Increased expression of cysteinyl leukotriene receptor-1 in the brain mediates neuronal damage and astrogliosis after focal cerebral ischemia in rats.

Cysteinyl leukotrienes are potent pro-inflammatory mediators. Cysteinyl leukotriene receptor 1 is one of the two cysteinyl leukotriene receptors cloned. We recently reported that cysteinyl leukotriene receptor 1 antagonists protected against cerebral ischemic injury, and an inducible expression of cysteinyl leukotriene receptor 1 was found in neuron- and glial-appearing cells after traumatic injury in human brain. To determine the role of cysteinyl leukotriene receptor 1 in ischemic brain injury, we investigated the temporal and spatial profile of cysteinyl leukotriene receptor 1 expression in rat brain from 3 h to 14 days after 30 min of middle cerebral artery occlusion, and observed the effect of pranlukast, a cysteinyl leukotriene receptor 1 antagonist, on the ischemic injury. We found that cysteinyl leukotriene receptor 1 mRNA expression was up-regulated in the ischemic core both 3-12 h and 7-14 days, and in the boundary zone 7-14 days after reperfusion. In the ischemic core, cysteinyl leukotriene receptor 1 was primarily localized in neurons 24 h, and in macrophage/microglia 14 days after reperfusion; while in the boundary zone it was localized in proliferated astrocytes 14 days after reperfusion. Pranlukast attenuated neurological deficits, reduced infarct volume and ameliorated neuron loss in the ischemic core 24 h after reperfusion; it reduced infarct volume, ameliorated neuron loss and inhibited astrocyte proliferation in the boundary zone 14 days after reperfusion. Thus, we conclude that cysteinyl leukotriene receptor 1 mediates acute neuronal damage and subacute/chronic astrogliosis after focal cerebral ischemia.

Unilateral low-frequency stimulation of central piriform cortex delays seizure development induced by amygdaloid kindling in rats.

Low-frequency stimulation of the kindling site interferes with the course of kindling epileptogenesis. The present study examined the effect of unilateral low-frequency stimulation of the central piriform cortex on seizure development induced by amygdaloid kindling in rats. The ipsilateral or contralateral central piriform cortex received low-frequency stimulation (15 min train of 0.1 ms pulses at 1 Hz and 50-150 muA) immediately after termination of once daily kindling stimulation (2 s train of 1 ms pulses at 60 Hz and 150-300 microA) in the right amygdala for 30 days. Low-frequency stimulation of either the ipsilateral or contralateral central piriform cortex significantly suppressed the progression of seizure stages and reduced afterdischarge duration throughout the course of amygdaloid kindling. The marked suppression induced by low-frequency stimulation of the central piriform cortex on either side was predominantly due to the significant retardation of progression from stage 0 to stage 1 and stage 3 to stage 4 seizures. In addition, the suppressive effect of low-frequency stimulation did not disappear when the stimulation was stopped; it could persist for at least 10 days. These findings indicate that brain areas other than the kindling focus, such as the central piriform cortex on both sides, can also be used as reasonable targets for low-frequency stimulation to retard seizure development induced by amygdaloid kindling. Secondly, like the ipsilateral central piriform cortex, the contralateral central piriform cortex may also participate in the progression and secondary generalization of focal seizures. The study suggests that unilateral low-frequency stimulation of the central piriform cortex may have a significant antiepileptogenic effect, and may be helpful for exploring effective and long-lasting therapies for human temporal lobe epilepsy.

NAMPT inhibitor and metabolite protect mouse brain from cryoinjury through distinct mechanisms.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD). In the brain, NAMPT is primarily expressed in neurons and can prevent neuronal degeneration. NAMPT is also highly expressed in inflammatory cells, and is responsible for their activation. Since inflammation following traumatic brain injury enhances neuronal damage, we assessed the effects of nicotinamide mononucleotide (NMN), the direct NAMPT metabolite, and FK866, a potent NAMPT inhibitor, on brain injury in a cryoinjury mouse model. Twenty-four hours after brain cryoinjury, the density of neuron and the level of NAD decreased. Both NMN and FK866 alleviated the neuronal loss and decreased the lesion volume. NMN prevented the cryoinjury-induced decrease of NAD level, and FK866 decreased it further. On day 14 after cryoinjury, further neuronal loss occurred, astrocytes and Iba1-positive macrophage/microglia activated, and the NAD level increased. At this time-point, NAMPT expression was strongly induced in Iba1-positive macrophages/microglia in the lesion core. NMN and FK866 also alleviated the neuronal loss and decreased the lesion volume. In addition, FK866 significantly attenuated the activation of astrocytes and Iba1-positive macrophages/microglia, and decreased the NAD, while NMN had no such effects. Taken together, both FK866 and NMN attenuate traumatic brain injury. However, FK866 acts via the inhibition of the NAMPT activity in inflammatory cells resulting in the inhibition of inflammation, whereas NMN is effective via replenishing NAD.

HAMI 3379, a CysLT2R antagonist, dose- and time-dependently attenuates brain injury and inhibits microglial inflammation after focal cerebral ischemia in rats.

Cysteinyl leukotrienes (CysLTs) induce inflammatory responses by activating their receptors, CysLT1R and CysLT2R. We have reported that CysLT2R is involved in neuronal injury, astrocytosis, and microgliosis, and that intracerebroventricular (i.c.v.) injection of the selective CysLT2R antagonist HAMI 3379 protects against acute brain injury after focal cerebral ischemia in rats. In the present study, we clarified features of the protective effect of intraperitoneally-injected HAMI 3379 in rats. We found that HAMI 3379 attenuated the acute brain injury 24 h after middle cerebral artery occlusion (MCAO) with effective doses of 0.1-0.4 mg/kg and a therapeutic window of ∼1h. It attenuated the neurological deficits, and reduced infarct volume, brain edema, and neuronal loss and degeneration 24 and 72h after MCAO. RNA interference with i.c.v. injection of CysLT2R short hairpin RNA (shRNA) attenuated the acute injury as well. Also, HAMI 3379 inhibited release of the cytokines IL-1ß, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) into the serum and cerebrospinal fluid 24h after MCAO. Moreover, HAMI 3379 ameliorated the microglial activation and neutrophil accumulation in the ischemic regions, but did not affect astrocyte proliferation 72h after MCAO. In comparison, the CysLT1R antagonist pranlukast did not affect microglial activation and IFN-γ release, but inhibited astrocyte proliferation and reduced serum IL-4. Thus, we conclude that HAMI 3379 has a protective effect on acute and subacute ischemic brain injury, and attenuates microglia-related inflammation. CysLT2R antagonist(s) alone or in combination with CysLT1R antagonists may be a novel class of therapeutic agents in the treatment of ischemic stroke.

A mechanistic study of proliferation induced by Angelica sinensis in a normal gastric epithelial cell line.

It has been reported that an extract from Angelica sinensis mainly consisting of polysaccharides (95%) prevented ethanol- or indomethacin-induced gastric mucosal damage (Cho CH et al. Planta Med 2000;66:348-51). However, it is not known whether Angelica sinensis has a direct stimulatory effect on the healing of gastric mucosal lesions. To study the hypothesis that Angelica sinensis has a direct mucosal healing effect in rats and in isolated gastric epithelial cells, we assessed the wound repair in both animals and normal cell culture (RGM-1), as well as [3H]thymidine incorporation, ornithine decarboxylase (ODC) activity, and ODC protein and c-Myc protein expression after different treatments in RGM-1 cells. We found that Angelica sinensis crude extract (ASCE) dose-dependently enhanced gastric ulcer healing in rats and promoted wound repair in RGM-1 cells. It also significantly stimulated [3H]thymidine incorporation and ODC activity in RGM-1 cells in a concentration-dependent manner. ODC and c-Myc protein expression was also increased as a result of this process. DL-alpha-difluoromethyl-ornithine repressed the [3H]thymidine incorporation and ODC activity induced by ASCE. Pretreatment with c-Myc antisense oligodeoxynucleotides blocked the stimulatory action of ASCE on [3H]thymidine incorporation and ODC protein expression. These data suggest that ASCE has a direct mucosal healing effect on gastric epithelial cells, while ODC and c-Myc are closely associated with this effect.

Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes.

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.

Activation of calcium ion-dependent proteinases by bradykinin in dental pulp of the rat.

The present study was aimed to examine whether BANA-degrading enzyme activities could be enhanced by bradykinin(BK) in dental pulp of the rat in vitro. The results showed that BK(0.1-10 microM) dose-dependently enhanced BANA-degrading enzyme activity at pH 7.4. The effects of BK(1 microM) were found to be most effective at both pH 7 and 8, with enhancement of the enzyme activities at a wide range of pH. The BK effects at both the pH were not inhibited by FOY-305(0.1 microM), an inhibitor of trypsin-like enzymes, differing from that at pH 6 in adrenal medulla of the rat. On the other hand, the effects of BK at both the pH were remarkably inhibited by EGTA (2 mM), followed by reversal with calcium ion (2.42 mM). These results suggested as follows: 1) there might be two kinds of BANA-degrading enzymes activated by BK in the pulp. 2) it was conceivable that BANA-degrading enzymes activated by BK were quite different from serine proteinases and were interfered with them in the pulp. 3) calcium ion might play a role in BK-induced enhancement of BANA-degrading enzyme activities which were regarded as met-enkephalin (ME) processing enzyme activities in the pulp.

Enhancement of proteinase activities by bradykinin in adrenal medulla of the rat.

In the present study, a significant positive correlationship was found between the contents of bradykinin (BK)-like and met-enkephalin(ME)-like peptides in adrenal medulla of the rat with cavity-formed incisors in vivo, and the production of ME-like peptides was increased by BK in adrenal medulla of the rat in vitro. Influence of BK on the degradation of BANA, a synthetic substrate for trypsin, by the tissue enzymes was also studied. It was found that BK (0.1-10 microM) enhanced the enzyme activities in a dose-dependent manner, and the effect of BK(1 microM) was most effective at pH 6 and 8. The BK effect was inhibited by FOY-305, an inhibitor of serine proteinases, at pH 6, but not at pH 8. However, E-64, an inhibitor of cysteine proteinases, reduced the BK effects at both pH 6 and 8. These results suggested that 1) BK was an activator for BANA-degrading enzymes which were thought as processing proteinases of ME-like peptides in adrenal medulla of the rat, and 2) there may be, at least, two kinds of BANA-degrading enzymes activated by BK, one might be a serine proteinase with optimal pH at 6, and the others might be cysteine proteinases with optimal pH at both 6 and 8.

[A new non-invasive method for measurement of airway responsiveness in sedated rats].

Sprague-Dawley rats sedated with intraperitoneal injection of diazepam (7.5 mg/kg) were placed in a plethysmograph to measure the changes in spontaneous respiration. Inhalation of methacholine (MCh) or acetylcholine (ACh) aerosol did not alter the volume of breathing, but increased respiratory frequency (RF) to the same extent in a concentration-dependent manner. On the other hand, the tachypnea effect of MCh lasted 11 min, and that of ACh only 3 min. Urethane anesthesia inhibited spontaneous respiration and the response to MCh. Atropine, salbutamol and aminophylline inhibited MCh-induced tachypnea. In sensitized rats, the response to MCh was potentiated 6 h after inhalation of ovalbumin aerosol. The results indicate that sedation with diazepam and inhalation of MCh aerosol used in this report are suitable for measuring airway responsiveness in terms of degree of increase of respiratory frequency.

[Inhibitory effects of tachykinin receptor antagonists on leukotriene C4-induced cardiovascular responses in guinea pigs].

This study is to determine whether sensory neuropeptides are involved in the cardiovascular effects of leukotriene C4 (LTC4). LTC4 (0.8 nmol.kg-1, i.v.) caused hypotensive response and increased Evans blue extravasation from the atria and ventricles in anaesthetized guinea pigs. CP-96345 (2.06 mumol.kg-1, i.v.), a tachykinin NK-1 receptor antagonist, and SR-48968 (1.66 mumol.kg-1, i.v.), an NK-2 receptor antagonist, partially inhibited LTC4-induced increase (46.6% and 37.5%, respectively) of dye extravasation from the atria of guinea pigs. Combination of CP-96345 and SR-48968 markedly inhibited LTC4-induced hypotension and increase of microvascular leakage in both atria and ventricles (58.1% and 54.1%, respectively), similar to the inhibition by ONO-1078 (0.06 mumol.kg-1, i.v.), a specific leukotriene antagonist. These results suggest that NK-1 and NK-2 receptors may be involved in the hypotension and the inflammation of heart induced by LTC4.

[The anti-anaphylactic action of potassium channel openers and its mechanism].

The anti-anaphylactic action of potassium channel openers was studied and reported in this paper. Minoxidil(Min) was shown to inhibit passive cutaneous anaphylaxis in rats. Diazoxide (Dia) and Min were found to inhibit antigen-induced guinea-pig ileum smooth muscle contraction in vitro. Min was shown to antagonize 5-HT-induced capillary permeability in rat skin. Dia was demonstrated to inhibit histamine release from rat peritoneal mast cells induced by A23187 and compound 48/80, but it failed to antagonize guinea-pig ileum smooth muscle contraction induced by histamine in vitro. These results provide evidence that potassium channel openers may be a new group of inhibitors of histamine release and indicate that the mechanism of its anti-anaphylactic action may be related to its potassium channel opening effect. As a result of this effect, Ca2+ influx to the mast cells decreases and Ca2+ release from calcium storage was inhibited, thus inhibiting histamine release.

[Effects of tachykinin receptor antagonists on allergic asthma in guinea pigs].

In conscious sensitized guinea pigs, CP-96345 (2.06 mumol.kg-1, i.p.), a specific antagonist for tachykinin NK-1 receptors, SR-48968 (1.66 mumol.kg-1, i.p.), an NK-2 receptor antagonist, and the combination of both agents decreased the wheezing percentage and the mortality from anaphylactic shock induced by 0.25% ovalbumin (OA, for 0.5 or 2 min) aerosol inhalation. In the anesthetized guinea pigs, SR-48968 attenuated OA (5 mg.kg-1, i.v.)-induced bronchoconstriction, while CP-96345 inhibited OA-induced Evans blue extravasation in bronchi and intrapulmonary airways. In the isolated tracheal and bronchial smooth muscle preparations of guinea pigs, SR-48968 concentration-dependently inhibited OA (10 micrograms.ml-1)-induced contraction both in trachea and in bronchi, while CP-96345 only attenuated the contraction of bronchi. Pretreatment with capsaicin, a depleting agent of sensory neuropeptides from sensory nerve C-fibers, attenuated the OA-induced contractions both in trachea and in bronchi. The results indicate that (1) tachykinins in the airways are involved in the pathogenesis of allergic asthma; (2) tachykinin receptor antagonists have inhibitory effects on the allergic asthmatic responses, which is at least partly through the inhibition of antigen-induced contraction of airway smooth muscles (NK-2 receptor effect) and airway microvascular leakage (NK-1 receptor effect).

[Effect of a leukotriene antagonist, ONO-1078, on electric stimulation of vagus (ESV)-induced bronchoconstriction and microvascular leakage in guinea pigs].

In atropine-pretreated guinea pigs, electric stimulation of vagus (ESV, 10 Hz, 5 ms, 2 V or 10 V, for 90 s) increased intrapulmonary pressure (IPP), and Evans blue extravasation in trachea, main bronchi, peripheral and distal intrapulmonary airways in a voltage-dependent manner. ONO-1078, a noval leukotriene antagonist, (0.03 and 0.1 mg.kg-1, iv) showed no remarkable inhibiting effect on ESV-induced increase of IPP. However, the agent significantly inhibited ESV-induced increase of Evans blue extravasation in the airways, especially in lower potency of stimulation (2 V). The results suggest that leukotrienes may be involved in airway microvascular leakage in response to neurogenic inflammation.

[Protective effect of ONO-1078, a leukotriene antagonist, on focal cerebral ischemia in mice].

AIM: To determine whether ONO-1078 [pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy) benzoyl-amino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate], a potent leukotriene antagonist, has protective effect on focal cerebral ischemia in mice. METHODS: Focal cerebral ischemia was induced by permanent middle cerebral artery (MCA) occlusion in mice. ONO-1078 (0.01, 0.05, 0.10 mg.kg-1), dexamethasone (0.5 mg.kg-1), nimodipine (0.2 mg.kg-1) or saline (control) were injected i.p. once daily for 3 days, and 30 min before MCA occlusion. Twenty-four hours after cerebral ischemia, the neurological scores were evaluated, infarct volumes and areas of the right and left cerebral hemispheres were measured by computer imaging analysis. RESULTS: ONO-1078, dexamethasone and nimodipine reduced the neurological scores. ONO-1078 and dexamethasone reduced the ratio of right/left hemisphere area, indicating inhibition of brain edema, while nimodipine showed no effect. ONO-1078 dose-dependently reduced infarct size, and dexamethasone and nimodipine showed the same effect. CONCLUSION: ONO-1078 showed protective effect on focal cerebral ischemia. This may represent a novel approach to the treatment of acute cerebral ischemia.

[Inhibition of chemically induced microvascular leakage by ONO-1078, a leukotriene antagonist, in rat skin].

This study is to determine whether ONO-1078, a potent leukotriene antagonist, influences chemically induced rat skin microvascular leakage which is considered to be, at least in part, due to stimulation of sensory nerve ending and release of sensory neuropeptides. Evans blue dye was used as a tracer for plasma leakage. Intradermal injections of chemical stimuli, histamine (10 micrograms), capsaicin (10 micrograms) and formalin (0.5 mg), evoked Evans blue dye extravasation in rat skin. Intraperitoneal ONO-1078 dose-dependently inhibited the dye extravasation induced by these stimuli, with ID5v values of 1.98 mg.kg-1 for histamine, 1.78 mg.kg-1 for capsaicin, and 2.23 mg.kg-1 for formalin. In contrast to chlorpheniramine, a H1 receptor antagonist, the inhibitory effect of ONO-1078 was weaker on histamine, but more potent on capsaicin and formalin. The inhibitory effect of dexamethasone was more potent than that of ONO-1078 on these stimuli. On the other hand, ONO-1078 inhibited the dye extravasation induced by leukotriene D4 (0.05 micrograms), but showed no effect on those induced by substance P (0.5 micrograms, a sensory neuropeptide), a larger dose of histamine (100 micrograms), and bradykinin (1 microgram). These results suggest that inhibition of chemically induced skin microvascular leakage by ONO-1078 may be mediated by inhibiting the release of sensory neuropeptides from capsaicin-sensitive sensory fibers.

[Effect of SR-140333, a tachykinin NK-1 antagonist, on antigen-induced airway hyperresponsiveness in sensitized rats].

In the present study, the effects of SR-140333, ((S)-1-(2-[3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin- 3yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane.chloride), a nonpeptide antagonist for tachykinin NK-1 receptor, on the antigen-induced airway response to methacholine (MCh) aerosol and airway inflammation in sensitized SD rats were investigated. The baseline respiratory frequencies, tachypnea response to methacholine(MCh), the -log PC30 values of MCh and the leukocyte counts in bronchoalveolar lavage significantly increased after inhalation of 1% oval albumin(OA) aerosol. SR-140333 (152 nmol.kg-1, i.p.) or dexamethasone(368 nmol.kg-1, i.p.), bid x 3 d inhibited these responses. SR-140333 at a low dose of 0.01 mg.kg-1 showed an incomplete inhibition. From these results, we conclude that antigen challenge causes airway hyperresponsiveness and airway inflammation and that tachykinin NK-1 receptor antagonist inhibits these responses.

The new P2Y-like receptor G protein-coupled receptor 17 mediates acute neuronal injury and late microgliosis after focal cerebral ischemia in rats.

G protein-coupled receptor 17 (GPR17), the new P2Y-like receptor, is phylogenetically related to the P2Y and cysteinyl leukotriene receptors, and responds to both uracil nucleotides and cysteinyl leukotrienes. GPR17 has been proposed to be a damage sensor in ischemic stroke; however, its role in brain inflammation needs further detailed investigation. Here, we extended previous studies on the spatiotemporal profiles of GPR17 expression and localization, and their implications for brain injury after focal cerebral ischemia. We found that in the ischemic core, GPR17 mRNA and protein levels were upregulated at both 12-24 h and 7-14 days, but in the boundary zone the levels increased 7-14 days after reperfusion. The spatiotemporal pattern of GPR17 expression well matched the acute and late (subacute/chronic) responses in the ischemic brain. According to previous findings, in the acute phase, after ischemia (24 h), upregulated GPR17 was localized in injured neurons in the ischemic core and in a few microglia in the ischemic core and boundary zone. In the late phase (14 days), it was localized in microglia, especially in activated (ED1-positive) microglia in the ischemic core, but weakly in most microglia in the boundary zone. No GPR17 was detectable in astrocytes. GPR17 knockdown by a small interfering RNA attenuated the neurological dysfunction, infarction, and neuron loss at 24 h, and brain atrophy, neuron loss, and microglial activation at 14 days after reperfusion. Thus, GPR17 might mediate acute neuronal injury and late microgliosis after focal cerebral ischemia.

Cysteinyl leukotriene receptor 2 is spatiotemporally involved in neuron injury, astrocytosis and microgliosis after focal cerebral ischemia in rats.

Cysteinyl leukotrienes (CysLTs), potent inflammatory mediators, are released from ischemic brain, and may regulate ischemic injury through activating CysLT1 and CysLT2 receptors. The CysLT1 receptor is closely associated with ischemic injury and post-ischemic repair; however, the CysLT2 receptor-mediated responses remain unknown. Here, we investigated the spatiotemporal profiles and implications of CysLT2 receptor expression and localization in rat brain after focal cerebral ischemia. CysLT2 receptors were normally localized in astrocytes in the cortex and around the ventricles. After focal cerebral ischemia, CysLT2 receptor expression was up-regulated in concert with neuronal and glial responses. In the acute phase (6-24 h), up-regulated CysLT2 receptors were restricted to injured neurons in the ischemic core; while in the late phase (3-28 days), the up-regulation was restricted to hypertrophic microglia (ischemic core) and mainly localized in hypertrophic astrocytes (boundary zone). Thus, the spatiotemporal profiles of CysLT2 receptor expression suggest that it plays regulatory roles in acute neuron injury, and astrocytosis and microgliosis in the late phase.

Haemodynamic brain response to visual sexual stimuli is different between homosexual and heterosexual men.

The underlying neurobiological factors involved in sexual orientation are largely unknown. This study investigated whether neural circuits or different cognitive processes accounted for differences in brain activation in 14 heterosexual and 14 homosexual males. Brain scans were undertaken in each subject using functional magnetic resonance imaging while they viewed different sexual stimuli, i.e. heterosexual couple stimuli (HCS), gay couple stimuli (GCS), lesbian couple stimuli (LCS) and neutral stimuli (NS). Ratings of sexual attractiveness of the stimuli were assessed. Subjective sexual arousal was induced by HCS and GCS in heterosexual and homosexual men, respectively. Sexual disgust was induced by GCS and LCS in heterosexual and homosexual men, respectively. Compared with viewing NS, viewing sexual stimuli induced significantly different brain activations, most of which had the characteristics of cognitive processes. These observations suggest that different cognitive patterns may be the major cause of different subjective responses to sexual stimuli between heterosexual and homosexual men.