Hirohito Shiomi

Prostaglandin E2 protects cultured cortical neurons against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity

The effects of prostaglandin (PG) E2 on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to either glutamate or N-methyl-D-aspartate (NMDA) then incubated with normal medium for 1 h. Similar cytotoxicity was observed with the brief application of ionomycin, a calcium ionophore, and S-nitrosocysteine, a nitric oxide (NO)-generating agent. PGE2 at concentrations of 0.01-1 microM dose-dependently ameliorated the glutamate-induced cytotoxicity. PGE1, butaprost, an EP2 receptor agonist, and 8-bromo-cAMP were also effective in protecting cultures against glutamate cytotoxicity. By contrast, neither 17-phenyl-omega-trinor-PGE2, an EP1 receptor agonist, nor M&B 28767, an EP3 receptor agonist, affected glutamate-induced cytotoxicity. NMDA-induced cytotoxicity was ameliorated by PGE2, butaprost, MK-801, N-omega-nitro-L-arginine, a NO synthase inhibitor, and hemoglobin, which binds NO. These agents excluding MK-801 ameliorated the ionomycin-induced cytotoxicity. The cytotoxicity induced by S-nitrosocysteine was prevented only by hemoglobin but not by the other agents including PGE2. These findings indicate that PGE2 protects cultured cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via EP2 receptors. EP2 receptor stimulation may suppress a step in NO formation triggered by Ca(2+)-influx through NMDA receptors.

Attenuation by PACAP of glutamate-induced neurotoxicity in cultured retinal neurons

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs: PACAP27, PACAP38) on glutamate-induced neurotoxicity were examined using cultured retinal neurons obtained from 3- to 5-day old Wistar rats. Cell viability was evaluated by double staining with fluorescein diacetate and propidium iodide. Effects of PACAPs on the increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in retinal neurons was investigated using the Ca(2+) image analyzing system with fura-2. The cAMP contents and the mitogen-activated protein (MAP) kinase activity in retinal cultures were measured by radioimmunoassay. Concomitant application of PACAPs (10 nM-1 microM) with glutamate (1 mM) for 10 min inhibited the delayed death of retinal neurons, which was observed 24 h after glutamate (1 mM) treatment in a dose-dependent manner. Protection by PACAPs (100 nM) against glutamate-induced neurotoxicity was antagonized by PACAP6-38 (1 microM), a PACAP antagonist, and H-89 (1 microM), a protein kinase A (PKA) inhibitor. However, PACAPs did not affect the glutamate-induced increase in [Ca(2+)](i), but PACAPs (1-100 nM) increased the cAMP levels in a dose-dependent manner. In addition, activation of MAP kinase by PACAP38 (1 microM) was inhibited by simultaneous application with H-89 (1 microM). These findings suggest that PACAPs attenuate glutamate-induced delayed neurotoxicity in cultured retinal neurons by activating MAP kinase through the activation of cAMP-stimulated PKA.

Mechanisms of cholecystokinin-induced protection of cultured cortical neurons against N-methyl-d-aspartate receptor-mediated glutamate cytotoxicity

The protective effects of cholecystokinin (CCK) against glutamate-induced cytotoxicity were examined using cultured neurons obtained from the rat cerebral cortex. Cell viability was significantly reduced when the cultures were briefly exposed to glutamate or N-methyl-D-aspartate (NMDA) and then incubated with normal medium for 60 min. A 60-min exposure to kainate also reduced cell viability. CCK protected cortical neurons against glutamate-, NMDA- and kainate-induced cytotoxicity. Glutamate- and NMDA-induced cytotoxicity was also reduced by N omega-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor. However, CCK did not prevent the cytotoxic effects of sodium nitroprusside (SNP) which spontaneously releases NO. Moreover, CCK did not affect NMDA-induced Ca2+ influx measured with rhod-2, a fluorescent Ca2+ indicator. Therefore, release of a NO-like factor from the cerebral cortex was assayed using the thoracic artery in vitro. When the artery was incubated with minced cerebral tissues, glutamate elicited marked relaxation. SNP also elicited relaxation of the smooth muscle. CCK inhibited glutamate-induced relaxation but did not affect that induced by SNP. These results indicate that CCK prevents NMDA receptor-mediated cytotoxicity without reducing the Ca2+ influx. It is suggested that CCK inhibits NO-formation triggered by NMDA receptor activation.

Phase-specific central regulatory systems of hibernation in Syrian hamsters.

The central body temperature (T(b)) regulation system during hibernation was investigated in Syrian hamsters of either sex. Hibernation induced in Syrian hamsters by housing them in a cold room under short day-light/dark cycle was confirmed by marked reductions in the heart rate, T(b) and respiratory rate. The hibernation of hamsters was classified into (i) entrance, (ii) maintenance and (iii) arousal phases according to T(b) changes. In hibernating hamsters, T(b) elevations were phase-selectively elicited by intracerebroventricular (ICV) injection of 8-cyclopenthyltheophylline (CPT; a selective A1-adenosine receptor antagonist) and naloxone (a non-selective opioid receptor antagonist) during the entrance and maintenance phases, respectively. Moreover, a similar T(b) elevation tendency during the maintenance phase was also induced by ICV naloxonazine, (a selective mu1-opioid receptor antagonist), although such was not the case for naltrindole (a selective delta-opioid receptor antagonist) or nor-binaltorphimine (nor-BNI, a selective kappa-opioid receptor antagonist). Furthermore, T(b) elevations in hibernating hamsters were similarly induced with ICV thyrotropin-releasing hormone (TRH) during the entrance and maintenance phases. Furthermore, ICV injection of the anti-TRH antibody ameliorated the T(b) elevations induced by tactile stimulation. These results suggest that activation of the A1-receptor by adenosine is important for the generation of hypothermia in the entrance phase, and that activation of the mu1-opioid receptor by opioid peptides is required for perpetuation of hypothermia in the maintenance phase. In addition, TRH is a key endogenous substance involved in T(b) elevations during the arousal phase of hibernating hamsters.

Involvement of endogenous nitric oxide in the mechanism of bradykinin‐induced peripheral hyperalgesia

1 When NG‐nitro‐L‐arginine methyl ester (L‐NAME, 0.1–10 nmol) or NG‐monomethyl‐L‐arginine (L‐NMMA, 10 nmol‐1 μmol) was intradermally administered with bradykinin (BK, 3 nmol) into the instep of rat hind‐paws, a dose‐related suppression of BK‐induced hyperalgesia, assessed by the paw‐pressure test, was produced. 2 L‐Arginine (1 μmol) but not D‐arginine (1 μmol) reversed the suppressive effects of L‐NAME (10 nmol) and L‐NMMA (1 μmol) on BK‐induced hyperalgesia. 3 Concomitant intradermal administration of BK (3 nmol) with haemoglobin (1 nmol) significantly suppressed BK‐induced hyperalgesia in the paw‐pressure test. The BK‐induced hyperalgesia was abolished by concomitant intradermal administration of either a guanylate cyclase inhibitor, methylene blue (10 nmol), or LY83583 (1 nmol). In addition, KT5823 (1 nmol) or Rp‐8‐bromoguanosine‐3′:5′‐cyclic monophosphothioate (Rp‐8‐Br‐cGMPS; 1 nmol), an inhibitor of cyclic GMP‐dependent protein kinase, also significantly suppressed BK‐induced hyperalgesia. 4 The carrageenin‐induced hyperalgesia was significantly attenuated by L‐NAME in a dose‐dependent manner. 5 L‐Arginine (1 μmol), sodium nitroprusside (1 μmol), dibutyryl cyclic GMP (1 μmol) or 8‐bromo cyclic GMP (1 μmol) all failed to produce any significant relieving effect on the nociceptive threshold of rodent hind‐paws. Concomitant administrations of each agent with a sub‐threshold dose (0.1 nmol) of BK induced significant hyperalgesia. 6 Rp‐adenosine 3′:5′‐cyclic monophosphothioate (Rp‐cAMPS; 1 nmol), an inhibitor of cyclic AMP‐dependent protein kinase, significantly suppressed BK‐induced mechanical hyperalgesia. Concomitant administration of forskolin (1 nmol) with 8‐bromo cyclic GMP (100 nmol) induced significant hyperalgesia. 7 In the superfusion experiment of a blister base on the instep of rodent hind‐paws, intradermally administered BK (3 nmol) significantly increased the outflow of both cyclic GMP and cyclic AMP from the blister base. Concomitant administrations of L‐NAME (10 nmol) with BK significantly reduced the BK‐induced outflow of cyclic GMP without affecting the cyclic AMP content. 8 These results suggest that the NO‐cyclic GMP pathway is involved in the mechanism of BK‐induced hyperalgesia, and an activation of both cyclic GMP‐and cyclic AMP‐second messenger system plays an important role in the production of peripherally induced mechanical hyperalgesia.

Protective effects of vasoactive intestinal peptide against delayed glutamate neurotoxicity in cultured retina

The effects of vasoactive intestinal peptide (VIP) on glutamate-induced delayed death were examined using the primary cultures of rat retinal neurons. Effects of VIP on glutamate-induced neurotoxicity were evaluated by double staining with fluorescein diacetate and propidium iodide. Glutamate (1 mM) was applied to the culture for 10 min in the presence and absence of VIP, and visible cells enumerated 24 h after culture in normal medium. Effects of VIP on increase in the intracellular Ca2+ concentration and currents induced by glutamate in retinal neurons were investigated using the Ca2+ image analyzing system with fura-2 and whole-cell patch-clamp recording, respectively. The cAMP contents in retinal cultures were measured by radioimmunoassay. VIP (10 nM-1 microM) dose-dependently protected against glutamate-induced neurotoxicity in cultured retinal neurons. Protection by VIP (100 nM) against glutamate (1 mM)-induced neurotoxicity was antagonized by VIP6-28 (1 microM), a VIP antagonist, and H-89 (100 nM and 1 microM), a protein kinase A inhibitor. However, VIP had no effect on glutamate-induced inward currents nor glutamate-induced increase in the intracellular Ca2+ concentration. A 10-min exposure of VIP (100 nM) with glutamate (1 mM) resulted in an increase in the cAMP level to 446+/-58 from 22+/-1 pmol/mg protein. These findings suggest that VIP protects against the glutamate-induced neurotoxicity in retinal cultures by elevating the cAMP level via VIP receptors and thereby activating protein kinase A.

Thyrotropin-releasing hormone induced thermogenesis in Syrian hamsters: Site of action and receptor subtype

Early work in our laboratory has revealed the important role played by thyrotropin-releasing hormone (TRH) in the arousal from hibernation in Syrian hamsters. In the present study, we investigated the thermogenic mechanism of TRH in Syrian hamsters. Six to 10 female Syrian hamsters were used in the respective experiments. Intracerebroventricular (icv) injection of TRH elevated the intrascapular brown adipose tissue (IBAT) temperature (T(IBAT)) and rectal temperature (T rec) in Syrian hamsters. Thermogenic response of icv TRH was suppressed by bilateral denervation of the sympathetic nerve. Icv injection of TRH increased the norepinephrin (NE) turnover rate in IBAT without affecting the total serum triiodothyronine (T3) level. Moreover, TRH microinjections into the dorsomedial hypothalamus (DMH), preoptic area (PO), anterior hypothalamus (AH) and ventromedial hypothalamus (VMH) induced T(IBAT) and T(rec) increases. However, neither T(IBAT) nor T rec was affected by similar TRH administrations into the lateral hypothalamus and posterior hypothalamus. Interestingly, although TRH-induced hyperthermia was suppressed by pretreatment of anti-TRH-R1 antibodies, no changes were induced by anti-TRH-R2 antibodies. These results suggest that the sites of action of TRH associated with thermogenesis are probably localized in the DMH, PO, AH and VMH. In addition, TRH-induced thermogenesis is probably elicited by facilitation of the sympathetic nerve system via the central TRH-R1 irrelevant of T3.

Mechanisms of cholecystokinin-induced protection of cultured cortical neurons against receptor-mediated glutamate cytotoxicity

The protective effects of cholecystokinin (CCK) against glutamate-induced cytotoxicity were examined using cultured neurons obtained from the rat cerebral cortex. Cell viability was significantly reduced when the cultures were briefly exposed to glutamate or N-methyl-D-aspartate (NMDA) and then incubated with normal medium for 60 min. A 60-min exposure to kainate also reduced cell viability. CCK protected cortical neurons against glutamate-, NMDA- and kainate-induced cytotoxicity. Glutamate- and NMDA-induced cytotoxicity was also reduced by N omega-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor. However, CCK did not prevent the cytotoxic effects of sodium nitroprusside (SNP) which spontaneously releases NO. Moreover, CCK did not affect NMDA-induced Ca2+ influx measured with rhod-2, a fluorescent Ca2+ indicator. Therefore, release of a NO-like factor from the cerebral cortex was assayed using the thoracic artery in vitro. When the artery was incubated with minced cerebral tissues, glutamate elicited marked relaxation. SNP also elicited relaxation of the smooth muscle. CCK inhibited glutamate-induced relaxation but did not affect that induced by SNP. These results indicate that CCK prevents NMDA receptor-mediated cytotoxicity without reducing the Ca2+ influx. It is suggested that CCK inhibits NO-formation triggered by NMDA receptor activation.

Neuroprotective effects of hibernation-regulating substances against low-temperature-induced cell death in cultured hamster hippocampal neurons.

The neuroprotective effects of hibernation-regulating substances (HRS) such as adenosine (ADO), opioids, histamine and thyrotropin-releasing hormone (TRH) on low-temperature-induced cell death (LTCD) were examined using primary cultured hamster hippocampal neurons. LTCD was induced when cultures were maintained at <22 degrees C for 7 days. ADO (10-100 microM) protected cultured neurons from LTCD in a dose-dependent manner. The neuroprotective effects of ADO were reversed by both 8-cyclopenthyltheophilline (CPT; A(1) receptor antagonist) and 3,7-dimethyl-1-propargylxanthine (DMPX; A(2) receptor antagonist). Morphine (a non-selective opioid receptor agonist) was also effective in attenuating LTCD at an in vitro dose range of 10-100 muM. The neuroprotective effects of morphine were antagonized by naloxone (a non-selective opioid receptor antagonist). In addition, although [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin (DAMGO; mu-opioid receptor agonist), [D-Pen(2,5)]-enkephalin (DPDPE; delta-opioid receptor agonist) and U-69593 (kappa-opioid receptor agonist) were also effective, LTCD of cultured hippocampal neurons was not affected by TRH. Furthermore, histamine produced hypothermia in Syrian hamsters and protected hippocampal neurons in vitro at 100 microM. The neuroprotective effect of histamine was reversed by pyrilamine (H(1) receptor antagonist). Apoptosis was probably involved in LTCD. These results suggest that ADO protected hippocampal neurons in vitro via its agonistic actions on both A(1) and A(2) receptors, whereas morphine probably elicited its neuroprotective effects via agonistic effects on the mu-, delta- and kappa-opioid receptors. In addition, histamine also protected hippocampal neurons via its agonistic action on the H(1) receptor. Thus, HRS-like adenosine-, opioid- and histamine-like hypothermic actions would most likely induce neuroprotective effects against LTCD in vitro.

Delta-sleep-inducing peptide (DSIP) stimulates the release of immunoreactive Met-enkephalin from rat lower brainstem slices in vitro

We studied whether delta-sleep-inducing peptide (DSIP) acted on opioid receptor directly or indirectly. DSIP did not have binding activity to any subtype of opioid receptors. DSIP at doses of 1 pM-1 nM significantly stimulated the release of immunoreactive Met-enkephalin (iME) from superfused slices of the rat lower brainstem. The DSIP-induced release of iME was calcium-dependent. These results show that DSIP acts on opioid receptor indirectly by stimulating the release of iME in producing antinociceptive effects.