The role of TRPA1 in muscle pain and mechanical hypersensitivity under inflammatory conditions in rats.

Transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is expressed in muscle afferents and direct activation of these receptors induces acute mechanical hypersensitivity. However, the functional role of TRPA1 under pathological muscle pain conditions and mechanisms by which TRPA1 mediate muscle pain and hyperalgesia are not clearly understood. Two rodent behavioral models validated to assess craniofacial muscle pain conditions were used to study ATP- and N-Methyl-D-aspartate (NMDA)-induced acute mechanical hypersensitivity and complete Freund's adjuvant (CFA)-induced persistent mechanical hypersensitivity. The rat grimace scale (RGS) was utilized to assess inflammation-induced spontaneous muscle pain. Behavioral pharmacology experiments were performed to assess the effects of AP18, a selective TRPA1 antagonist under these conditions. TRPA1 expression levels in trigeminal ganglia (TG) were examined before and after CFA treatment in the rat masseter muscle. Pre-treatment of the muscle with AP18 dose-dependently blocked the development of acute mechanical hypersensitivity induced by NMDA and α,ß-methylene adenosine triphosphate (αßmeATP), a specific agonist for NMDA and P2X3 receptor, respectively. CFA-induced mechanical hypersensitivity and spontaneous muscle pain responses were significantly reversed by post-treatment of the muscle with AP18 when CFA effects were most prominent. CFA-induced myositis was accompanied by significant up-regulation of TRPA1 expression in TG. Our findings showed that TRPA1 in muscle afferents plays an important role in the development of acute mechanical hypersensitivity and in the maintenance of persistent muscle pain and hypersensitivity. Our data suggested that TRPA1 may serve as a downstream target of pro-nociceptive ion channels, such as P2X3 and NMDA receptors in masseter afferents, and that increased TRPA1 expression under inflammatory conditions may contribute to the maintenance of persistent muscle pain and mechanical hyperalgesia. Mechanistic studies elucidating transcriptional or post-translational regulation of TRPA1 expression under pathological pain conditions should provide important basic information to further advance the treatment of craniofacial muscle pain conditions.

The role of TRPM2 in hydrogen peroxide-induced expression of inflammatory cytokine and chemokine in rat trigeminal ganglia.

Trigeminal ganglia (TG) contain neuronal cell bodies surrounded by satellite glial cells. Although peripheral injury is well known to induce changes in gene expression within sensory ganglia, detailed mechanisms whereby peripheral injury leads to gene expression within sensory ganglia are not completely understood. Reactive oxygen species (ROS) are an important modulator of hyperalgesia, but the role of ROS generated within sensory ganglia is unclear. Since ROS are known to affect transcription processes, ROS generated within sensory ganglia could directly influence gene expression and induce cellular changes at the soma level. In this study, we hypothesized that peripheral inflammation leads to cytokine and chemokine production and ROS generation within TG and that transient receptor potential melastatin (TRPM2), a well known oxidative sensor, contributes to ROS-induced gene regulation within TG. The masseter injection of complete Freund's adjuvant (CFA) resulted in a significantly elevated level of ROS within TG of the inflamed side with a concurrent increase in cytokine expression in TG. Treatment of TG cultures with H2O2 significantly up-regulated mRNA and protein levels of cytokine/chemokine such as interleukin 6 (IL-6) and chemokine (C-X-C motif) ligand 2 (CXCL2). TRPM2 was expressed in both neurons and non-neuronal cells in TG, and pretreatment of TG cultures with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of TRPM2, or siRNA against TRPM2 attenuated H2O2-induced up-regulation of IL-6 and CXCL2. These results suggested that activation of TRPM2 could play an important role in the modulation of cytokine/chemokine expression within TG under oxidative stress and that such changes may contribute to amplification of nociceptive signals leading to pathological pain conditions.

Peripheral G protein-coupled inwardly rectifying potassium channels are involved in δ-opioid receptor-mediated anti-hyperalgesia in rat masseter muscle.

BACKGROUND: Although the efficacy of peripherally administered opioid has been demonstrated in preclinical and clinical studies, the underlying mechanisms of its anti-hyperalgesic effects are poorly understood. G protein-coupled inwardly rectifying potassium (GIRK) channels are linked to opioid receptors in the brain. However, the role of peripheral GIRK channels in analgesia induced by peripherally administered opioid, especially in trigeminal system, is not clear. METHODS: Expression of GIRK subunits in rat trigeminal ganglia (TG) was examined with reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. Chemical profiles of GIRK-expressing neurons in TG were further characterized. Behavioural and Fos experiments were performed to examine the functional involvement of GIRK channels in δ-opioid receptor (DOR)-mediated anti-hyperalgesia under an acute myositis condition. RESULTS: TG expressed mRNA and proteins for GIRK1 and GIRK2 subunits. Majority of GIRK1- and GIRK2-expressing neurons were non-peptidergic afferents. Inhibition of peripheral GIRK using Tertiapin-Q (TPQ) attenuated antinociceptive effects of peripherally administered DOR agonist, [D-Pen(2), D-Pen(6) ]-enkephalin (DPDPE), on mechanical hypersensitivity in masseter muscle. Furthermore, TPQ attenuated the suppressive effects of peripheral DPDPE on neuronal activation in the subnucleus caudalis of the trigeminal nucleus (Vc) following masseteric injection of capsaicin. CONCLUSIONS: Our data indicate that peripheral DOR agonist-induced suppression of mechanical hypersensitivity in the masseter muscle involves the activity of peripheral GIRK channels. These results could provide a rationale for developing a novel therapeutic approach using peripheral GIRK channel openers to mimic or supplement the effects of peripheral opioid agonist.

4-Week repeated intravenous dose toxicity study of a new camptothecin anticancer agent CKD-602 in dogs.

CKD-602 is a new camptothecin derivative antitumor agent with a formula (7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin) developed by Chong Kun Dang Pharmaceutical Company in Korea. In the present study, the subacute toxicity of CKD-602 was investigated after 4-week repeated intravenous administration of the test chemical in beagle dogs. The test chemical was administered intravenously at dose levels of 0, 0.001, 0.005, or 0.01 mg/kg/day for 4 weeks to male and female dogs (n = 3 for male and female dogs for each dose). During the test period, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights and histopathology were examined. In the high dose group, an increase in the incidence of abnormal clinical signs and a decrease in food and water intake and body weight gain were observed in both sexes. Hematological investigations revealed decreased white blood cells (WBC) in both sexes and reduced red blood cells (RBC), hemoglobin and hematocrit in females. Histopathological examinations revealed an increase in the incidence of atrophy of the sternal and femoral marrow and spleen in both sexes and atrophy of the thymus and mesenteric lymph node in males. No treatment-related adverse effects were observed in both sexes of the low and middle dose groups. In conclusion, the 4-week repeated intravenous dose of CKD-602 to beagle dogs caused increases in the clinical signs and histopathological changes, and decreases in the body weight gain, food and water intake, RBC, hemoglobin, hematocrit and WBC at the dose level of 0.01 mg/kg/day. In the present experimental conditions, the target organs were determined to be bone marrow, blood cells, spleen, thymus, and mesenteric lymph node. The no-observed-adverse-effect levels (NOAEL) for males and females were considered to be 0.005 mg/kg/day, respectively.

Developmental toxicity assessment of the new fluoroquinolone antibacterial DW-116 in rabbits.

DW-116 is a newly developed fluoroquinolone antibacterial with a broad spectrum against both Gram-positive and Gram-negative bacteria. We have reported recently that DW-116 is embryotoxic and teratogenic in rats. The present study was conducted to investigate the teratogenicity of DW-116, together with maternal toxicity and developmental toxicity using New Zealand White rabbits. The test chemical was administered by gavage to pregnant rabbits from gestational day (GD) 6 through to GD 18 at dose levels of 0, 5, 19.5 and 76.1 mg kg(-1) day(-1). All does were subjected to caesarean section on day 28 of gestation and their foetuses were examined for external, visceral and skeletal abnormalities. In the 76.1 mg kg(-1) group, a minimal maternal toxicity, as evidenced by decreased body weight gain during treatment period, was observed in pregnant rabbits. Significant embryo-foetal toxicity, including increased number of foetal deaths and delayed foetal ossification, was seen. However, no treatment-related morphological changes were detected in foetal external, visceral and skeletal examinations. There were no adverse effects on either pregnant dams or embryo-foetal development at 19.5 and 5 mg kg(-1). It was concluded that administration of DW-116 during the major organogenetic period in rabbits produced decreased maternal body weight gain, increased number of foetal deaths and foetal developmental delay but no evidence of teratogenicity. The no-observed-adverse-effect levels (NOAELs) of DW-116 are considered to be 19.5 mg kg(-1) day(-1) for does and embryo-foetuses, respectively.

Subacute toxicity evaluation of a new camptothecin anticancer agent CKD-602 administered by intravenous injection to rats.

The subacute toxicity of a new camptothecin anticancer agent, CKD-602, was investigated after 4-week repeated intravenous administration of the chemical in Sprague-Dawley rats. The test chemical was administered intravenously to rats at dose levels of 0, 0.003, 0.013, or 0.067 mg/kg/day for males and 0, 0.004, 0.018, or 0.089 mg/kg/day for females. At the end of the treatment period, 10 rats/sex/group were sacrificed. The remaining 5 rats/sex in the vehicle control and high dose groups continued the study without treatment for 2 weeks (recovery period). During the test period, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights, and histopathology were examined. In both sexes of the high dose group, an increase in the incidence of abnormal clinical signs and paleness of the eyes, a reduction in the body weight gain, food consumption and urine protein, and an increase in the water consumption were observed. Hematological investigations revealed a decrease in the red blood cells, hemoglobin and hematocrit and an increase in the mean corpuscular volume, mean corpuscular hemoglobin, platelets, and reticulocytes in a dose-dependent manner. Serum total cholesterol and total protein values were lower in females than those of controls, but not in males. An increase in the heart and liver weights and a decrease in the thymus weight were also found. Histopathological alterations included an increase in the incidence of atrophy of the sternal marrow, atrophy, fibrosis and mast cell hyperplasia of the femoral marrow, atrophy of the white pulp and extramedullary hematopoiesis of the spleen, atrophy of the thymus, auricular hypertrophy of the heart, extramedullary hematopoiesis and centriacinar telangiectasis of the liver, follicular degeneration of the ovary, and inflammation of the tail. The major treatment-related effects were not recovered at the end of 2-week recovery period. There were no adverse effects in the low and middle dose groups of both genders. In the present experimental conditions, the target organs were determined to be bone marrow, blood cells, spleen, liver, thymus, and heart. The no-observed-adverse-effect level was considered to be 0.013 mg/kg/day for males and 0.018 mg/kg/day for females.

Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation.

Atrial fibrillation (AF), the most common chronic arrhythmia, increases the risk of stroke and is an independent predictor of mortality. Available pharmacological treatments have limited efficacy. Once initiated, AF tends to self-perpetuate, owing in part to electrophysiological remodeling in the atria; however, the fundamental mechanisms underlying this process are still unclear. We have recently demonstrated that chronic human AF is associated with increased atrial oxidative stress and peroxynitrite formation; we have now tested the hypothesis that these events participate in both pacing-induced atrial electrophysiological remodeling and in the occurrence of AF following cardiac surgery. In chronically instrumented dogs, we found that rapid (400 min(-1)) atrial pacing was associated with attenuation of the atrial effective refractory period (ERP). Treatment with ascorbate, an antioxidant and peroxynitrite decomposition catalyst, did not directly modify the ERP, but attenuated the pacing-induced atrial ERP shortening following 24 to 48 hours of pacing. Biochemical studies revealed that pacing was associated with decreased tissue ascorbate levels and increased protein nitration (a biomarker of peroxynitrite formation). Oral ascorbate supplementation attenuated both of these changes. To evaluate the clinical significance of these observations, supplemental ascorbate was given to 43 patients before, and for 5 days following, cardiac bypass graft surgery. Patients receiving ascorbate had a 16.3% incidence of postoperative AF, compared with 34.9% in control subjects. In combination, these studies suggest that oxidative stress underlies early atrial electrophysiological remodeling and offer novel insight into the etiology and potential treatment of an enigmatic and difficult to control arrhythmia. The full text of this article is available at http://www.circresaha.org.