Transient Receptor Potential Ankyrin 1 in Taste Nerve Contributes to the Sense of Sweet Taste in Mice.

Transient receptor potential (TRP) channels play a significant role in taste perception. TRP ankyrin 1 (TRPA1) is present in the afferent sensory neurons and is activated by food-derived ingredients, such as Japanese horseradish, cinnamon, and garlic. The present study aimed to investigate the expression of TRPA1 in taste buds, and determine its functional roles in taste perception using TRPA1-deficient mice. In circumvallate papillae, TRPA1 immunoreactivity colocalised with P2X2 receptor-positive taste nerves but not with type II or III taste cell markers. Behavioural studies showed that TRPA1 deficiency significantly reduced sensitivity to sweet and umami tastes, but not to salty, bitter, and sour tastes, compared to that in wild-type animals. Furthermore, administration of the TRPA1 antagonist HC030031 significantly decreased taste preference to sucrose solution compared to that in the vehicle-treated group in the two-bottle preference tests. TRPA1 deficiency did not affect the structure of circumvallate papillae or the expression of type II or III taste cell and taste nerve markers. Adenosine 5'-O-(3-thio)triphosphate evoked inward currents did not differ between P2X2- and P2X2/TRPA1-expressing human embryonic kidney 293T cells. TRPA1-deficient mice had significantly decreased c-fos expression in the nucleus of the solitary tract in the brain stem following sucrose stimulation than wild-type mice. Taken together, the current study suggested that TRPA1 in the taste nerve contributes to the sense of sweet taste in mice.

Expression profile of the zinc transporter ZnT3 in taste cells of rat circumvallate papillae and its role in zinc release, a potential mechanism for taste stimulation.

Zinc is an essential trace element, and its deficiency causes taste dysfunction. Zinc accumulates in zinc transporter (ZnT)3-expressing presynaptic vesicles in hippocampal neurons and acts as a neurotransmitter in the central nervous system. However, the distribution of zinc and its role as a signal transmitter in taste buds remain unknown. Therefore, we examined the distribution of zinc and expression profiles of ZnT3 in taste cells and evaluated zinc release from isolated taste cells upon taste stimuli. Taste cells with a spindle or pyriform morphology were revealed by staining with the fluorescent zinc dye ZnAF-2DA and autometallography in the taste buds of rat circumvallate papillae. Znt3 mRNA levels were detected in isolated taste buds. ZnT3-immunoreactivity was found in phospholipase-ß2-immunopositive type II taste cells and aromatic amino acid decarboxylase-immunopositive type III cells but not in nucleoside triphosphate diphosphohydrolase 2-immunopositive type I cells. Moreover, we examined zinc release from taste cells using human transient receptor potential A1-overexpressing HEK293 as zinc-sensor cells. These cells exhibited a clear response to isolated taste cells exposed to taste stimuli. However, pretreatment with magnesium-ethylenediaminetetraacetic acid, an extracellular zinc chelator - but not with zinc-ethylenediaminetetraacetic acid, used as a negative control - significantly decreased the response ratio of zinc-sensor cells. These findings suggest that taste cells release zinc to the intercellular area in response to taste stimuli and that zinc may affect signaling within taste buds.

Intracellular labile zinc is a determinant of vulnerability of cultured astrocytes to oxidative stress.

Recently, we found that treatment of cultured mouse astrocytes of ddY-strain mice (ddY-astrocytes) with 400 µM H2O2 for 24 h increased the intracellular labile zinc level without cell toxicity. In contrast, 170 µM H2O2 for 12 h is reported to kill mouse astrocytes obtained from C57BL/6-strain mice (C57BL/6-astrocytes) with an increase in intracellular labile zinc. To clarify this discrepancy, we compared the intracellular zinc levels and cell toxicity in H2O2-treated ddY- and C57BL/6-astrocytes. Exposure of C57BL/6-astrocytes to 170 or 400 µM H2O2 for 12 h dose-dependently decreased the cell viability and administration of plasma membrane-permeable zinc chelator TPEN prevented the 170 µM H2O2-induced astrocyte death, while neither concentration of H2O2 killed ddY-astrocytes. The intracellular zinc level in H2O2-treated C57BL/6-astrocytes was higher than that in H2O2-treated ddY-astrocytes, and this increase was suppressed by TPEN. There was no apparent difference in the expression levels of zinc transporters ZIPs and ZnTs between the two types of astrocytes, while expression of zinc releasable channel TRPM7 was found on the plasma membrane in ddY-astrocytes, but not in C57BL/6-astrocytes, although the total cellular expression levels were almost the same. In addition, a TRPM7 blocker, 2-aminoethoxydiphenyl borate, increased the intracellular zinc level in H2O2-treated ddY-, but not C57BL/6-astrocytes. Collectively, it is suggested that vulnerability of astrocytes to oxidative stress depends on an increased level of intracellular labile zinc, and TRPM7 localized on the plasma membrane contributes, at least in part, to ameliorate the cell injury by decreasing the zinc level.

Transient receptor potential vanilloid 4 mediates sour taste sensing via type III taste cell differentiation.

Taste buds are comprised of taste cells, which are classified into types I to IV. Transient receptor potential (TRP) channels play a significant role in taste perception. TRP vanilloid 4 (TRPV4) is a non-selective cation channel that responds to mechanical, thermal, and chemical stimuli. The present study aimed to define the function and expression of TRPV4 in taste buds using Trpv4-deficient mice. In circumvallate papillae, TRPV4 colocalized with a type IV cell and epithelial cell marker but not type I, II, or III markers. Behavioural studies showed that Trpv4 deficiency reduced sensitivity to sourness but not to sweet, umami, salty, and bitter tastes. Trpv4 deficiency significantly reduced the expression of type III cells compared with that in wild type (WT) mice in vivo and in taste bud organoid experiments. Trpv4 deficiency also significantly reduced Ki67-positive cells and ß-catenin expression compared with those in WT circumvallate papillae. Together, the present results suggest that TRPV4 contributes to sour taste sensing by regulating type III taste cell differentiation in mice.

Ergothioneine ameliorates oxaliplatin-induced peripheral neuropathy in rats.

AIMS: Oxaliplatin (l-OHP) is a key drug in therapeutic regimens for metastatic or advanced-stage colorectal cancer, but causes peripheral neuropathy as a dose-limiting adverse effect. It is reported that this peripheral neuropathy results from l-OHP accumulation in dorsal root ganglion (DRG) neurons, and that one of the transporters responsible for the accumulation in DRG neurons is organic cation transporter novel (OCTN) 1. Here, we examined whether co-administration of ergothioneine, a substrate/inhibitor of OCTN1, with l-OHP could prevent this peripheral neuropathy. MAIN METHODS: l-OHP (4 mg/kg, i.p., twice/week, for 6 weeks) and ergothioneine or l-carnitine (1.5 or 15 mg/kg, i.v., twice per l-OHP administration) were administered to rats, and tissue/cellular platinum concentrations and peripheral neuropathy were determined. Expression of transporters in DRG neuronal cells was evaluated by real-time PCR and immunocytochemistry. KEY FINDINGS: On administration of l-OHP to rats, it accumulated in DRG neurons and their mitochondria, while negligible accumulation was found in Schwann cells. Expression of OCTN1 was observed in DRG neurons, especially in small- and medium-sized ones, which are responsible for the nociceptive response. In rats repeatedly administered l-OHP, co-administration of ergothioneine (15 mg/kg), but not l-carnitine, a substrate/inhibitor of OCTN2, decreased l-OHP accumulation in DRGs and development of the mechanical allodynia. SIGNIFICANCE: These results indicated that l-OHP-induced peripheral neuropathy was ameliorated by co-administration of ergothioneine, at least in part, via a decrease in its accumulation in DRG neurons. Plant diets contain ergothioneine, and thus their consumption might offer relief to patients suffering from l-OHP-induced peripheral neuropathy.

ATP metabolizing enzymes ENPP1, 2 and 3 are localized in sensory neurons of rat dorsal root ganglion.

In dorsal root ganglion (DRG) neurons, ATP is an important neurotransmitter in nociceptive signaling through P2 receptors (P2Rs) such as P2X2/3R, and adenosine is also involved in anti-nociceptive signaling through adenosine A1R. Thus, the clearance system for adenine nucleotide/nucleoside plays a critical role in regulation of nociceptive signaling, but there is little information on it, especially ectoenzyme expression profiles in DRG. In this study, we examined expression and localization of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPs), by which ATP is metabolized to AMP, in rat DRG. The mRNA expression levels of ENPP2 were greater than those of ENPP1 and ENPP3 in rat DRGs. On immunohistochemical analysis, ENPP1, 2 and 3 were found in soma of DRG neurons. Immunopositive rate of ENPP3 was greater than that of ENPP1 and ENPP2 in all DRG neurons. ENPP3, as compared with ENPP1 and ENPP2, was expressed mainly by isolectin B4-positive cells, and slightly by neurofilament 200-positive ones. In this way, the expression profile of ENPP1, 2 and 3 was different in DRGs, and they were mainly expressed in small/medium-sized DRG neurons. Moreover, ENPP1-, 2- and 3-immunoreactivities were colocalized with P2X2R, P2X3R and prostatic acid phosphatase (PAP), as an ectoenzyme for metabolism from AMP to adenosine. Additionally, PAP-immunoreactivity was colocalized with equilibrative nucleoside transporter (ENT) 1, as an adenosine uptake system. These results suggest that the clearance system consisted of ENPPs, PAP and ENT1 plays an important role in regulation of nociceptive signaling in sensory neurons.

Oxidative stress-induced increase of intracellular zinc in astrocytes decreases their functional expression of P2X7 receptors and engulfing activity.

Neuron-glia communication mediated by neuro- and glio-transmitters such as ATP and zinc is crucial for the maintenance of brain homeostasis, and its dysregulation is found under pathological conditions. It is reported that under oxidative stress-loaded conditions, astrocytes exhibit increased intra- and extra-cellular labile zinc, the latter triggering microglial M1 activation, while the pathophysiological role of the former remains unrevealed. In this study, we examined whether the oxidative stress-induced increase of intracellular labile zinc is involved in the P2X7 receptor (P2X7R)-mediated regulation of astrocytic engulfing activity. The exposure of cultured astrocytes to sub-lethal oxidative stress through their treatment with 400 µM H2O2 increased intracellular labile zinc, of which the concentration reached a peak level of approximately 2 µM at 2 h after the treatment. In astrocytes under sub-lethal oxidative stress, the uptake of YO-PRO-1 and latex beads as markers for P2X7R channel/pore activity and astrocytic engulfing activity, respectively, was decreased, and these decreased activities were accompanied by decreased expression of P2X7R at the plasma membrane via intracellular labile zinc-mediated translocation of it. With the oxidative stress, the expression level of full length P2X7R relative to that of its splice variants in astrocytes was decreased, leading to a decrease of the relative expression of the trimer consisting of full length P2X7R. Collectively, sub-lethal oxidative stress induces an astrocytic modal shift from the normal resting engulfing mode to the activated astrogliosis mode via an intracellular labile zinc-mediated decrease of the functional expression of P2X7R.

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

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.

Bortezomib alters sour taste sensitivity in mice.

Chemotherapy-induced taste disorder is one of the critical issues in cancer therapy. Bortezomib, a proteasome inhibitor, is a key agent in multiple myeloma therapy, but it induces a taste disorder. In this study, we investigated the characteristics of bortezomib-induced taste disorder and the underlying mechanism in mice. Among the five basic tastes, the sour taste sensitivity of mice was significantly increased by bortezomib administration. In bortezomib-administered mice, protein expression of PKD2L1 was increased. The increased sour taste sensitivity induced by bortezomib returned to the control level on cessation of its administration. These results suggest that an increase in protein expression of PKD2L1 enhances the sour taste sensitivity in bortezomib-administered mice, and this alteration is reversed on cessation of its administration.

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.

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.

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±2.46µM. 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 48.3µM, respectively. In contrast, magnesium and manganese at concentrations of up to 1500 and 200µM, 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.

Expression of Prostatic Acid Phosphatase in Rat Circumvallate Papillae.

ATP and its metabolites are important for taste signaling in taste buds, and thus a clearance system for them would play critical roles in maintenance of gustatory function. A previous report revealed that mRNAs for ecto-5'-nucleotidase (NT5E) and prostatic acid phosphatase (PAP) were expressed by taste cells of taste buds, and NT5E-immunoreactivity was detected in taste cells. However, there was no information on PAP-immunoreactivity in taste buds. In this study, we examined the expression profile of PAP in rat taste buds. In the isolated rat taste buds, we detected expression of mRNA for PAP, but NT5E was not detected differing from the case of mouse ones (Dando et al., 2012, J Neuroscience). On immunohistochemical analysis, PAP-immunoreactivity was found predominantly in NTPDase2-positive type I and SNAP25-positive type III taste cells, while there were no apparent signals of it in PLC-ß2-positive type II, α-gustducin-positive type II, AADC-positive type III and 5HT-positive type III ones. As for NT5E, we could not detect its immunoreactivity in rat taste buds, and co-localization of it with any taste cell markers, although mouse taste buds expressed NT5E as reported previously. These findings suggest that PAP expressed by type I and one of type III taste cells of rats may contribute to metabolic regulation of the extracellular levels of adenine nucleotides in the taste buds of circumvallate papillae, and the regulating mechanisms for adenine nucleotides in taste buds might be different between rats and 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.

Characterization of zinc uptake by mouse primary cultured astrocytes and microglia.

To elucidate the regulatory mechanism for extracellular zinc in the CNS, we examined the zinc uptake characteristics in mouse astrocytes and microglia. Zinc was taken up into the two cell types time-dependently, and the cell-to-medium concentration (C/M) ratio in the initial uptake phase in astrocytes was significantly smaller than that in microglia, while in the steady state phase, there was no difference in their C/M ratios. In both astrocytes and microglia, the zinc uptake was mediated, at least in part, by high- and low-affinity systems. There were no differences for both in the Km values for zinc uptake between astrocytes and microglia, and those for the low-affinity system in both cell types were the same as that for mouse ZIP1 reported previously. On the other hand, the Vmax values for both systems were greater in microglia than in astrocytes. Among ZIP isoforms, expression of ZIP1 was high in astrocytes and microglia. Nickel, a competitive inhibitor of ZIP1, and ZIP1 knock-down decreased zinc uptake by both types of cells. Overall, it is demonstrated that astrocytes and microglia had a similar uptake system for zinc including ZIP1, and the differences found in their uptake profiles imply that they play different roles in the regulation of extracellular zinc to maintain brain homeostasis.

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

BACKGROUND: Pregabalin, 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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.

Astrocytes, but not neurons, exhibit constitutive activation of P2X7 receptors in mouse acute cortical slices under non-stimulated resting conditions.

We previously demonstrated that the P2X7 receptor (P2X7R), a purinergic receptor, expressed by mouse cultured cortical astrocytes is constitutively activated without any exogenous stimulus, differing from the case of neurons. It is well known that astrocytic morphology differs between in vitro and in vivo situations, implying different functionalities. Brain acute slices are widely accepted as an in vitro experimental system that reflects in vivo cell conditions better than in vitro cell culture ones. We examined whether astrocytic P2X7Rs exhibited constitutive activation in mouse cortical slices. In acute cortical slices, P2X7R-immunoreactivity was detected in both glial fibrillary acidic protein-immunopositive astrocytes and microtubule-associated protein 2-immunopositive neurons. Astrocytic, but not neuronal, spontaneous uptake of propidium iodide, an indicator of P2X7R channel/pore activity, was inhibited by representative antagonists of P2X7R, but they had no effect on the uptake by astrocytes in membrane-permeabilized fixed slices. These findings indicate that astrocytes, but not neurons, in acute cortical slices exhibit constitutive activation of P2X7Rs under non-stimulated resting conditions as in the case of cell culture systems.

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.

Expression of adenosine A2b receptor in rat type II and III taste cells.

We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 µM adenosine or 10 µM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats.

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.