In mice pretreated intracerebroventricularly (i.c.v.) with either saline (1 μl/mouse), pertussis (1 μg/mouse) or cholera (2.5 μg/mouse) toxins, effect of κ-opioid receptor agonists on the colonic temperature and charcoal meal transit time were assessed. The κ-opioid receptor agonist, trans-(+)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide methane sulfonate hydrate (U-50488H, 50, 100 and 200 μg/mouse, i.c.v.) produced dose dependent hypothermia. Pertussis toxin pretreatment (72 and/or 144 h before) antagonized (P < 0.05) the hypothermic effect of U-50488H (100 μg/mouse) and (±)-trans-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl[benz[b]-thio-phene-4-acetamide (PD 117302, 30 μg/mouse). In contrast, cholera toxin pretreatment (48 and/or 96 h before) did not antagonize the hypothermic effect of the κ-opioid receptor agonists. Moreover, both i.c.v. and intrathecal (i.t.) administration of κ-opioid receptor agonists, U-50488H, {[5R-(5α,7α,8β)]-(±)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspirol[4,5]dec-8-yl]-benzeneacetamide} (U-65593) and PD 117302, produced dose dependent inhibition of the charcoal meal transit. Cholera toxin pretreatment (48 and 96 h before) augmented (P < 0.05) the antitransit effect of i.c.v. administered U-50488H (100 μg/mouse), U-69593 (100 μg/mouse) and PD 117302 (50 μg/mouse). However, pertussis toxin previous pretreatment did not affect the gastrointestinal inhibitory effect of the κ-opioid receptors agonists. The present results extend our previous results on the affect of κ-selective agonists on gastrointestinal motility and indicate, like the prototype opiate agonist morphine, κ-opioid receptor agonists are effective in inhibiting the gastrointestinal motility when administered either by intrathecal or intracerebroventricular routes. Thus, for the antitransit effect of κ-opioid receptor agonists, both spinal and supra spinal site could be implicated. Furthermore, these results also suggest that pertussis and cholera toxin-sensitive transducer G-proteins may be involved in the central κ-opioid receptor mediated hypothermia and gastrointestinal transit inhibition respectively.
A rat cytomegalovirus infection model for use in immunotoxicity testing has been developed. In resistance against viruses, natural killer cells and cytotoxic T-cells play an important role. Therefore, this model complements other rat host resistance models for immunotoxicity testing, i.e. existing bacterial and parasitic infection models in which cytotoxic T-cells and natural killer cells play a minor role. Host resistance against cytomegalovirus infections in the rat was determined by titrating infectious virus levels in organs after cytomegalovirus infection in an in vitro infectivity test denoted as the Plaque Forming Unit (PFU) Test. In this test, homogenates of different organs were investigated for infectious virus titers on rat embryonic cell monolayers. We demonstrated that in the salivary gland, the major target organ for rat cytomegalovirus, virus was detectable from 8 days onward after intraperitoneal infection. To show that this model is suitable for the detection of immunotoxicity four different methods for immunosuppression were investigated: 1. γ-irradiation, 2. congenitally athymic rats, 3. chemically induced immunosuppression, 4. ultraviolet-B (UVB) irradiation. Rat cytomegalovirus titers in the salivary glands of irradiated (500 rad 1 day prior to infection) or congenitally athymic rats were significantly increased as compared to non-irradiated rats and euthymic control rats respectively. In TOX-Wistar rats, given 20 or 80 mg bis(tri-n-butyltin)oxide (TBTO) per kg food beginning 6 weeks before cytomegalovirus infection, a regimen known to have immunotoxic effects, cytomegalovirus titers in the salivary glands were significantly increased as compared to non-TBTO-treated cytomegalovirus infected rats. Exposure to a suberythemal doses of UVB, which is known to induce immunotoxic effects, induced a significant increase in cytomegalovirus titers in the salivary gland as compared to non-UVB-exposed rats. Therefore it is concluded that this infection model is suitable for the assessment of immunotoxic effects induced by enviromental components.
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-cyclopropanecarbonyl-8,1 1-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 activation of glutamate receptors by endogenous glutamate has been implicated in the processes that underlie cell loss associated with ischemia and trauma and in the development of some neurodegenerative diseases. The antagonism of NMDA-sensitive glutamate receptors may therefore have therapeutic applications. The present study compared the side effects and neuroprotective potency of 1-aminoadamantane hydrochloride (amantadine), 1-amino-3,5-dimethyladamantane hydrochloride (memantine), and (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate ((+)-MK-801) against NMDA injected directly into the nucleus basalis manocellularis of rats. Each drug significantly attenuated the loss of nucleus basalis magnocellularis cholinergic cells. The ED50s were respectively 0.077, 2.81 and 43.5 mg/kg for (+)-MK-801, memantine and amantadine, giving a relative potency ratio of 1:36:565. The ration of the ED50 for the side effects observed, including ataxia, myorelaxation and stereotypy, and the ED50 for neuroprotective ability, was highest for memantine and the lowest for (+)-MK-801. The results suggest that a potential neuroprotective action of NMDA receptor antagonists, memantine and amantadine in particular, can be seen at low doses lacking side effects.
We studied the mechanism of antagonism of p-chloroamphetamine-induced neurotoxicity by dextromethorphan. The pretreatment with potent and selective σ receptor ligands, 4-phenyl-4-(1-phenylbutyl)piperidine (4-PPBP) and N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100), did not alter the reduction of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the cerebral cortex by repeated administration of p-chloroamphetamine. These results suggest that σ receptors might not play a significant role in the antagonism of p-chloroamphetamine-induced neurotoxicity by dextromethorphan.
The porphyrinogenic action of hexafluorobenzene was investigated and compared to that of hexachlorobenzene. Metabolite patterns in the urine of exposed rats were determined to quantify the extent of metabolism through cytochrome P450 catalysed oxidation and glutathione conjugation. Results obtained demonstrate an almost similar extent of formation of phenolic metabolites. However, in the urine of hexachlorobenzene exposed rats significantly higher levels of the N-acetyl-S-(pentahalophenyl)cysteine were observed than in the urine of hexafluorobenzene exposed rats. Hexafluorobenzene exposure did not result in induction of porphyria, whereas exposure to hexachlorobenzene did result in significantly elevated levels of urinary as well as liver porphyrins. Together these results indicate that if the reactive intermediate is indeed formed in the cytochrome P450 catalysed initial oxidative dehalogenation, the extent of its formation as well as its subsequent reactivity and reaction pathways vary with the type of the halogen substituents. Furthermore, the results seem to indicate that the extent of metabolism of hexahalogenated benzenes into urinary metabolites resulting from glutathione conjugation is a better indication of their porphyrinogenic action than their extent of metabolism to phenolic metabolites. Two explanations for this observation are presented.
Interleukin-2 (15 μg/mouse, i.p. twice daily for 4 days and once on the 5th day) significantly lowered cytochrome P-450 and heme content and increased heme oxygenase mRNA accumulation; the activities of 7-ethoxycoumarin O-deethylase, ethoxy- and pentoxyphenoxazone O-dealkylases were decreased. The activity of the type O form of hepatic xanthine oxidase increased, but there was no increase in lipid peroxide, expressed in terms of microsomal malondialdehyde. In vivo inactivation of xanthine oxidase activity by feeding mice with tungstate did not substantially change the degree of interleukin-2-induced cytochrome P-450 depression, suggesting that the two processes are not causally linked. Induction of tolerance to endotoxin by a 4-day pretreatment with lipopolysaccharide resulted in 50% protection against this depression despite inhibition of the interleukin-2 induced formation of tumor necrosis factor. This suggests that the release of tumor necrosis factor per se does not fully account for the depression of cytochrome P-450. Dexamethasone, already used in patients to reduce toxicity of interleukin-2 therapy, provided full protection against the cytochrome P-450 depression.
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 x 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 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.
Trimellitic anhydride is a cause of occupational asthma in humans. We have previously found that tracheal instillation of trimellitic anhydride conjugated to guinea pig serum albumin induces acute bronchoconstriction and airway plasma exudation in sensitised animals, responses mediated primarily via histamine release. In the present study, neural mechanisms mediating bronchoconstriction and goblet cell secretion were determined in trimellitic anhydride-sensitised guinea pigs using the ganglionic blocker hexamethonium to eliminate efferent reflex mechanisms, pretreatment with capsaicin to eliminate afferent mechanisms, or cimetidine and mepyramine to eliminate histamine-mediated mechanisms. The magnitude of secretion of intracellular mucus from tracheal goblet cells was quantified morphometrically as a mucus score which is inversely related to the degree of discharge. Guinea pigs were injected intradermally either with 0.1 ml 0.3% trimellitic anhydride in corn oil or with corn oil alone as control. Fourteen to eighteen days later all sensitised animals had developed specific immunoglobulin (Ig) G1 antibodies whereas the controls had not. Tracheal instillation of conjugated trimellitic anhydride in anaesthetised animals significantly increased airway lung resistance (RL) 24-fold in sensitised guinea pigs (34.3 +/- 7.9 cm H2O.ml-1.s) compared with controls (1.4 +/- 0.1 cm H2O.ml-1.s). Mucus score was significantly reduced by 51% (indicating goblet cell secretion) in sensitised guinea pigs (183 +/- 22 mucus score units) compared with controls (372 +/- 41 mucus score units). The antihistamines significantly inhibited conjugated trimellitic anhydride-induced bronchoconstriction by 89%, but did not significantly affect goblet cell discharge. Hexamethonium alone did not significantly affect conjugated trimellitic anhydride-induced bronchoconstriction or goblet cell secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
We investigated the regulation of the protein expression of the α isozymes of Na+,K+-ATPase in reference to the enzyme activity in the heart, brain skeletal muscle of rats during deoxycorticosterone acetate (DOCA)-salt hypertension. Treatment of rats with DOCA and salt for 28 days produced a significant increase in systolic blood pressure compared to the control groups which remained normotensive. Rats treated with DOCA expressed greater amounts of the immunoreactive α-1 isoform than untreated controls in whole heart membranes. However, the DOCA-induced increase in the α-1 isoform did not occur during DOCA-salt hypertension. There was a parallel change in the enzyme activity of the Na+,K+-ATPase and the protein expression of the α-1 isoform as a result of these treatments. We have also demonstrated that the hearts of DOCA-salt hypertensive rats expressed less of the α-2 isoform compared to the controls. We could not detect any alteration in the α-1 and α-2 isoforms of the skeletal muscle and α-1, α-2 and α-3 isoforms of the whole brain Na+,K+-ATPase during salt or DOCA treatments alone or DOCA-salt hypertension. Furthermore, the Na+,K+-ATPase activity was unaltered in these tissues during these treatments. In conclusion, cardiac Na+,K+-ATPase α-subunit protein expression appears to be regulated during DOCA-salt hypertension. In the skeletal muscle and brain, tissues not subjected directly to increased pressure, this regulation of the Na+,K+-ATPase was not apparent.
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 (QO(3)) and ATP production per unit oxygen (ADP/O ratio) were decreased by ischemia. The decreases in QO(3) 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 (QO(4)) 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 low level exposure of rats to 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) on their immune system was investigated. Dietary administration to young adult male Leeds strain rats of a total dose of 3 μg/kg body weight of TCDD resulted in an exposure duration-dependent reduction of in vitro lipopolysaccharide-induced production of interleukin (IL)-1 in cultures of their splenic macrophages. A 30-day exposure produced approximately 30% suppression and 180-day exposure produced approximately 52% suppression. This reduction did not negatively influence lipopolysaccharide-induced proliferation of B cells, instead an enhancement of B cell proliferation was observed after 30 days exposure. A 180 day exposure significantly suppressed the generation of IL-2 by either concanavalin A or phorbol myristate acetate/calcium ionophore stimulation, and reduced the lectin-induced proliferation of splenic T cells. The 30-day TCDD exposure showed no such immunotoxicity. TCDD at both exposure durations suppressed the expression of the α chain of the IL-2 receptor in concanavalin A-activated T cells, without affecting the CD4+/CD8+ ratio. The results suggest that exposure to a low dietary dose of TCDD suppresses the functions of several T cell subsets, some of the immunotoxic effects being produced early, while others require a longer exposure period. The effect of TCDD on B cells appears to be of transient nature, with less potentially serious consequences. Such exposure also down-regulates the IL-1 production function of macrophages. A common mechanism of TCDD immunotoxicity may be on the multifunctional signal transduction pathways downstream to the activation of protein kinase C and Ca2+ flux.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a ligand for the arylhydrocarbon receptor (Ah receptor), abundant in the murine thymus. In the thymus immunocompetent T cells develop. Upon exposure of murine fetal thymi in organ cultures to TCDD the distribution of mature and immature thymocytes is skewed towards apparently mature, prospective cytotoxic cells of the CD4−CD8+T cell receptor+ phenotype. The normally abundant CD4+CD8+ cells are decreased. Proliferation of the most immature thymocyte subpopulations is inhibited and maturation of thymocytes appears accelerated by TCDD. Eventually the thymocyte number is significantly decreased. Selective treatment of stroma cells showed them to be the primary target cells of TCDD action. Thymus stroma plays a pivotal role in thymocyte maturation and is indispensable for the selection of thymocytes bearing T cell receptors specific for foreign antigen in the context of self. We tested whether the effects of TCDD on thymocyte differentiation and maturation has further consequences for the selection processes by analysing (a) the repertoire of Vβ genes used as a measure for negative selection and (b) the expression of CD69 and bcl-2 by thymocytes as a parameter for positive selection. Our data indicate that TCDD does not cause gross disturbance of negative selection but provide evidence for more cells auditioning for positive selection by TCDD exposure.
We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.
The protein glycosylation inhibitor tunicamycin protected male BALB/c mice from tumor necrosis factor alpha-induced liver failure. Tunicamycin also inhibited tumor necrosis factor-induced cell death in primary hepatocyte cultures with a median inhibitory concentration of 8 nM, but not in the tumor cell line WEHI 164 clone 13. Hepatocyte death in our culture system was characterized by DNA fragmentation and apoptotic changes. These two characteristic signs of programmed cell death were also inhibited by tunicamycin treatment. These data suggest that protein glycosylation is an early and causal event of tumor necrosis factor (TNF)-induced parenchymal cell death in the liver.
Evidence from our previous work suggests that neurogenic mediators contribute to the inflammation following ultraviolet (UV) irradiation of the skin. We have investigated whether calcitonin gene-related peptide (CGRP), substance P and nitric oxide (NO) participate in the cutaneous inflammatory reaction of the rat hind paw and ear to UV irradiation. Skin blood flow was measured by laser Doppler technique. Oedema was quantified using a spring loaded micrometer to measure ear thickness. UV irradiation of the rat skin lead to a long lasting increase in skin blood flow. This increase was dose dependently attenuated by the CGRP receptor antagonist CGRP-(8–37) (0.15 nmol in 25 μl to 6.0 nmol in 25 μl, s.c.) up to 51% with a maximum of effectiveness at 24 h post irradiation. The inhibitor of NO synthase NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, 25 nmol in 25 μl, s.c.) attenuated skin blood flow by 38%. Concurrent injections s.c. of CGRP-(8–37) (1.5 nmol in 12.5 μl) and L-NAME (25 nmol in 12.5 μl) demonstrated an augmentive effect in attenuating skin blood flow. The tachykinin NK1 receptor antagonist CP-96,345 (6.0 nmol in 25 μl, s.c.) attenuated skin blood flow by 27%. NG-Nitro-D-arginine methyl ester hydrochloride (D-NAME) and CP-96,344 showed no effects on skin blood flow after UV irradiation. CGRP-(8–37) (0.6 nmol in 10 μl) i.d. and L-NAME (10 nmol in 10 μl) i.d. had no effect of oedema formation after UV irradiation. Furthermore, post UV irradiation enhanced CGRP- and NO synthase-immunoreactivity in nerve fibres in the exposed skin area were visible. Taken these findings together we suggest the involvement of the neuropeptides CGRP and substance P and of neuronal NO on the vasodilatory component of the UV-induced inflammatory reaction of the rat skin. CGRP contributing to UV-induced vasodilation acts in an endothelial NO-independent manner.
A monoclonal antibody against tetrodotoxin was produced. Tetrodotoxin coupled with keyhole limpet hemocyanin was used as an immunogen to BALB/c mice. These mice had no clinical signs for the toxicity of tetrodotoxin during the immunization. The reason may be that the guanidyl group of tetrodotoxin which is an important group for the toxicity was hidden by coupling with keyhole limpet hemocyanin. The monoclonal antibody was highly specific for tetrodotoxin and had no cross-reaction to tetrodotoxin derivatives, paralytic shellfish toxins, keyhole limpet hemocyanin and crude proteins from various organs of puffer fish. Also, tetrodotoxin was neutralized in vitro by this antibody. From the fact that the structural difference between tetrodotoxin and anhydro-tetrodotoxin is recognized by this antibody, it was suggested that this antibody reacted with the OH- groups on C-4 and/or C-9 of tetrodotoxin. In addition, the results from immunization and neutralization tests demonstrated that tetrodotoxin became non-toxic even when one of the active groups of tetrodotoxin was coupled by a molecule.
To determine if an Ag+-induced contracture is associated with the Ca2+-induced Ca2+ release mechanism in the sarcoplasmic reticulum, effects of Ca2+-induced Ca2+ release modulators on the Ag+-induced contracture were studied with single fibers of frog toe skeletal muscle. The fiber treated with 1 mM caffeine contracted significantly much more than controls without caffeine at Ag+ concentrations below 1 μM. Procaine shifted the Ag+ concentration-tension curve to the right, dose-dependently. When 10 mM procaine was applied to contracting fibers not treated with caffeine, the duration of 5 μM Ag+-induced contracture was shortened with a little decrease in tension amplitude, that was different from the effect of procaine on caffeine contracture. In caffeine solution, 0.5 μM Ag+ caused a long-lasting contracture with sometimes two peaks. 2 mM procaine led to disappearance of such two peaks, resulting in shortening of the contracture. K+ contracture was potentiated by 1 mM caffeine only at lower concentrations of K+, and inhibited by 10 mM procaine. These results suggest that the Ag+-induced contracture is composed of two components: Ca2+-induced Ca2+ release-dependent and -independent. 5 μM Ag+-induced contracture slowly relaxed with a wavy tension pattern to the resting level when 0.05 mM dithiothreitol was applied around peak of the tension. This relaxation was accelerated by procaine application. These findings may be explained by attributing a portion of Ag+-induced contracture to the effect of Ca2+ released through the Ca2+-induced Ca2+ release mechanism in the sarcoplasmic reticulum.
At which time-point and to what extent do N-methyl-d-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors and L-type voltage-sensitive Ca2+ channels (VSCC) contribute to glutamate-induced neuronal injury? To address this question, we induced glutamate neurotoxicity in two neuronal culture systems, chick telencephalic neurons and rat hippocampal neurons, and tested selective antagonists for their neuroprotective activity when administered either during the excitotoxic insult (acute treatment) or during the recovery period (posttreatment). In cultured chick telencephalic neurons exposed to 1 mM l-glutamate for 60 min, both the NMDA receptor antagonist dizocilpine (MK-801; 0.1 μM) and the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 μM) completely blocked glutamate-induced neuronal injury when applied concomitantly with glutamate. If the antagonists were applied during the recovery period, dizocilpine at concentrations up to 10 μM only moderately increased cell viability, whereas CNQX showed a neuroprotective activity comparable to that observed in the case of the acute treatment. In cultured rat hippocampal neurons, excitotoxic injury was induced by a 30-min exposure to 1 mM glutamate. Treatment with dizocilpine during the glutamate exposure could rescue the hippocampal neurons from the excitotoxic insult, whereas acute treatment with the AMPA/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxalime (NBQX) or the L-type VSCC blocker nimodipine showed no protection. In contrast, all three drugs showed neuroprotective activity when applied 30, 60 or 120 min after the glutamate exposure. Surprisingly, when the onset of the treatment was delayed for even 240 min, only NBQX and nimodipine led to a reduction in excitotoxic neuronal injury. We conclude that activation of AMPA/kainate receptors and L-type VSCC is critically involved in a late stage of glutamate neurotoxicity, thereby allowing pharmacological intervention at a time when blockade of NMDA receptors becomes less efficacious.
A V79 Chinese hamster cell line was constructed for stable expression of human cytochrome P450 2E1 (CYP2E1) by integration of a SV40 Early promoter recombinant CYP2E1 cDNA into the chromosomal DNA. The cDNA encoded CYP2E1 was effectively expressed and enzymatically active, as shown by hydroxylation of chlorzoxazone and of p-nitrophenol, at rates of about 70 pmol x mg(-1) total protein X min(-1). CYP2E1 content and activity was increased upon cultivation in the presence of ethanol indicating a substrate mediated stabilization effect. A similar stabilizing effect was also observed for inhibitors of CYP2E1, e.g. imidazole, 4-methylpyrazole, and isoniazid. The feasibility of the newly established cell line V79MZh2E1 for toxicological studies was shown by CYP2E1-mediated activation of N-nitrosodimethylamine and p-nitrophenol and a dose-dependent cytotoxic and mutagenic effect.
