Carbon tetrachloride-induced eicosanoid synthesis and enzyme release from rat peritoneal leucocytes

When rat peritoneal leucocytes were incubated with carbon tetrachloride, a PLA2 was activated, eicosanoids were generated and lysosomal and cytoplasmic enzymes were released. The predominant eicosanoid generated was TXB2 with lesser amounts of PGE2, 6-keto PGF1 alpha and LTB4. Preincubation of the cells with two structurally unrelated thromboxane synthetase inhibitors reduced PLA2 activity and enzyme release and also reduced the total amounts of eicosanoids liberated. An anti-PGI2 antibody partially reversed the effects of thromboxane synthetase inhibitors indicating a role for endogenous PGI2 generation in the cytoprotective effects of these agents in this system. Exogenous PGI2 was also cytoprotective but the timing of its administration was critical. The cytoprotective effect of PGI2 was potentiated by a phosphodiesterase inhibitor, indicating a possible pivotal role of cAMP in cell protection.     

Inhibiting or potentiating effects of flavonoids on carbon tetrachloride-induced toxicity in isolated rat hepatocytes

Carbon tetrachloride (CCl4) added to isolated rat hepatocytes produces toxic effects which were assessed by monitoring the release of aspartate aminotransferase (ASAT). CBrCl3 was equitoxic with CCl4, while CHCl3 was inactive, suggesting solvent properties not to be involved. The CCl4-mediated toxicity was markedly decreased by carbon monoxide, indicating possible activation by cytochrome P 450. 55 flavonoid compounds were tested for their ability to interfere with CCl4-induced release of ASAT. The compounds are cianidanol, 3-ethers and 3-esters thereof, flavanones, flavanolols, flavones and flavanols. The more hydrophilic compounds inhibit the CCl4-induced toxicity, while the lipophilic derivatives are potentiators. No other structure-activity relationships are apparent. The results are discussed in terms of the mechanisms of action of the compounds and of the validity of the technique as a screening test for hepatotropic agents.     

Effect of calcium on dextran-induced histamine release from isolated mast cells

Phosphatidyl serine potentiation of dextran-induced histamine release from isolated rat mast cells, was found to be calcium-dependent.     

Effect of tributyltin chloride on the release of calcium ion from intracellular calcium stores in rat hepatocytes.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).     

The effect of ionophore A23187 and calcium on carbon tetrachloride-induced toxicity in cultured rat hepatocytes

The aromatic monoisocyanates phenylisocyanate, o-toluene isocyanate, and p-toluene isocyanate and aliphatic isocyanates 1,6-hexamethylene diisocyanates and hexylisocyanate were investigated using an animal bioassay to determine their time-response and concentration-response relationships as sensory irritants. The results were compared with those with toluene diisocyanate. The results indicated that the level of response was dependent on both duration of exposure as well as exposure concentration. The recovery rate was extremely slow following a 3-hr exposure for all isocyanates. The aromatic and aliphatic diisocyanates had comparable potency while the aromatic monoisocyanates were slightly less potent and the aliphatic monoisocyanate was found to be the least potent.     

Phalloidin poisoning of isolated hepatocytes: Lack of enzyme release

Summary Enzyme release due to phalloidin was studied with isolated hepatocytes and with perfused livers from the rat. In isolated cells the spontaneous loss of lysosomal, mitochondrial and soluble enzymes (E.C. 3.1.1.31; E.C. 3.1.3.2; E.C. 2.6.1.1.; E.C. 2.6.1.2) was not significantly altered by phalloidin whereas the peptide triggers the release of high amounts of the enzymes from perfused livers. The results are discussed in connexion with the failure of phalloidin to induce swelling and vacuolization in isolated hepatocytes.This work was supported by the Deutsche Forschungsgemeinschaft.     

Uptake,accumulation and release of ouabain by isolated rat hepatocytes

Summary We investigated uptake of ouabain into isolated rat hepatocytes and release of ouabain from preloaded hepatocytes, thus assessing separately the two membrane transport steps, involved in biliary elimination of the drug. The following results were obtained:Uptake of ouabain was saturable (K _m=263±61M, V=3.3±1.0 nmol/mg prot.×min), energy-dependent, sensitive to dinitrofluorobenzene and temperature-dependent (E=80–96 kJ/mol). It had no pH-optimum in the physiological pH-range and was independent of the extracellular cation composition.Uptake of ouabain was competitively inhibited by the cardiac glycoside digitoxin (K _i=0.3 M).Uptake was not inhibited in the presence of the glycosidic sugar l-rhamnose, but it was competitively inhibited by the steroid taurocholate (K _i=6.3 M).Ouabain was accumulated within hepatocytes 170-fold. The predominant fraction of intracellular ouabain beeing unbound.Release of ouabain from preloaded cells was energyindependent, independent of the Na⁺-concentration and not susceptible to inhibition by dinitrofluorobenzene or taurocholate.It is concluded, first that hepatocellular uptake of ouabain is mediated by a carrier for steroids and second that the pathway of release is distinct from that of uptake. We assume, that the high bile/plasma concentration-gradient of ouabain in vivo is generated during active uptake into the cell and not during release into bile.     

Effect of chlordecone on carbon tetrachloride-induced increase in calcium uptake in isolated perfused rat liver

Intracellular accumulation of Ca2+ can occur in the livers of animals poisoned with a toxic dose of CCl4. We have reported even greater accumulation of cytosolic Ca2+ in animals treated with an ordinarily nontoxic dose of CCl4 in combination with prior exposure to chlordecone (CD). Present studies were designed to examine if intact perfused livers obtained from animals receiving either CCl4 (100 microliter/kg, ip) alone or in combination with prior dietary exposure to 10 ppm CD for 15 days accumulated 45Ca from the perfusate. Livers obtained at 0, 1, 4, 6, 12, 24, and 36 hr after a single CCl4 injection were perfused with Krebs-Ringer bicarbonate buffer containing erythrocytes, bovine serum albumin, and dextrose. After a 15-min equilibration, 45Ca was added to the perfusate, and the perfusion was continued for 30 min. Hepatic 45Ca accumulation in CCl4-treated animals in the whole liver or in the subcellular organelles obtained from perfused liver was not significantly different from corn oil controls. In the CD + CCl4 combination treatment, 45Ca accumulation followed a biphasic pattern with the first increase at 1 hr and then a progressive rise starting at 12 hr after CCl4 administration. Mitochondrial, microsomal, and cytosolic fractions from perfused liver showed a progressive rise in 45Ca at late time periods, which is indicative of unregulated and irreversible influx of extracellular Ca2+ into these livers. These studies demonstrate that cytosolic Ca2+ progressively increases as a result of the unregulated entry of extracellular Ca2+.     

Ethanol-induced stimulation of phosphoinositide turnover and calcium influx in isolated hepatocytes

Ethanol has been shown to mobilize intracellular calcium in isolated rat hepatocytes by activation of phosphoinositide-specific phospholipase C. However, addition of ethanol to 32P-labeled hepatocytes resulted in a rapid increase in the level of [32P]phosphatidylinositol 4-phosphate over a period of 2 min, concomitant with a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate and an increase in [32P]phosphatidic acid levels. These results indicate that polyphosphatidylinositol metabolism was stimulated by ethanol simultaneously with the activation of phospholipase C. Ethanol also caused a transient increase in the influx of extracellular calcium into quin 2-loaded hepatocytes over a similar period of time. The results demonstrate that ethanol, in common with calcium-mobilizing hormones, directly or indirectly stimulated polyphosphoinositide regeneration and allowed for increased movement of calcium across the hepatocyte plasma membrane.     

Potentiation of carbon tetrachloride-induced lipid peroxidation by trichloroethylene in isolated rat hepatocytes: no role in enhanced toxicity

Hepatocytes isolated from Sprague-Dawley rats were exposed to carbon tetrachloride together with various concentrations of trichloroethylene over a 40-fold range. A potentiation of carbon tetrachloride-induced lipid peroxidation by trichloroethylene and an enhanced toxicity on combined exposure were clearly demonstrated. Additionally, rats were treated 2.5 hr before isolation of hepatocytes, which were then exposed to carbon tetrachloride. Lipid peroxidation and potassium ion leakage were increased in these cells. Some incubations included the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) while others contained dithiothreitol (DTT), a thiol reducing compound. DPPD inhibited lipid peroxidation while DTT did not. Neither, however, was able to inhibit the toxicity. Assays to estimate total and nonprotein bound sulfhydryl groups were also performed. There was no indication of a causative role for cellular sulfhydryl groups in the enhanced toxicity. Therefore, our data show that lipid peroxidation is not responsible for the trichloroethylene-induced enhancement of toxicity in hepatocytes due to carbon tetrachloride. Furthermore, there is no evidence to indicate a role for sulfhydryl groups in this response.     

Mechanism of histamine release from isolated mast cell granules by calcium with phosphatidyl serine

Histamine is released from isolated mast cell granules with intact membranes by calcium (10 mM) in presence of phosphatidyl serine (25-50 micrograms/ml). The release occurs both in Krebs-Ringer solution and in sucrose solution without monovalent cations, but the release in Krebs-Ringer solution is somewhat higher. The histamine release is associated with increased calcium uptake. But calcium is taken up much faster, within 5 sec, while it takes several minutes before histamine release is completed. The observations suggest a rapid uptake of calcium to the granule membrane, from which it may be more slowly released to the matrix, displacing histamine from its binding sites. Phosphatidyl serine with calcium could also conceivably change the membrane permeability causing increased influx of sodium ions, thus accounting for the mild enhancement of the release in Krebs-Ringer solution.     

Effects of bipyridylium compounds on calcium release from triadic vesicles isolated from rabbit skeletal muscle.

1. The effects of 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP), a viologen for electrochromic memory display agent, on calcium release and ryanodine binding were studied with triad-rich sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscle. 2. DHBP inhibited the calcium release induced by 2 mM caffeine and 2 micrograms ml-1 polylysine with an IC50 value of 5 micrograms ml-1 and 4 micrograms ml-1 respectively. 3. DHBP inhibited [3H]-ryanodine binding in a dose-dependent manner with an IC50 of 2.5 micrograms ml-1 and 90-100% inhibition at 20-30 micrograms ml-1. 4. Calcium uptake by SR was inhibited in the presence of caffeine and this inhibition was antagonized by concomitant addition of DHBP. 5. The effect of DHBP on muscle twitches was studied on the mouse diaphragm. Muscle twitches elicited by direct electrical muscle stimulation and contractions induced by either 10 mM caffeine or 1 microM ryanodine were blocked by pretreatment with DHBP. 6. Data from this study provided evidence that DHBP blocked the calcium release from SR by direct interaction with the calcium release channel, also known as the ryanodine receptor. A possible use of this agent as a specific inhibitor for calcium release and as a muscle relaxant was suggested.     

Lack of protection against chemically induced injury to isolated hepatocytes by omission of calcium from the incubation medium

Recent evidence has renewed interest in the hypothesis that Ca plays a central role in cell death. It was previously found that Cd and CuCl2 cause loss of viability of isolated hepatocytes. It was therefore of interest to determine whether Ca was intimately involved with the toxic effect of these metals. Some of the chemicals that were previously shown to be toxic through a mechanism involving Ca (amphotericin B, lysolecithin, and Ca ionophore A23187) were also included in the study. Hepatocytes were incubated with one of these chemicals and samples taken at various time points up to 120 min for estimation of cell viability (intracellular K+ and leakage of aspartate aminotransferase) and lipid peroxidation. The toxic effects due to Cd or Cu were not ameliorated on omission of Ca from the incubation medium. Furthermore, of the other three chemicals investigated, only the toxic properties of the Ca ionophore were effectively blocked by incubation in a Ca-free medium. The results of this study do not support the hypothesis that Ca plays a ubiquitous role in the death of liver cells.     

Nicotine-induced noradrenaline release from the isolated rat stomach by activation of L- and N-type calcium channels

We examined the effect of nicotine on the release of endogenous noradrenaline (NA) from the isolated, vascularly perfused rat stomach. The stomach was perfused via the coeliac artery with Krebs-Ringer solution containing 10 microM pargyline at a constant flow rate of 4 ml per minute. Nicotine was once applied in the perfusion medium for 2 min. Nicotine (10(-6) - 10(-4) M) evoked NA release in a concentration-dependent manner. The nicotine (3 x 10(-5) M)-evoked NA release was abolished by hexamethonium and tetrodotoxin. Diltiazem and isradipine [blockers of L-type voltage-activated calcium channel (VACC)] and omega-conotoxin GVIA (a blocker of N-type VACC) also abolished this nicotine-evoked NA release. Previously we reported that N-type, but not L-type, VACCs are located on the gastric postganglionic sympathetic nerve terminals, since the NA release evoked by electrical stimulation of periarterial nerves around the left gastric artery (postganglionic sympathetic nerves) was abolished by omega-conotoxin GVIA, but not by diltiazem (Yokotani et al., Jpn. J. Pharmacol. 78, 75- 77, 1998). From these results, it was suggested that nicotine activates nicotinic acetylcholine receptors located on the sympathetic ganglia, thereby evoking NA release by activation of L-type VACC located on the gastric sympathetic ganglia and N-type VACC probably located on the sympathetic nerve terminals in the rat stomach.     

Effect of calcium channel antagonists on calcium uptake and release by isolated rat cardiac mitochondria

The effects of calcium channel antagonists on Ca2+ uptake and Na+-induced Ca2+ release were studied in isolated rat cardiac mitochondria. Diltiazem, nitrendipine and nimodipine were more effective inhibitors of Na+-induced Ca2+ release (IC50 = 19-100 microM) than of Ca2+ uptake (IC50 = 0.2-1 mM). Nitrendipine and nimodipine had virtually identical IC50 values for inhibiting Ca2+ uptake, but nitrendipine was 3-4 times more potent than nimodipine at inhibiting Na+-induced Ca2+ release. If these calcium channel antagonists achieve intracellular concentrations in the range of 10(-5)-10(-4) M, our results suggest that calcium channel antagonists would preferentially inhibit mitochondrial calcium release more than mitochondrial calcium uptake.     

Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice

The asialoglycoprotein (ASGP) receptor is an abundant hepatocyte-specific receptor involved in receptor-mediated endocytosis. This receptor's abundance and function is decreased by chronic ethanol administration prior to the appearance of pathology such as necrosis or inflammation. Hence, this study aimed to determine if ASGP receptor function is required to protect against liver injury by utilizing a knockout mouse model lacking functional ASGP receptor in the setting of carbon tetrachloride (CCl₄) hepatotoxicity. Briefly, ASGP receptor-deficient (RD) mice and wild-type (WT) mice were injected with 1 ml/kg body weight of CCl₄. In the subsequent week, mice were monitored for liver damage and pathology (aspartate transaminase (AST), alanine transaminase (ALT) and light microscopy). The consequences of CCl₄ injection were examined by measuring α-smooth muscle actin (α-SMA) deposition, contents of malondialdehyde and the percentage of apoptotic hepatocytes. After CCl₄ injection, RD mice showed increased liver pathology together with significantly increased activities of AST and ALT compared to that in WT mice. There were also significantly more apoptotic bodies, lipid peroxidation and deposition of α-SMA in RD mice versus WT mice following CCl₄ injection. Since these two mouse strains only differ in whether or not they have the ASGP receptor, it can be concluded that proper ASGP receptor function exerted a protective effect against CCl₄ toxicity. Thus, receptor-mediated endocytosis by the ASGP receptor could represent a novel molecular mechanism that is responsible for subsequent liver health or injury.     

Metabolism of the dihydropyridine calcium channel blockers mebudipine and dibudipine by isolated rat hepatocytes

The prototype 1,4-dihydropyridine (1,4-DHP) nifedipine, indicated for the management of hypertension and angina pectoris, has drawbacks of rapid onset of vasodilating action and a short half-life. Several newer analogues have been designed to offset these problems and these include mebudipine and dibudipine. These analogues contain t-butyl substituents that have been selected to alter the fast metabolism without altering pharmacological activity. In this study, the metabolism of mebudipine and dibudipine by isolated rat hepatocytes has been investigated. These compounds were extensively metabolized in 2 h by oxidative pathways, analogous to those known for nifedipine, and by O-glucuronidation after hydroxylation of the t-butyl substituents. The in-vitro half-lives of mebudipine (22 +/- 7.1 min) and dibudipine (40 +/- 9.8 min) were significantly longer than that of nifedipine (5.5 +/- 1.1 min), which was investigated in parallel in this study. These newer 1,4-DHPs address the problem of the short half-life of nifedipine and have potential for further development in view of their comparable potency to nifedipine.     

Effects of calcium on the release of serotonin from isolated sites within the diencephalon of the cat

In the unanesthetized cat, isolated sites in the hypothalamus and regions adjacent to this diencephalic structure were perfused with a Krebs solution by means of concentric push-pull cannulae. At 20 sites a substance with serotonin-like activity was released spontaneously at an average rate of 0.33 ± 0.05 ng/30 min perfusion interval. An anatomical mapping revealed that the sites of release of a substance with serotonin-like activity were distributed widely throughout the hypothalamus. When the Krebs solution contained an excess in calcium ions, the rate of release of the substance increased at 12 of 19 sites, decreased at 3 others and remained unchanged at 4 perfusion loci. In those regions in which the output of the substance with serotonin-like activity increased in comparison to its average rate of release, 2.6 mM excess calcium evoked an increase of 1.06 ± 0.28 ng/30 min, whereas 10.4 mM excess calcium in the perfusate caused an increase in the output of the substance to 1.65 ± 0.34 ng/30 min.