tert.-Butyl hydroperoxide metabolism and stimulation of the pentose phosphate pathway in isolated rat hepatocytes

The metabolism of tert.-butyl hydroperoxide (TBHP) by the glutathione peroxidase/reductase system in isolated hepatocytes results in the rapid depletion of reduced glutathione and NADPH. The regeneration of NADPH can occur through the pentose phosphate pathway, but only when the pathway is stimulated, for example, by NADP+ and possibly oxidized glutathione, both of which can be elevated in hepatocytes exposed to TBHP. TBHP is a cytotoxicant and the role of NADPH and the pentose phosphate pathway in protecting hepatocytes from TBHP-induced injury is unknown. Isolated rat hepatocytes exposed to TBHP (0.5 mM) for 30 min metabolized more [1-14C]glucose to 14CO2 than control (638.2 +/- 96.2 vs 306.9 +/- 69.5 dpm/10(6) cells) whereas 14CO2 evolution from [6-14C]glucose was unchanged, indicating that TBHP increases the activity of the pentose phosphate pathway and not glycolysis. TBHP (0.25 mM) metabolism also resulted in a rapid oxidation of hepatocyte NADPH from 2.85 +/- 0.32 to 0.55 +/- 0.24 nmol/10(6) cells which rapidly returned to 3.58 +/- 0.27 nmol NADPH/10(6) cells. Inhibition of the pentose phosphate pathway with 6-aminonicotinamide (70 mg/kg; 5 hr prior to hepatocyte isolation) inhibited TBHP-stimulated 14CO2 evolution from [1-14C]glucose and decreased the rate of NADP+ reduction. Hepatocytes isolated from 6-aminonicotinamide-treated animals were more susceptible to TBHP-induced cell injury than were control hepatocytes. These data demonstrate the following: The metabolism of TBHP by isolated hepatocytes stimulated the activity of the pentose phosphate pathway; and inhibition of the pentose phosphate pathway with 6-aminonicotinamide potentiated the toxicity of TBHP to isolated rat hepatocytes. These results suggest that the regeneration of NADPH by the pentose phosphate pathway may play a significant role in protecting hepatocytes from TBHP-induced damage.     

Effects of carbon tetrachloride on calcium homeostasis. A critical reconsideration

The incubation of isolated rat hepatocytes with 0.172 mM carbon tetrachloride caused a rapid decrease in the calcium content of both mitochondrial and extramitochondrial compartments. However, the release of Ca2+ from the intracellular stores was not associated with an increase in the cytosolic Ca2+ levels as measured by activation of phosphorylase alpha or by Quin-2 fluorescence. A rapid rise in hepatocyte free calcium was only observed with concentrations of CCl4 higher than 0.172 mM. The lack of activation of phosphorylase alpha was not due to the inhibition of the enzyme by CCl4, since in CCl4-treated hepatocytes the phosphorylase activity could be stimulated by glucagon, butyryl--cAMP or by the increase of cell calcium induced by the addition of A23187. Ca2+-dependent ATPase of plasma membranes was only slightly affected in the early phases of poisoning with CCl4 when both mitochondrial and extramitochondrial calcium pools were already lowered. This led to the conclusion that calcium released from intracellular organelles could be extruded from the cells in sufficient amounts to prevent the increase of the cytosolic levels. A rise in hepatocyte free calcium was observed during the second hour of incubation with CCl4, concomitantly with the appearance of both LDH leakage and plasma membrane blebbing. The addition of EGTA to the medium prevented both the increase in cytosolic Ca2+ and the blebbing suggesting that they were a consequence of an influx of calcium into the cells. However, neither EGTA nor the addition of inhibitors of calcium-dependent phospholipase A2 or non-lysosomal proteases were able to protect against cell death. These latter results suggested that the alterations of calcium distribution induced by CCl4 in isolated hepatocytes were not a primary cause of the toxic effects, although they did not exclude that a sustained rise in cytosolic Ca2+ could contribute in the progression of cell injury.     

Metabolism of pyridine nucleotides in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide.

The alterations in the metabolism of pyridine nucleotides, as well as the role such changes play in the genesis of lethal cell injury, were explored in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). The loss of NADPH, NADH, and NAD equalled the increase in NADP, with little if any change in the total content of pyridine nucleotides. Identical alterations occurred in the presence of N,N'-diphenyl-p-phenylenediamine, an antioxidant that prevented the death of the cells. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced the extent of the increase in NADP and the decrease in NADPH. At the same time, BCNU increased the cell killing. Depletion of ATP with oligomycin reduced the loss of NAD and the accumulation of NADP. Treatment of the hepatocytes with the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide had no effect on the depletion of NAD. Thus, all of the alterations in pyridine nucleotides that accompany the exposure of cultured hepatocytes to TBHP can be dissociated from the development of lethal cell injury. The changes do suggest, however, a rapid interconversion of the respective species. The initial response reflects activation of glutathione reductase with the consequent oxidation of NADPH to NADP. The conversion of NADH to NAD and then NAD to NADP, the latter by nicotinamide adenine dinucleotide kinase, can account for the increase in NADP over the resulting from the oxidation of NADPH by glutathione reductase. Finally, there was no evidence in cultured hepatocytes treated with TBHP for changes in NAD that reflect the activation of poly(ADP-ribose) polymerase.     

Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress

To investigate the relationship between alterations of cytosolic Ca2+ concentration and development of cytotoxicity, isolated rat hepatocytes were loaded with the fluorescent indicator Quin-2 AM and then incubated with non-toxic or toxic levels of menadione (2-methyl-1,4-naphthoquinone) or tert-butyl hydroperoxide (t-BH). The resulting changes in cytosolic Ca2+ concentration were compared to those seen upon exposure of the hepatocytes to an alpha 1-adrenergic agonist, phenylephrine, as well as to those induced by menadione and t-BH in hepatocytes pretreated with agents that modify their toxicity. Exposure of hepatocytes to phenylephrine or non-toxic levels of menadione caused a moderate and transient increase in cytosolic Ca2+ (less than or equal to 0.7 microM), whereas a toxic concentration of menadione produced a marked, sustained increase in Ca2+ which fully saturated the binding capacity of Quin-2 (greater than 1.5 microM). Treatment of the hepatocytes with the protective agent, dithiothreitol, prevented both the increase in cytosolic Ca2+ and the cytotoxicity induced by menadione. On the other hand, pretreatment of cells with diethylmaleate to deplete intracellular glutathione made otherwise non-toxic concentrations of menadione cause both a sustained increase in cytosolic Ca2+ and cytotoxicity. Similarly, toxic concentrations of t-BH also caused a sustained increase in cytosolic Ca2+. The iron chelator, desferrioxamine, and dithiothreitol (DTT), which protected the cells from t-BH toxicity, also prevented the sustained elevation of cytosolic Ca2+. Our findings provide further support for the hypothesis that a perturbation of intracellular Ca2+ homeostasis is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress.     

tert-Butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals

tert-Butyl hydroperoxide (TBHP) mobilizes arachidonic acid (AA) from membrane phospholipids in rat hepatocytes under cytotoxic conditions, thus leading to an increase in intracellular AA, which precedes cell death. In the present work, the involvement of lipid peroxidation, thiol status, and reactive oxygen species (ROS) in the intracellular AA accumulation induced by 0.5 mM TBHP was studied in rat hepatocytes. Cells treated with TBHP maintained viability and energy status at 10 min. However, TBHP depleted GSH, as well as inducing lipid peroxidation and ROS formation, detected by dichlorofluorescein (DCF) fluorescence. TBHP also significantly increased (32.5%) the intracellular [14C]-AA from [14C]-AA-labelled hepatocytes. The phospholipase A(2) (PLA(2)) inhibitor, mepacrine, completely inhibited the [14C]-AA response. The addition of antioxidants to the cell suspensions affected the TBHP-induced lipid response differently. The [14C]-AA accumulation correlated directly with ROS and negatively with endogenous GSH. No correlation between [14C]-AA and lipid peroxidation was found. Promethazine prevented lipid peroxidation and did not affect the [14C]-AA increase. We conclude that TBHP stimulates the release of [14C]-AA from membrane phospholipids through a PLA(2)-mediated mechanism. Endogenous GSH and ROS play a major role in this effect, while lipid peroxidation-related events are unlikely to be involved. Results suggest that specific ROS generated in iron-dependent reactions, different from lipid peroxyl radicals, are involved in PLA(2) activation, this process being important in TBHP-induced hepatocyte injury.     

Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes

Organic hydroperoxides such as tert-butyl hydroperoxide (TBHP) are cytotoxic to suspensions of isolated hepatocytes. The exact mechanism of toxicity is unknown but may involve peroxidation of cellular lipids, alkylation of cellular macromolecules, or alterations in cellular calcium homeostasis. These studies were designed to examine lipid peroxidation as a mechanism of organic hydroperoxide-induced cell death. Hepatocytes isolated from mice were more susceptible to the cytotoxic effects of TBHP than were rat hepatocytes. TBHP-induced cell death was preceded by malondialdehyde formation which was also greater in mouse than rat hepatocytes. Species differences in lipid peroxidation were due to intrinsic properties of hepatocyte membranes as lipids isolated from mouse liver and peroxidized with iron/ascorbate formed approximately eightfold more malondialdehyde than lipids isolated from rat liver. Initiation of lipid peroxidation in mouse and rat hepatocytes with iron/ascorbate caused the formation of malondialdehyde equal to that seen with TBHP and a slight depletion of cellular GSH. As with TBHP, malondialdehyde formation induced by iron/ascorbate was greater in mouse than in rat hepatocytes. However, iron/ascorbate had no effect on hepatocyte viability or morphology from either species. Furthermore, TBHP-induced malondialdehyde and ethane formation in isolated rat hepatocytes were completely blocked by promethazine whereas cell toxicity was altered only slightly. Therefore, these data do not support a role for lipid peroxidation in the acute cytotoxicity of TBHP to suspensions of isolated rat hepatocytes.     

Cytosolic calcium after carbon tetrachloride, 1,1-dichloroethylene, and phenylephrine exposure. Studies in rat hepatocytes with phosphorylase a and quin2

Carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE), both hepatotoxins, inhibit sequestration of Ca2+ by rat liver endoplasmic reticulum (ER) both in vivo and in vitro. It is possible that, as a result, cytosolic Ca2+ concentrations rise in liver cells. In experiments presented here, isolated hepatocytes were exposed to CCl4, DCE, and phenylephrine (PE), a non-hepatotoxic alpha 1-adrenergic agent that mobilizes Ca2+. Cytoplasmic Ca2+ concentrations were evaluated by two methods: indirectly by assaying the activity of glycogen phosphorylase a, and directly by monitoring the fluorescence of quin2. In primary hepatocyte cultures, CCl4, DCE, and PE exposure increased the activity of phosphorylase a at 5 min from 39 +/- 2 to 130 +/- 12, 80 +/- 13, and 97 +/- 10 nmoles PO4(3-)/mg protein/min respectively. In rat hepatocyte suspensions loaded with quin2 and exposed to CCl4, DCE, or PE, cytosolic Ca2+ concentrations were elevated within 20 sec to 0.83 +/- 0.13, 0.59 +/- 0.06 and 0.99 +/- 0.14 microM Ca2+ respectively. Basal Ca2+ levels in these cells averaged 0.25 +/- 0.03 microM. Thus, CCl4 and PE apparently increased cytosolic Ca2+ levels to approximately the same extent, whereas DCE was somewhat less effective. The durations of the effects of CCl4 and PE were examined further by determining their time courses of elevated phosphorylase a activity. In hepatocyte cultures, increased phosphorylase a activity persisted through at least 60 min following CCl4 exposure. In contrast, phosphorylase a activity returned to basal levels by 20 min after PE. Increases in cytoplasmic Ca2+ levels that are sustained rather than transient may distinguish these hepatotoxic chlorinated aliphatic hydrocarbons from non-toxic hormonal agents.     

Antioxidant roles of cellular ubiquinone and related redox cycles: potentiated resistance of rat hepatocytes having stimulated NADPH-dependent ubiquinone reductase against hydrogen peroxide toxicity

Protective effect of the cellular ubiquinone (UQ) reducing system linked to cytosolic NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) against hydrogen peroxide (H2O2)-induced lipid peroxidation was investigated using UQ and control hepatocytes freshly isolated from rats injected with UQ-10 and the vehicles 14 d in advance, respectively. The UQ hepatocytes had higher levels of ubiquinol (UQH2)-10 content and NADPH-UQ reductase activity than the control hepatocytes but did not differ in other antioxidant factors from the latter cells. The UQ hepatocytes exhibited higher cell viability and lower release of lactate dehydrogenase than the control hepatocytes when they were exposed to H2O2 of up to 100 mM for 1 h at 37 degrees C. Furthermore, the formation of thiobarbituric acid reactive substances (TBARS) by H2O2 was almost completely inhibited in the UQ hepatocytes. Decreases in UQH2 and alpha-tocopherol contents and NADPH-UQ reductase activity by H2O2 exposure were observed in both types of the hepatocytes, but those levels in the UQ hepatocytes after the exposure were still higher than in the control hepatocytes. The decreases in ascorbic acid, reduced glutathione and protein thiol contents and DT-diaphorase activity by H2O2 were not different between in the two types of hepatocytes. Antioxidant enzyme activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase and glutathione reductase in the hepatocytes were not inhibited by H2O2. From these results, it was concluded that the cellular UQ reducing system linked to cytosolic NADPH-UQ reductase functions mainly as an antioxidant defense for cellular membranes.     

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.     

2-Bromohydroquinone-induced toxicity to rabbit renal proximal tubules: evidence against oxidative stress

2-Bromohydroquinone (BHQ) plays an important role in bromobenzene-induced nephrotoxicity and is a model toxic hydroquinone. Since BHQ has a quinone nucleus and various quinones have been shown to produce cytotoxicity via oxidative stress, the goal of this study was to determine whether BHQ produced cytotoxicity in a suspension of rabbit renal proximal tubules via oxidative stress. t-Butyl hydroperoxide (TBHP), an agent known to produce cytotoxicity via oxidative stress in this preparation, was used as a positive control. BHQ decreased tubular glutathione disulfide content whether glutathione reductase was inhibited or not. Inhibition of glutathione reductase did not result in the potentiation of BHQ-induced mitochondrial dysfunction or cell death. In contrast, TBHP increased tubular glutathione disulfide content. TBHP-induced increases in glutathione disulfide content, mitochondrial dysfunction, and cell death were potentiated when glutathione reductase was inhibited. Unlike TBHP, BHQ did not initiate lipid peroxidation nor was the antioxidant butylated hydroxytoluene protective. However, BHQ and TBHP both increased sodium cyanide-insensitive oxygen consumption. These results suggest that BHQ may undergo "redox cycling," but BHQ-induced mitochondrial dysfunction and cell death are not due to oxidative stress.     

Evidence that neomycin inhibits plasma membrane Ca2+ inflow in isolated hepatocytes

The effects of neomycin on Ca2+ fluxes and inositol polyphosphates in hepatocytes were investigated since it has been proposed that this antibiotic inhibits inositol 1,4,5-triphosphate formation in fibroblasts [D. H. Carney, D. L. Scott, E. A. Gordon and E. F. LaBelle, Cell 42, 479 (1985)]. In hepatocytes incubated at 1.3 mM extracellular Ca2+ (Ca2+o) neomycin (2 mM) inhibited 45Ca2+ exchange both in the presence or absence of vasopressin. At 1.3 mM Ca2+o, but not at higher concentrations of Ca2+o, the antibiotic (2 mM) inhibited the increase in glycogen phosphorylase a activity observed at late but not at early times after addition of vasopressin. The antibiotic also inhibited the increase in phosphorylase activity caused by the subsequent addition of 1.3 mM Ca2+o to cells previously incubated in the presence of vasopressin and in the absence of added Ca2+o. The concentration of the antibiotic (2 mM) which gave half-maximal inhibition of phosphorylase activation by vasopressin had no effect on the activation of phosphorylase by glucagon or the release of Ca2+ from intracellular stores induced by vasopressin. At a concentration of 10 mM, neomycin caused a 50% inhibition of the formation of [3H]inositol polyphosphates induced by vasopressin. It is concluded that neomycin, at concentrations which inhibit phosphoinositide-specific phospholipase C in other types of cells inhibits the inflow of Ca2+ across the plasma membrane but does not inhibit inositol trisphosphate formation in hepatocytes.     

On the role of mitochondria in cell injury caused by vanadate-induced Ca2+ overload

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca2+ in the intracellular compartments. Vanadate- induced Ca2+ accumulation was related to inhibition of the plasma membrane Ca2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca2+ or the enhanced operation of a putative Na+/Ca2+ exchanger. After an initial rise in the cytosolic free Ca2+ concentration, as revealed by phosphorylase activation, Ca2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca2+ overload.     

Glycogen phosphorylase activation by two different alpha 1-adrenergic receptor subtypes: methoxamine selectively stimulates a putative alpha 1-adrenergic receptor subtype (alpha 1a) that couples with Ca2+ influx

We compared the effects of methoxamine on alpha 1-adrenergic receptor-mediated phosphorylase activation in rat hepatocytes and rabbit aorta. Although methoxamine is a potent agonist in activating phosphorylase of rabbit aorta, it had little effect in rat hepatocytes. Using the phenoxybenzamine inactivation method, we found that the quantitative relationship between 125I-BE2254 (125I-BE) binding capacity and maximal norepinephrine-stimulated phosphorylase activation was nonlinear in rabbit aorta, whereas it was linear in rat hepatocytes. The potency of methoxamine in inhibiting specific 125I-BE binding is significantly (p less than 0.05) higher in rabbit aorta (Kd, 96.4 +/- 7.7 microM), compared with rat hepatocytes (Kd, 283 +/- 16 microM). However, these quantitative differences could not fully explain the blunted [Ca2+]c and phosphorylase responses to methoxamine in rat hepatocytes. Treatment with chlorethylclonidine dose dependently suppressed 125I-BE binding sites and norepinephrine-induced phosphorylase activation in rat hepatocytes, whereas in rabbit aorta it resulted in only a 31% decrease in 125I-BE binding sites, with little effect on phosphorylase activation. Furthermore, alpha 1-adrenergic receptor-mediated cellular events of phosphatidylinositol (PI) hydrolysis and phosphorylase activation were unaffected by the removal of extracellular Ca2+ in rat hepatocytes, whereas both responses were markedly attenuated in rabbit aorta. The results indicate that two different alpha 1-adrenergic receptor subtypes activate glycogen phosphorylase, through different mechanisms for increasing [Ca2+]c in the two systems. In rat hepatocytes, alpha 1 receptors are closely linked to PI hydrolysis and Ca2+ release from intracellular stores and cause phosphorylase activation. In rabbit aorta, on the other hand, activation of alpha 1 receptors increases [Ca2+]c by Ca2+ influx from the extracellular fluid as well as by Ca2+ release, and both PI hydrolysis and phosphorylase activation are caused mainly by the Ca2+ entry. Methoxamine interacts with both chlorethylclonidine-sensitive and -insensitive alpha 1 receptor subtypes but selectively stimulates the alpha 1 receptor subtype that closely couples with the Ca2+ influx.     

Paraquat-induced oxidative stress and dysfunction of the glutathione redox cycle in pulmonary microvascular endothelial cells

Oxidative stress and changes in the antioxidant defense system that include the glutathione redox cycle in cultured pulmonary microvascular endothelial cells after exposure to paraquat at 0.1 and 0.5 mM were examined as a function of time. Cell viability was substantially lost 72 h after exposure to 0.5 mM paraquat, but not 0.1 mM paraquat. Viability loss was accompanied by increased glutathione-protein mixed disulfide formation, as well as a loss in glyceraldehyde-3-phosphate dehydrogenase activity, indicating a low defense potential. At 4 h after exposure to paraquat at both doses, however, a marked loss in NADPH was found, together with a decrease in aconitase activity. With 0.5 mM paraquat, increased NADP(+) accompanied by NADPH loss diminished constantly after 48 h without recovery of lost NADPH, suggesting destruction of pyridine nucleotides under oxidative stress. NAD(+) decreased 72 h after exposure to 0.5 mM paraquat, but NADH was not influenced. 3-Aminobenzamide did not protect the loss in NADP(+) or NAD(+) and cell viability. Although oxidized glutathione did not increase by exposure to paraquat at both doses through a 96-h exposure period, reduced glutathione increased at 48 to 72 h, with an increase in glutathione disulfide reductase activities. In contrast, a marked loss in glutathione peroxidase activity was produced 48 h after exposure to 0.5 mM paraquat, preceding cell injury. Mercaptosuccinate, an inhibitor of glutathione peroxidase, distinctly hastened viability loss by paraquat. These results indicate that the reduced ability of the glutathione redox cycle, leading to high oxidative stress, is closely associated with paraquat-induced cytotoxicity.     

Modulation of the hepatic alpha 1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca(2+)-dependent and independent responses.

1. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic alpha 1-adrenoceptor-mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the alpha 1-agonist-induced stimulation of gluconeogenesis or phosphorylase 'a' activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic alpha 2-adrenoceptor-mediated effects indicates that its actions are alpha 1-specific. 2. Colchicine enhances the acute alpha 1-adrenoceptor-mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the alpha 1-adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3. The lack of effect of colchicine in altering the alpha 1-adrenoceptor ligand binding affinity suggests that it might interact with some receptor-coupled regulatory element(s). 4. The acuteness of the colchicine effect and the ability of its isomer beta-lumicolchicine to prevent all the alpha 1-adrenoceptor-mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin-induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6. It is concluded that there are at least two major alpha 1-adrenoceptor signalling pathways. One is colchicine-sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C-dependent; the other one is colchicine-insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C-independent.     

Influences of glutathione status on different cytocidal responses of monolayer rat hepatocytes exposed to aflatoxin B1 or acetaminophen

In short-term primary monolayer cultures of rat hepatocytes, aflatoxin B1 (AFB1) causes a characteristic prelethal cytomorphological response in which peripheral attached cytoplasm contracts segmentally to form finger-like blebs. This response precedes lethal injury as detected by release of lactate dehydrogenase (LDH) into culture medium. We compared the influences of various modifiers of cellular glutathione (GSH) status on cytocidal responses of Fischer 344 rats hepatocytes exposed to AFB1 or acetaminophen (AAP), a hepatotoxin which does not produce segmental cytoplasmic contraction. N-Acetylcysteine (4 mM) reduced the degree of LDH release by AAP (4 to 16 mM) but was not protective against cell killing by AFB1, although it slightly reduced the percentage of hepatocytes with segmental cytoplasmic contraction at 6 hr. BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) at 40 microM markedly inhibited glutathione reductase and also strongly potentiated cell killing by AAP but did not significantly influence segmental cytoplasmic contraction or LDH release in response to AFB1. Diethylmaleate (40 to 160 microM), a depletor of hepatocellular GSH, and buthionine-D,L-sulfoximine (4 mM), an inhibitor of GSH synthesis, each did not alter hepatocyte killing by AFB1 but were strong potentiators of toxicity of AAP. AAP inhibited glutathione reductase but AFB1 did not. Total GSH concentrations at 6 and 18 hr were reduced by AAP and to a lesser extent by AFB1 in comparison with control cultures. These findings demonstrate that, in contrast to AAP toxicity, the characteristic mode of hepatocyte killing by AFB1 in monolayer cultures is substantially independent of induced alterations in GSH. These results indicate that GSH-dependent detoxification mechanisms do not play a major role in removing necrogenic metabolites of AFB1 in Fischer 344 rat hepatocytes. They further suggest that prelethal responses of AFB1-injured hepatocytes are not affected by GSH-dependent cytoprotective mechanisms.     

Dual effects of tetrandrine on cytosolic calcium in human leukaemic HL-60 cells: intracellular calcium release and calcium entry blockade.

1. Tetrandrine (TET, a Ca2+ antagonist of Chinese herbal origin) and thapsigargin (TSG, an endoplasmic reticulum Ca2+ pump inhibitor) concentration-dependently mobilized Ca2+ from intracellular stores of HL-60 cells, with EC50 values of 20 microM and 0.8 nM, respectively. After intracellular Ca2+ release by 30 nM TSG, there was no more discharge of Ca2+ by TET (100 microM), and vice versa. 2. Pretreatments with 100 nM rauwolscine (alpha 2-adrenoceptor antagonist), 100 nM prazosin (alpha 1-adrenoceptor antagonist), 10 nM phorbol myristate acetate (PMA, a protein kinase C activator) or 100 nM staurosporine (a protein kinase C inhibitor) had no effect on 100 microM TET-induced intracellular Ca2+ release. 3. After intracellular Ca2+ release by 30 nM TSG in Ca(2+)-free medium, readmission of Ca2+ caused a substantial and sustained extracellular Ca2+ entry. The latter was almost completely inhibited by 100 microM TET (IC50 of 20 microM) added just before Ca2+ readmission. In Ca(2+)-containing medium, 30 nM TSG caused a sustained phase of cytosolic Ca2+ elevation, which could be abolished by 100 microM TET. TET was also demonstrated to retard basal entry of extracellular Mn2+ and completely inhibit TSG-stimulated extracellular Mn2+ entry. 4. TSG-induced extracellular Ca2+ entry was insensitive to the L-type Ca2+ channel blocker, nifedipine (1 microM), but was completely inhibited by the non-selective Ca2+ channel blocker La3+ (300 microM). Depolarization with 100 mM KCl did not raise the cytosolic Ca2+ level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of glutathione reductase during menadione-induced NADPH oxidation in isolated rat hepatocytes

Metabolism of menadione (2-methyl-1,4-naphthoquinone) results in the rapid oxidation of NADPH within isolated rat hepatocytes. The glutathione redox cycle is thought to play a major role in the consumption of NADPH during menadione metabolism, chiefly through glutathione reductase (GSSG-reductase). This enzyme reduces oxidized glutathione (GSSG), formed via the glutathione-peroxidase reaction, with the concomitant oxidation of NADPH. To explore the relationship between GSSG-reductase and the consumption of NADPH during menadione metabolism, isolated rat hepatocyte suspensions were exposed to non-lethal and lethal menadione concentrations (100 and 300 microM respectively) following the inhibition of GSSG-reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Menadione produced a concentration-related depletion of GSH (measured as non-protein sulfhydryl content) which was potentiated markedly by BCNU. Menadione toxicity was potentiated at either concentration by BCNU based on lactate dehydrogenase leakage at 2 hr. In addition, the NADPH content of isolated hepatocytes rapidly declined following exposure to either concentration of menadione. However, at the lower menadione concentration (100 microM), the NADPH content returned to control values or above by 60 min, whereas the NADPH content of cells exposed to 300 microM menadione with or without BCNU remained depressed for the duration of the incubation. These data suggest that, although NADPH is required by GSSG-reductase for the reduction of GSSG to GSH during quinone-induced oxidative stress, this pathway does not appear to be the major route by which NADPH is consumed during the metabolism of menadione in isolated hepatocytes.     

The metabolism of steroids, toxins and drugs by 11β-hydroxysteroid dehydrogenase 1

11β-Hydroxysteroid dehydrogenase isoform 1 (11β-HSD1) is a member of the alcohol short-chain family enzyme, and catalyzes the interconversion between active glucocorticoid cortisol (in human) and inactive cortisone. The redox ratio of NADP+/NADPH determines its direction with NADPH favoring its reductase activity and NADP+ favoring its oxidase activity. In many tissues such as the liver and lung, 11β-HSD1 behaves primarily as a reductase because of the intracellular enzyme coupling with hexose-6-phosphate dehydrogenase, which uses the NADP+ as cofactor to supply NADPH driving 11β-HSD1 to function as a reductase. 11β-HSD1 catalyzes the metabolism of 7- or 11-keto steroids as well as many toxins and drugs. Some steroids, drugs and toxins are metabolically activated. The present review discusses the steroid, drug and toxin metabolism by 11β-HSD1.