Neurotropin inhibits neuronal activity through potentiation of sustained Kv currents in primary cultured DRG neurons

Neurotropin (NTP) is a Japanese analgesic agent for treating neuropathic pain; however, its method of action remains unclear. This study examined the effects of NTP on the activity of small dorsal root ganglion (DRG) neurons using whole-cell patch clamp recordings. After 3 days of treatment, NTP decreased current injection-induced firing activity of cultured DRG neurons by raising the current threshold for action potential generation. Additionally, NTP increased the sustained component of voltage-gated potassium (Kv) channel currents without affecting other K⁺ currents. These results suggest that NTP inhibits the firing activity of DRG neurons through augmentation of sustained Kv current

Sphingosine-1-phosphate induces Ca²⁺ signaling and CXCL1 release via TRPC6 channel in astrocytes.

A biologically active lipid, sphingosine‐1‐phosphate (S1P) is highly abundant in blood, and plays an important role in regulating the growth, survival, and migration of many cells. Binding of the endogenous ligand S1P results in activation of various signaling pathways via G protein‐coupled receptors, some of which generates Ca²⁺ mobilization. In astrocytes, S1P is reported to evoke Ca²⁺ signaling, proliferation, and migration; however, the precise mechanisms underlying such responses in astrocytes remain to be elucidated. Transient receptor potential canonical (TRPC) channels are Ca²⁺‐permeable cation channels expressed in astrocytes and involved in Ca²⁺ influx after receptor stimulation. In this study, we investigated the involvement of TRPC channels in S1P‐induced cellular responses. In Ca²⁺ imaging experiments, S1P at 1 μM elicited a transient increase in intracellular Ca²⁺ in astrocytes, followed by sustained elevation. The sustained Ca²⁺ response was markedly suppressed by S1P₂ receptor antagonist JTE013, S1P₃ receptor antagonist CAY10444, or non‐selective TRPC channel inhibitor Pyr2. Additionally, S1P increased chemokine CXCL1 mRNA expression and release, which were suppressed by TRPC inhibitor, inhibition of Ca²⁺ mobilization, MAPK pathway inhibitors, or knockdown of the TRPC channel isoform TRPC6. Taken together, these results demonstrate that S1P induces Ca²⁺ signaling in astrocytes via Gq‐coupled receptors S1P₂ and S1P₃, followed by Ca²⁺ influx through TRPC6 that could activate MAPK signaling, which leads to increased secretion of the proinflammatory or neuroprotective chemokine CXCL1

MrgprB4 in trigeminal neurons expressing TRPA1 modulates unpleasant sensations

Gentle touch such as stroking of the skin produces a pleasant feeling, which is detected by a rare subset of sensory neurons that express Mas-related G protein-coupled receptor B4 (MrgprB4) in mice. We examined small populations of MrgprB4-positive neurons in the trigeminal ganglion and the dorsal root ganglion, and most of these were sensitive to transient receptor potential ankyrin 1 (TRPA1) agonist but not TRPV1, TRPM8, or TRPV4 agonists. Deficiency of MrgprB4 did not affect noxious pain or itch behaviors in the hairless plantar and hairy cheek. Although behavior related to acetone-induced cold sensing in the hind paw was not changed, unpleasant sensory behaviors in response to acetone application or sucrose splash to the cheek were significantly enhanced in Mrgprb4-knockout mice as well as in TRPA1-knockout mice. These results suggest that MrgprB4 in the trigeminal neurons produces pleasant sensations in cooperation with TRPA1, rather than noxious or cold sensations. Pleasant sensations may modulate unpleasant sensations on the cheek via MrgprB4

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176

G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N-glycosylated. Peptide-N-glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N-glycosylation. Using a heterologous expression system, we show that N-glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N-glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N-glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N-glycosylated. Importantly, missense variations in the conserved N-glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N-glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N-glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176

Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice

「しびれ」による痛みのメカニズムを解明 -糖尿病や血流障害によるしびれ治療薬の開発に期待-. 京都大学プレスリリース. 2016-03-18.Dysesthesia is an unpleasant abnormal sensation, which is often accompanied by peripheral neuropathy or vascular impairment. Here, we examined the roles of transient receptor potential ankyrin 1 (TRPA1) in dysesthesia-like behaviours elicited by transient hindlimb ischemia (15–60 min) by tightly compressing the hindlimb, and reperfusion by releasing the ligature. The paw-withdrawal responses to tactile stimulation were reduced during ischemia and lasted for a while after reperfusion. Hindlimb ischemia/reperfusion elicited spontaneous licking of the ischemic hindpaw that peaked within 10 min. The licking was inhibited by reactive oxygen species (ROS) scavengers, a TRPA1 antagonist, or TRPA1 deficiency, but not by TRPV1 deficiency. In human TRPA1-expressing cells as well as cultured mouse dorsal root ganglion neurons, the H[2]O[2]-evoked TRPA1 response was significantly increased by pretreatment with hypoxia (80 mmHg) for 30 min. This hypoxia-induced TRPA1 sensitisation to H[2]O[2] was inhibited by overexpressing a catalytically-inactive mutant of prolyl hydroxylase (PHD) 2 or in a TRPA1 proline mutant resistant to PHDs. Consistent with these results, a PHD inhibitor increased H[2]O[2]-evoked nocifensive behaviours through TRPA1 activation. Our results suggest that transient hindlimb ischemia/reperfusion-evoked spontaneous licking, i.e. painful dysesthesia, is caused by ROS-evoked activation of TRPA1 sensitised by hypoxia through inhibiting PHD-mediated hydroxylation of a proline residue in TRPA1

Administration of micafungin as prophylactic antifungal therapy in patients undergoing allogeneic stem cell transplantation

Objective: Invasive fungal infection is one of the major causes of death in neutropenic patients undergoing allogeneic stem cell transplantation (SCT). Although prophylactic antifungal therapy with fluconazole (FLCZ) has become the standard care for these patients, there remains a need for more effective and cost-beneficial alternative drugs. Patients and Methods: We conducted a prospective study to evaluate the usefulness of the administration of micafungin (MCFG) as a prophylactic antifungal therapy for patients undergoing allogeneic SCT. The results were compared with previous data for patients who had received FLCZ. Results: A total of 44 patients who underwent allogeneic SCT were enrolled in the study. Data from 29 patients who received allogeneic SCT using prophylactic FLCZ before this study were used as historical control data. Underlying diseases included acute leukemia (n=16), non-Hodgkin’s lymphoma (n=11), myelodysplastic syndrome (n=6), and others (n=11) in the MCFG group and acute leukemia (n=18), chronic myelogenous leukemia (n=6), and others (n=5) in the FLCZ group. The median durations of administration of MCFG and FLCZ were 36 and 34 days, respectively. Prophylactic success, defined as the absence of proven, probable, and possible invasive fungal infection (IFI) until the end of prophylactic therapy was achieved in 36 (87.8%) of the 41 evaluated patients in the MCFG group and in 65.5% of the patients in the FLCZ group (p=0.038). No patients in the MCFG group showed proven or probable IFI, whereas proven or probable IFI was observed in 3 patients in the FLCZ group. Four patients in the MCFG group required dose escalation due to febrile neutropenia. Although one patient in the MCFG group required the discontinuation of MCFG due to allergic skin eruption (grade 2), none of the other patients in either group required dose reduction due to adverse effects. Conclusions: Although the study design was not a prospective randomized trial, our results indicate that the administration of MCFG at a daily dose of 100 mg is promising for prophylactic antifungal therapy in patients undergoing allogeneic SCT

Transient receptor potential canonical 3 inhibitor Pyr3 improves outcomes and attenuates astrogliosis after intracerebral hemorrhage in mice.

Background and Purpose—Intracerebral hemorrhage (ICH) stems from the rupture of blood vessels in the brain, with the subsequent accumulation of blood in the parenchyma. Increasing evidence suggests that blood-derived factors induce excessive inflammatory responses that are involved in the progression of ICH-induced brain injury. Thrombin, a major blood-derived factor, leaks into the brain parenchyma on blood–brain barrier disruption and induces brain injury and astrogliosis. Furthermore, thrombin dynamically upregulates transient receptor potential canonical 3 channel, which contributes to pathological astrogliosis through a feed-forward upregulation of its own expression. The present study investigated whether Ethyl-1-(4-(2, 3, 3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a specific transient receptor potential canonical 3 inhibitor, can improve functional outcomes and attenuate astrogliosis after ICH in mice.　Methods—Male C57BL6 mice received an intracerebral infusion of collagenase or autologous blood to induce ICH. Pyr3 was given both intracerebroventricularly and intraperitoneally after ICH induction. ICH-induced brain injury was evaluated by quantitative assessment of neurological deficits, brain swelling, and injury volume after ICH. Astrocyte activation was evaluated by immunohistochemical assessment of changes in S100 protein expression.　Results—Neurological deficits, neuronal injury, brain edema, and astrocyte activation were all significantly improved by administration of Pyr3. Moreover, delayed administration of Pyr3 at 6 hours or 1 day after blood or collagenase infusion, respectively, also improved the symptoms.　Conclusions—Pyr3, a specific inhibitor of transient receptor potential canonical 3, reduced the perihematomal accumulation of astrocytes and ameliorated ICH–induced brain injury. Therefore, transient receptor potential canonical 3 provides a new therapeutic target for the treatment of hemorrhagic brain injury

Clinical impact of cycling the administration of antibiotics for febrile neutropenia in Japanese patients with hematological malignancy

Despite the availability of newer classes of antibiotics, infection with multi-drug-resistant bacteria is a serious problem. To suppress the appearance of multi-drug-resistant bacteria and to avoid severe infection derived from febrile neutropenia (FN), we conducted cycling administration of antibiotics for FN in patients with hematological malignancy. The treatment protocol consisted of the administration of four antibiotics each for three months in one year. The above regimen was repeated for 4 years. A total of 193 patients were registered in the protocol. Mean duration of administration of cycling antibiotics was 5.9 days (range: 1-16 days). Frequency of FN before the study and during the study was unchanged until the third year but decreased significantly in the fourth year. Frequency of detection of multi-drug-resistant bacteria in the first year was the same as that before the study was started but dramatically decreased after the second year. Bacteriological treatment success rates were similar in each trimester and each year. Effective rate was not statistically different in each trimester and each year. We conclude that cycling administration of antibiotics in patients with FN is useful for suppressing the appearance of multi-drug-resistant bacteria and for obtaining excellent clinical efficacy