Rectification of ATP-gated current of rat P2X2 and P2X7 receptors depends on the cytoplasmic N-terminus.

The phenotypes of ATP-gated currents thought ionotropic P2X channels depend on the composition of the oligomeric receptor. We constructed chimeric P2X2/P2X7 receptors to study the effect of cytoplasmic domains on rectification of current flow through the open channel. We found that the identity of the N-terminus determines the pattern of rectification, with chimeric receptors containing the N-terminus of the P2X2 receptor displaying inward rectification, and chimeric receptors containing the N-terminus of the P2X7 receptor displaying slightly outward rectification. In contrast, rectification of current through chimeric receptors with swapped C-termini always mimicked the wild-type receptor. Thus, our findings suggest that the N-terminus of P2X receptors regulate ion flow through the channel pore and are responsible in part for determining current rectification.

Bortezomib Increased Vascular Permeability by Decreasing Cell-Cell Junction Molecules in Human Pulmonary Microvascular Endothelial Cells.

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell-cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and ß-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and ß-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell-cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

Association of T cell infiltration and morphological change of thymus gland with the aggravation of pulmonary emphysema in testosterone deficiency.

Chronic obstructive pulmonary disease is an inflammatory lung disease characterized by chronic bronchitis and emphysema. Our previous study revealed that testosterone depletion induced T cell infiltration in the lungs and aggravated pulmonary emphysema in orchiectomized (ORX) mice exposed to porcine pancreatic elastase (PPE). However, the association between T cell infiltration and emphysema remains unclear. The aim of this study was to determine whether thymus and T cells are involved in the exacerbation of PPE-induced emphysema in ORX mice. The weight of thymus gland in ORX mice was significantly greater than that of sham mice. The pretreatment of anti-CD3 antibody suppressed PPE-induced thymic enlargement and T cell infiltration in the lungs in ORX mice, resulting in improved expansion of the alveolar diameter, a marker of emphysema exacerbation. These results suggest that increased thymic function due to testosterone deficiency and the associated increased pulmonary infiltration of T cells may trigger the development of emphysema.

Impact of Concomitant Use of Azoles on Bortezomib-related Adverse Drug Reactions Using JADER.

BACKGROUND/AIM: Azoles are widely used for prophylaxis in patients with haematologic malignancies and are well known as selective cytochrome P450 isoenzyme 3A4 inhibitors. Although the interaction between bortezomib and azoles has been reported, most previous studies were case reports or small clinical studies. Hence, we conducted a pharmacoepidemiological study to elucidate the impact of azoles on bortezomib-related adverse reactions, using the Japanese adverse drug event report database (JADER). PATIENTS AND METHODS: We extracted 19,567 reports on patients prescribed bortezomib and/or azoles. We classified cases into three groups, namely bortezomib, bortezomib and azoles, and azoles groups. We estimated the odds ratios (OR) for the impact of concomitant azole use on five bortezomib-related adverse drug reactions (peripheral neuropathy, thrombocytopenia, neutropenia, leukopenia, and interstitial lung disease) using logistic regression. RESULTS: The OR for peripheral neuropathy in the 'bortezomib and azoles' group was higher than that in the bortezomib group [OR=2.02, 95% confidence interval (CI)=1.32-3.08]. The magnitude of the interaction was stronger with itraconazole than that with fluconazole (itraconazole, OR=3.22, 95% CI=1.78-5.70; fluconazole, OR=1.56, 95% CI=0.86-2.72). CONCLUSION: We found an association between concomitant administration of azoles with bortezomib and peripheral neuropathy. Azoles may enhance bortezomib-induced peripheral neuropathy based on their pharmacokinetic properties.

Occlusal disharmony transiently decrease cognition via cognitive suppressor molecules and partially restores cognitive ability via clearance molecules.

Occlusal disharmony has been reported to be affected not only by cytokine and steroid hormone secretion and sympathetic activation in peripheral organs, but also by neurotransmitter release in the central nervous system. However, little is known about whether occlusal disharmony can decrease cognitive ability. We hypothesized that hyperocclusion decreases cognition via Alzheimer's disease-associated molecule expression in the brain. The present study is aimed to elucidate the relationships among occlusal disharmony, cytokine and cognitive-regulated molecule expression in the brain, and the impairment of learning and memory cognition. We examined the effect of hyperocclusion on the relationships among cytokine expression, cognitive suppressor molecules in the hippocampus, and cognition in behavior using a hyperocclusion mouse model. Hyperocclusion dramatically increased interleukin-1ß expression in the serum and hippocampus 1 week after hyperocclusal loading in 2-month-old mice, but no effects in 12-month-old mice. The social and long-term cognitive abilities of the 2-month-old mice were transiently downregulated close to the level of the 12-month-old mice 1 week after hyperocclusion and recovered to close to basal level via the expression of cognitive suppressor clearing proteins. The expression levels of amyloid-ß and phosphorylated tau were significantly upregulated 1 week after hyperocclusal loading in the hippocampus of 2-month-old mice but were constant in 12-month-old mice. Occlusal disharmony-induced interleukin-1ß expression may contribute to accumulation of cognitive suppressor molecules such as amyloid-ß and phosphorylated tau and activate their clearance proteins, resulting in protection against transient dementia in young but not older individuals.

Testosterone deficiency promotes the development of pulmonary emphysema in orchiectomized mice exposed to elastase.

Testosterone deficiency is commonly observed in male patients with chronic obstructive pulmonary disease (COPD), which is characterized by chronic inflammation of the airways and pulmonary emphysema. Although clinical trials have indicated that testosterone replacement therapy can improve respiratory function in patients with COPD, the role of testosterone in the pathogenesis of COPD remains unclear. The aim of this study was to explore the effect of testosterone deficiency on the development of pulmonary emphysema in orchiectomized (ORX) mice exposed to porcine pancreatic elastase (PPE). ORX mice developed more severe emphysematous changes 21 d after PPE inhalation than non-ORX mice. Testosterone propionate supplementation significantly reduced PPE-induced emphysematous changes in ORX mice. PPE exposure also increased the number of neutrophils and T cells in bronchoalveolar lavage fluid (BALF) of mice that had undergone ORX and sham surgery. T cell counts were significantly higher in the BALF of ORX mice than of sham mice. Testosterone supplementation reduced the infiltration of T cells into BALF and alleviated emphysematous changes in the lungs of ORX mice. Our findings suggest that testosterone, a male-specific hormone, may suppress the development of pulmonary emphysema through the regulation of T cell-mediated immunity.

Bortezomib enhances G-CSF-induced hematopoietic stem cell mobilization by decreasing CXCL12 levels and increasing vascular permeability.

Bortezomib (BTZ) is known to enhance the mobilization of hematopoietic stem and progenitor cells (HSPCs) induced by granulocyte colony-stimulating factor (G-CSF). However, the most effective time at which to administer BTZ to produce this enhancing effect remains debatable, and the precise mechanism underlying the effect of BTZ is poorly understood. We addressed these questions in this article by performing animal experiments. First, in agreement with previous studies, BTZ administration 12 hours before blood collection was most effective for HSPC mobilization; in contrast, BTZ administration 3 days before blood collection negatively affected HSPC harvesting. Next, in terms of the mechanism of action, G-CSF, but not BTZ, downregulated the expression of very late antigen-4 on HSPCs and vascular cell adhesion molecule-1 on bone marrow (BM) stromal cells; however, intriguingly, both G-CSF and BTZ downregulated CXCL12 chemokine expression in BM. Notably, BTZ treatment also increased BM vascular permeability. These results suggest that the pro-mobilization effect of BTZ could involve the dissociation of HSPCs from BM stromal cells triggered by G-CSF, vascular hyperpermeability elicited by BTZ, and downregulation of CXCL12 concomitantly induced by G-CSF and BTZ.

Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice.

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.

FF-10501 induces caspase-8-mediated apoptotic and endoplasmic reticulum stress-mediated necrotic cell death in hematological malignant cells.

FF-10501 is a novel inhibitor of inosine monophosphate dehydrogenase (IMPDH). Clinical trials of FF-10501 for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are currently being conducted in the United States. Although it has been shown that FF-10501 induces apoptosis in hematological malignant cells, the intracellular mechanisms of this effect have not been characterized. We conducted an in vitro study to elucidate the mechanisms of FF-10501-induced cell death using 12 hematological malignant cell lines derived from myeloid and lymphoid malignancies. FF-10501 suppressed the growth of each cell line in a dose-dependent manner. However, the clinically relevant dose (40 µM) of FF-10501 induced cell death in three cell lines (MOLM-13, OCI-AML3, and MOLT-3). Investigation of the cell death mechanism suggested that FF-10501 induces both apoptotic and necrotic cell death. FF-10501-induced apoptosis was mediated by caspase-8 activation followed by activation of the mitochondrial pathway in MOLM-13 and MOLT-3 cells. FF-10501 induced necrotic cell death via endoplasmic reticulum stress in OCI-AML3 cells. The present study is the first to identify intracellular pathways involved in FF-10501-induced cell death.

Effects of geldanamycin on neurite outgrowth-related proteins and kinases in nerve growth factor-differentiated pheochromocytoma 12 cells.

Heat shock protein 90 (HSP90) antagonists are currently being evaluated as potential anticancer drugs. However, adverse effects related to these drugs, such as fatigue and pain, suggest that they affect neurons. Therefore, to understand the influence of HSP90 inhibitors on neurons, we investigated the effects of geldanamycin, an HSP90 antagonist, on nerve growth factor (NGF)-differentiated pheochromocytoma 12 (PC12) cells, particularly, on the expression and phosphorylation of proteins and kinases in the NGF pathway. Geldanamycin significantly inhibited NGF-induced neurite outgrowth and phosphorylation of Akt and extracellular signal-related kinase 1/2 in PC12 cells. Furthermore, geldanamycin inhibited the phosphorylation of collapsin response mediator protein 2 and the expression of cyclin-dependent kinase 5 in the presence of NGF, but did not significantly affect the expression of glycogen synthase kinase 3ß. These results suggest that geldanamycin influences microtubule-binding proteins and kinases relating to neurite outgrowth, thereby inducing neuronal impairment.

Morphological Pattern and Classification of the Superficial Middle Cerebral Vein by Cadaver Dissections: An Embryological Viewpoint.

In this study, we used 45 adult cadaveric cerebral hemispheres to investigate the anatomical classification of the superficial middle cerebral vein (SMCV) based on the number of stems, course, and anastomosis at the distal portion. We classified the SMCVs into five types based on embryological concept. Type A (18 cases, 40.0%) is that the frontosylvian veins (FSVs) merge with the vein of Trolard (VT) and the vein of Labbé (VL) at the distal portion of the sylvian fissure. Type B (5 cases, 11.1%) is that the temporosylvian veins (TSVs) merge with the VT and the VL at the distal portion. Type C (13 cases, 28.9%) is that no vein merge with the VT and the VL at the distal portion. The VT merges with the SMCV from the FSV and the VL merges with the SMCV from the TSV. They course along the sylvian fissure and merge at the proximal portion. In Type D (eight cases: 17.8%), the VT and the VL merge at the distal portion, and the SMCV from the FSV and the SMCV from the TSV join their confluence without merging. Type E (one case, 2.2%) show an undeveloped SMCV. Formation rate of intravenous anastomoses or bridging veins(BVs) at the distal portion between the frontosylvian trunk (FST) and the temporosylvian trunk (TST), between the FST and the temporal lobe, and between the TST and the frontal lobe was very low, because these formation may be difficult to occur during the embryological process in which the SMCV is formed from the telencephalic vein.

Stimulating muscarinic M1 receptors in the anterior cingulate cortex reduces mechanical hypersensitivity via GABAergic transmission in nerve injury rats.

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

Cholinesterase inhibitor rivastigmine enhances nerve growth factor-induced neurite outgrowth in PC12 cells via sigma-1 and sigma-2 receptors.

Rivastigmine (Riv) is a potent and selective cholinesterase (acetylcholinesterase, AChE and butyrylcholinesterase, BuChE) inhibitor developed for the treatment of Alzheimer's disease (AD). To elucidate whether Riv causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. At concentrations of 0-100 µM, Riv was non-toxic in PC12 cells. Riv caused dose-dependent (10-100 µM) enhancement of NGF-induced neurite outgrowth, which was completely inhibited by the TrkA antagonist GW-441756. By contrast, Riv-mediated enhancement of neurite outgrowth was not blocked by the acetylcholine receptor antagonists, scopolamine and hexamethonium. However, the sigma-1 receptor (Sig-1R) antagonist NE-100 and sigma-2 receptor (Sig-2R) antagonist SM-21 each blocked about half of the Riv-mediated enhancement of NGF-induced neurite outgrowth. Interestingly, the simultaneous application of NE-100 and SM-21 completely blocked the enhancement of NGF-induced neurite outgrowth by Riv. These findings suggest that both Sig-1R and Sig-2R play important roles in NGF-induced neurite outgrowth through TrkA and that Riv may contribute to neuronal repair via Sig-1R and Sig-2R in AD therapy.

Involvement of GABAB receptor in the antihypersensitive effect in anterior cingulate cortex of partial sciatic nerve ligation model.

The role of the GABAB receptor in the anterior cingulate cortex (ACC) of neuropathic pain is unclear. Injection of a GABAB receptor antagonist CGP35348 into the ACC induced mechanical hypersensitivity in normal rats. Activation of the GABAB receptor injected by a GABAB receptor agonist baclofen into the ACC attenuated mechanical hypersensitivity in partial sciatic nerve ligation (PSNL) rats. Co-microinjection of CGP35348 with a muscarinic M1 receptor agonist McN-A-343 into the ACC significantly inhibited McN-A-343-induced antihypersensitivity in PSNL rats. These results suggest that the GABAB receptor in the ACC contributes to mechanical hypersensitivity and is involved in muscarinic M1 receptor-mediated antihypersensitivity.

The Effect of Irrigation Solutions on Recurrence of Chronic Subdural Hematoma: A Consecutive Cohort Study of 234 Patients.

Chronic subdural hematomas (CSDHs) occur often in elderly persons and can occur with mild head trauma. With burr-hole irrigation as standard treatment, symptoms usually improve and can be cured, and outcomes are good, but postoperative recurrences are a common problem. This study investigated the effectiveness and recurrence rates when using artificial cerebrospinal fluid (ACF) instead of normal saline (NS) as an irrigation solution for burr-hole irrigation in patients with CSDH. This prospective study included 234 consecutive patients who underwent initial surgical treatment by burr-hole irrigation for a CSDH between April 2008 and June 2015. The irrigation solution used was changed from NS to ACF in June 2011. Factors examined with regard to recurrence included age, sex, unilateral or bilateral surgery, computed tomography (CT) findings, antiplatelet or anticoagulant drug use, past history, and irrigation solution (NS or ACF). These were analyzed by univariate and multivariate analyses. Univariate analyses (chi-square test) with a significance level <5% showed that recurrence rates were significantly lower in the ACF group than in the NS group (P = 0.003). Multivariate analysis (multiple logistic regression analysis) showed that the risk of recurrence was reduced 3.14-fold in the ACF group compared to the NS group (odds ratio, 3.143; 95% confidence interval, 0.1504-0.6733; P = 0.0028). None of the other factors were significantly different. In burr-hole irrigation for CSDH, the use of ACF instead of NS as an irrigation solution significantly reduces recurrence rates.

Propofol Anesthesia Is Reduced in Phospholipase C-Related Inactive Protein Type-1 Knockout Mice.

The GABA type A receptor (GABAA-R) is a major target of intravenous anesthetics. Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABAA-R phosphorylation and membrane trafficking. In this study, we investigated the role of PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate, and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO) mice by measuring the latency and duration of loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR). The effect of pretreatment with okadaic acid (OA), a protein phosphatase 1/2A inhibitor, on propofol- and etomidate-induced LORR was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1 could determine the potency of the anesthetic action of propofol. Pretreatment with OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA injection enhanced phosphorylation of cortical the GABAA-R ß3 subunit in PRIP-1 KO mice. These results suggest that PRIP-1-mediated GABAA-R ß3 subunit phosphorylation might be involved in the general anesthetic action induced by propofol but not by etomidate or pentobarbital.

Activations of muscarinic M1 receptors in the anterior cingulate cortex contribute to the antinociceptive effect via GABAergic transmission.

Background Cholinergic systems regulate the synaptic transmission resulting in the contribution of the nociceptive behaviors. Anterior cingulate cortex is a key cortical area to play roles in nociception and chronic pain. However, the effect of the activation of cholinergic system for nociception is still unknown in the cortical area. Here, we tested whether the activation of cholinergic receptors can regulate nociceptive behaviors in adult rat anterior cingulate cortex by integrative methods including behavior, immunohistochemical, and electrophysiological methods. Results We found that muscarinic M1 receptors were clearly expressed in the anterior cingulate cortex. Using behavioral tests, we identified that microinjection of a selective muscarinic M1 receptors agonist McN-A-343 into the anterior cingulate cortex dose dependently increased the mechanical threshold. In contrast, the local injection of McN-A-343 into the anterior cingulate cortex showed normal motor function. The microinjection of a selective M1 receptors antagonist pirenzepine blocked the McN-A-343-induced antinociceptive effect. Pirenzepine alone into the anterior cingulate cortex decreased the mechanical thresholds. The local injection of the GABAA receptors antagonist bicuculline into the anterior cingulate cortex also inhibited the McN-A-343-induced antinociceptive effect and decreased the mechanical threshold. Finally, we further tested whether the activation of M1 receptors could regulate GABAergic transmission using whole-cell patch-clamp recordings. The activation of M1 receptors enhanced the frequency of spontaneous and miniature inhibitory postsynaptic currents as well as the amplitude of spontaneous inhibitory postsynaptic currents in the anterior cingulate cortex. Conclusions These results suggest that the activation of muscarinic M1 receptors in part increased the mechanical threshold by increasing GABAergic transmitter release and facilitating GABAergic transmission in the anterior cingulate cortex.

[A Case of Aphasia after Neck Clipping of a Ruptured Aneurysm at the Origin of the Duplicated Middle Cerebral Artery].

We report a case of aphasia after neck clipping of a ruptured aneurysm at the origin of the duplicated middle cerebral artery(DMCA). A 60-year-old woman had a sudden onset of headache and nausea. A computed tomography(CT)scan revealed diffuse subarachnoid hemorrhage. Head three-dimensional CT angiography(3D-CTA)showed a left DMCA with a saccular aneurysm at the origin. She became aphasic on the third day after aneurysmal neck clipping. A CT scan revealed a low-density area in the anterior portion of the left temporal lobe, which is perfused by the DMCA. The DMCA was patent on 3D-CTA, but the angle between the ICA and the DMCA changed steep. It is suspected that the clip changed the branching angle at the DMCA origin, which may have led to decreased blood flow in the DMCA. She received linguistic rehabilitation for dysnomia and was discharged with slight difficulty in naming objects. Six months later, she recovered from the aphasia. One year later, the DMCA was patent on 3D-CTA. We should pay attention to ischemic complications in clipping because DMCAs are easily deformed.

HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus.

P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus.