Aberrant TRPV1 expression in heat hyperalgesia associated with trigeminal neuropathic pain.

Trigeminal neuropathic pain is a facial pain syndrome associated with trigeminal nerve injury. However, the mechanism of trigeminal neuropathic pain is poorly understood. This study aimed to determine the role of transient receptor potential vanilloid 1 (TRPV1) in heat hyperalgesia in a trigeminal neuropathic pain model. We evaluated nociceptive responses to mechanical and heat stimuli using a partial infraorbital nerve ligation (pIONL) model. Withdrawal responses to mechanical and heat stimuli to vibrissal pads (VP) were assessed using von Frey filaments and a thermal stimulator equipped with a heat probe, respectively. Changes in withdrawal responses were measured after subcutaneous injection of the TRP channel antagonist capsazepine. In addition, the expression of TRPV1 in the trigeminal ganglia was examined. Mechanical allodynia and heat hyperalgesia were observed in VP by pIONL. Capsazepine suppressed heat hyperalgesia but not mechanical allodynia. The number of TRPV1-positive neurons in the trigeminal ganglia was significantly increased in the large-diameter-cell group. These results suggest that TRPV1 plays an important role in the heat hyperalgesia observed in the pIONL model.

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts.

OBJECTIVE: Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E2 (PGE2) are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE2 by HGFs were examined. METHODS: HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE2 levels were evaluated by ELISA. RESULTS: H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE2 production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE2 production. Up to 10 µg/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at 100 µg/ml. Similarly, 0.01 µg/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at concentrations of 0.1 and 1 µg/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE2 production. CONCLUSION: These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE2 production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 µg/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.

A novel animal model of hearing loss caused by acute endoplasmic reticulum stress in the cochlea.

Many stimuli such as ischemia, hypoxia, heat shock, amino acid starvation, and gene mutation, exhibit a cellular response called endoplasmic reticulum (ER) stress. ER stress induces expression of a series of genes, leading to cell survival or apoptosis. Previously, we found that in an animal model of hearing loss caused by acute mitochondrial dysfunction, several ER stress markers including C/EBP homologous protein were induced in the cochlear lateral wall. To elucidate the mechanism of hearing loss caused by ER stress, we established a novel animal model of hearing loss by perilymphatic perfusion of tunicamycin, an ER stress activator that inhibits N-acetylglucosamine transferases. Subacute and progressive hearing loss was observed at all sound frequencies studied, and stimulation of ER stress marker genes was noted in the cochlea. The outer hair cells were the most sensitive to ER stress in the cochlea. Electron microscopic analysis demonstrated degeneration of the subcellular organelles of the inner hair cells and nerve endings of the spiral ganglion cells. This newly established animal model of hearing loss from ER stress will provide additional insight into the mechanism of sensorineural hearing loss.

Pharmacological evidences for the stimulation of calcium-sensing receptors by nifedipine in gingival fibroblasts.

OBJECTIVE: To investigate pharmacologically whether CaSRs are involved in the Ca(2+) antagonist-induced [Ca(2+)]i elevation in gingival fibroblasts. MATERIALS AND METHODS: Gin-1 cells, normal human gingival fibroblasts, were used as the material. The [Ca(2+)] i was measured with fura-2/AM, a Ca(2+)-sensitive fluorescent dye. RESULTS: At first, we confirmed the existence of CaSRs in these cells by showing that [Ca(2+)] i was elevated by high concentrations of extracellular Ca(2+) and by prototypic agonists of the CaSR such as gentamicin. The action of gentamicin was antagonized by inhibitors of phospholipase C (PLC), inositol trisphosphate (IP(3)) receptors, NSCCs, and, importantly, by the CaSR antagonist, NPS2390. Furthermore, the action of gentamicin was potentiated by activators of PLC and protein kinase C (PKC). This confirmed the pathway components mediating Ca(2+) responses to a known agonist of the CaSR. We then investigated whether nifedipine (an L-type Ca(2+) channel blocker) stimulates CaSRs to elevate [Ca(2+)] i via a similar mechanism. Nifedipine Ca(2+) responses were dose-dependently blocked by NPS2390 and by the same inhibitors of PLC, IP(3) receptors, and NSCCs that disrupted the action of gentamicin. Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca(2+) release from stores) also inhibited the nifedipine-induced [Ca(2+)] i elevation. CONCLUSION: These findings suggest that CaSRs are involved in the nifedipine-induced [Ca(2+)] i elevation in gingival fibroblasts.

Preventive effects of a kampo medicine, orento on inflammatory responses in lipopolysaccharide treated human gingival fibroblasts.

In the present study, we investigated the effects of a Kampo medicine Orento (TJ-120) on the production of prostaglandin E(2) (PGE(2)), interleukin (IL)-6 and IL-8 by human gingival fibroblasts (HGFs) treated with lipopolysaccharide from Porphyromonas gingivalis (PgLPS). HGFs proliferation was dose-dependently decreased with Orento at days 3 and 7. However, treatment with PgLPS (10 ng/ml), Orento (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Orento suppressed PgLPS-induced PGE(2) production in a dose-dependent manner but did not alter basal PGE(2) level. In contrast, Orento did not alter PgLPS-induced IL-6 and IL-8 productions. These alterations by Orento were similar to those by a mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059. A Orento showed no effect on cyclooxygenase (COX)-1 and COX-2 activities, and increased cytoplasmic phospholipase A(2) (cPLA(2)) expression and increased PgLPS-induced COX-2 expression. Orento suppressed PgLPS-induced mobility retardation of cPLA(2) band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, that is cPLA(2) phosphorylation and its activation, while Orento alone did not alter cPLA(2) phosphorylation. Orento suppressed PgLPS-induced extracellular signal-regulated kinase (ERK) phosphorylation, which is known to lead to ERK activation and cPLA(2) phosphorylation. These results suggest that Orento decreased PGE(2) production by inhibition of cPLA(2) phosphorylation and its activation via inhibition of ERK phosphorylation, and also that Orento may be useful to improve gingival inflammation in periodontal disease.

Enhanced expression of C/EBP homologous protein (CHOP) precedes degeneration of fibrocytes in the lateral wall after acute cochlear mitochondrial dysfunction induced by 3-nitropropionic acid.

We previously reported that treatment of the rat cochlea with a mitochondrial toxin, 3-nitropropionic acid (3-NP), causes temporary to permanent hearing loss depending on the amount of the drug. Furthermore, apoptosis of cochlear lateral wall fibrocytes, which are important for maintaining the endolymph, is a predominant pathological feature in this animal model. 3-NP is known to induce oxidative stress as well as neuronal apoptosis. C/EBP homologous protein gene (chop) is one of the marker genes induced during endoplasmic reticulum (ER) stress, and is also considered to be involved in apoptosis. To elucidate the molecular mechanism of cochlear fibrocyte apoptosis induced by 3-NP, we studied spatiotemporal expression of C/EBP homologous protein (CHOP) and other signaling molecules related to ER stress as well as the appearance of apoptotic cells in the cochlear lateral wall after 3-NP treatment. Quantitative real-time PCR revealed that chop and activating transcription factor 4 gene (atf-4) showed marked increase within 6h, whereas expression of other ER stress-responsive genes such as grp78 and grp94 did not change. Immunohistochemistry showed that 3-NP treatment caused up-regulation of CHOP, especially in type II and type IV fibrocytes, followed by the appearance of terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL)-positive apoptotic cells in the same confined area. Thus, apoptosis of lateral wall fibrocytes induced by 3-NP is likely to be mediated by induction of CHOP. These results contribute clarification of pathological mechanism of cochlear fibrocytes and may lead to development of novel therapeutic strategy for hearing loss.

Preventive effects of a Kampo medicine, Shosaikoto, on inflammatory responses in LPS-treated human gingival fibroblasts.

In the present study, we investigated the anti-inflammatory effects of a Kampo medicine Shosaikoto (TJ-9) using in vitro periodontal disease model, in which human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) produce IL-6, IL-8 and prostaglandin E2 (PGE2). Treatment with PgLPS (10 ng/ml), TJ-9 (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Moreover, TJ-9 did not alter LPS-induced IL-6 and IL-8 productions. However, TJ-9 significantly suppressed LPS-induced PGE2 production in a dose-dependent manner but TJ-9 alone did not affect basal PGE2 level. Western blotting demonstrated that TJ-9 decreased cyclooxygenase-2 (COX-2) expression in a dose-dependent manner but not phospholipase A2. Moreover, TJ-9 selectively and dose-dependently inhibited COX-2 activity. These results suggest that TJ-9 decreased PGE2 production by inhibition of both COX-2 expression and activity and that TJ-9 may be useful to improve gingival inflammation in periodontal disease.

Caspase inhibitor facilitates recovery of hearing by protecting the cochlear lateral wall from acute cochlear mitochondrial dysfunction.

We recently showed that acute energy failure in the rat cochlea induced by local administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) causes hearing loss mainly due to degeneration of cochlear lateral-wall fibrocytes. In the present study, we analyzed the effect of the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-VAD-FMK) on 3-NP-induced hearing loss in a model showing temporary threshold shifts at low frequencies and permanent threshold shifts at high frequencies. The model rats received an intraperitoneal injection of either Z-VAD-FMK or vehicle for 3 days starting 1 day prior to 3-NP treatment. One day after the administration of 3-NP, the auditory brain-stem response (ABR) threshold at 20 kHz was elevated to 70 dB in the Z-VAD-FMK group and to 85 dB in controls. The Z-VAD-FMK group completely recovered to the preoperative level within 14 days, whereas in the controls, the ABR threshold remained elevated at 50 dB even 28 days after the administration of 3-NP. Treatment with Z-VAD-FMK also improved recovery of hearing at 8 kHz but did not change recovery at 40 kHz. Histological examination demonstrated that treatment with Z-VAD-FMK inhibited progressive degeneration of the lateral-wall fibrocytes in the cochlear basal turn, as well as apoptosis of these fibrocytes. These results clearly indicate that caspase-dependent apoptosis of fibrocytes in the cochlear lateral wall plays an important role in hearing loss in the present animal model. Moreover, the results of the present study suggest that systemic administration of a caspase inhibitor may be an effective therapy for sensorineural hearing loss caused by acute energy failure such as that observed in cochlear ischemia.

Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.

Cochlear fibrocytes play important roles in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Recently, we developed a novel rat model of acute sensorineural hearing loss attributable to fibrocyte dysfunction induced by a mitochondrial toxin. In this model, we demonstrate active regeneration of the cochlear fibrocytes after severe focal apoptosis without any changes in the organ of Corti. To rescue the residual hearing loss, we transplanted mesenchymal stem cells into the lateral semicircular canal; a number of these stem cells were then detected in the injured area in the lateral wall. Rats with transplanted mesenchymal stem cells in the lateral wall demonstrated a significantly higher hearing recovery ratio than controls. The mesenchymal stem cells in the lateral wall also showed connexin 26 and connexin 30 immunostaining reminiscent of gap junctions between neighboring cells. These results indicate that reorganization of the cochlear fibrocytes leads to hearing recovery after acute sensorineural hearing loss in this model and suggest that mesenchymal stem cell transplantation into the inner ear may be a promising therapy for patients with sensorineural hearing loss attributable to degeneration of cochlear fibrocytes.

Fos-B expression is required for polyamine-induced increase in nuclear activator protein-1 DNA binding in discrete structures of murine brain.

Spermidine (SPD) and spermine (SPN) have been shown to be endogenous agonists for N-methyl-D-aspartate (NMDA) receptors that could lead to expression of the nuclear transcription factor activator protein-1 (AP1) complex in the mammalian central nervous system both in vitro and in vivo. In nuclear extracts of murine whole brain, AP1 DNA binding increased significantly in a concentration-dependent manner with the addition of either SPD or SPN at a concentration range of 50-500 microM. Similarly, the nuclear proteins histone and dephosphorylated casein, but not phosphorylated casein, significantly increased AP1 DNA binding alone but in the presence of either SPD or SPN did not increase further binding. By contrast, another endogenous polyamine, putrescine, significantly prevented AP1 DNA binding increases by histone and dephosphorylated casein, but did not by itself significantly alter binding. Invariably, SPD and SPN effected significantly increased AP1 DNA binding in neocortex, hippocampus, striatum, midbrain, hypothalamus and cerebellum, but not in medulla-pons and spinal cord. Supershift and Western blotting analyses revealed relatively high constitutive expression of Fos-B protein in neocortex and hippocampus, but not in medulla-pons and spinal cord. Immunoprecipitation of Fos-B led to complete abolition of the ability of SPN and SPD to increase AP1 DNA binding in neocortical and hippocampal nuclear extracts. These results suggest that expression of Fos-B protein may be required for modulation of nuclear gene transcription by both SPD and SPN through stimulation DNA-binding activity of AP1 complex in murine central structures.

Transcription factor activator protein-1 expressed by kainate treatment can bind to the non-coding region of mitochondrial genome in murine hippocampus.

We have demonstrated previously that the transcription factor activator protein-1 (AP-1) complex is translocated into mitochondria into the nucleus in murine hippocampus after systemic kainate injection (Ogita et al. [2002] J. Neurosci. 22:2561-2570). The present study investigates whether the mitochondrial AP-1 complex translocated in response to kainate treatment binds to AP-1-like sites located at the non-coding region of the mitochondrial genome in mouse hippocampus. There are 10 sites with sequences similar to the nuclear AP-1 site in the non-coding region. Of 10 pieces (MT-1-MT-10) of synthesized double-stranded oligonucleotides, each containing a mitochondrial AP-1-like site, MT-3, MT-4, and MT-9 were effective in inhibiting mitochondrial AP-1 DNA binding enhanced by kainate. Electrophoresis mobility shift analysis using radiolabeled MT-3 and MT-9 probes demonstrated marked enhancement with binding of these 2 probes in hippocampal mitochondrial extracts prepared 2-6 hr after kainate treatment. Unlabeled AP-1 probe was more potent than unlabeled MT-9 probe in inhibiting the mitochondrial MT-9 binding. Supershift analysis revealed participation of particular Fos/Jun family proteins, such as c-Fos, Fos-B, c-Jun, Jun-B, and Jun-D, in MT-9 binding in hippocampal mitochondrial extracts prepared 4 hr after kainate treatment. Immunoprecipitation analysis using anti-c-Fos antibody demonstrated that c-Fos associated with the mitochondrial genome in hippocampal mitochondria prepared from kainate-treated animals. These results suggest that the AP-1 complex expressed by in vivo kainate treatment would bind to AP-1-like sites in the non-coding region of the mitochondrial genome after translocation into mitochondria from murine hippocampus.

Localization of activator protein-1 complex with DNA binding activity in mitochondria of murine brain after in vivo treatment with kainate.

To elucidate mechanisms underlying mitochondrial dysfunctions induced by glutamate, we have examined the effects of in vivo treatment with the ionotropic glutamate receptor agonist kainate on localization of the transcription factor activator protein-1 (AP-1) in mitochondria as well as nuclei of murine brain. A systemic administration of kainate dramatically enhanced AP-1 DNA binding in both mitochondrial and nuclear extracts of mouse cerebral cortex and hippocampus 1 hr to 3 d later. Unlabeled AP-1 probe selectively competed for AP-1 DNA binding in mitochondrial extracts of cortex and hippocampus obtained from mice injected with kainate. Supershift and immunoblotting analyses revealed participation of c-Fos, Fos-B, and Jun-B proteins in potentiation by kainate of mitochondrial AP-1 DNA binding in cortex and hippocampus. An immunohistochemical study demonstrated marked expression by kainate of c-Fos protein in the pyramidal and dentate granular layers, whereas an immunoelectron microscopic analysis showed localization of c-Fos protein within mitochondria, as well as nuclei, of the CA1 pyramidal and dentate granular cells in hippocampus obtained 2 hr after the administration of kainate. Mitochondrial AP-1 DNA binding was inhibited by particular unlabeled oligonucleotides containing sequences similar to the AP-1 site found in the noncoding region of mitochondrial DNA. Kainate markedly potentiated binding of radiolabeled oligonucleotide probes containing sequences effective in competing for AP-1 DNA binding in hippocampal mitochondrial extracts. These results suggest that kainate may facilitate expression of the AP-1 complex and subsequent translocation into mitochondria to participate in mechanisms associated with transcriptional regulation of mitochondrial DNA in murine hippocampus.