Akihiro Ohishi

Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death.

J. Neurochem. (2012) 122, 1118–1128.

P2X7 receptors regulate engulfing activity of non-stimulated resting astrocytes.

We previously demonstrated that P2X7 receptors (P2X7Rs) expressed by cultured mouse astrocytes were activated without any exogenous stimuli, but its roles in non-stimulated resting astrocytes remained unknown. It has been reported that astrocytes exhibit engulfing activity, and that the basal activity of P2X7Rs regulates the phagocytic activity of macrophages. In this study, therefore, we investigated whether P2X7Rs regulate the engulfing activity of mouse astrocytes. Uptake of non-opsonized beads by resting astrocytes derived from ddY-mouse cortex time-dependently increased, and the uptaken beads were detected in the intracellular space. The bead uptake was inhibited by cytochalasin D (CytD), an F-actin polymerization inhibitor, and agonists and antagonists of P2X7Rs apparently decreased the uptake. Spontaneous YO-PRO-1 uptake by ddY-mouse astrocytes was reduced by the agonists and antagonists of P2X7Rs, but not by CytD. Down-regulation of P2X7Rs using siRNA decreased the bead uptake by ddY-mouse astrocytes. In addition, compared to in the case of ddY-mouse astrocytes, SJL-mouse astrocytes exhibited higher YO-PRO-1 uptake activity, and their bead uptake was significantly greater. These findings suggest that resting astrocytes exhibit engulfing activity and that the activity is regulated, at least in part, by their P2X7Rs.

Regulation of activity of P2X7 receptor by its splice variants in cultured mouse astrocytes

Of purinergic receptors, P2X7 receptor (P2X7R, defined as a full‐length receptor) has unique characteristics, and its activation leads to ion channel activity and pore formation, causing cell death. Previously, we demonstrated that P2X7R expressed by nonstimulated astrocyte cultures obtained from SJL‐strain mice exhibits constitutive activation, implying its role in maintenance of cellular homeostasis. To obtain novel insights into its physiological roles, we examined whether constitutive activation of P2X7R is regulated by expression of its splice variants in such resting astrocytes, and whether their distinct expression profiles in different mouse strains affect activation levels of astrocytic P2X7Rs. In SJL‐ and ddY‐mouse astrocytes, spontaneous YO‐PRO‐1 uptake, an indicator of pore activity of P2X7R, was detected, but the uptake by the formers was significantly greater than that by the latter. Between the two mouse strains, there was a difference in their sensitivity of YO‐PRO‐1 uptake to antagonists, but not in the expression levels and sequences of P2X7R and pannexin‐1. Regarding expression of splice variants of P2X7R, expression of P2X7R variant‐3 (P2X7R‐v3) and ‐4 (P2X7R‐v4), but not variant‐2 and ‐k, was lower in SJL‐mouse astrocytes than in ddY‐mouse ones. On transfection of P2X7R‐v3 and ‐v4 into SJL‐mouse astrocytes, the pore activity was attenuated as in the case of the HEK293T cell‐expression system. These findings demonstrate that basal activity of P2X7R expressed by resting astrocytes is negatively regulated by P2X7R‐v3 and ‐v4, and that their distinct expression profiles result in the different activation levels of astrocytic P2X7Rs in different mouse strains. GLIA 2014;62:440–451

Species Difference in Sensitivity of Human and Mouse P2X7 Receptors to Inhibitory Effects of Divalent Metal Cations

P2X7 receptor (P2X7R), a purinergic receptor, is involved in pathophysiological events such as inflammation and cell death, and thus is an attractive target for therapeutic approaches. It is reported that divalent metal cations (DMCs) inhibit P2X7R activation and that there are species differences in their inhibitory effects. To extrapolate the findings in experimental animals to humans, these species differences have to be clarified, but species differences in the sensitivity of P2X7R to DMCs between man and mouse have not been demonstrated. Here we performed direct comparison of the inhibitory effects of DMCs on human and mouse P2X7R activation. Cell lines constitutively expressing human and mouse P2X7R were used, and their P2X7R activation was evaluated as means of YO-PRO-1 dye uptake. MgCl2, NiCl2, ZnCl2, CuCl2 and CaCl2 dose-dependently decreased agonist-induced YO-PRO-1 uptake via both human and mouse P2X7Rs. Apparent differences in the inhibitory profiles for NiCl2 and CaCl2 between them were found, and the IC50 values of DMCs were in the order of CaCl2>MgCl2>NiCl2≈ZnCl2>CuCl2 for both human and mouse P2X7Rs. In this study, we demonstrate that human P2X7R exhibits different sensitivity to nickel and calcium compared with the case of the mouse one, while there is no species difference in the sensitivity of their P2X7Rs to magnesium, zinc and copper, suggesting that the effects of magnesium, zinc and copper on P2X7R-associated pathophysiological events in humans might be predicted from those in mice.

Oxaliplatin Alters Expression of T1R2 Receptor and Sensitivity to Sweet Taste in Rats

As one of the adverse effects of oxaliplatin, a key agent in colon cancer chemotherapy, a taste disorder is a severe issue in a clinical situation because it decreases the quality of life of patients. However, there is little information on the mechanism underlying the oxaliplatin-induced taste disorder. Here, we examined the molecular and behavioral characteristics of the oxaliplatin-induced taste disorder in rats. Oxaliplatin (4-16 mg/kg) was administered to Sprague-Dawley (SD) rats intraperitoneally for 2 d. Expression levels of mRNA and protein of taste receptors in circumvallate papillae (CP) were measured by real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry, respectively. Taste sensitivity was assessed by their behavioral change using a brief-access test. Morphological change of the taste buds in CP was evaluated by hematoxyline-eosin (HE) staining, and the number of taste cells in taste buds was counted by immunohistochemical analysis. Among taste receptors, the expression levels of mRNA and protein of T1R2, a sweet taste receptor subunit, were increased transiently in CP of oxaliplatin-administered rats on day 7. In a brief-access test, the lick ratio was decreased in oxaliplatin-administered rats on day 7 and the alteration was recovered to the control level on day 14. There was no detectable alteration in the morphology of taste buds, number of taste cells or plasma zinc level in oxaliplatin-administered rats. These results suggest that decreased sensitivity to sweet taste in oxaliplatin-administered rats is due, at least in part, to increased expression of T1R2, while these alterations are reversible.

Opioid analgesics increase incidence of somnolence and dizziness as adverse effects of pregabalin: a retrospective study

BackgroundPregabalin, a gabapentinoid, is an adjuvant analgesic for treatment of neuropathic pain, but it has serious adverse effects such as somnolence and dizziness, particularly in elderly patients. Although decreased renal function is considered to the contributing factor for high frequency of these adverse effects in elder patients, only a few systematic clinical investigations, especially for hospitalized patients, have been performed on factors that might affect the incidence of its adverse effects. In this study, we performed a retrospective study on the effect of concomitant drugs on induction of somnolence and dizziness as adverse effects of pregabalin in hospitalized patients.MethodsThe subjects were all pregabalin-administered patients in Shiga University of Medical Science Hospital from September 2010 to September 2012, and the subject number was 195. Multivariate logistic regression analysis was performed to determine predictors of the adverse effects, creatinine clearance, duration of pregabalin therapy, initial and maintenance doses of pregabalin, and concomitant drugs, including hypoglycemic drugs, anti-hypertensive ones, non-steroidal anti-inflammatory ones, opioids and central nervous system depressants, being used as independent variables.ResultsThe median initial doses of pregabalin in each renal function group were the same with the case of the defined dose. Although renal function is a well-known factor for prediction of development of adverse effects of pregabalin, we did not detect significant contribution of it. Alternatively, it was demonstrated that concomitant administration of opioids was the significant factor of the incidence of somnolence and dizziness. The first onset date of the adverse effects was frequently detected in the early days of the pregabalin therapy.ConclusionsThe fine tuning of pregabalin dosage schedule based on the renal function appeared to be critical for prevention of development of its adverse effects. Adverse effects tended to develop in the initial phase of pregabalin therapy. Concomitant administration of opioids with pregabalin has the potential to increase the incidence of adverse effects, and thus much more careful attention has to be paid especially in those situations.

The effect of divalent metal cations on zinc uptake by mouse Zrt/Irt-like protein 1 (ZIP1)

AIM In this study, we evaluated the effect of divalent metal cations on zinc uptake via mouse Zrt/Irt-like protein 1 (mZIP1), a ubiquitously expressed zinc transporter, which plays a role in maintenance of cellular zinc homeostasis, using HEK293T cells overexpressing it. MAIN METHODS mZIP1 cDNA, which was cloned from mouse microglia, was transfected into HEK293T cells by a lipofection method, and its functional expression was confirmed by Western blotting and immunocytochemical analyses, and (65)Zn ((65)ZnCl2) uptake. KEY FINDINGS (65)Zn uptake by mZIP1 cDNA-transfected cells time-dependently increased compared with that by mock cells, indicating functional expression of mZIP1. mZIP1-mediated (65)Zn uptake showed clear saturable kinetics consisting of a single component with a Michaelis constant of 5.88 μM. FeCl2 and NiCl2 competitively inhibited the (65)Zn uptake, the inhibition constants (Ki) being estimated to be 0.92 and 28.6 μM, respectively. In addition, CoCl2 and CdCl2 showed non-competitive inhibition of mZIP1-mediated (65)Zn uptake, the Ki values being 219 and 32.5 μM, respectively. On the other hand, CuCl2 also significantly decreased the uptake, but the inhibition mode could not evaluate because of its low solubility, while MnCl2 and MgCl2 had no effect on (65)Zn uptake via mZIP1. SIGNIFICANCE Iron, nickel, cobalt and cadmium act as inhibitors of mZIP1, the affinity order being iron>zinc>nickel=cadmium>cobalt, and copper might also act as an inhibitor, while manganese and magnesium are not recognized by mZIP1. These findings provide valuable information as to the contribution of mZIP1 to total cellular zinc transport.

Liposomalization of oxaliplatin induces skin accumulation of it, but negligible skin toxicity

&NA; Liposomalization causes alteration of the pharmacokinetics of encapsulated drugs, and allows delivery to tumor tissues through passive targeting via an enhanced permeation and retention (EPR) effect. PEGylated liposomal doxorubicin (Doxil®, Lipo‐DXR), a representative liposomal drug, is well‐known to reduce cardiotoxicity and increase the anti‐tumor activity of DXR, but to induce the hand‐foot syndrome (HFS) as a result of skin DXR accumulation, which is one of its severe adverse effects. We have developed a new liposomal preparation of oxaliplatin (l‐OHP), an important anti‐tumor drug for treatment of colorectal cancer, using PEGylated liposomes (Lipo‐l‐OHP), and showed that Lipo‐l‐OHP exhibits increased anti‐tumor activity in tumor‐bearing mice compared to the original preparation of l‐OHP. However, whether Lipo‐l‐OHP causes HFS‐like skin toxicity similar to Lipo‐DXR remains to be determined. Administration of Lipo‐l‐OHP promoted accumulation of platinum in rat hind paws, however, it caused negligible morphological and histological alterations on the plantar surface of the paws. Administration of DiI‐labeled empty PEGylated liposomes gave almost the same distribution profile of dyes into the dermis of hind paws with DXR as in the case of Lipo‐DXR. Treatment with Lipo‐l‐OHP, Lipo‐DXR, DiI‐labeled empty PEGylated liposomes or empty PEGylated liposomes caused migration of CD68+ macrophages into the dermis of hind paws. These findings suggest that the skin toxicity on administration of liposomalized drugs is reflected in the proinflammatory characteristics of encapsulated drugs, and indicate that Lipo‐l‐OHP with a higher anti‐cancer effect and no HFS may be an outstanding l‐OHP preparation leading to an improved quality of life of cancer patients. Graphical abstract Figure. No caption available. HighlightsPEGylated liposomal oxaliplatin induced negligible skin toxicity on rat hind paws.Hand‐foot syndrome‐like skin toxicity was caused by PEGylated liposomal doxorubicin.PEGylated liposomes distributed into rat hind paw skin tissues, especially dermis.Macrophages migrated to dermis to engulf the drug‐ and vehicle‐containing liposomes.The skin toxicity depends on proinflammatory characteristics of encapsulated drugs.

Prophylactic Oral Administration of Magnesium Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice through a Decrease of Colonic Accumulation of P2X7 Receptor-Expressing Mast Cells

The number of patients with colitis has been increasing year by year. Recently, intestinal inflammation, as one of the factors for its onset, has been demonstrated to be induced by P2X7 receptor-mediated activation of colonic immune cells such as mast cells. Activation of P2X7 receptor (P2X7R) is known to be inhibited by divalent metal cations such as magnesium, but whether or not magnesium administration prevents/relieves colitis is unknown so far. Here, we report that oral (per os (p.o.)) administration of MgCl2 and ingestion of commercially available magnesium-rich mineral hard water relieves dextran sulfate sodium (DSS)-induced colitis in mice. Colitis was induced through ingestion of a 3% (w/v) DSS solution ad libitum for 10 d. Brilliant blue G (BBG, a P2X7R antagonist), MgCl2 or magnesium-rich mineral hard water was administered p.o. to mice via gastric intubation once a day or ad libitum from a day before DSS administration for 11 times or 11 d, respectively. DSS-treated mice exhibited a low disease activity index, a short colon and a high histological score compared to in control mice. As BBG (250 mg/kg, p.o.), administration of a MgCl2 solution (100 or 500 mg/kg, p.o.) and ad libitum ingestion of the magnesium-rich mineral hard water (212 ppm as magnesium) partially, but significantly, attenuated the severity of colitis by decreasing the accumulation of P2X7R-immunopositive mast cells in the colon. Therefore, prophylactic p.o. administration/ingestion of magnesium is considered to be partially effective to protect mice against DSS-induced colitis by inhibiting P2X7R-mediated activation/accumulation of colonic mast cells.

Inhibitory effect of divalent metal cations on zinc uptake via mouse Zrt-/Irt-like protein 8 (ZIP8)

Aims: There is controversy regarding the substrate specificity of ZIP8, a ZIP isoform, involved in regulation of extra‐ and intracellular zinc levels. Here, we investigated the inhibitory effects of divalent metal cations on zinc uptake via mouse ZIP8 (mZIP8). Main methods: mZIP8 cDNA was transfected into HEK293T cells by a lipofection method, and its functional expression was evaluated by immunocytochemistry, Western blotting and 65Zn (65ZnCl2) uptake measurement. Key findings: Transfection of mZIP8 cDNA into HEK293T cells induced expression of mZIP8 in the cells, and increased zinc uptake. mZIP8‐mediated zinc uptake depended on extracellular bicarbonate, and the Michaelis constant for the uptake was estimated to be 8.48 ± 2.46 &mgr;M. In the inhibition study, iron and cadmium competitively, and cobalt, nickel and copper non‐competitively inhibited the mZIP8‐mediated zinc uptake, the inhibition constants being calculated to be 3.37, 55.5, 80.6, 198 and 48.3 &mgr;M, respectively. In contrast, magnesium and manganese at concentrations of up to 1500 and 200 &mgr;M, respectively, had no inhibitory effect on the zinc uptake via mZIP8. Significance: In this study, we reveal that the inhibition profiles of divalent metal cations as to zinc uptake via mZIP8 apparently differ from those for mZIP1, especially in the affinity and inhibition manner of nickel. These findings should contribute to identification of ZIP isoforms involved in total cellular zinc transport.