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Introduction: We investigated the localization of a ganglioside, N-acetylgalactosaminyl GD1a (GalNAc-GD1a), in peripheral nerves with an IgG anti–GalNAc-GD1a antibody, which was produced in rabbits immunized with GalNAc-GD1a. Methods: Teased fibers from ventral and dorsal roots and hemidiaphragm sections of rats were assessed using fluorescent double- and triple-labeling methods. Results: The nodal and paranodal regions of teased fibers from ventral roots were immunostained with IgG anti–GalNAc-GD1a antibodies. After collagenase treatment, no staining was seen with IgG anti–GalNAc-GD1a or anti-NF200 antibodies, whereas α-bungarotoxin selectively stained nerve terminals. In cross-sectional and longitudinal sections of rat hemidiaphragm, IgG anti–GalNAc-GD1a antibodies overlapped with α-BuTx and anti-NF200 antibodies, indicating that GalNAc-GD1a is localized to the nerve terminal. IgG anti–GalNAc-GD1a antibody staining also overlapped with that of AChR clusters and syntaxin-positive presynaptic nerve terminals. Conclusion: GalNAc-GD1 is localized in both pre- and postsynaptic nerve terminals of neuromuscular junctions. Muscle Nerve 46: 705–710, 2012

It is known that psychostimulants stimulate dopamine transmission in the nucleus accumbens. In the present study, we examined the effects of systemically administered beta-phenylethylamine (beta-PEA), a psychomotor-stimulating trace amine, on dopamine concentrations in the nucleus accumbens and prefrontal cortex in freely moving rats, using an in vivo microdialysis technique. Intraperitoneal administration of beta-PEA (12.5 and 25 mg/kg) significantly increased extracellular dopamine levels in the nucleus accumbens shell. The observed increase in the dopamine concentration in nucleus accumbens shell dialysate after intraperitoneal administration of 25 mg/kg beta-PEA was inhibited by pre-treatment with a dopamine uptake inhibitor, GBR12909 (10 mg/kg, i.p.). In contrast, beta-PEA (25 mg/kg, i.p.) did not affect dopamine release in the nucleus accumbens core. Although a high dose of beta-PEA (50 mg/kg) significantly increased dopamine levels in the nucleus accumbens core, the dopamine increasing effect of beta-PEA was more potent in the nucleus accumbens shell. Systemic administration of 12.5 and 25 mg/kg beta-PEA also increased extracellular dopamine levels in the prefrontal cortex of rats. However, systemic 25 mg/kg beta-PEA-induced increases in extracellular dopamine levels were not blocked by GBR12909 within the prefrontal cortex. These results suggest that beta-PEA has a greater effect in the shell than in the core and low-dose beta-PEA stimulates dopamine release in the nucleus accumbens shell through uptake by a dopamine transporter. Similarly, beta-PEA increased extracellular dopamine levels in the prefrontal cortex. Thus, beta-PEA may increase extracellular dopamine concentrations in the mesocorticolimbic pathway.

To investigate the pathophysiological mechanisms of immune-mediated peripheral neuropathies, we studied the effects of sera from patients with Guillain-Barré syndrome (GBS) on the Cav2.1 voltage-dependent calcium channel (VDCC) current in Purkinje cells. Using the whole-cell recording technique, Cav2.1 VDCC current was measured in cerebellar Purkinje cells in the presence of serum from GBS patients with acute motor axonal neuropathy (AMAN) or acute inflammatory demyelinating polyneuropathy (AIDP). The AMAN patient sera significantly inhibited the Cav2.1 VDCC current compared with healthy volunteer sera, and this inhibition was fully reversible by washing out the AMAN serum. Similarly, IgG purified from AMAN sera also inhibited the Cav2.1 VDCC current. However, the activation and inactivation kinetics of the Cav2.1 VDCC currents were not affected by serum from an AMAN patient. Moreover, the VDCC current of Purkinje cells was also inhibited by IgG anti-GM1 monoclonal antibody (anti-GM1 mAb). In an immunocytochemical study using double fluorescence staining, Purkinje cells were stained by monoclonal IgG anti-GM1 mAb. In contrast, AIDP patient and healthy volunteer sera did not affect the Cav2.1 VDCC current. These results suggest that in some case of GBS, particularly of AMAN patients with IgG anti-GM1 mAb, muscle weakness may be induced by dysfunction of Cav2.1 VDCC functioning at the motor nerve terminals.

We investigated the expression and localization of Kv1 channels in dorsal spinal roots (DRs) and ventral spinal roots (VRs) in rats. Among Kv1.1-1.6 tested by RT-PCR, mRNAs of Kv1.1, 1.2, and 1.5 were moderately expressed, those of Kv1.3 and Kv1.6 were weakly expressed, and that of Kv1.4 was hardly expressed at all in both DRs and VRs, whereas all six mRNAs were detected in spinal cord. Western blotting revealed that the major immunoreactive proteins were Kv1.1 and Kv1.2 in both DRs and VRs. Quantitative analysis indicated that levels of Kv1.1 and Kv1.2 protein were significantly higher in DRs than VRs. Immunohistochemical examination showed that Kv1.1 and Kv1.2 were colocalized in juxtaparanodal regions of axons in both DRs and VRs. Finally, immunoprecipitation experiments revealed that Kv1.1 and Kv1.2 were coassembled. These findings indicate that Kv1 subtypes in DRs and VRs are somewhat different from those in spinal cord, and that the numbers of Kv1.1 and Kv1.2 channels are higher in DRs than VRs.

Oxaliplatin is a chemotherapeutic agent that is effective against various types of cancer including colorectal cancer. Acute cold hyperalgesia is a serious side effect of oxaliplatin treatment. Although the therapeutic drug pregabalin is beneficial for preventing peripheral neuropathic pain by targeting the voltage-dependent calcium channel α2δ-1 (Cavα2δ-1) subunit, the effect of oxaliplatin-induced acute cold hypersensitivity is uncertain. To analyze the contribution of the Cavα2δ-1 subunit to the development of oxaliplatin-induced acute cold hypersensitivity, Cavα2δ-1 subunit expression in the rat spinal cord was analyzed after oxaliplatin treatment. Behavioral assessment using the acetone spray test showed that 6 mg/kg oxaliplatin-induced cold hypersensitivity 2 and 4 days later. Oxaliplatin-induced acute cold hypersensitivity 4 days after treatment was significantly inhibited by pregabalin (50 mg/kg, p.o.). Oxaliplatin (6 mg/kg, i.p.) treatment increased the expression level of Cavα2δ-1 subunit mRNA and protein in the spinal cord 2 and 4 days after treatment. Immunohistochemistry showed that oxaliplatin increased Cavα2δ-1 subunit protein expression in superficial layers of the spinal dorsal horn 2 and 4 days after treatment. These results suggest that oxaliplatin treatment increases Cavα2δ-1 subunit expression in the superficial layers of the spinal cord and may contribute to functional peripheral acute cold hypersensitivity.

Peripheral neuropathy is a common adverse effect of paclitaxel treatment. The major dose-limiting side effect of paclitaxel is peripheral sensory neuropathy, which is characterized by painful paresthesia of the hands and feet. To analyze the contribution of substance P to the development of paclitaxel-induced mechanical hyperalgesia, substance P expression in the superficial layers of the rat spinal dorsal horn was analyzed after paclitaxel treatment. Behavioral assessment using the von Frey test and the paw thermal test showed that intraperitoneal administration of 2 and 4mg/kg paclitaxel induced mechanical allodynia/hyperalgesia and thermal hyperalgesia 7 and 14 days after treatment. Immunohistochemistry showed that paclitaxel (4mg/kg) treatment significantly increased substance P expression (37.6±3.7% on day 7, 43.6±4.6% on day 14) in the superficial layers of the spinal dorsal horn, whereas calcitonin gene-related peptide (CGRP) expression was unchanged. Moreover, paclitaxel (2 and 4mg/kg) treatment significantly increased substance P release in the spinal cord on day 14. These results suggest that paclitaxel treatment increases release of substance P, but not CGRP in the superficial layers of the spinal dorsal horn and may contribute to paclitaxel-induced painful peripheral neuropathy.

Guillain-Barre syndrome with antibodies against ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a) is characterized by a rapid onset of predominantly distal pure motor neuropathy. However, the pathogenic role of anti- GalNAc-GD1a antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. We investigated the effects of IgM anti-GalNAc-GD1a monoclonal antibody (IgM anti-GalNAc-GD1a mAb) on spontaneous muscle action potentials (SMAP) in a rat spinal cord–muscle co-culture system and the localization of IgM anti-GalNAc- GD1a mAb and calcium channel binding in the rat hemi-diaphragm. Immunohistochemistry of the rat hemidiaphragm showed that IgM anti-GalNAc-GD1a mAb binding overlapped with anti-neurofilament 200 antibody and α- bungarotoxin staining, demonstrating that IgM anti-GalNAc-GD1a mAb was localized at the motor nerve terminal. Moreover, IgM anti-GalNAc-GD1a mAb binding overlapped with anti-Cav2.1 antibody in the nerve terminal. We suggest that the inhibitory effect of IgM anti-GalNAc-GD1a mAb on SMAP is related to the GalNAc-GD1a epitope on P/Q-type calcium channels in motor nerve terminals at NMJs.

We performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a coculture of rat muscle and spinal cord by exposure to serum, as well as purified IgG, from patients with the acute motor axonal neuropathy (AMAN) variant of Guillain-Barré syndrome (GBS). However, exposure to serum from two patients with the acute inflammatory demyelinating polyneuropathy (AIDP) form of GBS had no effect. Using a whole-cell recording technique, we then investigated the effects of serum and purified IgG from patients with GBS on voltage-dependent calcium channel (VDCC) currents in nerve growth factor-differentiated PC12 cells. Serum from patients with GBS (AMAN) inhibited VDCC currents in PC12 cells, which was fully reversible by washing with the bath solution. Similarly, purified IgG from the serum of two patients with GBS (AMAN) also inhibited VDCC currents in PC12 cells. In contrast, sera from patients with AIDP and healthy volunteers did not affect VDCC currents in PC12 cells. These results suggest that muscle weakness in some patients with GBS might be induced by inhibition of Ca2+ channel currents within motor nerve terminals.

Voltage-gated K(+) channels contain six membrane spanning segments and a pore-forming domain. We used site-directed mutation to examine the role of specific amino acids in the extracellular region of the pore in Kv1.2. When expressed in CHO cells, a K(+) current was not observed for mutants S356A, S360A, T383A and T384A. However, coexpression of the Kvbeta2 subunit and the S360A mutant resulted in a robust peak current. Immunocytochemistry for Kv1.2 showed staining throughout the cytoplasm in cells coexpressing the beta2 and S360A, whereas only the perinuclear region was stained in cells expressing the S360A mutant. Western blotting revealed that the major immunoreactive protein in wild-type- and mutant-expressing cells is 60-kDa, but 87-kDa bands were also detected in cells expressing wild-type Kv1.2 and cells coexpressing beta2and S360A. These results suggest that amino acids in the pore region help regulate ion permeability or cellular trafficking by affecting glycosylation of Kv1.2.

Exposure of mice to the anesthetic gas nitrous oxide (N 2 O) produces a marked antinociceptive effect. Protein kinase C is a key regulatory enzyme that may be targeted by general anesthetics. However, a relationship between N 2 O-induced antinociception and protein kinase C has yet to be established. The present study was conducted to identify whether protein kinase C might influence N 2 O-induced antinociception in mice. Regular exposure (11 min) to N 2 O produced concentration-dependent antinociception in mice, as determined using the abdominal constriction test. N 2 O-induced antinociception was attenuated by i.c.v. pretreatment with phorbol 12,13-dibutyrate, a protein kinase C activator. This phorbol 12,13-dibutyrate antagonism of N 2 O-induced antinociception was reversed by i.c.v. pretreatment with calphostin C, a protein kinase C inhibitor. Long-term exposure (41 min in total, including 30 min prior to, and 11 min of analgesic testing) to 70% N 2 O produced reduced analgesic effects, compared with regular exposure to 70% N 2 O, thus indicating acute tolerance to N 2 O-induced antinociception. However, mice pretreated with calphostin C, chelerythrine, which is another protein kinase C inhibitor, and phorbol 12,13-dibutyrate, did not develop acute tolerance. Regarding activation of protein kinase C, regular exposure to 70% N 2 O did not increase protein kinase C within the membrane fraction of brain tissue, as determined by immunoblot analysis, but long-term exposure to 70% N 2 O did. The i.c.v. pretreatment with calphostin C and phorbol 12,13-dibutyrate prevented the increase in protein kinase C observed with long-term exposure to 70% N 2 O. These results suggest that brain protein kinase C negatively regulates the antinociceptive effect of N 2 O, and that activation of brain protein kinase C is related to the development of acute tolerance to N 2 O-induced antinociception in mice.

We investigated the localization of GalNAc-GD1a biochemically in the human and bovine peripheral nervous system (PNS). The high-performance thin-layer chromatography (HPTLC)-overlay method with rabbit IgG polyclonal antibody against GalNAc-GD1a (anti-GalNAc-GD1a antibody) revealed expression of GalNAc-GD1a in the ventral spinal nerve roots (VRs) but not in the dorsal spinal nerve roots (DRs) of both species. The amount of GalNAc-GD1a in the human and bovine VRs was 2.22 +/- 0.35 microg/g wet tissue and 7.71 +/- 0.49 microg/g wet tissue, respectively. These results suggest that IgG anti-GalNAc-GD1a antibody may be involved in disturbance of peripheral motor nerves and in the pathogenesis of pure motor neuropathy.

The effects of systemic administration of beta-phenylethylamine (beta-PEA) and microiontophoretically applied beta-PEA on the spontaneous discharge of dopamine (DA) neurons in the ventral tegmental area (VTA) of the anesthetized rat were examined. Intravenous administration of beta-PEA (1.0, 2.5, and 5.0 mg/kg) and microiontophoretic applications of beta-PEA caused inhibitory responses in DA neurons. Systemic administration and microiontophoretic applications of beta-PEA induced dose- or current-dependent responses. The systemic beta-PEA-induced inhibitory responses were reversed by pretreatment with the DA D(2) receptor antagonists haloperidol (0.5 mg/kg i.p.) and sulpiride (10 mg/kg i.p). Pretreatment with reserpine (5 mg/kg i.p. 24 h earlier) did not completely block the systemic administration of beta-PEA (2.5 mg/kg) inhibition. A microdialysis study of freely moving rats demonstrated that the extracellular DA level increased significantly in response to local application of beta-PEA (100 muM) in the VTA via a microdialysis probe, and local application of beta-PEA-stimulated somatodendritic DA release in the VTA. The beta-PEA-induced release of DA was calcium ion-independent and was enhanced by pretreatment with pertussis toxin. These findings indicate that beta-phenylethylamine inhibits DA neuron activity via DA D(2) autoreceptors in the rat VTA and that this inhibitory effect is mediated by the somatodendritic DA release.

Anti-ganglioside antibodies were investigated in plasma exchange solutions (PEs) from two patients with acute and chronic inflammatory demyelinating neuropathies (AIDP and CIDP). Both cases show markedly elevated antibody titers against the lacto-series gangliosides, GM3, GD3, and GT3. In the CIDP patient, the IgG antibody titer to GD3 was remarkably elevated (titer, 1:10,000), indicating maximal avidity to the tetrasaccharide epitope (-NeuAcalpha2-8NeuAcalpha2-3Galbeta1-4Glc-). There were also activities toward GM4 and GM2 with the affinity higher to GM4 than to GM2, indicating that the antibody activity was not highly specific. In contrast, the antibody activities in the AIDP patient showed similar avidity to GM3, GD3, and GT3. These two patients are very rare cases that have not previously encountered in GBS. The effects on co-cultured cells of rat spinal cord and muscle differed according to which PE was used. PE from the AIDP patient produced an inhibitory effect (reduction to 26.8%) on the spontaneous muscle action potential of the neuromuscular junction (NMJ), but the PE from the CIDP patient did not. Thus, in AIDP, the common epitope of GM3, GD3, or GT3 may be shared with certain antigens localized in the peripheral nervous system (PNS) and may participate in a component of conduction-related molecules in the NMJ. High titers of anti-GD3 antibody and the distortion of antibody recognition found in CIDP seem to have no immediate effect on electrophysiologic function in the PNS.

We investigated the epitopes and functional role of IgG anti-GalNAc-GD1a antibodies appearing in serum from a patient with Guillain-Barre syndrome (GBS) and IgG anti-GalNAc-GD1a antibody that was produced by immunization of a rabbit with GalNAc-GD1a. Both sera blocked neuromuscular transmission in muscle–spinal cord co-culture cells. The acetylcholine-induced potential did not reduce by adding sera, suggesting that the blockade is presynaptic. The effect was complement-independent and purified IgG from serum of the patient or the rabbit had the same effects. The epitope with both anti-GalNAc-GD1a antibodies was observed in the soma of large neurons in the anterior horns of the adult rat spinal cord and their motor axons of rat ventral roots. Both anti-GalNAc GD1a antibodies reacted strongly with the motor nerve terminals in rats. The anti-GalNAc-GD1a antibodies may block neuromuscular transmission by attacking on presynaptic motor axon, probably affecting the ion channels in the presynaptic motor axon.

Cardanol (ginkgol) extracted from Ginkgo biloba leaves and cashew nutshell liquid enhances the growth of NSC-34 immortalized motor neuron-like cells and, when chronically administered to young rats, improves working memory-related learning ability as assessed by eight-arm radial maze tasks. These findings suggest that cardanol is one of the components in Ginkgo biloba leaves that improves cognitive learning ability.

We examined the roles of prostaglandin receptors regulating tumor necrosis factor-alpha (TNFα) and IL-10 production by zymosan-stimulated murine peritoneal macrophages. During the stimulation with zymosan for 5 h, the presence of PGE2 or carbacyclin suppressed the TNFα production, whereas it increased that of IL-10; and indomethacin caused the reverse effects, suggesting that endogenous prostaglandins may have regulatory effects on the cytokine production. Among the EP subtype-selective synthetic agonists used, EP2 and EP4 agonists caused down-regulation of the TNFα production, but up-regulation of the IL-10 production; whereas EP1 and EP3 agonists showed no effect. Macrophages harvested from IP, EP2 or EP4 receptor-deficient mice lacked the up- and down-regulatory effects on the cytokine production by each corresponding agonist. These effects of prostaglandins agreed well with the mRNA expression of TNFα and IL-10 when mRNA levels were examined by the RT-PCR method. Thus, the results suggest that endogenous PGI2 and PGE2 may exert their anti-inflammatory influence on the cytokine production through IP, EP2, and EP4 receptors.

Psilocin (3-[2-(dimethylamino)ethyl]-1H-indol-4-ol) is a hallucinogenic component of the Mexican mushroom Psilocybe mexicana and a skeletal serotonin (5-HT) analogue. Psilocin is the active metabolite of psilocybin (3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate). In the present study, we examined the effects of systemically administered psilocin on extracellular dopamine and 5-HT concentrations in the ventral tegmental area (VTA), nucleus accumbens, and medial prefrontal cortex of the dopaminergic pathway in awake rats using in vivo microdialysis. Intraperitoneal administration of psilocin (5, 10 mg/kg) significantly increased extracellular dopamine levels in the nucleus accumbens. Psilocin did not affect the extracellular 5-HT level in the nucleus accumbens. Conversely, systemic administration of psilocin (10 mg/kg) significantly increased extracellular 5-HT levels in the medial prefrontal cortex of rats, but dopamine was decreased in this region. However, neither extracellular dopamine nor 5-HT levels in the VTA were altered by administration of psilocin. Behaviorally, psilocin significantly increased the number of head twitches. Thus, psilocin affects the dopaminergic system in the nucleus accumbens. In the serotonergic system, psilocin contribute to a crucial effect in the medial prefrontal cortex. The present data suggest that psilocin increased both the extracellular dopamine and 5-HT concentrations in the mesoaccumbens and/or mesocortical pathway.

We carried out behavioral, pathological, and biochemical studies in order to determine whether the stereo-structure of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) affects the onset of Parkinson’s disease-like symptoms, which are induced by 1,2,3,4-tetrahydroisoquinoline (TIQ) in mice. Pretreatment with (R)-1-MeTIQ or its racemate (RS)-1-MeTIQ prevented the TIQ-induced bradykinesia. Pretreatment with a combination of L-DOPA and carbidopa significantly prevented subsequent TIQ-induced bradykinesia. Furthermore, the pathological study demonstrated that either (R)-1-MeTIQ or its racemate protected against TIQ-induced loss of tyrosine hydroxylase-positive cells of the substantia nigra pars compacta. (R)-1-MeTIQ and its racemate also prevented the TIQ-induced reduction in the levels of dopamine and its metabolites in the striatum. Serotonin and its metabolite were not affected by repeated administration of (RS)-1-MeTIQ or its derivatives. On the other hand, (S)-1-MeTIQ induced moderate but significant bradykinesia, whereas (R)-1-MeTIQ did not induce this behavioral abnormality at all. In addition, (S)-enantiomer prevented the onset of TIQ-induced bradykinesia, though to a lesser extent than did either (R)-enantiomer or its racemate. However, (S)-enantiomer did not prevent the loss of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. We concluded that (R)-1-MeTIQ, and not (S)-enantiomer, plays a crucial role in protection against TIQ-induced parkinsonism, a fact which suggests that enantiomeric biochemical events such as 1-MeTIQ biosynthesis may participate in the pathogenesis of Parkinson’s disease.

We administered 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ; 80 mg/kg, i.p.), an endogenous neurotoxin known to cause bradykinesia, the Parkinson’s disease-like symptom, in order to obtain biochemical and pathological evidence of behavioral abnormalities. Immunohistochemical analysis demonstrated that 1-BnTIQ did not decrease the number of tyrsosine hydroxylase-positive cells in the substantia nigra. Biochemical analysis demonstrated significantly increased striatal dopamine (DA) content, while DA metabolites in the striatum remained at control levels. We concluded that the 1-BnTIQ-induced bradykinesia has a different mechanism of action than that underlying the MPTP-induced depletion of striatal DA neurons.

We previously reported that ciliary neurotrophic factor (CNTF) increased the serum-free cell survival of immortalized motor neuron-like cells (NSC-34), and addition of the exogenous ganglioside GalNAcβ4(Neu5Acα3)Galβ4GlcCer (GM2) facilitated cell survival together with CNTF. Moreover β 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity increased in NSC-34 cells cultured with CNTF. We now have examined whether CNTF-induced cell survival is associated with the collaboration between GM2 and the CNTF receptor (CNTF-R). Despite the presence of CNTF (50 ng/ml), anti-CNTF-R antibody caused cell death and prevented the up-regulation of GM2 synthase expression. The addition of GM2 (1 to 20 μM) abrogated the anti-CNTF-R antibody effect which shortened cell survival and blocked GM2 synthase activation. Use of [125I]CNTF showed the specificity of CNTF binding in NSC-34 cells in situ. GM2 produced a 5-fold increase in the CNTF binding affinity per cell but did not change the binding site number. The study by metabolic labeling with [1−14C]N-acetyl-D-galactosamine ([14C]GalNAc) showed that biosynthesized GM2 was involved in the immunoprecipitation of CNTF-R. These findings indicate that up-regulated GM2 synthesis induces functional conversion of CNTF-R to the activated state, in which it has affinity for CNTF. We conclude that GM2 is a bio-regulating molecule of CNTF-R in motor neurons.

Defensins released by neutrophils are able to kill a broad spectrum of microbes. They also induce leukocyte migration in vitro and elicit inflammatory leukocyte responses at s.c. injection sites in mice. In vitro experiments showed that human defensins enhanced concanavalin A-stimulated murine spleen cell proliferation and IFN-gamma production. This led us to examine the effects of human defensins on specific immune responses in vivo. BALB/c mice were immunized with 50 microg of keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide and administered with defensins in aqueous solution. Intraperitoneal administration of defensins significantly increased the production of KLH-specific IgG1, IgG2a and IgG2b antibodies 14 days after immunization. In vitro splenic KLH-specific proliferative responses were higher in mice treated with KLH and defensins than in those treated with KLH alone. Increased IFN-gamma and, to a lesser extent, IL-4 production were also detected in the supernatants of ex vivoKLH-activated spleen cells from mice treated with defensins. Finally, defensins significantly enhanced the antibody response to a syngeneic tumor antigen, lymphoma Ig idiotype and also augmented resistance to tumor challenge. These results indicate that defensins act as potent immune adjuvants by inducing the production of lymphokines, which promote T cell-dependent cellular immunity and antigen-specific Ig production. Thus, defensins appear to function as neutrophil-derived signals that promote adaptive immune responses.

Levels of inflammatory cytokines tumour necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and cytokine-induced neutrophil chemoattractant (CINC), which is a member of the alpha-chemokine family in rats, were measured in the pleural exudates during zymosan-induced pleurisy to examine the relationship between the local production of cytokines and the inflammatory reaction. All four cytokine levels in the pleural exudate began to increase after 1-2 h, preceding the influx of neutrophils, and peaked after 4-5 h. Thereafter, these cytokine levels declined after 24 h, whereas the exudate volume still continued to increase and leukocyte number reached a plateau. Concomitant injection of actinomycin D (10 microg) with zymosan markedly suppressed the neutrophil infiltration, parallel with CINC production in the pleural exudate at 4 h. A transient elevation of IL-6 level, peaking at 5 h, and subsequent rise in the level of an acute-phase protein, T-kininogen, were also observed in the plasma. When recombinant human TNF-alpha (rhTNF-alpha) (20 000 U) was intrapleurally injected a rapid increase in pleural CINC level, followed by neutrophil infiltration, and a sharp rise in IL-6 level in the plasma, followed by an increase in T-kininogen, were demonstrated. These results suggest that CINC produced in the pleural exudate may participate in neutrophil infiltration, that IL-6 induced in the plasma stimulates T-kininogen production, and that endogenous TNF may be partly involved in the induction of CINC and IL-6 in this zymosan inflammation.

The neutrophil chemotactic cytokine, IL-8, has been reported to also chemoattract T lymphocytes in vitro and in vivo. Previously we showed that freshly isolated T cells migrated in response to IL-8, but incubation of T cells at 37 degrees C resulted in progressively decreased levels of IL-8 binding sites on T cells in association with reduced chemotactic responses. However, this reduced binding and migration of cultured T cells in response to IL-8 can be prevented by the presence of mononuclear cells in the culture. In order to define the factor(s) responsible for the restoration of T cell binding and migration in response to IL-8, we examined the effects of various cytokines. Addition of IFN-gamma in cultured T cells maintained both the CXC chemokine receptor CXCR1 and CXCR2 binding sites for IL-8 on these cells to the level comparable to that expressed on freshly purified T cells accompanied by an almost complete restoration of their chemotactic response to IL-8. The results suggest that Th1 cytokine, IFN-gamma, produced by mononuclear cells stimulated by proinflammatory signals may play an important role in regulating IL-8 receptor expression on T cells and in sustaining the function of these cells in response to IL-8.

To assess roles of cytokines in the inflammatory process, we examined local production of proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1, a member of the CXC chemokine in rats), in the exudates of carrageenin induced pleurisy of rats. All four cytokine levels began to increase at 1-2h, and their production preceded the influx of neutrophils, and peaked at 3-5h, whereas the exudate volume and leukocyte number still continued to increase. When recombinant human (rh) TNFα or rhlL-1α was injected into the pleural cavity, the pleural CINC-1 level rapidly increased, preceding the neutrophil infiltration. A sharp rise in IL-6 level in the plasma, followed by an increase in T-kininogen, an acute-phase protein, was also observed. Intrapleural injection of rhIL-8 or CINC-1 caused neutrophil infiltration in a protein synthesis independent manner. Treatment with indomethacin, an anti-inflammatory drug, increased TNF and IL-1 levels but suppressed IL-6 level in the exudate of rats with pleurisy, whereas dexamethasone suppressed all these cytokine levels. Prostaglandins (PGs) and dibutyryl cAMP reduced the production of TNF and IL-1, while they increased that of IL-6 and CINC-1 from rat pleural resident cells. The above results clearly indicate that proinflammatory cytokines, TNF, IL-1, IL-6 and CINC-1 were produced locally. Moreover, these results suggest that these proinflammatory cytokine production could be regulated by inflammatory stimuli and also by other mediators such as PGE2 and PGI2 concomitantly produced in the inflammatory sites.

The C-C chemokine RANTES, a T lymphocyte chemoattractant, is considered an important mediator of inflammation, allergy, and host defense against HIV-1 infection. In this study, we investigated the modulation of binding of RANTES to T lymphocytes. Human peripheral blood CD3+ T cells, when freshly isolated from buffy-coat blood, expressed a considerable number of high-affinity binding sites for RANTES. These cells also showed significant chemotactic migration in response to RANTES in vitro. After 6-15 h incubation at 37 degrees C, the binding of RANTES, but not of macrophage inflammatory protein-1 alpha (MIP-1 alpha) or of monocyte chemotactic protein-3 (MCP-3), consistently increased. Scatchard analyses indicated that the number of binding sites for RANTES increased about threefold by 15 h without any change in the affinity. The increase in RANTES binding was no longer detected by 24 h. This increase in the specific binding was mainly attributable to CD4+ T cells and was not associated with increased chemotactic activity of these cells in response to RANTES. Incubation with anti-CD3 antibody for 15 h markedly reduced the binding capability of T cells for RANTES and was associated with decreased chemotactic activity. On the other hand, when T cells were incubated with interleukin-2 (IL-2) for 1 week, the specific binding for all three C-C chemokines, RANTES, MIP-1 alpha, and MCP-3 was markedly increased in comparison to cells cultured in the absence of IL-2. These results suggest that the expression of binding sites on T cells for RANTES is differentially modulated, indicating the existence of novel receptors for RANTES that do not bind MIP-1 alpha.

1. To assess in vivo chemotactic activity of tumour necrosis factor (TNF), interleukin-1 (IL-1), IL-8, and cytokine-induced neutrophil chemoattractant (CINC), we injected these cytokines into the pleural cavity of rats. 2. CINC (0.1-1 microgram) and recombinant human IL-8 (rhIL-8, 0.2-5 micrograms) caused neutrophil infiltration into the rat pleural cavity in a dose-dependent fashion, peaking at 3 h. The number of leukocytes in the peripheral blood did not change significantly. 3. RhTNF alpha and rhIL-1 alpha also induced neutrophil accumulation. The dose response curves of rhTNF alpha (0.67 ng-6.7 micrograms) and rhIL-1 alpha (0.45 ng-4.5 micrograms) at 3 h were bell shaped. On the other hand, unlike CINC and rhIL-8, rhTNF alpha and rhIL-1 alpha caused transient marked leukopenia at 3 h in a simple dose-dependent fashion. 4. Concomitant injection of actinomycin D dose-dependently and completely at 10 micrograms inhibited neutrophil infiltration induced by rhTNF alpha (0.67 microgram) and rhIL-1 alpha (0.45 microgram) at 3 h. However, that induced by CINC or rhIL-8 was not affected by actinomycin D. 5. Peaking at 1 h, CINC production in the pleural cavity was found after intrapleural injection of rhTNF alpha (0.67 microgram) or rhIL-1 alpha (0.45 microgram), but not after that of rhIL-8 (5 micrograms). The CINC production induced by rhTNF alpha or rhIL-1 alpha and the neutrophil infiltration was suppressed by concomitant injection of actinomycin D (1 and 10 micrograms). 6. These results indicate that CINC and IL-8 themselves are direct chemoattractants for neutrophils, whereas TNF and IL-1 induce neutrophil infiltration indirectly via newly synthesized mRNA for chemotactic protein including CINC, which may be involved in neutrophil emigration at local inflammatory sites in rats.

We investigated the localization of a ganglioside, N-acetylgalactosaminyl GD1a (GalNAc-GD1a), in peripheral nerves with an IgG anti-GalNAc-GD1a antibody, which was produced in rabbits immunized with GalNAc-GD1a.Teased fibers from ventral and dorsal roots and hemidiaphragm sections of rats were assessed using fluorescent double- and triple-labeling methods.The nodal and paranodal regions of teased fibers from ventral roots were immunostained with IgG anti-GalNAc-GD1a antibodies. After collagenase treatment, no staining was seen with IgG anti-GalNAc-GD1a or anti-NF200 antibodies, whereas α-bungarotoxin selectively stained nerve terminals. In cross-sectional and longitudinal sections of rat hemidiaphragm, IgG anti-GalNAc-GD1a antibodies overlapped with α-BuTx and anti-NF200 antibodies, indicating that GalNAc-GD1a is localized to the nerve terminal. IgG anti-GalNAc-GD1a antibody staining also overlapped with that of AChR clusters and syntaxin-positive presynaptic nerve terminals.GalNAc-GD1 is localized in both pre- and postsynaptic nerve terminals of neuromuscular junctions.

Peripheral neuropathy is a common adverse effect of paclitaxel treatment. To analyze the contribution of transient receptor potential vanilloid 1 (TRPV1) in the development of paclitaxel-induced thermal hyperalgesia, TRPV1 expression in the rat dorsal root ganglion (DRG) was analyzed after paclitaxel treatment. Behavioral assessment using the tail-flick test showed that intraperitoneal administration of 2 and 4 mg/kg paclitaxel induced thermal hyperalgesia after days 7, 14, and 21. Paclitaxel-induced thermal hyperalgesia after day 14 was significantly inhibited by the TRP antagonist ruthenium red (3 mg/kg, s.c.) and the TRPV1 antagonist capsazepine (30 mg/kg, s.c.). Paclitaxel (2 and 4 mg/kg) treatment increased the expression of TRPV1 mRNA and protein in DRG neurons. Immunohistochemistry showed that paclitaxel (4 mg/kg) treatment increased TRPV1 protein expression in small and medium DRG neurons 14 days after treatment. Antibody double labeling revealed that isolectin B4-positive small DRG neurons co-expressed TRPV1. TRPV1 immunostaining was up-regulated in paw skin day 14 after paclitaxel treatment. Moreover, in situ hybridization histochemistry revealed that most of the TRPV1 mRNA-labeled neurons in the DRG were small or medium in size. These results suggest that paclitaxel treatment increases TRPV1 expression in DRG neurons and may contribute to functional peripheral neuropathic pain.

The effect of anti-inflammatory drugs on cytokine production at local inflammatory sites was investigated in a carrageenin-induced rat pleurisy model. Exudate volume and leukocyte number in the pleural cavity at 3 h after the carrageenin injection were significantly reduced by the pretreatment with indomethacin or dexamethasone. Both drugs also reduced the prostaglandin E2 level in the exudate. However, production of tumor necrosis factor (TNF) and interleukin-1 in the pleural exudate was significantly enhanced by the pretreatment with indomethacin, whereas the interleukin-6 level was reduced. Pretreatment with dexamethasone markedly suppressed all these cytokine levels. When resident pleural cells were stimulated with lipopolysaccharide in vitro, the presence of exogenous prostaglandin E2 reduced the production of TNF and interleukin-1, while it increased that of interleukin-6 in a dose-dependent manner. These results suggest that prostaglandin E2 could be a regulating factor involved in cytokine production at the inflammatory site. Dexamethasone may express a direct suppressive action on cytokine production rather than an indirect regulatory action through prostaglandin E2 level.

In order to assess a role of inflammatory cytokines for infiltration of leukocytes, we injected 4 cytokines into the pleural cavity of rats. Intrapleural injection of recombinant human interleukin-8 (rhIL-8) and cytokine-induced neutrophil chemoattractant (CINC) caused neutrophil infiltration in the pleural cavity dose dependently, and both, showed a peak at 3hr. Recombinant human tumor necrosis factor (rhTNF) and rhIL-1α also caused neutrophil infiltration in the cavity but lower extent than those by rhIL-8 and CINC. Neutrophil infiltration by rhTNF and rhIL-1α was suppressed significantly by the pretreatment with dexamethasone at a dose of 0.5 mg/kg.These results suggest that IL-8 and CINC may act directly in the recruitment of neutrophils at local inflammatory sites, whereas TNF and IL-1 may express their activities for neutrophil infiltration through the induction of CINC in rats.

J. Neurochem. (2009) 112, 913–923. Abstract The Kv1.4 potassium channel is reported to exhibit higher cell surface expression than the Kv1.1 potassium channel when expressed as a homomer in cell lines. Kv1.4 also shows highly efficient trans-Golgi glycosylation whereas Kv1.1 is not glycosylated. The surface expression and glycosylation of Kv1.2 is intermediate between those of Kv1.1 and Kv1.4. Amino acid determinants controlling the surface expression of Kv1 channels were localized to the highly conserved pore region and both positive and negative determinants of Kv1.1 and Kv1.4 trafficking have been reported. In this study, we analyzed the effect of substituting amino acids in the pore region of Kv1.2 with the corresponding amino acid present in Kv1.1 or Kv1.4 on glycosylation and trafficking of Kv1.2. Mutations in the outer pore region of Kv1.2 of Arg354 to Pro (corresponding to Kv1.4) and to Ala (corresponding to Kv1.1) enhanced and reduced, respectively, cell surface expression of Kv1.2. Mutations in a different outer pore region of Val381 to Lys (Kv1.4) and Tyr (Kv1.1) both reduced the cell surface expression. In contrast, mutation in the deep pore region of Ser371 to Thr (Kv1.4) markedly enhanced cell surface expression. These results suggest that the cell surface expression of Kv1.2 is regulated by specific amino acids in the pore region in a similar manner to Kv1.1 and Kv1.4, and that the cell surface expression of Kv1.2, a channel intermediate between Kv1.1 and Kv1.4, can be attributed to these specific residues.

Miller-Fisher syndrome (MFS), which is known to be associated with anti-GQ1b antibodies and to cause ataxia, is a variant of an acute inflammatory neuropathy. However, the pathogenic role of anti-GQ1b antibodies remains unclear. In this study, we investigated the effects of mouse IgM anti-GQ1b monoclonal antibody (IgM anti-GQ1b mAb) on the spontaneous muscle action potential of a rat spinal cord-muscle co-culture system and on the voltage-dependent calcium channel (VDCC) current in cerebellar granule cells and Purkinje cells using the whole-cell patch clamp technique. The frequency of spontaneous muscle action potential of the innervated muscle cells was transiently increased by IgM anti-GQ1b mAb and then was blocked completely, which was the same finding as reported previously. Moreover, the cerebellar granule cell VDCC current was decreased by 30.76+/-7.60% by 5 microg/mL IgM anti-GQ1b mAb, whereas IgM anti-GQ1b mAb did not affect the VDCC current in cerebellar Purkinje cells. In immunocytochemistry, IgM anti-GQ1b mAb stained the whole cell surface of cerebellar granule cells, but not that of Purkinje cells. Therefore, the clinical symptoms of Miller-Fisher syndrome, such as cerebellar-like ataxia, may be explained by the inhibitory effects of anti-GQ1b antibodies on VDCC current in cerebellar granule cells.

We performed a combined neurochemical and behavioral study to determine the effects of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) on the extracellular dopamine concentrations in the striatum. Single dose administration of 1-BnTIQ (20, 40, and 80 mg/kg i.p.) increased striatal dopamine extracellular levels in a dose-dependent manner when an in vivo microdialysis technique was used to assess dopamine levels in the striatum of rats. Enhancement of striatal dopamine levels by systemic administration of a single dose of 1-BnTIQ was suppressed by perfusion of tetrodotoxin and a calcium ion-free solution into the striatum. This 1-BnTIQ-induced increase in extracellular dopamine concentration was also inhibited by pre-treatment with a dopamine uptake inhibitor, GBR12909 (1-(2-[bis(4-Fluorophenyl)-4-(3-phenylpropyl)piperazine dihydrochloride). Local application of 1-BnTIQ into the striatum via a dialysis probe failed to enhance the extracellular concentration of dopamine. However, microinjection of 1-BnTIQ into the substantia nigra pars compacta increased the extracellular dopamine levels in the striatum. Locomotor activity was increased by systemic administration of a single dose of 1-BnTIQ in a dose-dependent manner. This 1-BnTIQ-induced locomotor activity was attenuated by pre-treatment with SCH23390 (R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlodride) and raclopride, D(1) and D(2) dopaminergic receptor antagonists, respectively. Moreover, 1-BnTIQ induced ipsilateral rotational behavior in 6-hydroxydopamine-lesioned rats. These results suggest that systemic administration of a single dose of 1-BnTIQ increases striatal extracellular dopamine concentration through activation of dopaminergic nigra striatal neurons via the dopamine transporter.

Prolonged exposure to nitrous oxide (N 2 O) results in development of acute tolerance to its antinociceptive effect. Cross-tolerance to N 2 O-induced antinociception is also observed in morphine-tolerant animals. Despite increasing evidence of tolerance development to N 2 O-induced antinociception, the details of the mechanisms that underlie this tolerance remain unknown. The present study was conducted to investigate the involvement of brain protein kinase C (PKC) isoform in these two types of tolerance to N 2 O-induced antinociception in mice. Prolonged exposure (41 min in total, including 30 min pre-exposure and 11 min of antinociceptive testing) to 70% N 2 O produced a reduction in N 2 O-induced antinociception, indicating development of acute tolerance. The prolonged exposure to 70% N 2 O caused an activation of PKCγ isoform in the brain, but not the PKCe isoform. Pretreatment with a PKCγ-antisense oligonucleotide but not the corresponding mismatch oligonucleotide (i.c.v.) prevented the development of acute tolerance to N 2 O-induced antinociception. Chronic morphine treatment (10 mg/kg, s.c., b.i.d. for 5 days) resulted in development of tolerance to morphine-induced antinociception and cross-tolerance to N 2 O-induced antinociception. The development of tolerance to morphine and cross-tolerance to N 2 O were both inhibited by pretreatment with PKC inhibitor, chelerythrine (1 nmol, i.c.v.). Morphine-tolerant mice showed an activation of PKC within the brain, which was suppressed by pretreatment with chelerythrine (1 nmol, i.c.v.). Thus, activation of brain PKC, in particular, the PKCγ isoform, appears to play an important role in the development of both acute tolerance and cross-tolerance to N 2 O-induced antinociception in mice.

Background Peripheral cold neuropathic pain is a serious side effect of oxaliplatin treatment. However, the mechanism of oxaliplatin-induced cold hyperalgesia is unknown. In the present study, we investigated the effects of oxaliplatin on transient receptor potential ankyrin 1 (TRPA1) in dorsal root ganglion (DRG) neurons of rats. Results Behavioral assessment using the acetone spray test showed that 3 and 6 mg/kg oxaliplatin (i.p.) induced acute cold hypersensitivity after 1, 2, 4, and 7 days. Real-time PCR showed that oxaliplatin (6 mg/kg) significantly increased TRPA1 mRNA expression in DRGs at days 1, 2, and 4. Western blotting revealed that oxaliplatin significantly increased TRPA1 protein expression in DRGs at days 2, 4, and 7. Moreover, in situ hybridization histochemistry revealed that most TRPA1 mRNA-labeled neurons in the DRGs were small in size. Oxaliplatin significantly increased co-localization of TRPA1 expression and isolectin B4 binding in DRG neurons. Oxaliplatin induced a significant increase in the percent of TRPA1 mRNA-positive small neurons in DRGs at days 1, 2, and 4. In addition, we found that intrathecal administration of TRPA1 antisense, but not TRPA1 mismatched oligodeoxynucleotides, knocked down TRPA1 expression and decreased oxaliplatin-induced cold hyperalgesia. Double labeling showed that p-p38 mitogen-activated protein kinase (MAPK) was co-expressed in TRPA1 mRNA-labeled neurons at day 2 after oxaliplatin administration. Intrathecal administration of the p38 MAPK inhibitor, SB203580, significantly decreased oxaliplatin-induced acute cold hypersensitivity. Conclusions Together, these results demonstrate that TRPA1 expression via activation of p38 MAPK in DRG neurons, at least in part, contributes to the development of oxaliplatin-induced acute cold hyperalgesia.

We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

Using an in vivo intra-striatal microdialysis technique, we examined the effects of systemically administered beta-phenylethylamine (beta-PEA), a psychomotor stimulating trace amine, on striatal acetylcholine release in freely moving rats. Infusion of N-methyl-D-aspartic acid (NMDA; 10(-5) M) significantly increased acetylcholine release. In addition, locally applied amino-3-hydroxy-5-methylisozasole-4-propionic acid (AMPA; 10(-5) M) significantly increased acetylcholine release in the striatum. Intra-striatal application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M), an AMPA-type glutamatergic receptor antagonist, had little effect on acetylcholine release, while application of MK-801 (10(-5) M, 10(-6) M), an NMDA-type glutamatergic receptor antagonist, significantly reduced acetylcholine release. Acetylcholine within striatal perfusate was significantly increased by intraperitoneal administration of beta-PEA in a dose-dependent manner. This increase in acetylcholine release was completely blocked by application of CNQX (10(-5) M) through the microdialysis probe into the striatum. However, increased acetylcholine response to systemic beta-PEA was unaltered by addition of MK-801 to the perfusion medium. These results suggest a regulatory function of beta-PEA, mediated by AMPA-type glutamatergic receptors, on the release of acetylcholine in the rat striatum.

Parkinson's disease involves the progressive degeneration of dopaminergic neurons in the substantia nigra. However, the etiology of the disease remains to be elucidated. Endogenous amines, such as 1,2,3,4-tetrahydroisoquinoline (TIQ) derivatives present in the mammalian brain, are known to participate in the pathogenesis of Parkinson's disease. These endogenous neurotoxins have been extensively studied because of their structural resemblance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an agent widely used for generating animal models of Parkinson's disease-like symptoms. Investigations of the synthesis and pharmacological properties of TIQ derivatives are expected to contribute to the development of new therapeutic agents for treating Parkinson's disease. In the present study, we describe more efficient synthesis methods for TIQ derivatives via Pummerer-type cyclization of the substrate N-acyl sulfoxide. Furthermore, the modified Pummerer reaction provided a convenient and efficient method for synthesizing various TIQs. TIQ and its derivative, 1-benzyl-TIQ, can induce parkinsonism in primates and rodents. On the other hand, one TIQ derivative, 1-methyl-TIQ, has been shown to prevent MPTP, TIQ, and 1-benzyl-TIQ induced behavioral abnormalities. Therefore, TIQ derivatives are considered to play an important role in both the onset and prevention of Parkinson's disease. In this article, we focus on the synthesis and pharmacological aspects of 1,2,3,4-tetrahydroisoquinoline derivatives in Parkinson's disease.

This study investigated the effects of the calcium channel blockers nicardipine, calcicludine, omega-conotoxin GVIA, omega-agatoxin IVA, SNX-482, and NiCl on spontaneous muscle action potential of a rat spinal cord-muscle co-culture system. Spontaneous muscle action potential of the innervated muscle cells was blocked by d-tubocurarine (1 microM), but was not significantly affected by the L-type channel blocker nicardipine (100 nM). The neuronal L-type calcium channel blocker, calcicludine (50 and 100 nM), also had no effect on the frequency of spontaneous muscle action potential. However, nicardipine (100 nM) and calcicludine (100 nM) significantly increased the amplitude of muscle action potential. Application of the N-type calcium channel blocker, omega-conotoxin GVIA (50 and 100 nM), and the P/Q-type calcium channel blocker, omega-agatoxin IVA (10, 30, 50, and 100 nM), blocked the frequency and amplitude of spontaneous muscle action potential of the spinal cord-muscle co-cultured cells. In contrast, spontaneous muscle action potential was not affected by the R-type calcium channel blockers SNX-482 (100 nM) or NiCl (500 nM). These results indicate that blockers of N- and P/Q-type voltage-dependent calcium channels inhibit transmitter release at neuromuscular junctions in the rat spinal cord-muscle co-culture system.

We produced anti-asialo-GM1 (GA1) polyclonal antibodies by sensitizing New Zealand rabbits with GA1 and investigated the epitopes and pathogenic role of anti-GA1 antibodies that appeared in serum. The serum blocked neuromuscular transmission, but not acetylcholine (ACh)-induced potentials, in muscle-spinal cord cocultured cells. The effect was complement independent. The antibodies inhibited voltage-gated Ca2+ channel (VGCC). The epitopes recognized by the antibodies were located in the outer membrane of Schwann cells and motor axons of Wistar rat ventral roots and on motor axons extended from spinal cord to muscle cells in muscle-spinal cocultured cells. The ACh-induced potential was not reduced by the addition of sera, suggesting the blockade is presynaptic. Thus, anti-GA1 antibodies may block neuromuscular transmission by suppressing VGCC on axonal terminals of motor nerves.

The present study investigated the role of muscarinic receptor subtypes in the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha of the rat rostral ventrolateral medulla in morphine-induced antinociception. The antinociceptive effects of morphine were evoked by systemic administration or microinjection into the nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha. Administration of morphine produced antinociception for hot plate and tail immersion responses to noxious heat stimuli. These effects were antagonized by prior exposure to naloxone and inhibited by mecamylamine pretreatment. Morphine-induced antinociception was significantly inhibited by atropine in a dose-dependent manner. Muscarinic toxin-1 and pirenzepine inhibited morphine-induced antinociception for both the hot plate and tail immersion tests. At a dose of 5 nmol/site, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) also inhibited morphine-induced antinociception, although low doses of this drug did not significantly affect hot plate test response latency when morphine was systemically administered. These results suggest that the antinociceptive effects induced by morphine in part involve the muscarinic M(1) and M(3) receptors of the rat nucleus reticularis gigantocellularis/nucleus reticularis gigantocellularis alpha.

We examined the effects of cutaneous noxious heat as well as the intrathecal administration of morphine on the oxidation current of peaks 1 and 2 in the dorsal raphe nucleus (DRN) of anesthetized rats. Differential normal pulse voltammetry with carbon fiber electrodes identified distinct oxidation currents at +120 mV (peak 1: catechol signals) and +280 mV (peak 2: 5-hydroxyindole signals). The catechol signal was significantly increased by 22.9 +/- 4.2% after applying cutaneous noxious heat at 52 degrees C. The 5-hydroxyindole signal was decreased by 39.8 +/- 4.3 and by 25.2 +/- 4.7% after stimulation with cutaneous noxious heat at 52 and 45 degrees C, respectively. A low dose of morphine (2.5 microg) potentiated the increase in the catechol signal and the decrease in the 5-hydroxyindole signal induced by noxious heat, and a high dose (10.0 microg) attenuated both. The effects of morphine at low (2.5 microg) and high doses (10.0 microg) were antagonized by naloxone (0.5 mg/kg, i.p.). These results indicate that noxious heat stimulation increased the catechol signal and decreased the 5-hydroxyindole signal in the DRN. The intrathecal administration of morphine affects the noxious stimulation-induced activity of noradrenergic and serotonergic neurotransmission in the DRN.

Verotoxin receptor is a globotriaosylceramide (Gb3) present on vascular endothelial cells, in which the toxin causes hemolytic uremic syndrome. We established a sensitive method of immunohistochemical staining in serial sections using monoclonal antibodies (mAbs) against verotoxin and glycolipids that includes Gb3 and galactosylGb3 (GalGb3). With it, we showed co-localization of the verotoxin receptor and Gb3 in dorsal root ganglia (DRG) of humans, rabbits, rats and mice. We also used a very simple method to identify the presence of lipofuscin-like autofluorescence which complicates fluorescence microscopy observation of aged human nervous tissues.

To know which receptors of prostaglandins are involved in the regulation of TNFalpha and interleukin 10 (IL-10) production, we examined the production of these cytokines in murine peritoneal macrophages stimulated with zymosan. The presence of PGE(2) or the PGI(2) analog carbacyclin in the medium reduced the TNFalpha production to one-half, whereas IL-10 production increased several fold; and indomethacin caused the reverse effects, suggesting that endogenous prostaglandins may have a regulatory effect on the cytokine production. Among prostaglandin E (EP) receptor-selective synthetic agonists, EP2 and EP4 agonists caused down-regulation of the zymosan-induced TNFalpha production, but up-regulation on the IL-10 production; while EP1 and EP3 agonists showed no effect. Macrophages harvested from prostaglandin I (IP) receptor-deficient mice showed the up- and down-regulatory effects on the cytokine production by the EP2 and EP4 agonists or PGE(2), but no effect was obtained by carbacyclin. On the contrary, macrophages from EP2-deficient mice showed the effect by PGE(2), carbacyclin, and the EP4 agonist, but not by the EP2 agonist; and the cells from EP4-deficient mice showed the effect by PGE(2), carbacyclin, and EP2 agonist, but not by the EP4 agonist. These functional effects of prostaglandins well accorded with the mRNA expression of TNFalpha and IL-10 when such expression was examined by the RT-PCR method. The peritoneal macrophages from normal mice expressed IP, EP2, and EP4 receptors, but not EP1 and EP3, when examined by RT-PCR. Thus the results suggest that PGI(2) and PGE(2) generated simultaneously with cytokines by macrophages treated with zymosan may influence the cytokine production through IP, EP2, and EP4 receptors.

We use immunohistochemistry to show the existence of verotoxin receptor in small sensory neurons in DRG of human, rabbit, rat and mouse. In capillary in nervous system, the verotoxin receptor exists in human and rabbit, but the receptor could not be demonstrated in rat and mouse, by this method. The receptors in sensory neuron of rat and in capillary in rabbit brain are determined as galactosylglobotriaosylceramide (GalGb3) and globotriaosylceramide (Gb3,), respectively. Although verotoxin was reported to bind to glycolipid receptors that possess the terminal disaccharide Galα1-4Galβ (galactobiose), the binding to toxin to galabiosylceramide was half of that of GalGb3 which has galactobiose internally.

Prostaglandins E1, prostaglandin E2, 3-oxa-methano-prostaglandin I1 (SM-10906), a stable prostaglandin I2 analog, and dibutyryl cyclic AMP suppressed the production of tumor necrosis factor and interleukin-1 in lipopolysaccharide-stimulated rat pleural resident monocytic cells, whereas they enhanced the production of interleukin-6 and cytokine-induced neutrophil chemoattractant (CINC), a rat interleukin-8-like chemokine, in these cells. SM-10906 also inhibited the in vivo production of tumor necrosis factor and interleukin-1 in pleural exudates, when injected into the rat pleural cavity concomitantly with carrageenin. The cyclic AMP (cAMP) level in the lipopolysaccharide-stimulated resident cells was increased when the cells were incubated in the presence of prostaglandin E1, prostaglandin E2 or SM-10906. Prostaglandin I2 showed only slight effects. The addition of pentoxifylline, a phosphodiesterase inhibitor, to the incubation mixture increased the cAMP level and also enhanced the effect of prostaglandins, indicating that these regulating actions of prostaglandins may be exerted partly through a mechanism involving an increased intracellular cAMP level.

The Brown Norway Katholiek (B/N-Ka) strain rat is the only animal strain that demonstrates deficiency in plasma HMW- and LMW-kininogens with a low level of prekallikrein. We developed an RIA for rat HMW-kininogen, LMW-kininogen, and T-kininogen, and using them measured these proteins in B/N-Ka and normal strain (B/N-Ki) rats. Plasma level of immunoreactive as well as kinin-releasing HMW-kininogen and LMW-kininogen in B/N-Ka rats was either around 3% of their levels in the normal B/N-Ki rats. The cause of the plasma deficiency of kininogens in the B/N-Ka strain was examined by 35S-methionine uptake of primary cultures of hepatocytes from the B/N-Ki and B/N-Ka strains. The results indicated that the kininogens were synthesized in the B/N-Ka liver but not secreted into the medium. Northern blot analysis of poly A(+)RNA extracted from the livers of both strains demonstrated that the band corresponding to mRNA of HMW-kininogen was present in the mRNA from B/N-Ka liver as well as in that from the B/N-Ki one. The band was similar in size and intensity in both cases. This result confirmed the data that immunoreactive HMW-kininogen was found in the liver of B/N-Ka rats (12). Thus, the cause of plasma deficiency of HMW-kininogen in the mutant appears to be secretory defect in nature. The B/N-Ka rats showed less reactivity to the inflammatory stimulus, such as carrageenin or kaolin, but the strain expressed almost the same response as normal rats to phorbol ester (PMA) or zymosan for pleurisy induction. These results indicate that kinin may play an important role in exudation in carrageenin- and kaolin-induced edema but not in that induced by PMA or zymosan. The deficient rat strain could be useful for differentiation of the inflammatory model which shows involvement of the kinin system.