Role of peripheral sigma-1 receptors in ischaemic pain: Potential interactions with ASIC and P2X receptors.

BACKGROUND: The role of peripheral sigma-1 receptors (Sig-1Rs) in normal nociception and in pathologically induced pain conditions has not been thoroughly investigated. Since there is mounting evidence that Sig-1Rs modulate ischaemia-induced pathological conditions, we investigated the role of Sig-1Rs in ischaemia-induced mechanical allodynia (MA) and addressed their possible interaction with acid-sensing ion channels (ASICs) and P2X receptors at the ischaemic site. METHODS: We used a rodent model of hindlimb thrombus-induced ischaemic pain (TIIP) to investigate their role. Western blot was performed to observe changes in Sig-1R expression in peripheral nervous tissues. MA was measured after intraplantar (i.pl.) injections of antagonists for the Sig-1, ASIC and P2X receptors in TIIP rats or agonists of each receptor in naïve rats. RESULTS: Sig-1R expression significantly increased in skin, sciatic nerve and dorsal root ganglia at 3 days post-TIIP surgery. I.pl. injections of the Sig-1R antagonist, BD-1047 on post-operative days 0-3 significantly attenuated the development of MA during the induction phase, but had no effect on MA when given during the maintenance phase (days 3-6 post-surgery). BD-1047 synergistically increased amiloride (an ASICs blocker)- and TNP-ATP (a P2X antagonist)-induced analgesic effects in TIIP rats. In naïve rats, i.pl. injection of Sig-1R agonist PRE-084 alone did not produce MA; but it did induce MA when co-administered with either an acidic pH solution or a sub-effective dose of αßmeATP. CONCLUSION: Peripheral Sig-1Rs contribute to the induction of ischaemia-induced MA via facilitation of ASICs and P2X receptors. Thus, peripheral Sig-1Rs represent a novel therapeutic target for the treatment of ischaemic pain.

Anti-inflammatory effects of glutamine on LPS-stimulated human dental pulp cells correlate with activation of MKP-1 and attenuation of the MAPK and NF-κB pathways.

AIM: To evaluate the anti-inflammatory effects of glutamine and the underlying signal pathway mechanisms in lipopolysaccharide (LPS)-stimulated human dental pulp cells (HDPCs). METHODS: Human dental pulp cells were exposed to 10 µg mL(-1) LPS and various concentrations of glutamine for 24 h. The production of PGE2 and nitric oxide was determined by enzyme-linked immunosorbent assay (ELISA) and Griess reagent kit, respectively. Cytokines were examined by ELISA, reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. iNOS and COX protein expression as well as signal pathways were accessed by Western blot. The data were analysed by anova with Bonferroni's test (α = 0.05). RESULTS: Glutamine reduced LPS-induced iNOS and COX-2 protein expression as well as production of NO and PGE2 in a dose-dependent fashion. Additionally, glutamine suppressed the production and mRNA expression of inflammatory cytokines including interleukin-1ß (IL-1ß), TNF-α, and IL-8. Furthermore, glutamine attenuated phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK) and IκB-α, and nuclear translocation of NF-κB p65, but enhanced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in LPS-treated HDPCs. CONCLUSION: Glutamine exerted an anti-inflammatory effect via activation of MKP-1 and inhibition of the NF-κB and MAPK pathways in LPS-treated HDPCs.

σ1 receptors activate astrocytes via p38 MAPK phosphorylation leading to the development of mechanical allodynia in a mouse model of neuropathic pain.

BACKGROUND AND PURPOSE: Spinal astrocytes have emerged as important mechanistic contributors to the genesis of mechanical allodynia (MA) in neuropathic pain. We recently demonstrated that the spinal sigma non-opioid intracellular receptor 1 (σ1 receptor) modulates p38 MAPK phosphorylation (p-p38), which plays a critical role in the induction of MA in neuropathic rats. However, the histological and physiological relationships among σ1, p-p38 and astrocyte activation is unclear. EXPERIMENTAL APPROACH: We investigated: (i) the precise location of σ1 receptors and p-p38 in spinal dorsal horn; (ii) whether the inhibition of σ1 receptors or p38 modulates chronic constriction injury (CCI)-induced astrocyte activation; and (iii) whether this modulation of astrocyte activity is associated with MA development in CCI mice. KEY RESULTS: The expression of σ1 receptors was significantly increased in astrocytes on day 3 following CCI surgery. Sustained intrathecal treatment with the σ1 antagonist, BD-1047, attenuated CCI-induced increase in GFAP-immunoreactive astrocytes, and the treatment combined with fluorocitrate, an astrocyte metabolic inhibitor, synergistically reduced the development of MA, but not thermal hyperalgesia. The number of p-p38-ir astrocytes and neurons, but not microglia was significantly increased. Interestingly, intrathecal BD-1047 attenuated the expression of p-p38 selectively in astrocytes but not in neurons. Moreover, intrathecal treatment with a p38 inhibitor attenuated the GFAP expression, and this treatment combined with fluorocitrate synergistically blocked the induction of MA. CONCLUSIONS AND IMPLICATIONS: Spinal σ1 receptors are localized in astrocytes and blockade of σ1 receptors inhibits the pathological activation of astrocytes via modulation of p-p38, which ultimately prevents the development of MA in neuropathic mice.

Suppression of adrenal gland-derived epinephrine enhances the corticosterone-induced antinociceptive effect in the mouse formalin test.

BACKGROUND: There is both clinical and experimental evidence to support the application of corticosterone in the management of inflammation and pain. Corticosterone has been used to treat painful inflammatory diseases and can produce antinociceptive effects. Epinephrine is synthesized from norepinephrine by the enzyme phenylethanolamine N-methyltransferase (PNMT) and works as an endogenous adrenoceptor ligand secreted peripherally by the adrenal medulla. It is currently unclear whether corticosterone's antinociceptive effect is associated with the modulation of peripheral epinephrine. METHODS: We first determined whether exogenous corticosterone treatment actually produced an antinociceptive effect in a formalin-induced pain model, and then examined whether this corticosterone-induced antinociceptive effect was altered by suppression of adrenal-derived epinephrine, using the following three suppression methods: (1) inhibition of the PNMT enzyme; (2) blocking peripheral epinephrine receptors; and (3) adrenalectomy. RESULTS: Exogenous treatment with corticosterone at a high dose (50 mg/kg), but not at lower doses (5, 25 mg/kg), significantly reduced pain responses in the late phase. Moreover, injection of 2,3-dichloro-a-methylbenzylamine, a PNMT enzyme inhibitor, (10 mg/kg) before corticosterone treatment caused a leftward shift in the dose-response curve for corticosterone and injection of propranolol (5 mg/kg), but not phentolamine, also shifted the dose-response curve to the left during the late phase. Chemical sympathectomy with 6-hydroxydopamine had no effect on corticosterone-induced antinociceptive effect, but injection of a low dose of corticosterone produced an antinociceptive effect in adrenalectomized animals. CONCLUSIONS: These results demonstrate that suppression of epinephrine, derived from adrenal gland, enhances the antinociceptive effect of exogenous corticosterone treatment in an inflammatory pain model.

Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice.

BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.

Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants.

AIM: This study has been aimed (i) to isolate and identify diazotrophs from Korean rice varieties; (ii) to examine the long-term effect of N and compost on the population dynamics of diazotrophs and (iii) to realize the shot-term inoculation effect of these diazotrophs on rice seedlings. METHODS AND RESULTS: Diazotrophic and heterotrophic bacterial numbers were enumerated by most probable number method and the isolates were identified based on morphological, physiological, biochemical and 16s rDNA sequence analysis. Long-term application of fertilizer N with compost enhanced both these numbers in rice plants and its environment. Bacteria were high in numbers when malate and azelaic acids were used as carbon source, but less when sucrose was used as a carbon substrate. The combined application promoted the association of diazotrophic bacteria like Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Gluconacetobacter diazotrophicus and Pseudomonas spp. in wetland rice plants. Detection of nifD genes from different diazotrophic isolates indicated their nitrogen fixing ability. Inoculation of a representative isolate from each group onto rice seedlings of the variety IR 36 grown in test tubes indicated the positive effect of these diazotrophs on the growth of rice seedlings though the percentage of N present in the plants did not differ much. CONCLUSIONS: Application of compost with fertilizer N promoted the diazotrophic and heterotrophic bacterial numbers and their association with wetland rice and its environment. Compost application in high N fertilized fields would avert the reduction of N(2)-fixing bacterial numbers and their association was beneficial to the growth of rice plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of high N fertilization on diazotrophic bacterial numbers could be reduced by the application of compost and this observation would encourage more usage of organic manure. This study has also thrown light on the wider geographic distribution of G. diazotrophicus with wetland rice in temperate region where sugarcane (from which this bacterium was first reported to be associating and thereon from other plant species) is not cultivated.

The anti-inflammatory effect of bee venom stimulation in a mouse air pouch model is mediated by adrenal medullary activity.

Cutaneous electrical or chemical stimulation can produce an anti-inflammatory effect, which is dependent on adrenal medullary-sympathetic activation. We have previously shown that peripheral injection of bee venom (BV) also produces a significant anti-inflammatory effect that is neurally mediated. In the present study, we examined whether this anti-inflammatory effect is also dependent on the adrenal gland using the mouse inflammatory air pouch model. Subcutaneous (s.c.) BV injection produced a marked suppression of leucocyte migration and tumour necrosis factor (TNF)-alpha concentration induced by zymosan injection into the air pouch. The role of the adrenal gland in this suppression was evaluated in adrenalectomized mice. Adrenalectomy significantly reversed the suppression of leucocyte migration and TNF-alpha elevation caused by BV. Serum concentrations of corticosteroid were increased in mice with zymosan-induced air-pouch inflammation and this increase was reduced by BV administration, suggesting that adrenal corticosteroid release is not involved in mediating the anti-inflammatory effects of BV. To test this hypothesis, the corticosteroid receptor antagonist (RU486) was administered and found not to affect the BV-induced inhibition of leucocyte migration. By contrast, pretreatment with the beta-adrenergic antagonist propranolol reversed the BV-induced inhibitory effect on leucocyte migration. These results suggest that the anti-inflammatory effect of s.c. BV administration is mediated in part by the release of catecholamines from the adrenal medulla.

Distribution of nociceptin-like immunoreactivity in the central nervous system of the Mongolian gerbil: an immunohistochemical study.

This study is designed to demonstrate the distribution of nociceptin, endogenous ORL1 receptor ligand, in the central nervous system of the Mongolian gerbil. To intensify the nociceptin-like immunoreactivity (NOC-LI), colchicine was administered into the lateral ventricle, at 48 h prior to the transcardiac perfusion. In the group without colchicine treatment, NOC-LI was observed in the fibres of the spinal dorsal horn, specifically in the superficial layers. However, the NOC-LI in the superficial layers disappeared after the administration of colchicine. In the brain, NOC-LI was prominent in the hypothalamus, hippocampus, cerebral peduncle, substantia nigra, dorsal raphe, periaqueductal grey, locus coeruleus and trapezoid nucleus. Colchicine treatment markedly intensified the NOC-LI in the somata of the central nervous system, whereas the untreated sections were too weak to observe and analyse. The distribution of NOC-LI provides informative data for studies of the neuronal circuit that nociceptin may be involved in.

The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell.

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.

Transcriptional regulation of BMP-4 in the Xenopus embryo: analysis of genomic BMP-4 and its promoter.

Recent experiments in the Xenopus embryo suggest that proper regulation of BMP-4 signaling is critical to the dorsal ventral specification of both mesoderm and ectoderm. Regulation of BMP-4 signaling is known to occur extracellularly by direct binding with chordin, noggin, and follistatin, and intracellularly through the antagonistic signal interaction with dorsalizing TGF-beta family member activin. However, tight repressional regulation of BMP transcription may also be required to sustain the dorsal and neural status of the induced cells. Here we demonstrate that the dominant negative mutant of the BMP receptor (DN-BR) or the BMP-4 antagonizers, chordin and noggin, negatively regulate BMP-4 transcription in animal cap explants. We suggest that repression of BMP-4 transcription is important in the maintenance of dorsal fate and that continuous input of BMP-4 signaling is required to sustain the expression of BMP-4 transcription in the maintenance of epidermal/ventral fate. Consistent with this postulation, we found that the promoter region of the isolated BMP-4 genomic DNA includes several consensus binding sites for transcriptional regulators functioning under BMP-4 signaling such as GATA binding and ventralizing homeobox genes. In a functional assay we found that the GATA binding and ventral homeobox proteins can positively modulate BMP-4 promoter activity. We also observed that DN-BR decreases BMP-4 promoter activity. This was likely due to a repression of the above-mentioned transcription factors. The significance of these observations to embryonic patterning is discussed.