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Human immunoglobulin G (IgG) concentrates are immune-modulating, anti-inflammatory plasma-derived products. Clinical studies in recent years have suggested that IgG attenuates neuropathic pain. In this study, effects of sulphonated IgG on the development and maintenance of a mechanical allodynia-like response were examined in mice with neuropathic pain induced by a partial sciatic nerve ligation (PSL). When sulphonated IgG (400 or 1,000 mg/kg/day, i.p.) was administered for 5 days, from 1 day before surgery to post-operative day (POD) 3, the development of a mechanical allodynia-like response was attenuated. On the other hand, sulphonated IgG had little effect on the maintenance of a mechanical allodynia-like response when administered for 5 days, from POD 11 to POD 15, at which time a mechanical allodynia-like response had already been developed. To explore the mechanism of sulphonated IgG, the mRNA expression of inflammatory cytokines was evaluated in the injured sciatic nerve. Sulphonated IgG (1,000 mg/kg/day, i.p.) that was administered for 3 days, from 1 day before surgery to POD 1, significantly attenuated the up-regulation of tumor necrosis factor-α and monocyte chemotactic protein-1 mRNAs on POD 1. These results suggest that prophylactic treatment with sulphonated IgG attenuates the development of mechanical allodynia-like response by inhibition of inflammatory cytokine expression in mice with PSL.

The regulatory mechanism of Bcl-2 protein expression was investigated in SH-SY5Y cells, the human neuroblastoma cell line that expresses natively Bcl-2 proteins. WHen the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or retinoic acid, the level of Bcl-2 protein was increased compared with the control. These effects were inhibited by pretreatment with a protein kinase C (PKC) inhibitor, staurosporine or calphostin C. The level of Bcl-2 protein was also increased by treatment with carbachol, a muscarinic acetylcholine receptor (mAChR) agonist, and the effect were also inhibited by pretreatment with staurosporine or calphostin C. An addition, a carbachol-induced increase in Bcl-2 protein levels and a transient elevation of [Ca2+]i were inhibited by pretreatment with 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), an m3 mAChR antagonist. In contrast, the level of Bcl-2 protein was decreased by treatment with dibutyryl cAMP (diBu-cAMP), forskolin, or cholera toxin, and the effects of diBu-cAMP were inhibited by pretreatment with a protein kinase A (PKA) inhibitor, H-89. From these results, we suggest that the expression of Bcl-2 proteins is regulated by PKC and PKA in positive and negative manners, respectively, in SH-SY5Y cells. Furthermore, the nucleosomal DNA fragmentation induced by serum depletion for 4 h was observed in SH-SY5Y cells when the level of Bcl-2 protein was down-regulated by treatment with 1 mM diBu-cAMP for 3 days, although the DNA fragmentation by serum depletion for 4 h was not observed in nontreatment cells, indicating that Bcl-2 proteins whose expression is regulated by PKC and PKA play important roles in serum depletion-induced apoptosis.

When PC12 cells were treated with 1-methyl-4-phenylpyridinium ion (MPP+) at various concentrations for 1 week, the dopamine (DA) content was increased compared to the control at 1-30 microM but was decreased to less than the control at concentrations above 100 microM. Cell death was caused by 300 microM MPP+, indicating that decrease in DA content proceeds cell death. When the cells were treated with 100 microM MPP+ for various periods, DA content was transiently increased (6 h-2 days) and then gradually decreased below the control (4-7 days). These results suggest that MPP+ possesses biphasic effects on DA content, being dependent on both concentrations and treatment periods. Moreover, by treatment with 100 microM MPP+, tyrosine hydroxylase (TH) mRNA expression was also transiently increased and then gradually decreased below the control, suggesting that MPP+ also possesses biphasic effects on TH mRNA expression.

The Pig-a gene mutation assay using perpherial blood erythrocytes is being investigated as a screening tool for assessing mutagenicity in vivo. In this study, we evaluated two distinct approaches for performing the Pig-a assay in rats. We used antibodies to CD45 or the erythroid marker HIS49 to identify red blood cells (RBCs), and then monitored the kinetics of Pig-a mutant frequency, as measured by the frequency of CD59-deficient RBCs, in rats treated with the genotoxic chemicals, N-ethyl-N-nitrosourea, cyclophosphamide, 4-nitroquinoline-1-oxide, and ethylmethanesulfonate. In some instances, micronucleus frequency also was measured in the same animals. Time- and dose-related increases in Pig-a mutant frequency were found in all the chemical-treated groups, except for the groups treated with cyclophosphamide, which was a potent inducer of micronuclei. The two different approaches we employed were comparable for measuring induced mutant frequencies, but our historical data showed that the mean background frequencies for the CD45/CD59 method and the HIS49/CD59 method were 12.7 × 10(-6) and 5.5 ×10(-6), respectively. The relatively low, stable background mutant frequency associated with the HIS49/CD59 method indicates that it may have greater power for discriminating weak induced responses. These results suggest that the HIS49/CD59 method is a promising tool for measuring Pig-a mutant RBCs. In addition, differences in their manifestation kinetics and in their relative sensitivity for detecting different test compounds suggest that the combination of the Pig-a assay and the micronucleus assay may be effective in identifying in vivo genotoxicity.

Recent studies indicate that the Pig-a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig-a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid-specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 x 10(6) CD71-positive RETs could be evaluated by flow cytometry for Pig-a mutant frequency within 5 to 8 min. CD59-deficient RET and BME frequencies of more than 100 x 10(-6) and 80 x 10(-6) were detected 1 week after treating rats with 40 mg/kg N-ethyl-N-nitrosourea; by comparison, the frequency of CD59-deficient total RBCs in these rats was 13.2 x 10(-6). The frequency of spontaneous Pig-a mutant RETs and BMEs was less than 5 x 10(-6) and 15 x 10(-6), respectively. Since approximately 98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59-deficient Pig-a mutations in cells of erythroid lineage. Conducting concurrent Pig-a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.

Background— The acetylcholine-activated K + current ( I K,ACh ) is a novel candidate for atrial-specific antiarrhythmic therapy. The present study investigates the involvement of I K,ACh in atrial fibrillation (AF) using NTC-801, a novel potent and selective I K,ACh blocker. Methods and Results— The effects of NTC-801, substituted 4-(aralkylamino)-2,2-dimethyl-3,4-dihydro- 2H -benzopyran-3-ol, on I K,ACh and other cardiac ionic currents ( I Na , I CaL , I to , I Kur , I Kr , I Ks , I Kl , I KATP , and I f ) and on atrial and ventricular action potentials were examined in vitro. NTC-801 potently inhibited carbachol-induced I K,ACh in guinea pig atrial cells and the GIRK1/4 current in Xenopus oocytes with IC 50 values of 5.7 and 0.70 nmol/L, respectively. NTC-801 selectively inhibited I K,ACh >1000-fold over other cardiac ionic currents. NTC-801 (10 to 100 nmol/L) reversed the action potential duration (APD 90 ) shortened by carbachol or adenosine in atrial cells, whereas it did not affect APD 90 at 100 nmol/L in ventricular cells. Antiarrhythmic effects of NTC-801 were evaluated in 3 AF models in vivo. NTC-801 significantly prolonged atrial effective refractory period without affecting ventricular effective refractory period under vagal nerve stimulation. NTC-801 dose-dependently converted AF to normal sinus rhythm in both vagal nerve stimulation–induced (0.3 to 3 μg · kg −1 · min −1 IV) and aconitine-induced (0.01 to 0.1 mg/kg IV) models. In a rapid atrial pacing model, NTC-801 (3 μg · kg −1 · min −1 IV) significantly decreased AF inducibility with a prolonged atrial effective refractory period that was frequency-independent. Conclusions— A selective I K,ACh blockade induced by NTC-801 exerted anti-AF effects mediated by atrial-selective effective refractory period prolongation. These findings suggest that I K,ACh may be important in the development and maintenance of AF.

We examined the effects of progesterone on the elevation of tail skin temperature (TST) in ovariectomized rats and compared them with those of estradiol. Progesterone showed only insignificant effects on the TST elevation, whereas estradiol showed complete inhibition. The TST elevation induced by ovariectomy is caused by estradiol deficiency, but progesterone plays little or no role.

Apoptosis has been shown to be induced by some pathological stimuli. MPP+ is a neurotoxin and an inducer of parkinsonism. When SH-SY5Y cells, human neuroblastoma cell line, were treated with MPP+, cell death estimated by lactate dehydrogenase (LDH) leakage assay occurred. The cell death was associated with the DNA fragmentation into nucleosomal fragments at 180 bp, suggesting that MPP(+)-induced cell death of SH-SY5Y cells occurs through apoptosis. Although SH-SY5Y cells natively express Bcl-2 protein, which inhibits apoptosis, the level of Bcl-2 protein in SH-SY5Y cells increased with increases in the treatment periods of MPP+. MPP+ inhibits the mitochondrial respiratory chain. The other inhibitors of the mitochondrial respiratory chain, antimycin A and oligomycin, also caused cell death associated with DNA fragmentation, but did not increase the Bcl-2 protein level, suggesting that an MPP(+)-induced apoptosis may be due to the inhibition of the mitochondrial respiratory chain but the MPP(+)-induced increase in the Bcl-2 protein level is not due to it. A protein kinase inhibitor, staurosporine, inhibited the MPP(+)-induced increase in the Bcl-2 protein level, but not the MPP(+)-induced cell death. These results also suggest that the mechanism by which MPP+ increases the Bcl-2 protein level is different from that of MPP(+)-induced cell death.

Systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to damage the dopaminergic nigrostriatal system in C57BL/6 mice. We have investigated the effects of immunosuppressants, FK-506 and cyclosporin A (CsA), on MPTP-induced dopamine (DA) depletion in the striatum of young C57BL/6 mice. 10 days after MPTP treatment (25 mg/kg i.p. given daily, 5 days), DA in the striatum was depleted by 80%. However, pretreatment with FK-506, a novel immunosuppressant, significantly protected MPTP-induced DA depletion in the striatum, but FK-506 itself did not affect the DA content. CsA, another immunosuppressant, also protected MPTP-induced DA depletion. From these results can be seen that immunosuppressants seem to inhibit MPTP neurotoxicity toward nigrostriatal dopaminergic neurons of young C57BL/6 mice.

Three major subtypes of glutamate receptors that are coupled to cation channels—N-methyl-d-aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors—are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 ± 2.2 and 6.4 ± 2.3 μM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 μM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 μM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment. In conclusion, the rat hippocampus appears to contain a novel class of metabotropic receptors that prefer glutamate and NMDA and is coupled with adenylate cyclase in an inhibitory manner via pertussis toxin-sensitive GTP binding proteins.