Cytotoxicity of menadione and related quinones in freshly isolated rat hepatocytes: effects on thiol homeostasis and energy charge

The cytotoxic events in freshly isolated rat hepatocytes following exposure over 2 h to menadione (2-methyl-1,4-naphthoquinone) and two closely related quinones, 2,3-dimethyl-1,4-naphthoquinone (DMNQ) and 1,4-naphthoquinone (NQ), were examined. These quinones differ in their arylation capacity (NQ > menadione DMNQ) and in their potential to induce redox cycling (NQ menadione DMNQ). The glutathione status (reduced and oxidized glutathione) of the hepatocytes was determined using HPLC after derivatization with monobromobimane. Protein thiols were measured spectrophotometrically and the energy charge of the cells was determined with HPLC using ion pair chromatography. The leakage of lactate dehydrogenase was used as a marker for cell viability. All three quinones caused alterations of the glutathione status of the exposed cells but the effects were markedly different. Exposure to DMNQ resulted in a slow decrease of reduced glutathione and an increase of mixed disulfides. The other two quinones caused an almost complete depletion of reduced glutathione within 5 min. Hepatocytes exposed to NQ accumulated oxidized glutathione whereas menadione-exposed hepatocytes showed increased levels of mixed disulfides. We did not find any effects of DMNQ (200 M) on protein thiols, energy charge or cell viability. There was a clear difference in the effects of menadione and NQ on protein thiols, energy charge and cell viability: exposure to NQ resulted in a more extensive decrease of protein thiols and energy charge and an earlier onset of lactate dehydrogenase leakage. From our results we conclude that the arylation capacity of a quinone is a determining factor in the cytotoxic potential of such compounds and that the decrease of protein thiols and of the energy charge are critical events preceding loss of cell viability.     

Paracetamol toxicity in hamster isolated hepatocytes: The increase in cytosolic calcium accompanies,rather than precedes,loss of viability

Paracetamol is cytotoxic to hamster isolated hepatocytes by a mechanism that does not involve an early increase in [Ca²⁺]_i. Although an increase in [Ca²⁺]_i does occur, it accompanies rather than precedes, loss of viability. Studies with the ionophore, 4-bromo-A23187, suggest that although sustained elevations of [Ca²⁺]_i per se can initiate cell death, this occurs at levels of [Ca²⁺]_i only above 500 nM. This concentration was not achieved on exposure of cells to a cytotoxic concentration of paracetamol for 30 min. The [Ca²⁺]_i-response of hepatocytes to vasopressin stimulation was not altered by exposing the cells to toxic concentrations of paracetamol. This demonstrates that paracetamol does not cause any impairment in the mobilisation or redistribution of Ca²⁺. The role of elevated levels of [Ca²⁺]_i in mediating chemically-induced cell-killing requires re-evaluation.     

Allyl alcohol cytotoxicity in isolated rat hepatocytes: mechanism of cell death does not involve an early rise in cytosolic free calcium

 We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes. Received: 16 February 1994 / Accepted: 25 May 1994     

The effectiveness of N-acetylcysteine in isolated hepatocytes,against the toxicity of paracetamol,acrolein, and paraquat

The protective effect of N-acetylcysteine against the toxicity of paracetamol, acrolein, and paraquat was investigated using isolated hepatocytes as the experimental system. N-acetylcysteine protects against paracetamol toxicity by acting as a precursor for intracellular glutathione. N-acetylcysteine protects against acrolein toxicity by providing a source of sulfhydryl groups, and is effective without prior conversion. Paraquat toxicity can be decreased by coincubating the cells with N-acetylcysteine, but the mechanism for the protective effect is not as clear in this instance. It is probable that N-acetylcysteine protects against paraquat toxicity by helping to maintain intracellular glutathinone levels.     

冠心病患者外周血细胞黏附分子的表达及Ca~(2+)变化的研究

目的　观察冠心病患者外周血细胞黏附分子的表达及中性粒细胞内Ca2 +的变化 ,了解细胞黏附分子和细胞内Ca2 +与冠心病的发生及病情的关系。方法　采用流式细胞术检测细胞黏附分子的表达及细胞内Ca2 +的变化。结果　与对照组相比 ,冠心病患者外周血中性粒细胞黏附分子CD11b、CD18的表达显著上调 ,而CD6 2L的表达下调 (P <0 0 0 1) ;冠心病患者中性粒细胞内Ca2 +浓度增加 ,且与CD11b、CD18的表达呈正相关 ,与CD6 2L表达呈负相关 (P <0 0 0 1)。结论　黏附分子的表达及细胞内Ca2 +浓度变化与冠心病的发生发展和病情存在一定的关系     

Effects of paraquat,dinoseb and 2,4-D on intracellular calcium and on vasopressin-induced calcium mobilization in isolated hepatocytes

 The effects of the herbicides paraquat, dinoseb and 2,4-D on intracellular Ca²⁺ levels and on vasopressin-induced Ca²⁺ mobilization were investigated in intact isolated hepatocytes. Incubation of rat hepatocytes with paraquat (5 mM for 60 min) and dinoseb (10 μM) resulted in a time-dependent loss of viability by approximately 25%. Viability of cells treated with 2,4-D decreased significantly, dropping to about 20% at 10 mM and 60 min incubation. Exposure of hepatocytes to paraquat (1–10 mM) for 60 min had no effect on the basal level of [Ca²⁺] _i . Additionally, exposure to paraquat had no effect on the magnitude and on the duration of the [Ca²⁺] _i response to vasopressin. In the presence of 2,4-D (1–10 mM), basal [Ca²⁺] _i increases as a function of herbicide concentration. The magnitude of the Δ[Ca²⁺] _i response decreases from 256±8 nM in control to 220±5 nM, at 10 mM 2,4-D. Exposure of hepatocytes to dinoseb (1–10 μM) had no effect on the basal level of [Ca²⁺] _i . However, a strong concentration-dependent decrease in the magnitude of Δ[Ca²⁺] _i in response to vasopressin was noticed at 60 min incubation. Dinoseb markedly inhibited the stimulation of the production of inositol phosphates by vasopressin stimulus. The present study demonstrates that paraquat, 2,4-D and dinoseb cause cell death in hepatocytes by mechanisms not related to an early increase in [Ca²⁺] _i . Additionally, it has been shown for the first time that dinoseb disturbs the transduction mechanism promoted by vasopressin by inhibiting the formation of IP₃. Received: 11 October 1994/Accepted: 5 December 1994     

The role of extracellular K+-concentration in phalloidin effects on isolated hepatocytes and perfused rat livers

Summary Phalloidin is known to produce potassium loss and swelling in perfused livers. In isolated hepatocytes no volume increase was observed in the presence of the mushroom toxin; however, the cells lose potassium by treatment with phalloidin.Swelling and K⁺-balance were studied at various extracellular K⁺-concentrations. At high extracellular K⁺-concentrations (140 mM) the potassium loss from both isolated hepatocytes and perfused livers was higher in the controls than in experiments with phalloidin. In the presence of 10^–4 M ouabain the K⁺-loss from perfused livers, as caused by phalloidin, depends on the K⁺-gradient.This work was supported by the Deutsche Forschungsgemeinschaft.     

Toluene metabolism in isolated rat hepatocytes: effects of in vivo pretreatment with acetone and phenobarbital

Hepatocytes isolated from control, acetone- and phenobarbital-pretreated rats were used to study the metabolic conversion of toluene to benzyl alcohol, benzaldehyde, benzoic acid and hippuric acid at low (<100 M) and high (100–500 M) toluene concentrations. The baseline formation rates of toluene metabolites (benzyl alcohol, benzoic acid and hippuric acid) were 2.9±01.7 and 10.0±2.3 nmol/mg cell protein/60 min at low and high toluene concentrations, respectively. In vivo pretreatment of rats with acetone and phenobarbital increased the formation of metabolites: at low toluene concentrations 3- and 5-fold, respectively; at high toluene concentrations no significant increase (acetone) and 8-fold increase (phenobarbital). Apparent inhibition by ethanol, 7 and 60 mM, was most prominent at low toluene concentrations: 63% and 69%, respectively, in control cells; 84% and 91% in acetone-pretreated cells, and 32% (not significant) and 51% in phenobarbital-pretreated cells. Ethanol also caused accumulation of benzyl alcohol. The apparent inhibition by isoniazid was similar to that of ethanol at low toluene concentrations. Control and acetone-pretreated cells were apparently resistant towards metyrapone; the decrease was 49% and 64% in phenobarbital-pretreated cells at low and high toluene concentrations, respectively. In these cells, the decrease in presence of combined ethanol and metyrapone was 95% (low toluene concentrations). 4-Methylpyrazole decreased metabolite formation extensively in all groups. Benzaldehyde was only found in the presence of an aldehyde dehydrogenase inhibitor. Increased ratio benzoic/hippuric acid was observed at high toluene concentrations. These results demonstrate that toluene oxidation may be studied by product formation in isolated hepatocytes. However, the influence of various enzymes in the overall metabolism could not be ascertained due to lack of inhibitor specificity.     

Effect of fructose on the biochemical toxicity of hydrazine in isolated rat hepatocytes

Rat hepatocyte suspensions were incubated with various concentrations of hydrazine (0, 8, 12, 16, 20 mM) for 1, 2 and 3 h. In some experiments fructose (10 mM) was added either during the preincubation period, or 1 h after the start of hydrazine treatment. In certain experiments in which fructose was added, the glycolytic inhibitor sodium fluoride (3 mM) was also added during the preincubation period. Hepatocytes incubated with hydrazine alone demonstrated both a concentration- and time-dependent loss of cell viability as measured by increased Trypan blue uptake and lactate dehydrogenase (LDH) leakage. These parameters were reduced and delayed by fructose when added either before or 1 h after hydrazine treatment. There was also both a concentration- and time-dependent loss of ATP and reduced glutathione (GSH) content with hydrazine treatment. Moreover, fructose caused an initial rapid depletion of ATP but thereafter ATP levels were increased in control hepatocytes. Fructose reduced both the depletion of ATP and GSH in hydrazine- treated hepatocytes. Urea synthesis was inhibited by all concentrations of hydrazine studied but fructose treatment after 1 h did not alter this. This study also demonstrated that fluoride, an enolase inhibitor, abolished the protection against depletion of ATP levels provided by fructose, without affecting cell viability or GSH levels. These findings suggest that the cytotoxicity of hydrazine and its effects on urea synthesis and GSH levels are not a direct result of ATP depletion. The protective effects of fructose against the cytotoxicity may be due to a direct interaction with hydrazine.     

Respiratory effects of hexachlorocyclopentadiene on intact rainbow trout (Salmo gairdneri) and on oxidative phosphorylation of isolated trout heart mitochondria

Acclimated normal rainbow trout were exposed to 130 ppb hexachlorocyclopentadiene (HEX) in a flow-through well water circuit which was designed to permit measurements of oxygen consumption by the fish. Compared to preHEX values, HEX increased oxygen consumption rates by 186 ± 24% ($\text{x}\text{±}\text{SEM}$), with maximum oxygen consumption rates being reached in approximately 84 min after HEX exposure. Oxygen consumption subsequently decreased, and all HEX-exposed fish died within 6.5 hr of exposure. Fish exposed to HEX-free vehicle (acetone) showed no changes of oxygen consumption. When added to normal isolated trout heart mitochondria, HEX appeared to uncouple oxidative phosphorylation, with calculated respiratory control ratios being decreased 50% from control values at a HEX concentration of 0.41 μm. We postulate that one important mechanism of HEX intoxication in the intact animal may be due to increased oxygen consumption and impaired oxidative ATP synthesis due to the mitochondrial uncoupling action of the toxicant.     

Flow cytometric evaluation of synergistic pro-apoptotic effects of statins and clofibrates in IM-9 human lymphoblasts

1. In the present study, we evaluated fibrate-mediated potentiation of statin-induced apoptosis in IM-9 human lymphoblasts. 2. The pro-apoptotic effects of statin and fibrate were measured by flow cytometry with biotin-annexin V, followed by addition of avidin-fluorescein isothiocyanate and propidium iodide. Apoptosis was confirmed using karyopyknotic staining, as well as detection of DNA fragmentation and caspase 3 activation. 3. Incubation of IM-9 cells with both 0.1 micromol/L cerivastatin and 200 micromol/L clofibrate had a synergistic effect compared with 0.1 micromol/L cerivastatin alone or 200 micromol/L clofibrate alone. The magnitude of apoptosis induced by various combinations of statins and clofibrate were as follows: cerivastatin (0.1 micromol/L) + clofibrate (200 micromol/L) > atorvastatin (0.1 micromol/L) + clofibrate (200 micromol/L) > pravastatin (100 micromol/L) + clofibrate (200 micromol/L). Other fibrates (bezafibrate and clinofibrate) did not show any synergistic effect. Furthermore, karyopyknotic staining, caspase 3 activation and DNA fragmentation demonstrated synergistic pro-apoptotic effects of statin and fibrate. 4. The results of the present study suggest that simultaneous treatment with statins and clofibrate could provide improved therapeutic efficacy in leukaemia patients.     

In vitro characterization of cadmium and zinc uptake via the gastro-intestinal tract of the rainbow trout (Oncorhynchus mykiss): Interactive effects and the influence of calcium

An in vitro gut sac technique was employed to study whether Cd and Zn uptake mechanisms in the gastro-intestinal tract of the rainbow trout are similar to those at the gills, where both metals are taken up via the Ca transport pathway. Metal accumulation in surface mucus, in the mucosal epithelium, and transport into the blood space were assayed using radiolabelled Cd or Zn concentrations of 50micromolL(-1) in the luminal (internal) saline. Elevated luminal Ca (10 or 100mmolL(-1)versus 1mmolL(-1)) reduced Cd uptake into all three phases by approximately 60% in the stomach, but had no effect in the anterior, mid, or posterior intestine. This finding is in accordance with recent in vivo evidence that Ca is taken up mainly via the stomach, and that high [Ca] diets inhibit Cd accumulation from the food specifically in this section of the tract. In contrast, 10mmolL(-1) luminal Ca had no effect on Zn transport in any section, whereas 100mmolL(-1) Ca stimulated Zn uptake, by approximately threefold, into all three phases in the stomach only. There was no influence of elevated luminal Zn (10mmolL(-1)) on Cd uptake in the stomach or anterior intestine, or of high Cd (10mmolL(-1)) on Zn uptake in these sections. However, high [Zn] stimulated Cd transport into the blood space but inhibited accumulation in the mucosal epithelium and/or mucus-binding in the mid and posterior intestine, whereas high [Cd] exerted a reciprocal effect in the mid-intestine only. We conclude that Cd uptake occurs via an important Ca-sensitive mechanism in the stomach which is different from that at the gills, while Cd transport mechanisms in the intestine are not directly Ca-sensitive. Zn uptake does not appear to involve Ca uptake pathways, in contrast to the gills. These results are discussed in the context of other possible Cd and Zn transport pathways, and the emerging role of the stomach as an organ of divalent metal uptake.     

Effects of diltiazem on lactate, ATP, and cytosolic free calcium levels in ischemic hearts.

Changes in tissue lactate, ATP, and cytosolic free calcium (Cai) were examined in isolated, perfused rat hearts receiving 20 min of zero-flow global ischemia (37 degrees C). Addition of diltiazem before ischemia caused a concentration-dependent decrease in lactate accumulation. This effect was not mediated by modulation of norepinephrine release since depletion of catecholamines by reserpine did not alter lactate accumulation, and diltiazem treatment reduced lactate accumulation in catecholamine-depleted hearts. Diltiazem-treated hearts showed a concentration-dependent decrease in tissue ATP utilization that was associated with the decrease in tissue lactate during ischemia. Basal time averaged Cai, determined by fluorine NMR using 5FBAPTA, was 620 nM. Diltiazem (0.9 microM) decreased this value to 489 nM and reduced heart rate and maximum pressure developed (81.3 and 53.9% of control, respectively) before ischemia. Cai increased fourfold between 9 and 15 min of ischemia in hearts receiving no drug, while there was no increase in Cai in diltiazem-treated hearts. These results show that diltiazem reduces the use of ATP and therefore production of lactate during ischemia, and indicate a relationship between preservation of ATP and maintenance of Cai that may be important in the beneficial effects of diltiazem during myocardial ischemia.     

Influence of papaverine,D 600, and nifedipine on the effects of noradrenaline and calcium on the isolated aorta and mesenteric artery of the rabbit

Summary The effects of papaverine and of the organic calcium-antagonistic agents D 600 and nifedipine on the contraction induced by noradrenaline and calcium were studied on the isolated aorta and mesenteric artery. The affinities of both agonists, given as pD₂-values, were significantly higher on the aorta than on the mesenteric artery. Under our experimental conditions D 600, nifedipine and papaverine were found to act as antagonists against calcium and noradrenaline in a non-competitive fashion. In either vessel, the calcium-antagonistic activity of D 600 and nifedipine was about 1000-fold greater than that of papaverine, whereas their antagonistic activity against noradrenaline was about 1000-times weaker, i.e. D 600 as well as nifedipine were about equipotent with papaverine. The comparison between the calcium-and the noradrenaline-antagonistic activity offers the possibility to evaluate the specificity of calcium antagonistic agents.This work was supported by the Deutsche Forschungsgemeinschaft     

The toxicity of the mutagen ‘MX’ and its analogue, mucochloric acid, to rainbow trout hepatocytes and gill epithelial cells and to Daphnia magna

The cytotoxicity of the, in Salmonella, potent mutagenic compound, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and its structural analogue 3,4-dichloro-5-hydroxy-2[5H]-furanone (mucochloric acid, MCA), was studied in freshly isolated rainbow trout hepatocytes and gill epithelial cells by determining ⁸⁶Rb-leakage and decrease in fluorescence intensity in calcein AM-loaded cells. The acute toxicity of the compounds to Daphnia magna was studied by determining the concentration causing immobilization of the organism. MX proved to be more toxic than MCA both in the cellular assays and in the acute toxicity test with D. magna. MX was more toxic to hepatocytes than to gill epithelial cells. The uptake of [¹⁴C]MX was also much more efficient in hepatocytes than in gill epithelial cells. The uptake of [¹⁴C]MX in hepatocytes was not inhibited by taurocholic acid in excess, indicating that MX is not taken up by the carrier complex responsible for the uptake of taurocholate in the hepatocytes. Both the acute toxicity to D. magna and cytotoxicity of MX and MCA was rather low (EC₅₀ values > 0.1 mM) and we conclude that it is very unlikely that MX and MCA at concentrations occurring in recipients receiving chlorination effluents from pulp mills or chlorinated domestic sewage, as regards their acute toxicity, implies a risk for aquatic animals.     

The effects of haloalkene cysteine conjugates on cytosolic free calcium levels in LLC-PK(1) cells--studies utilising digital imaging fluorescence microscopy

The aim of this study was to examine the effect of haloalkene S-cysteine conjugates on cytosolic free Ca(2+) levels in renal epithelial cells using digital imaging fluorescence microscopy (DIFM). S-(1,2,3,4,4-pentachloro-1,3,-butadienyl)-L-cysteine (PCBC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) were both cytotoxic to LLC-PK(1) cells in culture. Prior treatment of the cells with aminooxyacetic acid (AOAA), an inhibitor of the enzyme cysteine conjugate beta-lyase, afforded complete protection against the toxicity at concentrations of PCBC up to 100 microM and DCVC up to 500 microM. The cytotoxicity produced by PCBC (100 microM) was time dependent with no loss of lactate dehydrogenase (LDH) into the medium being observed until 4 h after exposure, while removal of calcium from the medium prevented the toxicity. Addition of PCBC (100 microM) to LLC-PK(1) cells produced a small progressive increase in intracellular calcium ([Ca(2+)](i)) from 72+/-6 to 126+/-11 nM following 10 min of exposure. At this time there was a marked cellular heterogeneity in the calcium response with some cells showing marked increases in [Ca(2+)](i), while others cycled between low and high values and some just maintained basal levels. Exposure to PCBC (100 microM) for 1 h produced a more marked increase in [Ca(2+)](I), 469+/-46 nM, with all cells responding. The elevation in [Ca(2+)](i) was concentration-related with increases seen at concentrations of 5 microM PCBC and above. The increase in [Ca(2+)](i) produced by PCBC (100 microM) was prevented by treatment with AOAA, and markedly reduced by a nominally calcium free medium or the addition of the calcium chelator EGTA. DCVC (500 microM) also markedly elevated [Ca(2+)](i) following exposure for 1 h, this was also prevented by AOAA and a nominal calcium free medium. These findings indicate that elevation in [Ca(2+)](i) produced by PCBC in renal epithelial cells, is an early event in the cascade of signalling changes leading to renal cell death. The major source of calcium appears to be from increased influx although a small component is released from intracellular stores which my trigger a stress protein response.     

The effect of endothelin-2 (ET-2) on migration and changes in cytosolic free calcium of neutrophils

The effect of endothelin-2 (ET-2) on neutrophil migration and intracellular calcium was studied. Depending on the concentration, ET-2 enhanced or inhibited neutrophil migration. At low concentrations ET-2 caused a chemotactic stimulation of migration, in contrast with endothelin-1 (ET-1) which caused a chemokinetic stimulation of migration. At higher concentrations ET-2 inhibited formyl-methionylleucyl-phenylalanine(fMLP)-activated migration. Both activation and inhibition by ET-2 were completely dependent on extracellular Ca²⁺. Unlike ET-1 which caused an increase in cytosolic free Ca²⁺ at a concentration which stimulated migration, ET-2 caused a measurable increase of cytosolic free Ca²⁺ at a concentration which did not stimulate migration. This strongly suggests that there is no correlation between maximal stimulation of cytoplasmic free calcium, and maximal stimulation of migration. Influx of extracellular Ca²⁺ was required for both activation of migration and change in cytosolic free Ca⁺, because no effect was observed in the absence of extracellular Ca⁺, and because blockers of Ca²⁺-influx inhibited ET-2-activated migration. The ET_A-receptor antagonist cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), and the ET_B-receptor antagonist [Cys¹¹-Cys¹⁵]-endothelin-1(11–21) (IRL1038) antagonized the stimulatory effect of ET-2 on migration, and the inhibitory effect of high concentrations of ET-2 on fMLP-activated chemotaxis. This suggests that both the ET_A-receptor and the ET_B-receptor are involved in the stimulatory effect of low concentrations of ET-2, and in the inhibitory effect of high concentrations of ET-2.     

Effect of microcystin-LR on protein phosphatase activity and glycogen content in isolated hepatocytes of fed and fasted juvenile goldfish Carassius auratus L.

Toxic effects of microcystin-LR were studied in hepatocytes isolated from fed and fasted juvenile goldfish Carassius auratus (30 g body weight). The hepatocytes were incubated with 10 μg MC-LR l⁻¹ during 4 h. MC-LR induces no effect in terms of cell number and viability. The toxin accumulation pattern was different in fed and fasted treatments. MC-LR accumulated more rapidly in ‘fasted’ cells where the highest concentration was observed by 1 h of exposure. It was delayed to 4 h in the ‘fed’ cells.

MC-LR accumulation induced a severe decrease in hepatic protein phosphatase activity in both treatments. It was almost totally inhibited in both treatments during the first hour of exposure. The glycogen content was significantly reduced after 2 h of exposure in the fasting treatments, but not in the feeding one.     

The effects of cadmium on prolactin cell activity and plasma cortisol levels in the rainbow trout (Salmo gairdneri)

Treatment of rainbow trout with cadmium by intraperitoneal injection (4.4 and 7.7 mg·kg⁻¹), exposure in tank water (0.5, 1 and 10 mg·l⁻¹) or incubation of trout pituitary glands in medium containing cadmium (50 mg·l⁻¹) had no consistent effect on prolactin cell activity. Exposure of trout to 0.05 and 0.1 mg·l⁻¹ of cadmium in the tank water produced time-dependent changes in plasma cortisol levels which may reflect the alarm, resistance and exhaustion stages in the response of the fish to the cadmium.     

Compared effects of calcium entry blockers on calcium-induced tension in rat isolated cerebral and peripheral resistance vessels

Summary The effects of the calcium entry blockers verapamil (V), diltiazem (D), nifedipine (NF) and nicardipine (NC) have been studied on calcium concentration-effect curves elicited in depolarized (K⁺, 40 mmol/l) and in serotonin-exposed (6 mol/l) rat middle cerebral arteries (RMCA) in order to compare the relative potencies of the blockers against these two calcium channel activating mechanisms. In control conditions, Ca²⁺ sensitivity expressed as pD₂ and maximal active wall tension (AWT) were not significantly different in depolarized and in 5-HT-exposed vessels: pD₂: 3.39 ±0.08 vs 3.50 ± 0.06 and AWT: 0.93 ± 0.15 mN · mm^–1 vs 0.90 ± 0.16 mN · mm^–1 respectively. V, D, NF and NC displaced Ca²⁺ control curves to the right and depressed the maximum contractile response in the two experimental conditions, which suggests a noncompetitive type of antagonism. All the blockers were more potent inhibitors of Ca²⁺-induced contractions in depolarized than in serotonin-exposed middle cerebral arteries. The IC₅₀ values (concentration of blockers producing a 50% inhibition of maximal control contractile response) were (nmol/l) : V = 20, D = 120, NF = 0.4, NC = 1 and V = 400, D = 10000, NF = 20, NC = 7 in depolarized and serotonin-exposed arteries respectively. From these IC₅₀ values, the relative order of potency of the CEB's was not the same in the two experimental conditions suggesting that while serotonin and K⁺ both promote the entry of Ca²⁺ into vascular smooth muscle cells of RMCA, they either activate a different gating mechanism associated with a single common channel or perhaps distinct channels. Comparison of the results obtained in this study for depolarized rat middle cerebral arteries with those previously obtained in depolarized rat mesenteric resistance arteries (RMRA) revealed that while Ca²⁺-induced contractile responses were inhibited in a similar non-competitive manner by the four CEB's, the respective IC₅₀ values showed that potencies and rank of relative potency of the blockers were different in the two types of vessels. D and NC were equally potent in both preparations (IC₅₀ ratio = 2.5 and 3 respectively) but RMCA were more sensitive to V and NF than RMRA (IC₅₀ ratio = 6.5 and 11 respectively). These results are discussed and it is proposed that regional differencies in the conformation and/or the activation of the voltage-gated Ca²⁺ channels may exist in different vascular beds. Send offprint requests to J. L. Freston