European Journal of Pharmacology: Environmental Toxicology and Pharmacology最新文献

Alterations of cellular functions induced by recombinant human tumor necrosis factor α (TNFα) were compared in rat hepatocytes cultured under either periportal-equivalent (10 nM insulin; 10 nM glucagon; 13% O₂) or perivenous-equivalent conditions (10 nM insulin; 1 nM glucagon; 4% O₂). TNFα induced a time- and dose-dependent increase in nitric oxide (NO) production and an acute phase response (inhibition of albumin secretion and elevation of α₂-macroglobulin production) under both culture conditions. NO production was more pronounced in periportal cultures, while the acute phase response was stronger in pericentral cultures. This suggests that NO production and the acute phase response are controlled by different pathways. After exposure to TNFα, DNA content was measured fluorimetrically and biochemically. A marked decrease in nuclear DNA content was found exclusively in pericentral cultures after an 8-h exposure, followed by an elevation of lactic dehydrogenase (LDH) release after a 12-h exposure. Aurintricarboxylic acid (100 μM), an inhibitor of endonuclease, significantly inhibited the TNFα-induced decrease in nuclear DNA content but only partially inhibited the LDH release. This indicates that the loss of nuclear DNA content in pericentral cultures is due to an activation of endonuclease and the resulting DNA fragmentation and does not correlate with NO production. Furthermore, the release of LDH seems to be only partially associated with DNA damage. Dexamethasone (100 nM) completely inhibited both TNFα-induced DNA fragmentation and the elevation of LDH release. The results clearly indicate that the toxicity of TNFα is influenced by the metabolic state of hepatocytes. Accordingly, the preferential perivenous cell injury observed after exposure to endotoxins in vivo seems to be due to a higher sensitivity of the pericentrally localized hepatocytes towards TNFα rather than a TNFα concentration gradient.

In the present study, we asked whether platelet-activating factor (PAF) mediates the ozone-induced increase in airway microvascular leakage. To answer this question, we examined the effect of a PAF receptor antagonist on the ozone-induced increase in airway microvascular leakage quantified by the extravasation of Evans blue dye in the guinea pig trachea and main bronchi. Guinea pigs were pretreated with the PAF receptor antagonist, E6123 ((S)-(+)-6-(2-chlorophenyl)-3-cyclopropane-carbonyl-8,11- dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4′,3′:4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine) (0.01, 0.1 and 1.0 mg/kg i.v.) and then exposed to 3 ppm ozone for 30 min. The PAF receptor antagonist significantly reduced the ozone-induced increase in microvascular leakage in a dose-dependent manner in both the trachea and main bronchi. Our results indicate that PAF mediates the ozone-induced increase in airway microvascular leakage. We therefore suggest that PAF may be involved in ozone-induced airway inflammation.

The pharmacological and toxicological activity of pyridine was determined in rat thoracic aorta. Pyridine inhibited norepinephrine (3 μM)-induced phasic and tonic contractions in the thoracic aorta as well as the endothelium-denuded aorta of the rat. The tonic pre-contraction elicited by norepinephrine was also relaxed by the addition of pyridine and this relaxing effect was not affected by indomethacin (20 μM), $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine acetate}$ (50 μM) or methylene blue (50 μM). In high-K⁺ medium (80 mM), pyridine inhibited the Ca²⁺ concentration-dependent vasocontraction. Moreover, in Ca²⁺-free medium, the norepinephrine (3 μM)-induced phasic contraction was also suppressed by pyridine, while the caffeine (10 mM)-induced contraction remained unaffected. The cAMP and cGMP levels of rat aorta were not changed by pyridine. The ⁴⁵Ca²⁺ influx elicited by either norepinephrine or high-K⁺ was inhibited by pyridine in a concentration-dependent manner. All of these findings indicated that pyridine relaxes rat thoracic aorta by virtue of its Ca²⁺ channel-blocking properties in vascular smooth muscle.

The effect of low concentrations of ethanol on Na⁺, K⁺-ATPase activity, defined as ouabain-inhibitable ⁸⁶Rb⁺(K⁺) uptake, was investigated in a crude synaptosome preparation which was subject to minimal subcellular fractionation procedures. Moderate (20–30%) but potent (EC₅₀ = 3.8 mM) stimulation of total ouabain (1 mM)-inhibitable K⁺ uptake by ethanol was observed following incubation periods of up to 20 min. The activity of the ethanol-induced component of K⁺ uptake was antagonized by nanomolar concentrations of ouabain. Thus, the moderate stimulation of total ouabain-inhibitable K⁺ uptake by ethanol was attributable to the activation of a component of K⁺ uptake which was very sensitive (VS; IC₅₀ = 2.8×10⁻¹⁰ M) to inhibition by ouabain. Slightly higher concentrations of ouabain (10⁻⁹–10^−6.6 M) stimulated K⁺ uptake above control (no ethanol or ouabain) in both the absence and presence of ethanol. The selectivity of the VS-ethanol interaction was demonstrated by the lack of any ethanol effect on two other components of two other components of ouabain-inhibitable K⁺ uptake which accounted for inhibition of K⁺ uptake by concentrations of ouabain above 10^−6.6 M and were defined as sensitive (S; IC₅₀ = 10⁻⁶ M) and insensitive (I; IC₅₀ = 10⁻⁴ M) to ouabain. These results define the ethanol-inducible component of ouabain-inhibitable Na⁺,K⁺-ATPase activity and promote the view that changes in Na⁺,K⁺-ATPase-dependent ion translocation may contribute to ethanol intoxication in vivo.

The application of capsaicin (1 μM) produced a minor relaxant effect in endothelium-denuded rat aortae. However, capsaicin caused a greater relaxation of blood vessels precontracted with high K⁺ or phenylephrine. The effects of capsaicin on the ionic currents were also examined in A7r5 vascular smooth muscle cells. The tight-seal whole-cell voltage clamp technique was used. Capsaicin inhibited the Ba²⁺ inward current (I_Ba) through the voltage-dependent L-type Ca²⁺ channel in a concentration-dependent fashion, whereas calcitonin gene-related peptide and phenylephrine produced a minor increase in I_Ba. Capsaicin did not alter the overall shape of current-voltage relationship of I_Ba. However, capsaicin (3 μM) shifted the quasi-steady-state inactivation curve of I_Ba to more negative membrane potential by about 5 mV. These effects of capsaicin on I_Ba were reversible. In addition, capsaicin had inhibitory effects on voltage dependent K⁺ currents. These results suggest that inhibition of the voltage-dependent L-type Ca²⁺ channel is involved in the capsaicin-induced relaxation of the vascular smooth muscle, whereas capsaicin-induced inhibition of voltage-dependent K⁺ channels might produced an increase in cell excitability.

This study defined the ability of a large sample of heterogeneous pesticides and neurotoxins to interact with the [³H]tyramine-labelled vesicular transporter of dopamine in rat striatum. Botanical (with rotenone as the most potent), and organochlorine (Kepone) insecticides, as well as fungicides (Zineb), as a whole, consistently inhibited [³H]tyramine binding, with K_i values ranging from 5 nM to 10 μM. ATP/Mg²⁺-dependent [³H]tyramine uptake to purified striatal synaptic vesicles was also inhibited by rotenone. Organophosphate and carbamate insecticides, and miscellaneous herbicides poorly antagonized [³H]tyramine binding, yielding K_i values exceeding 10 μM. Several, though not all, of the best recognized central neurotoxins tested were major binding antagonists. Their rank order of potency was 1-methyl-4-phenylpyridinium ion (MPP⁺) > trimethyltin ≥ 6-hydroxydopamine > N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) > 1-methyl-4-pheny;-1,2,3,6-tetrahydropyridine (MPTP), with K_i values ranging from 35 nM to 3 μM. Overall, the potent interaction of selected pesticides and chemicals with the vesicular transporter for dopamine, although, by itself, not synonymous with neurotoxicity, would argue for a likely impairment of transmitter homeostasis, or the putative formation of neurodegenerative toxin pools.

The effect of the antioxidant $\text{N-}\text{acetyl-}\text{L}\text{-cysteine}$ was studied in a model of polymicrobial sepsis induced in CD-1 mice by cecal ligation and puncture. $\text{N-}\text{acetyl-}\text{L}\text{-cysteine}$ significantly improved survival during the 6 days following sepsis induction and caused lower liver toxicity. This effect was not related to free radicals generated by xanthine oxidase which was significantly induced in liver after cecal ligation and puncture. A specific inhibitor of xanthine oxidase, allopurinol, significantly reduced this enzyme and reduced the early survival rate. The effect of $\text{N-}\text{acetyl-}\text{L}\text{-cysteine}$ was not related either to a reduction in tumor necrosis factor production or to a modulation of nitrites or to liver glutathione content. These results show that the induction of xanthine oxidase is not deleterious in this model of sepsis and suggest that $\text{N-}\text{acetyl-}\text{L}\text{-cysteine}$ works as a direct antioxidant and scavenger of free radicals generated from other sources.

The aim of the present study was to examine the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on mitochondrial respiration in ischemic rat hearts, and to compare the effects between water-soluble pravastatin and lipid-soluble simvastatin. Either vehicle (0.5% carboxymethyl cellulose), pravastatin (2 or 4 mg/kg per day), or simvastatin (1 or 2 mg/kg per day) was orally administered for 3 weeks. Ischemia was induced by ligating the aorta for 60 min in anesthetized open chest rats under artificial respiration. The hearts were removed, mitochondria were isolated, and the respiration was determined by polarography using glutamate and succinate as substrates. When succinate was used as a substrate, the ADP-stimulated respiration (QO₃) and ATP production per unit oxygen (ADP/O ratio) were decreased by ischemia. The decreases in QO₃ and ADP/O ratio in the pravastatin-and simvastatin-treated groups appeared to be more prominent than those in the vehicle-treated group. This was especially true in the simvastatin-treated group. The ADP-limited respiration (QO₄) with succinate in the vehicle-treated heart was slightly increased by ischemia, while that in the pravastatin- or simvastatin-treated hearts was decreased. In conclusion, HMG-CoA reductase inhibitors may result in worsening of myocardial mitochondrial respiration during ischemia.

The effects of free radicals on voltage-gates Ca²⁺ currents (I_Ca) were investigated in single guinea-pig ventricular myocytes using the whole-cell clamp technique. I_Ca was measured in the baseline state and after the application of free radicals from cumene hydroperoxide or generated from the addition of purine to xanthine oxidase. I_Ca decreased from 846 ± 533 (S.D.) pA to 688 ± 444 pA (n = 7, P < 0.05) in the presence of 100 μM cumene hydroperoxide and from 708 ± 157 pA to 457 ± 163 pA(n = 5, P < 0.0001) in the presence of 500 μM cumene hydroperoxide. I_Ca also decreased from 1303 ± 560 pA to 965 ± 360 pA in the presence of the free radical generating system (2.3 mM purine plus 20 U/l xanthine oxidase). The reduced I_Ca could not be restored by washing for up to 5 min using normal recording solution. We conclude that I_Ca is decreased in the presence of cumene hydroperoxide and an oxygen-derived free radical generating system in single guinea-pig ventricular myocytes. The cellular Ca²⁺ overload observed in free radical mediated reperfusion injury is therefore unlikely to result from an increase in sacrolemmal Ca²⁺ entry via voltage-gated Ca²⁺ channels.