poor source of material for metabolism studies with the majority of the enzymes investigated.

Reductive metabolism of halothane by purified cytochrome P-450.

The reductive metabolism of halothane was determined using purified RLM2, PBRLM4 and PBRLM5 forms of rat liver microsomal cytochrome P-450. The metabolites, 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), were determined. All three forms of cytochrome P-450 produced CTE with relatively small differences in its production among the various forms.

Proton pump-generated electrochemical gradients in rat liver multivesicular bodies. Quantitation and effects of chloride.

Endocytic vesicles possess an electrogenic proton pump, and measurements of ATPase activity suggest that Cl- may stimulate proton pump activity. This study was undertaken to measure the steady-state pH, potential (delta psi), and total proton electrochemical gradients established by the rat liver multivesicular body (MVB) proton pump and to examine the effects of Cl- (0.5-140 mM) on these gradients.

Ursodeoxycholic acid choleresis: relationship to biliary HCO-3 and effects of Na+-H+ exchange inhibitors.

We have recently shown that substitution of Li+ for perfusate Na+ eliminates the HCO3(-)-rich choleresis produced by ursodeoxycholic acid (UDCA) in isolated perfused rat liver and that the increase in bile flow produced by both UDCA and taurocholic acid is partially inhibited by 1 mM amiloride. Although these findings are consistent with a role for Na+-H+ exchange in the choleresis produced by these bile acids, both Li+ substitution and amiloride affect other cellular processes, including Na+-K+-ATPase activity. We have now further explored both the relationship between UDCA-stimulated bile flow and biliary HCO3- secretion and the possible role of Na+-H+ exchange in this process by comparing the effects of amiloride with two of its more potent and presumably more specific analogues, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA).

Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes.

Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.

Subchronic inhalation toxicity of hexamethylenediamine in rats.

Four groups of 15 male and 15 female Sprague-Dawley-derived (CD) rats each were exposed to aqueous hexamethylenediamine (HMD) aerosols for 6 hr/day, 5 days/week for 13 weeks at mean analytical concentrations of 0, 12.8, or 51 mg/m3. Because of exposure-related deaths in a group of male and female rats similarly exposed to 215 mg/m3 HMD, this group was terminated during the seventh week of the study.

Isoflurane causes endothelium-dependent inhibition of contractile responses of canine coronary arteries.

The authors sought to determine if isoflurane would attenuate effects of three different types of vasoconstrictors on isolated segments of canine epicardial coronary arteries removed from healthy dogs. As the endothelium has a major role in regulating epicardial coronary artery tone, and as it modulates the effect of many vasoactive substances, experiments were conducted both on normal rings and on rings whose endothelium had been mechanically removed. In addition, the endothelium is thought to be damaged in human atherosclerosis.

Human liver and conjugation of catecholamines.

To investigate the role of the liver in conjugation of catecholamines we measured the concentrations of free and conjugated norepinephrine, epinephrine, and dopamine in plasma of patients with severe liver disease who were undergoing liver transplantation. Comparisons were made with catecholamine levels in plasma of euhepatic patients who were undergoing abdominal aortic aneurysmectomy. We were also able to determine the importance of the liver in conjugation of exogenous dopamine because this compound was given to both groups of patients.

Acidic vesicles in cultured rat hepatocytes. Identification and characterization of their relationship to lysosomes and other storage vesicles.

We and others recently have demonstrated adenosine triphosphate-dependent acidification in a variety of prelysosomal organelles isolated from liver including clathrin-coated vesicles, multivesicular bodies, and Golgi. Little is known, however, regarding the number or distribution of acidic compartments in intact hepatocytes. We therefore have utilized acridine orange, a fluorescent weak base, to study the number and distribution of acidic vesicles of rat hepatocytes in primary culture and compared these with the number and distribution of lysosomes and other storage vesicles.

Stabilization of the reduced halocarbon-cytochrome P-450 complex of halothane by N-alkanes.

Under anaerobic conditions, various halogenated compounds, when metabolized by cytochrome P-450, form complexes which are spectrally detectable. Previous studies have shown that halothane forms such a complex with cytochrome P-450, and the result is a strong absorption at 470 nm. Stabilization of this proposed intermediate carbanion complex has never been demonstrated in a biological system.

Effect of inhalation anesthetics on antipyrine pharmacokinetics of mice.

The effects of the volatile anesthetics, enflurane, isoflurane and halothane, on the pharmacokinetics of antipyrine were examined in mice. The administration of 0.75% isoflurane or 1.

Hepatic oxygen supply and consumption in rats exposed to thiopental, halothane, enflurane, and isoflurane in the presence of hypoxia.

Hepatic oxygen supply and uptake were assessed in phenobarbital-pretreated male Sprague-Dawley rats receiving subanesthetic doses of thiopental, halothane, enflurane, or isoflurane combined with hypoxia (approximately 0.5 MAC and 12% oxygen) for the purpose of evaluating the role of these combinations in hepatic blood flow alterations and the concomitant hepatic oxygen supply and uptake. Hepatic blood flow was measured using microspheres; hepatic oxygen supply and consumption was calculated from measured hepatic blood flow and oxygen content in hepatic arterial, portal venous, and hepatic venous blood.

Effects of Na+ replacement and amiloride on ursodeoxycholic acid-stimulated choleresis and biliary bicarbonate secretion.

In these studies, we have tested the hypothesis that bile acid-dependent bile formation is attributable, in part, to the stimulation of active bicarbonate secretion and have further explored the cellular mechanism(s) possibly involved in this process using the isolated perfused rat liver. Under control conditions, ursodeoxycholic acid (UDCA) infusion (3 mumol/min X 20 min) produced a 3.7-fold increase in bile flow and a 7.

Proton transport by hepatocyte organelles and isolated membrane vesicles.

It is apparent that proton transport plays an important role in many essential hepatocyte functions. Important unanswered issues include the location of the H+-ATPase and its role in hepatic functions, the regulators of Na+-H+ exchange, the exact role of Na+-H+ exchange in bile formation and in hepatic regeneration, and the role of bile acids such as UDCA and nor-UDCA in mediating transepithelial proton transport.

Anion inhibition of the proton pump in rat liver multivesicular bodies.

Rat liver multivesicular bodies (MVB), as well as other hepatic subcellular organelles, are acidified by an electrogenic ATP-dependent proton pump that requires Cl- for maximal acidification (Van Dyke, R. W., Hornick, C.

Fluid phase endocytosis by cultured rat hepatocytes and perfused rat liver: implications for plasma membrane turnover and vesicular trafficking of fluid phase markers.

Hepatocytes take up a variety of ligands via receptor-mediated endocytosis, yet little is known regarding either the volume of fluid or the amount of membrane internalized via endocytosis in liver cells. In these studies, we have utilized radiolabeled inulin to characterize fluid phase endocytosis by rat hepatocytes in primary culture and perfused rat liver. Uptake of inulin by cultured hepatocytes was nonlinear with time, occurring most rapidly during the first 2 min.

Rat pancreatic zymogen granules. An actively acidified compartment.

In this study, we looked for acidification in pancreatic zymogen granules as recently reported for other secretory vesicles. In intact dispersed acinar cells, acidic intracellular compartments identified by fluorescence microscopy using acridine orange corresponded exactly to the distribution of zymogen granules visualized by light microscopy. Acridine orange fluorescence in zymogen granules was reversibly dissipated by protonophores (carbonyl cyanide m-chlorophenylhydrazone, monensin) and NH4Cl; and the percentages of cytoplasmic area occupied by the acidic compartments and by zymogen granules were identical under fasting conditions and decreased in parallel after in vivo cholinergic stimulation.

Increased toxicity of the antitumor drug cyclophosphamide in mice in the presence of the volatile anesthetic agent halothane.

Exposure of mice to 0.5% halothane in air, which is close to a maintenance concentration in man, after an IP dose of cyclophosphamide produced an increase in the lethality of cyclophosphamide. The LD50 (30 day) for cyclophosphamide without halothane was 251 mg/kg; with 2 h subsequent exposure to halothane it was 152 mg/kg; and with 20 h subsequent exposure to halothane it was 158 mg/kg.

Uptake of [125I]iododeoxycytidine by cells infected with herpes simplex virus: a rapid screening test for resistance to acyclovir.

A rapid screening test for resistance to acyclovir, mediated by a lack of thymidine kinase (TK) activity in herpes simplex virus (HSV), was developed by utilizing the uptake of [125I]iododeoxycytidine (IdC) by infected Vero cells. Cells infected with TK+ virus demonstrate uptake of IdC within 3 hr of infection. The assay can detect as few as 690 pfu of virus.

Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies.

Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin.