Intradermal administration of magnesium sulphate and magnesium chloride produces hypesthesia to mechanical but hyperalgesia to heat stimuli in humans

<p>Abstract</p> <p>Background</p> <p>Although magnesium ions (Mg²⁺) are known to display many similar features to other 2+ charged cations, they seem to have quite an important and unique role in biological settings, such as NMDA blocking effect. However, the role of Mg²⁺in the neural transmission system has not been studied as sufficiently as calcium ions (Ca²⁺). To clarify the sensory effects of Mg²⁺in peripheral nervous systems, sensory changes after intradermal injection of Mg²⁺were studied in humans.</p> <p>Methods</p> <p>Magnesium sulphate, magnesium chloride and saline were injected into the skin of the anterior region of forearms in healthy volunteers and injection-induced irritating pain ("irritating pain", for short), tactile sensation, tactile pressure thresholds, pinch-pain changes and intolerable heat pain thresholds of the lesion were monitored.</p> <p>Results</p> <p>Flare formation was observed immediately after magnesium sulphate or magnesium chloride injection. We found that intradermal injections of magnesium sulphate and magnesium chloride transiently caused irritating pain, hypesthesia to noxious and innocuous mechanical stimulations, whereas secondary hyperalgesia due to mechanical stimuli was not observed. In contrast to mechanical stimuli, intolerable heat pain-evoking temperature was significantly decreased at the injection site. In addition to these results, spontaneous pain was immediately attenuated by local cooling.</p> <p>Conclusion</p> <p>Membrane-stabilizing effect and peripheral NMDA-blocking effect possibly produced magnesium-induced mechanical hypesthesia, and extracellular cation-induced sensitization of TRPV1 channels was thought to be the primary mechanism of magnesium-induced heat hyperalgesia.</p

Diverging Mechanisms of Activation of Chemokine Receptors Revealed by Novel Chemokine Agonists

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions

Structural biology of chemokine receptors

Chemokine receptors are G protein-coupled receptors that mediate migration and activation of leukocytes as an important part of a protective immune response to injury and infection. In addition, chemokine receptors are used by HIV-1 to infect CD4 positive cells. The structural bases of chemokine receptor recognition and signal transduction are currently being investigated. High-resolution X-ray diffraction and NMR spectroscopy of chemokines indicate that all these peptides exhibit a common folding pattern, in spite of its low degree of primary-sequence homology. Chemokines' functional motifs have been identified by mutagenesis studies, and a possible mechanism for receptor recognition and activation is proposed, but high-resolution structure data of chemokine receptors is not yet available. Studies with receptor chimeras have identified the putative extracellular domains as the major selectivity determinants. Single-amino acid substitutions in the extracellular domains produce profound changes in receptor specificity, suggesting that motifs in these domains operate as a restrictive barrier to a common activation motif. Similarly HIV-1 usage of chemokine receptors involves interaction of one or more extracellular domains of the receptor with conserved and variable domains on the viral envelope protein gp 120, indicating a highly complex interaction. Elucidating the structural requirements for receptor interaction with chemokines and with HIV-1 will provide important insights into understanding the mechanisms of chemokine recognition and receptor activation. In addition, this information can greatly facilitate the design of effective inmunomodulatory and anti-HIV-1 therapeutic agent

DNA damage-induced apoptosis in C3H mouse peritoneal resident macrophages

Previously we reported irradiation-induced apoptosis in peritoneal resident macrophages (PRM) of C3H mice but not other strains of mice. Studies with atm knockout, p53 knockout or scid mice suggested that a well-known DNA damage-induced apoptotic pathway involving the activation of ATM, DNA-PK and/or TP53 did not take part in irradiation-induced apoptosis in C3H mouse PRM. Furthermore, superoxide anion and the depletion of Mcl-1, an anti-apoptotic Bcl-2 family protein, through irradiation-induced arrest of global protein synthesis were identified as critical factors for the apoptosis. The present study was planed to reconfirm the involvement of reactive oxygen species but not DNA damage in irradiation-induced apoptosis in C3H mouse PRM. PRM were treated with paraquat (a superoxide inducer), hydrogen peroxide or cadmium at various concentrations to enhance intracellular level of reactive oxygen species or oxidative stress. As a selective indicator of irradiation-induced apoptosis in C3H mouse PRM, Mcl-1 level was examined by western blotting. Hydrogen peroxide and cadmium treatment did not affect the amount of Mcl-1. Paraquat slightly diminished Mcl-1 at extremely high concentrations where oxidative stress was elicited as severely as caspase 3 was completely inactivated. On the other hand, Mcl-1 was markedly depleted by gamma-irradiation or UV irradiation in C3H mouse PRM. PRM were treated with DNA topoisomerase II inhibitor, etoposide or introduced with a restriction endonuclease, Pvu II by means of HVJ envelope vector kit (GenomeONETM) to generate DNA double-strand breaks. Unexpectedly, these treatments induced marked apoptosis in C3H mouse PRM, but not in radioresistant B6 mouse PRM. Therefore, it is concluded that irradiation-induced apoptosis in C3H mouse PRM is attributable to irradiation-induced DNA damage, possibly DNA double-strand breaks.International Congress of Radiation Researc

Identification of Genes Responding to Low-Dose Arsenite Using HiCEP

Chronic ingestion of arsenic in polluted food and water cancause various human disorders including skin and lung cancers. Sensitivebiomarkers from human tissue/cells could help to prevent chronic intoxicationwith low-dose arsenite. Using High-Coverage Expression Profiling (HiCEP),an Amplified Fragment Length Polymorphism (AFLP)-based gene expressionprofiling technique, we analyzed the expression of approximately 11,000genes in human lung fibroblasts (HFLIII) and compared the profiles betweencells, treated and untreated with 1 uM sodium arsenite (NaAsO2). Hundredsof genes appeared upregulated and downregulated more two-fold 2 h after thetreatment. Marked induction was found (>4.4-fold) in a few genes includingHMOX1, INHBA, and ANKRD11. Induction of the HMOX1 was detectedwith a dose of arsenite at as low as 0.3 uM(0.04 ppm) and reached its maximumat 4 h after the treatment. The arsenite-induced HMOX1 expression wasattenuated by the promoted glutathione (GSH) synthesis by N-acetyl-L-cysteine(NAC). However, it was not affected by pretreating the cells with generalradical scavengers, consistent with the fact that ionizing radiation at eitherhigh- or low-doses has never induced HMOX1 in the same assay system. Thus,induction of HMOX1 gene is highly sensitive and also selective against arsenitein the cells. The present process could provide a useful strategy for exploringbiomarkers that might help in assessing the known and unknown risks of anynatural and artificial toxic reagents

Development of a risk assessment system of toxicants by HiCEP

In our previous study, we have indicated a novel expressionprofiling method called \u27high coverage expression profiling\u27(HiCEP) could detect an alteration in gene expression as small as1.5-fold and covers 70% to 80% of all transcripts. As HiCEP has agreat advantage over other common methods for detecting minimalalterations in gene expression as described above, our comprehensiveapproach using HiCEP method is thought to be a powerfulstrategy to develop the system of risk assessment of manychemicals.Arsenic is one of the typical toxic metals and is also known tobe carcinogen. Exposure to arsenic is supposed to increase the riskof occurrences of tumors (lung, skin, liver, bladder, and kidney). Inorder to investigate the efficiency of HiCEP method on developingthe system of risk assessment of arsenic in the environment, wecarried out HiCEP analysis of human lung embryonic fibroblasts(HFLIII) after 1 and 2 hours\u27 exposure to 1 micro M sodium arsenite.The most up-regulated gene is hemoxigenase 1 (HMOX1), which isknown to be induced by arsenic. We examined the effect of arsenicon the HMOX1 expression by quantitative PCR (qPCR) afterseveral periods of incubation time with 1-10 micro M sodium arsenite.HMOX1 expression reached to the peak value after 4 hours\u27incubation with 1-10 micro M arsenic. We also examined the effect oflow level of arsenic on HMOX1 expression. HMOX1 expressionreached to the peak value after 2 hours\u27 incubation with arsenic at0.1 and 0.3 micro M, and after 4 hours\u27 incubation with arsenic at 0.5micro M. Up-regulation of HMOX1 gene was observed in HFLIII cellstreated with arsenic at 0.3 and 0.5 micro M. We examined the effect ofarsenic concentration on cell viability. Arsenic at less than 1 micro Mdoes hardly affect the viability of HFLIII cells.We further explored the effect of ionizing radiation onHMOX1 expression by qPCR 0.5, 2, and 4 hours after X-irradiationat 2 and 4 Gy. At all of the period of time after X-irradiation at 2 and4 Gy, HMOX1 expression was not up-regulated. In HiCEP analysis,HMOX1 expression after low-dose X-irradiation was not upregulatedas well.We conclude HiCEP could be used to develop the system ofrisk assessment of many chemicals and HMOX1 could be a goodbiomarker for arsenic contamination in the environment.13th International Congress of Radiation Researc

Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice: selective involvement of superoxide anion, but not other reactive oxygen species

Remarkably, apoptosis was induced by gamma-ray irradiation in peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice. The mechanism of this strain-specific apoptosis induction was studied. Apoptosis in PRM was detected microscopically. Various radical scavengers were examined to identify the critical radicals involved in apoptosis induction. Intracellular peroxide levels were measured with a redox-sensitive dye, 2,7-dichlorofluorescin diacetate (DCFH). Superoxide dismutase or catalase was introduced into the cells using commercially available Hemagglutinating Virus of Japan (HVJ) envelope vector kit. The enzyme activity of superoxide dismutase was also measured. Radiation-induced apoptosis in C3H mouse PRM was significantly suppressed by treatment with a pharmacological scavenger of superoxide anion, Tiron, but not with other radical scavengers. Intracellular peroxide levels were not elevated by irradiation at doses high enough to induce apoptosis maximally. Radiation-induced apoptosis in C3H mouse PRM was markedly suppressed by superoxide disumutase introduced into the cells using the HVJ envelope vector, but not catalase. The enzyme activity of superoxide dismutase in C3H mouse PRM was comparable with that in B6 mouse PRM. It was concluded that superoxide played the major role in radiation-induced apoptosis in the C3H mouse PRM and that cellular responses downstream or unrelated to superoxide might be responsible for the strain difference in radiation-induced apoptosis of mouse PRM

Gene expression profiling in normal human fibroblasts following the irradiation with heavy ion particles by HIMAC

HiCEP (High-coverage expression profiling) is a novel comprehensive analysis method which is based on DNA finger printing and PCR amplification. It enables to detect any altered gene expression among 60-70% of all the actually transcribed genes in any eukaryotic cells and tissues. We previously applied HiCEP to a primary culture of normal human fibroblasts and observed gene expression responding to X-ray at the very low dose (10 mGy). As the result of screening approximately 23.000 transcripts, we have identified a set of CXC chemokines (CXCL1, CXCL2, CXCL6 and CXCL8) up-regulated by the 10 mGy X-rays in the normal human fibroblasts (HFL III) (Cancer Res 2005; 65: 10159-10163). Those genes have hardly been expected from the previous studies using the higher (>100 mGy) doses of radiation. Our observation indicated that different molecular mechanisms are involved in the response to ionizing radiation with different doses /dose rates, suggesting that different cellular responses could be induced by ionizing radiation with different LETs. Accelerated heavy ion particles (at high LET) provide promising effects for radiotherapy of certain types of malignancy, however, the molecular basis of its advantage to gamma rays is not fully understood. This time, we applied HiCEP to compare the gene expression profiles in normal human fibroblasts (HFL III) irradiated with radiation with different LET\u27s.International Congress of Radiation Researc

Mcl-1 Depletion in Apoptosis Elicited by Ionizing Radiation in Peritoneal Resident Macrophages of C3H Mice

Remarkably, apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice, to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. In order to determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins, Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1, but not Bcl-2, Bcl-XL, Bax, A1, or cIAP1. Mcl-1s depletion was suppressed by a proteasome inhibitor, but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation, and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand, Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific siRNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis