Heptachlor epoxide induces a non-capacitative type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE) and a related non-tumor promoting OC, delta-hexachlorocyclohexane (δ-HCH), on the dynamics of intracellular calcium (Ca²⁺) were investigated in mouse 1c1c7 hepatoma cells. HE induced a non-capacitative, Ca²⁺ entry-like phenomenon, which was transient and concentration-dependent with 10 and 50 μM HE. The plasma membrane Ca²⁺ channel blocker SKF-96365 antagonized this HE-induced Ca²⁺ entry. δ-HCH failed to induce Ca²⁺ entry, rather it antagonized the HE-induced Ca²⁺ entry. Both HE and δ-HCH induced Ca²⁺ release from endoplasmic reticulum (ER) at treatment concentrations as low as 10 μM; at 50 μM, the former induced 5× as much Ca²⁺ release as the latter. The HE-induced Ca²⁺ release from the ER was antagonized using the IP₃ receptor/channel blocker xestospongin C, suggesting that HE induces ER Ca²⁺ release through the IP₃ receptor/channel pore. These results show that the effect of HE on cellular Ca²⁺ mimics that of mitogens such as epidermal and hepatocyte growth factors. They also provide insight into the similarities and differences between tumorigenic and non-tumorigenic OCs, in terms of the mechanisms and the extent of the [Ca²⁺]_i increased by these agents.     

Catecholamine-induced cardiac hypertrophy uncouples β-adrenoceptors from slow calcium channels

Changes in the parameters of Ca²⁺-dependent slow action potentials (APs) and in their sensitivity to noradrenaline, forskolin, dibutryl-cAMP and extracellular Ca²⁺ concentration were studied and compared in left ventricular trageculae from normal control rats and rats with cardiac hypertophy. Cytochemical studies were also carried out to determine changes in the activity of membrane-bound adenylate cyclase. Hypertrophy was induced by administration of 5 mg/kg isoproterenol once daily for 7 days. In hypertrophied cardiac muscle, the overshoot of the slow APs was increased by 75%, the maximum rate of rise ( _max) increased by 76% and the AP duration at 50% repolarization (APD₅₀) prolonged by 56%. The _max, an indicator of the slow inward Ca²⁺ current, increased, in a dose-dependent manner, in response to the β-adrenoceptor agonist noradrenaline, the adenylate cyclase activator forskolin, the protein kinase aktivator cAMP and elevated Ca²⁺ concentration in normal control preparations, whereas in hypertrophied myocardium, the β-agonist noradrenaline and the adenylate cyclase activator forskolin had no effect. In cytochemical studies with ATP as substrate, adenylate cyclase activity was localized in the sarcolemma, and significantly fewer reaction products appeared on the outer side of the cell membrane in hypertrophied myocytes than in control myocytes. The results suggest that catecholamine-induced cardiac hypertrophy damages the catalytic subunit of membrane-bound adenylate cyclase, thus uncoupling β-adrenoceptors from slow Ca²⁺ channels in the transmembrane signalling process.     

Adenosine A2 receptor activation facilitates Ca uptake by rat brain synaptosomes

Adenosine has been shown to increase the release of neurotransmitters by stimulation of adenosine A₂ receptors. This effect probably depends on Ca²⁺ entry into presynaptic nerve terminals. In the present work the ability of the mixed adenosine A₁/A₂ agonist, 2-chloroadenosine, to stimulate Ca²⁺ uptake into rat brain synaptosomes was investigated. ⁴⁵Ca²⁺ uptake was induced by 20 μM veratridine. In the absence of other drugs, 2-chloroadenosine (1 μM) decreased ⁴⁵Ca²⁺ uptake into synaptosomes. Blocking the adenosine A₁ receptor with 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 2-chloroadenosine (1 μM) increased rather than decreased the uptake of ⁴⁵Ca²⁺ into synaptosomes. The excitatory effect of 2-chloroadenosine observed in the presence of DPCPX was reversed by 200 nM of ω-agatoxin-IVA, a specific P-type Ca²⁺ channel antagonist, but not by L-type (nifedipine, 100 nM to 1 μM; methoxyverapamil 1-10 μM) or N-type (ω-conotoxin GVIA, 500 nM) Ca²⁺ channel antagonists. The adenosine A_2A selective agonist, 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethyl-carboxamido-adenosine (CGS 21680), did not significantly modify Ca²⁺ uptake induced by veratridine. In contrast, the selective adenosine A₂ receptor agonist, N⁶-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), in concentrations ranging from 10 nM to 1 μM increased Ca²⁺ uptake induced by veratridine. The selective adenosine A₂ receptor antagonist 3,7-dimethyl-1-propargylxanthine (DPMX) at a concentration of 10 μM antagonized the stimulatory effect of DPMA (0.1 μM) on ⁴⁵Ca²⁺ uptake. In conclusion, activation of adenosine A₂ receptors increases Ca²⁺ uptake by synaptosomes depolarized by veratridine, which could explain the increase of neurotransmitter release observed when A₂ receptors are activated.     

A novel aspect of lindane testicular toxicity: in vitro effects on peritubular myoid cells

The in vitro effects of the insecticide lindane have been investigated in rat testis peritubular myoid cells (PMCs). Upon PMC exposure to lindane, polarity increase and decrease of dipole dynamics were seen at the membrane level (EC50 20 μM), leading to a partial dissipation of the membrane intrinsic dipole potential. The initial membrane depolarization was increased by Cl⁻ efflux and limited by Ca²⁺-activated repolarizing currents. Concomitantly, lindane produced an increase in [Ca²⁺]_i (EC50 125 μM) resulting from both Ca²⁺ release from an inositol 1,4,5-trisphosphate-sensitive intracellular store and a voltage-dependent Ca²⁺ influx from the extracellular medium. Of particular interest from a toxicologic point of view, insecticide concentrations well below those effective in altering ion homeostasis potently inhibited both [Ca²⁺]_i increase and contraction induced by the natural agonists vasopressin and endothelin-1 (IC50s < 10 μM). These data demonstrate that PMCs are highly susceptible to lindane and suggest that the insecticide may exert testicular toxicity by interfering with hormone-regulated PMC function.     

The Protective Action of Tetrodotoxin and (±)-Kavain on Anaerobic Glycolysis, ATP Content and Intracellular Na and Ca of Anoxic Brain Vesicles

Because recent reports point to Na⁺ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (±)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na⁺ and Ca²⁺ ([Na⁺]_i, [Ca²⁺]_i), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (τ ) af 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na⁺]_i and [Ca²⁺]_i increased persistently after anoxia by 22.1 mmol/l Na⁺ and 274.9 nmol/l Ca²⁺, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (τ = 5.1 min) and provoked a massive Na⁺ overload, which levelled off to 119 mmol/l Na⁺ within a few minutes. Concomitantly, [Ca²⁺]_i increased linearly with a rate of 355 nmol Ca²⁺/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na⁺ channel blockers TTX and (±)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na⁺]_i and [Ca²⁺]_i and prevented both the veratridine-induced increases of [Na⁺]_i and [Ca²⁺]_i and the inhibition of lactate production. The data indicate a considerable Na⁺ influx via voltage-dependent Na⁺ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca²⁺]_i. A massive Na⁺ and Ca²⁺ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition. © 1997 Elsevier Science Ltd. All rights reserved.     

Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca transport proteins and causing dysregulation of Ca homeostasis

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant (BFR) utilized to reduce the flammability of a variety of products. Studies have indicated that a number of BFRs are becoming widely distributed within the environment and are bio-accumulating within organisms. There has been much speculation that a variety of phenolic pollutants (including compounds chemically related to TBBPA, such as bisphenol A) may cause endocrine disruption and Ca²⁺ dysregulation in cells involved in spermatogenesis. In this study we therefore investigate the effects of TBBPA on mouse TM4 Sertoli cells (essential for sperm development). Results show that TBBPA increases Ca²⁺ within these cells in the 5–60 μM concentration range (EC₅₀, 21 μM). TBBPA also causes cell death (LC₅₀, 18 μM) partly via apoptosis, involving Ca²⁺-dependent mitochondrial depolarisation. Studies on intracellular Ca²⁺ transporters shows that TBBPA can inhibit sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases (SERCA) at low concentrations (IC₅₀, 0.4 to 1.2 μM) and also activate the Ryanodine receptor Ca²⁺ channel within the 0.4–4 μM concentration range. Therefore these studies suggest that the cytotoxic effects of TBBPA on cells is partly due to dysregulation of Ca²⁺ signalling, by directly affecting Ca²⁺ transport proteins.     

High-affinity inhibition of glutamate release from corticostriatal synapses by ω-agatoxin TK

To know which Ca²⁺ channel type is the most important for neurotransmitter release at corticostriatal synapses of the rat, we tested Ca²⁺ channel antagonists on the paired pulse ratio. ω-Agatoxin TK was the most effective Ca²⁺ channel antagonist (IC₅₀=127 nM; maximal EFFECT=211% (with >1 μM) and Hill COEFFICIENT=1.2), suggesting a single site of action and a Q-type channel profile. Corresponding parameters for Cd²⁺ were 13 μM, 178% and 1.2. The block of L-type Ca²⁺ channels had little impact on transmission, but we also tested facilitation of L-type Ca²⁺ channels. The L-type Ca²⁺ channel agonist, s-(−)-1,4 dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester (Bay K 8644 (5 μM)), produced a 45% reduction of the paired pulse ratio, suggesting that even if L-type channels do not participate in the release process, they may participate in its modulation.     

Sarcoplasmic reticulum Ca2+ release channel ryanodine receptor (RyR2) plays a crucial role in aconitine-induced arrhythmias

The present study established a model of RyR₂ knockdown cardiomyocytes and elucidated the role of RyR₂ in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR₂ expression. Reduction of RyR₂ expression was documented by RT-PCR, western blot, and immunofluorescence. Ca²⁺ signals were investigated by measuring the relative intracellular Ca²⁺ concentration, spontaneous Ca²⁺ oscillations, caffeine-induced Ca²⁺ release, and L-type Ca²⁺ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]_i remained at the low level. Exposure to 3 μM aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]_i and the level of caffeine-induced Ca²⁺ release were increased but the L-type Ca²⁺ currents were inhibited after application of 3 μM aconitine for 5 min. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]_i level; the level of caffeine-induced Ca²⁺ release was increased but L-type Ca²⁺ currents were inhibited. Alterations of RyR₂ are important consequences of aconitine-stimulation and activation of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca²⁺ leakage through the sarcoplasmic reticulum RyR₂ channel.     

Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca level and contraction in vascular smooth muscle

The effects of isoquinoline derivatives, HA1077 (1-[5-isoquinolinesulfonyl]-homopiperazine) and H-7 (1-[5-isoquinoline-sulfonyl]-2-methylpiperazine), on cytosolic Ca²⁺ levels ([Ca²⁺]_i) and muscle tension were examined in vascular smooth muscle of rat aorta. High K⁺ (72.7 mM) and norepinephrine (1 μM) induced a sustained contraction with a sustained increase in [Ca²⁺]_i. HA1077 and H-7 (3–10 μM) inhibited the increse in muscle tension more strongly than the increase in [Ca²⁺]_i. Verapamil (10 μM) completely inhibited the increase in [Ca²⁺]_i and the contraction induced by K⁺ whereas it inhibited the increase in [Ca²⁺]_i more strongly than the contraction due to norepinephrine. The verapamil-insensitive portion of the norepinephrine-induced contraction was inhibited by HA1077 or H-7. In Ca²⁺-free solution, 0.1 μM norepinephrine induced a transient increase in [Ca²⁺]_i and muscle tension. The transient contraction was inhibited by 10 μM HA1077 or 10 μM H-7 without inhibiting the increase in [Ca²⁺]_i. 12-Deoxyphorbol 13-isobutyrate (DPB) (1 μM) caused a sustained contraction, and this contraction was inhibited by HA1077 and H-7 at similar concentrations needed to inhibit the contractions induced by high K⁺ or norepinephrine. In rabbit mesenteric artery permeabilized with Staphylococcus aureus -toxin, 100 μM HA1077 and 100 μM H-7 inhibited the contraction induced by 0.3 μM Ca²⁺. These results suggest that the inhibitory effects of isoquinoline derivatives, HA1077 and H-7, are due to a decrease in [Ca²⁺]_i and in the Ca²⁺ sensitivity of contractile elemenst in vascular smooth muscle.     

Distinct effects of ω-toxins and various groups of Ca-entry inhibitors on nicotinic acetylcholine receptor and Ca channels of chromaffin cells

The effects of ω-toxins and various Ca²⁺ antagonist subtypes on the ⁴⁵Ca²⁺ entry into bovine adrenal medullary chromaffin cells stimulated via nicotinic acetylcholine receptors or via direct depolarization with K⁺, have been compared. The conditions selected to stimulate the ⁴⁵Ca²⁺ entry consisted of a 60-s period of exposure of cells to 100 μM of the nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium or to 70 mM K⁺. The N-type voltage-dependent Ca²⁺ channel blockers ω-conotoxin GVIA and MVIIA (1 μM) inhibited ⁴⁵Ca²⁺ entry stimulated by dimethylphenylpiperazinium or K⁺ by around 25–30%. The P-type Ca²⁺ channel blocker ω-agatoxin IVA (10 nM) did not affect the dimethylphenylpiperazinium nor the K⁺ responses; 1 μM (Q-channel blockade) inhibited both responses by around 50%. The N/P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC (1 μM) inhibited the K⁺ evoked ⁴⁵Ca²⁺ entry by 70%, while dimethylphenylpiperazinium was blocked by 50% (P<0.001). The L-type Ca²⁺ channel blockers nifedipine, furnidipine, diltiazem or verapamil (3 μM each) inhibited much more the dimethylphenylpiperazinium than the K⁺ response. The dimethylphenylpiperazinium signal was blocked 71, 88, 89, and 53%, respectively, by nifedipine, furnidipine, diltiazem and verapamil, and the K⁺ response by 38, 29, 22, and 10%. Combined ω-conotoxin MVIIC (1 μM) and furnidipine (3 μM) blocked 100% of the K⁺ evoked ⁴⁵Ca²⁺ entry. However, combined ω-conotoxin GVIA (1 μM), and furnidipine left unblocked 50% of the K⁺ response. The ‘wide spectrum' Ca²⁺ channel antagonists flunarizine or dotarizine (3 μM each) blocked the dimethylphenylpiperazinium and the K⁺ responses to a similar extent (50%); cinnarizine (3 μM) inhibited more the dimethylphenylpiperazinium (82%) than the K⁺ response (21%). At 3 μM, the highly lipophilic β-adrenoceptor antagonist (±)-propranolol, reduced by 68% the dimethylphenylpiperazinium signal and by 23% the K⁺ signal. Other high lipophilic β-adrenoceptor antagonists such as metoprolol and labetalol, reduced little the dimethylphenylpiperazinium and the K⁺ responses. The highly lipophilic agent penfluridol blocked the dimethylphenylpiperazinium response by 30% and the K⁺ response by 50%. One of the least lipophilic compounds tested, (+)-lubeluzole, blocked by 40% the dimethylphenylpiperazinium and the K⁺ responses. These data are compatible with the idea that the various ω-toxin peptides used to separate pharmacologically the different voltage-dependent Ca²⁺ channels expressed by neurones, do not block the neuronal nicotinic acetylcholine receptor ion channel. In contrast the L-type Ca²⁺ channel blockers do block the nicotinic acetylcholine receptor ionophore. Lipophilicity of the compounds is not a requirement for Ca²⁺ channel or nicotinic acetylcholine receptor blockade.     

Effects of Ca channel blockers on Ca loading induced by metabolic inhibition and hyperkalemia in cardiomyocytes

The effects of the L-type (nifedipine and verapamil) and the T-type (mibefradil) Ca²⁺ channel blockers on the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by NaCN metabolic inhibition and hyperkalemia were examined in chicken cardiomyocytes using fluorescence imaging with Fura-2. NaCN induced a slow and sustained rise in [Ca²⁺]_i, which was not affected by pretreating the cells for 5 min with nifedipine, verapamil, or mibefradil at 100 nM or 10 μM. Pretreatment of the cells with 10 μM nifedipine, verapamil, or mibefradil for 5 min remarkably inhibited the K⁺-induced increase in [Ca²⁺]_i. These inhibitory effects diminished after 48-h pretreatment with nifedipine or verapamil but not with mibefradil. Ryanodine also induces an increase in [Ca²⁺]_i, and this effect was enhanced by 48-h pretreatment of the cells with 10 μM verapamil but not with 10 μM mibefradil. We conclude that the NaCN-induced increase in [Ca²⁺]_i is independent of the Ca²⁺ influx though the L-type or T-type Ca²⁺ channels. Chronic inhibition of the L-type Ca²⁺ channels but not T-type channels may enhance the ryanodine receptor-mediated Ca²⁺ release, which may be responsible for the development of tolerance to their inhibitory effects on K⁺-induced increase in [Ca²⁺]_i.     

Ca dependence of the response of three adenosine type receptors in rat hepatocytes

The effect of three different receptor-specific adenosine agonists on the rate of ureagenesis by isolated rat hepatocytes and the dependence on the external free Ca²⁺ concentration ([Ca²⁺]_e) were investigated. In the presence of high [Ca²⁺]_e all adenosine receptor agonists increased ureagenesis to similar levels. However, with low [Ca²⁺]_e the effects of each agonist varied as follows: (i) the adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyl-adenosine, increased ureagenesis depending partially on [Ca²⁺]_e, (ii) the adenosine receptor A₂ agonist, 2-p-(-2-carboxy-ethyl) phenethylamino-5′-N-ethylcarboxyamido adenosine hydrochloride, increased ureagenesis independently of [Ca²⁺]_e and (iii) in contrast, the adenosine receptor A₃ agonist N⁶-2-(-4-aminophenyl) ethyladenosine, increased ureagenesis only in the presence of high [Ca²⁺]_e. The adenosine receptor A₁ antagonist, 1-allyl-3,7-dimethyl-8-phenyl xanthine, inhibited the effect of the adenosine receptor A₁ agonist on ureagenesis, but not the effect of the adenosine A₂ or A₃ receptor agonists. The adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine, inhibited only the effect of the adenosine A₂ receptor agonist. Thus, in addition to A₁ and A₂ type adenosine receptors, rat hepatocytes possess an A₃-like adenosine receptor which responds to the addition of an adenosine A₃ agonist by accelerating ureagenesis a [Ca²⁺]_e dependent manner. Moreover, it was observed that in the presence of extracellular Ca²⁺ each agonist increased [Ca²⁺]_i and this effect was inhibited by the appropriate specific antagonist.     

Mechanisms of pinacidil-induced vasodilatation

The mechanism of the vasodilator effect of pinacidil was examined. Pinacidil (0.1–100 μM) inhibited the increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and muscle tension due to norepinephrine in rat aorta. In contrast, a Ca²⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca²⁺]_i more strongly than the contraction. Higher concentrations of pinacidil (3–100 μM) inhibited the verapamil-insensitive portion of the contraction and [Ca²⁺]_i. An inhibitor of ATP-sensitive K⁺ channels, glibenclamide, antagonized the inhibitory effect of low concentrations ( 10 pM) of pinacidol. Pinacidil did not change the contraction induced by Ca²⁺ in vascular smooth muscle permeabilized with Staphylococcus aureus -toxin. Norepinephrine (in the presence of GTP), 12-deoxyphorbol 13-isobutyrate (in the absence of GTP), and treatment with GTP_γS potentiated the contraction of permeabilized smooth muscle induced by the addition of Ca²⁺. Pinacidil (100 μM) inhibited the potentiation due to GTP_γS or noepinephrine but not to phorbol ester. These results suggest that pinacidil has dual effects on vascular smooth muscle contraction. At lower concentrations (>0.1 μM), it decreases [Ca²⁺]_i, possibly by activating ATP-sensitive K+ channels. At higher concentrations (> 3 μM), it may additionally inhibit the receptor-mediated, GTP-binding protein-coupled phosphatidyl inositol turnover.     

Behavioural and neurochemical evidence that the antimicrobial agent oxolinic acid is a dopamine uptake inhibitor

The antimicrobial agent oxolinic acid, injected i.p. in mice, induced a dose dependent increase in locomotor activity. This stimulation culminated at the 32 mg/kg dose and became smaller for higher doses (64–128 mg/kg). When opposed to increasing doses (50–100–200 μg/kg i.p.) of haloperidol (D2 dopamine receptor antagonist), the stimulant locomotor effect of 32 mg/kg oxolinic acid was not significantly reversed. On the contrary increasing doses (7.5–15–30 μg/kg s.c.) of SCH 23390 (D1 dopamine receptor antagonist) inhibited the stimulant locomotor effect. In mice made completely akinetic by a pretreatment with reserpine (4 mg/kg s.c., 18 h before testing), dexamphetamine (2 mg/kg s.c.) reversed this akinesia and even displayed a stimulant activity, similar to that observed in mice not treated by reserpine. On the contrary, oxolinic acid (32 mg/kg) did not reverse the reserpine induced akinesia and even opposed the reversion induced by dexamphetamine. In a synaptosomal fraction prepared from striatum of rats, oxolinic acid inhibited the ³H dopamine uptake with an IC₅₀=4.3±0.6×10⁻⁶ M. Finally, in mice injected i.v. with a tracer dose of ³H WIN 35428 (1 μCi) (a dopamine uptake blocker), 32 mg/kg oxolinic acid, i.p. administered, reduced by about 50% the specific binding of the radioligand to striatal dopamine carriers. It is concluded that the stimulant locomotor effect of oxolinic acid depends on the blockade of the neuronal dopamine uptake complex.     

Antagonism of cyanide poisoning by dihydroxyacetone

Dihydroxyacetone (DHA) effectively antagonized the lethal effect of cyanide in mice and rabbits, particularly if administered in combination with thiosulfate. Oral DHA (2 and 4 g/kg) given to mice 10 min before injection (i.p.) of cyanide increased the LD50 values of cyanide from 5.7 mg/kg to 12 and 17.6 mg/kg, respectively. DHA prevented cyanide-induced lethality most effectively, if given orally 10-15 min before injection of cyanide. A combination of pretreatment with oral DHA (4 g/kg) and post-treatment with sodium thiosulfate (1 g/kg) increased the LD50 of cyanide by a factor of 9.9. Furthermore, DHA given intravenously to rabbits 5 min after subcutaneous injection of cyanide increased the LD50 of cyanide from 6 mg/kg to more than 11 mg/kg, while thiosulfate (1 g/kg) given intravenously 5 min after cyanide injection increased the LD50 of cyanide only to 8.5 mg/kg. DHA also prevented the convulsions that occurred after cyanide intoxication.     

The efficacy of alpha-ketoglutaric acid in the antagonism of cyanide intoxication

It has been reported that compounds containing carbonyl groups can readily react with cyanide. Pyruvic acid, an alpha-ketocarboxylic acid, has been shown to antagonize the lethal effects of cyanide. It is suggested that its mechanism of action rests in its ability to react with or "bind" cyanide. In this study, alpha-ketoglutaric acid, also an alpha-ketocarboxylic acid, was evaluated for its ability to counteract the lethal effects of cyanide. alpha-Ketoglutaric acid increased the LD50 value of cyanide (6.7 mg/kg) by a factor of five, a value statistically equivalent to that ascertained in mice pretreated with sodium thiosulfate and sodium nitrite. The combination of alpha-ketoglutaric acid and sodium thiosulfate increased the LD50 value of cyanide to 101 mg/kg. Addition of sodium nitrite to the alpha-ketoglutaric acid/sodium thiosulfate regimen increased the LD50 value of cyanide to 119 mg/kg. Unlike sodium nitrite, no induction of methemoglobin formation was observed with alpha-ketoglutaric acid pretreatment. It is apparent from these studies that the administration of alpha-ketoglutaric acid in conjunction with sodium thiosulfate resulted in fewer animal deaths than sodium nitrite and sodium thiosulfate without the dangerous formation of methemoglobin.     

Implication of alterations in intracellular calcium ion homoeostasis in the advent of paracetamol-induced cytotoxicity in primary mouse hepatocyte monolayer cultures

We have examined the fluctuation of free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fluorescent probe quin-2 during the cytotoxic response induced by low concentrations (100–250 μ ) of the model hepatotoxin paracetamol (APAP) in primary mouse hepatocyte cultures over 5 days. APAP-associated increases in [Ca²⁺]_i were recorded prior to APAP-associated cytotoxicity, and correlated with the subsequent loss of cell viability as measured by intracellular lactate dehydrogenase and K⁺ efflux. Co-incubation with promethazine (1 μ ) or ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic 0215 acid (4 m ) attenuated both the APAP-associated [Ca²⁺]_i changes and cytotoxicity. These results support the hypothesis that mobilization of intracellular Ca²⁺ may be an important early event in APAP-induced hepatotoxicity.     

Force-frequency relationship in intact mammalian ventricular myocardium: physiological and pathophysiological relevance

The force–frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. The FFR in most mammalian ventricular myocardium is positive; that is, an increase in contractile force in association with an increase in the amplitude of Ca²⁺ transients is induced by elevation of the stimulation frequency, which reflects the cardiac contractile reserve. The relationship is different depending on the range of frequency and species of animal. In some species, including rat and mouse, a ‘primary-phase’ negative FFR is induced over the low-frequency range up to approximately 0.5–1 Hz (rat) and 1–2 Hz (mouse). Even in these species, the FFR over the frequency range close to the physiological heart rate is positive and qualitatively similar to that in larger mammalian species, although the positive FFR is less prominent. The integrated dynamic balance of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is the primary cellular mechanism responsible for the FFR and is determined by sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ flux through the sarcolemma via L-type Ca²⁺ channels and the Na⁺-Ca²⁺ exchanger. Intracellular Na⁺ concentration is also an important factor in [Ca²⁺]_i regulation. In isolated rabbit papillary muscle, over a lower frequency range (<0.5 Hz), an increase in duration rather than amplitude of Ca²⁺ transients appears to be responsible for the increase in contractile force, while over an intermediate frequency range (0.5–2.0 Hz), the amplitude of Ca²⁺ transients correlates well with the increase in contractile force. Over a higher frequency range (>2.5 Hz), the contractile force is dissociated from the amplitude of Ca²⁺ transients probably due to complex cellular mechanisms, including oxygen limitation in the central fibers of isolated muscle preparations, while the amplitude of Ca²⁺ transients increases further with increasing frequency (‘secondary-phase’ negative FFR). Calmodulin (CaM) may contribute to a positive FFR and the frequency-dependent acceleration of relaxation, although the role of calmodulin has not yet been established unequivocally. In failing ventricular myocardium, the positive FFR disappears or is inverted and becomes negative. The activation and overexpression of cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) is able to reverse these abnormalities. Frequency-dependent alterations of systolic and diastolic force in association with those of Ca²⁺ transients and diastolic [Ca²⁺]_i levels are excellent indicators for analysis of cardiac excitation-contraction coupling, and for evaluating the severity of cardiac contractile dysfunction, cardiac reserve capacity and the effectiveness of therapeutic agents in congestive heart failure.     

Activity and gene expression profile of certain antioxidant enzymes to microcystin-LR induced oxidative stress in mice

Microcystins are cyclic heptapeptide toxins produced by certain strains of Microcystis aeruginosa and microcystin-LR (MC-LR) is the most toxic among the 70 variants isolated so far. These toxins have been implicated in both human and livestock mortality. In the present study we investigated the microcystin-LR induced oxidative stress in mice in terms of its effect on activity and gene expression profile of certain antioxidant enzymes and expression of heat shock protein-70 (HSP-70). Mice were treated with 0.5 LD₅₀ (38.31 μg/kg) and 1 LD₅₀ (76.62 μg/kg) and the biochemical variables were determined at 1, 3, 7 days and 15, 30, 60 and 120 min post-exposure for 0.5 and 1 LD₅₀ dose, respectively. A significant time-dependent increase in HSP-70 expression over control was observed at 1 LD₅₀ dose. The toxin induced significant increase in liver body weight index, hepatic lipid perxoidation and depletion of GSH levels at 1 LD₅₀ compared to control group. There was significant decrease in the activity of antioxidant enzymes glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione-S-transferase (GST) at 1 LD₅₀. Except catalase, there was no effect on other antioxidant enzymes at 0.5 LD₅₀ dose. In contrast to activity of antioxidant enzymes the gene expression profile did not show any significant difference compared to control at 1 LD₅₀. GR showed significant decrease in expression at 1, 3 and 7 days in animals dosed with 0.5 LD₅₀ MC-LR. The results of our in vivo study clearly show the oxidative stress induced by MC-LR, and a correlation with activity and regulation at gene expression level of antioxidant enzymes.     

Comparative study of the effects of salinomycin and monensin on electrophysiologic and contractile properties of canine myocardium

The effects of the monocarboxylic ionophore, salinomycin (K⁺-selective), on isometric twitcches, high K⁺-induced contracture and transmembrane action potentials were compared with those of the monocarboxylic ionophore, monensin (Na⁺-selective), in isolated canine right ventricular muscle. In a concentration (5 × 10⁻⁶) which did not produce changes in resting force, salinomycin increased peak active force ( P₀, + 170 ± 36%, mean % change from control ±S.D., P< 0.01). and relaxation and maximal rates of force development (dP/dt_max, + 123 ± 33%, P < 0.01) and relaxation (−dP/dt_max, + 180 ± 40%, P < 0.01) of the isometric twitch. A similar response pattern was found for 5 × 10⁻⁶ M monensin (P₀, + 90 ± 24%, P < 0.01; dP/dt_max, + 137 ± 19%, P < 0.01; −dP/dt_max, + 145 ± 20%, P < 0.01). In contrast to their effects on isometric twitches, salinomycin reduced peak K⁺ contracture force (P_c, −35 ± 14%, P < 0.01) whereas monensin increased it (P_c, +30 ± 12%, P < 0.02). Ventricular muscle action potential duration was shortened similarly by the ionophores. β-Adrenergic receptor blockade with nadolol diminished salinomycin's effects on the isometric twitch and K⁺ contracture, but not its effect to shorten the action potential. Monensin's actions were unaffected by nadolol. These results suggest that salinomycin's effects arise from both a direct modulation of K⁺ movement and the release of endogenous catecholamine. In contrast, monensin may act to alter intracellular Na⁺ which in turn leads to Na⁺---Ca²⁺ exchange and Ca²⁺ -mediated modulation of K⁺ movement.