Characterization of Motor Neuron Prostaglandin E2 EP3 Receptor Isoform in a Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with adult onset, characterized by progressive loss of motor neurons. Prostaglandin E2 (PGE2), a lipid mediator, exerts its biological functions by binding to four subtypes of E-prostanoid (EP1-4). Among them, EP3 has been shown to have multiple isoforms, EP3α, EP3ß, and EP3γ, produced by alternative splicing. Since PGE2 has been shown to have important pathophysiological roles in ALS, experiments were performed to identify EP3 receptor isoform(s) in spinal motor neurons of wild-type (WT) and ALS model (G93A) mice. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of adult mice demonstrated expression of EP3α and EP3γ mRNAs in the lumbar spinal cord, whereas EP3ß mRNA was barely detectable. Laser capture microdissection was used to dissect out motor neurons from frozen samples of lumbar spinal cord in these mice for analysis by real-time PCR. We found that expression of EP3γ mRNA was predominant in these neurons, whereas EP3α and EP3ß mRNAs were undetectable. At the early symptomatic stage, the mRNA expression profiles of these splice isoforms in G93A motor neurons were comparable to those in neurons from WT mice. These results suggest that the PGE2-to-EP3 signaling pathway is mediated mainly by the EP3γ isoform in the motor neurons of mice, and that modulation of the EP3γ isoform in motor neurons may be a promising new therapeutic approach for ALS.

mRNA expression and localization of LPS-induced ß-defensin isoforms in rat salivary glands.

ß-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of ß-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat ß-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. ß-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. ß-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce ß-defensins.

Immunolocalization and distribution of functional temperature-sensitive TRP channels in salivary glands.

Transient receptor potential (TRP) cation channels are unique cellular sensors involved in multiple cellular functions. Their role in salivary secretion remains to be elucidated. The expression and localization of temperature-sensitive TRP channels in salivary (submandibular, sublingual and parotid) glands were analyzed by immunohistochemistry and quantitative real-time reverse transcription plus the polymerase chain reaction (RT-PCR). The effects of various TRP channel agonists on carbachol (CCh)-induced salivary secretion in the submandibular gland and on the intracellular Ca(2+) concentration ([Ca(2+)]i) in a submandibular epithelial cell line were also investigated. Immunohistochemistry revealed the expression of TRP-melastatin subfamily member 8 (TRPM8) and TRP-ankyrin subfamily member 1 (TRPA1) in myoepithelial, acinar and ductal cells in the sublingual, submandibular and parotid glands. In addition, TRP-vanilloid subfamily member 1 (TRPV1), TRPV3 and TRPV4 were also expressed in myoepithelial, acinar and ductal cells in all three types of gland. Quantitative real-time RT-PCR results demonstrated the mRNA expression of TRPV1, TRPV3, TRPV4, TRPM8 and TRPA1 in acinar and ductal cells in these salivary glands. Perfusion of the entire submandibular gland with the TRPV1 agonist capsaicin (1 µM) via the submandibular artery significantly increased CCh-induced salivation, whereas perfusion with TRPM8 and TRPA1 agonists (0.5 µM WS12 and 100 µM allyl isothiocyanate) decreased it. Application of agonists for each of the thermosensitive TRP channels increased [Ca(2+)]i in a submandibular epithelial cell line. These results indicate that temperature-sensitive TRP channels are localized and distributed in acinar, ductal and myoepithelial cells in salivary glands and that they play a functional role in the regulation and/or modulation of salivary secretion.

Pregnenolone biosynthesis in the rat salivary gland and its inhibitory effect on secretion.

Pregnenolone (PRG), a major neurosteroid, suppressed carbachol-induced salivary secretion in perfused submandibular gland in rats. These effects were enhanced and depressed by agonistic muscimol (MUS) and antagonistic bicuculline to the γ-aminobutyric acid A receptor (GABA(A)-R), respectively. In contrast, PRG-sulfate, a sulfate-conjugated PRG metabolite, antagonized the suppressive effects of MUS, resulting in upregulation of salivary secretion. Quantitative RT-PCR and Western blotting revealed lesser expression of the PRG synthetase CYP11A1 protein and mRNA in the parotid, submandibular, and sublingual gland than in the cerebral cortex or adrenal gland as positive control organs. However, in response to methamphetamine withdrawal-induced stress, CYP11A1 production in each type of the salivary gland was highly upregulated to levels similar to those seen in the cerebral cortex. These results indicate that the salivary gland is capable of producing neurosteroids, as well as the brain. This suggests that steroid biosynthesis occurs in the salivary gland and is involved in the inhibitory regulation of salivary secretion in cooperation with GABA(A)-R. Further studies are needed to determine the pathophysiological significance of the biosynthesis of neurosteroids and their mechanisms of action via nuclear and membrane receptors.

Diazepam enhances production of diazepam-binding inhibitor (DBI), a negative saliva secretion regulator, localized in rat salivary gland.

Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase-activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

Diazepam Enhances Production of Diazepam-Binding Inhibitor (DBI), a Negative Saliva Secretion Regulator, Localized in Rat Salivary Gland.

Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase-activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

Proteomic analysis of lipopolysaccharide-treated submandibular gland in rat.

We investigated changes in the protein profile of submandibular gland (SMG) with inflammation induced by exposure to lipopolysaccharide (LPS) with the aim of identifying potential molecular markers of injured gland. Lipopolysaccharide (2.5µg) was directly administered into rat SMG unilaterally by retrograde ductal injection. At 12hr after treatment, the gland was excised and the proteins identified by two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Many proteins in the LPS-treated gland showed a marked change compared to those in the contralateral gland. Of particular note were increases in ubiquitin, a highly-conserved small regulatory protein and in calgranulin B, which has an immunological function in inflammation. Proteins related to apoptosis and stress also showed change in the inflamed gland. The results of this study suggest that the ubiquitin system of protein modification is involved in LPS-induced inflammation in salivary gland, and that a number of specific proteins might be applicable as molecular markers in the monitoring of inflamed or injured gland.

Multiple myeloma involving the thyroid cartilage.

Multiple myeloma, solitary plasmacytoma, and extramedullary plasmacytoma constitute a continuous disease spectrum of plasma cell neoplasms. In the larynx, although extramedullary plasmacytoma in the supraglottic region has been sometimes reported, plasma cell neoplasm with involvement of the thyroid cartilage is extremely rare. We report a case of multiple myeloma involving the thyroid cartilage. A 72-year-old male patient presented with a pathological fracture of the cervical vertebrae. CT scan revealed low-density areas within the thyroid cartilage, but the laryngeal mucosa and submucosal soft tissue were intact. Multiple myeloma was diagnosed, since the tumors in the thyroid cartilage and cervical vertebrae revealed plasmacytoma. Because no other lesion was found, irradiation of the larynx and cervical vertebrae was performed. Neither aggressive change of these lesions nor new lesions have been found over 3-year follow-up following the initial treatment without adjuvant therapy.

Localization and expression patterns of TRP channels in submandibular gland development.

OBJECTIVE: Expression of Transient receptor potential (TRP) channels: TRP canonical (TRPC)1, TRP vanilloid (TRPV)3, TRPV4 and TRP melastatin (TRPM)8 in adult rat salivary gland has recently been reported. The authors investigated expression of these TRP channels in the submandibular gland during early developmental stage in which the cell constitution is different, and discussed the function of TRP in the submandibular gland in early development. DESIGN: Using rat submandibular gland at embryonic days (E)18 and E20 and postnatal days (PN)0 and PN5 and PN28, expression of TRPV3, TRPV4, TRPC1 and TRPM8 was investigated using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: All TRP channels were expressed in cells constituting the submandibular gland in early developmental stage, but an increase in the expression level at PN5 on RT-PCR was significant compared with those at E18, PN0 and PN28 in TRPC1 and TRPV4 channels, whereas an increase was observed but not significant in the others. On immunohistochemical staining at PN5, whereas strong reactions of anti-TRPM8 antibody, anti-TRPV3 and anti-TRPV4 antibodies were observed in cells which proliferated from a terminal portion of cells arranged tubular structure which previously constituted mostly the submandibular gland. CONCLUSION: It was clarified that TRP channels are expressed in the rat submandibular gland in early developmental stage although cells constituting the submandibular gland are different from those in adult animals, suggesting that these TRP channels are involved in cell differentiation in at PN5 into the adult submandibular gland during early development.

Desmin and nerve terminal expression during embryonic development of the lateral pterygoid muscle in mice.

OBJECTIVE: In adults, the lateral pterygoid muscle (LPM) is usually divided into the upper and lower head, between which the buccal nerve passes. Recent investigations have demonstrated foetal developmental changes in the topographical relationship between the human LPM and buccal nerve. However, as few studies have investigated this issue, we clarified the expression of desmin and nerve terminal distribution during embryonic development of the LPM in mice. METHODS: We utilized immunohistochemical staining and reverse transcription chain reaction (RT-PCR) to clarify the expression of desmin and nerve terminal distribution. RESULTS: We observed weak expression of desmin in the LPM at embryonic day (ED) 11, followed by an increase in expression from embryonic days 12-15. In addition, starting at ED 12, we observed preferential accumulation of desmin in the vicinity of the myotendinous junction, a trend that did not change up to ED 15. Nerve terminal first appeared at ED 13 and formed regularly spaced linear arrays at the centre of the muscle fibre by ED 15. The results of immunohistochemical staining agreed with those of RT-PCR analysis. CONCLUSION: We found that desmin accumulated in the vicinity of the myotendinous junction starting at ED 12, prior to the onset of jaw movement. We speculate that the accumulation of desmin is due to factors other than mechanical stress experienced during early muscle contraction. Meanwhile, the time point at which nerve terminals first appeared roughly coincided with the onset of jaw movement.

Methamphetamine-withdrawal stress activates PACAP-DBI pathway in rat salivary gland, resulting in inhibition of salivary secretion.

The purpose of this study was to investigate activation of inhibitory regulation pathways by methamphetamine (METH)-withdrawal stress in rat salivary gland. Our previous study showed that METH-withdrawal stress activated steroid biosynthesis and that pregnenolone produced during the early stage of this process inhibited salivary secretion. However, how this type of stress inhibits salivary secretion and the activation pathway of steroid biosynthesis in salivary gland remain to be clarified. In the present study, using an in vivo cannulation method, METH-withdrawal stress decreased salivary secretion and increased expression of diazepam-binding inhibitor (DBI), an endogenous peripheral-type benzodiazepine receptor (PBR) agonist; Western blot and RT-PCR also showed increased expression of DBI mRNA in parotid, submandibular, and sublingual gland. In addition, METH-withdrawal stress also elicited an increase in pituitary adenylate cyclase-activating polypeptide (PACAP) and PBR mRNA, which is associated with DBI activity. These results suggest that METH-withdrawal stress activates a PACAP-DBI pathway in salivary gland, enhancing steroid genesis and inhibiting secretion.

Immunohistochemical study on GABAergic system in salivary glands.

Gamma-aminobutyric acid (GABA) and its receptors are found in the central nervous system and several peripheral tissues. The purpose of this study was to determine the expression and distribution of GABA and glutamate decarboxylase (GAD), a GABA biosynthetic enzyme, in rat salivary gland. Western blot and real time quantitative RT-PCR revealed that GAD67 was the major isoform of GAD in the salivary glands. Furthermore, both GABA and GAD were detected around the acinar cells in the submandibular glands by immunohistochemical analysis. When both sympathetic and parasympathetic nerves related to the submandibular glands were denervated, the immunoreactivities of GABA and GAD were dramatically depressed, and levels of GAD67 and GABA significantly decreased. However, no morphological changes in the glands were observed after denervation. These results indicate that GAD67 is present around acinar cells in the salivary glands, and suggest that the GABAergic system in the glands is closely related to the autonomic nervous system.

Chronic administration of methamphetamine increases the mRNA expression of diazepam binding inhibitor in rat brain.

Anxiety is one of the common features of withdrawal syndrome caused by abuse-inducing drugs such as methamphetamine (MAP). The neural pathways associated with anxiety are established within the network sustained by diencephalon, cerebral cortex, cerebellum and hippocampus. Diazepam binding inhibitor (DBI), a peptide consisting of 87 amino acids, serves as an inverse agonist for the type A receptor of the gamma-aminobutyric acid (GABAA receptor) with endogenous anxiogenic potential. We examined the effect of chronic administration of MAP on the mRNA expression of DBI and DBI-related proteins, such as alpha 2 subunit of GABAA receptor (GABA-α2), peripheral-type benzodiazepine receptor (PBR), and pituitary adenylate cyclase-activating polypeptide (PACAP) in seven regions (diencephalon, cerebral cortex, cerebellum, striatum, hippocampus, midbrain, and pons-medulla) of the rat brain. The mRNA expression of DBI increased significantly in all areas of the brain, especially diencephalon, after chronic administration of MAP. The mRNA expression of PBR, GABA-α2 and PACAP increased significantly in all areas of the brain, especially cerebral cortex, after chronic administration of MAP. These results suggest that anxiety is associated with the mRNA expression of DBI as well as DBI-related genes.

Acute administration of methamphetamine decreases the mRNA expression of diazepam binding inhibitor in rat brain.

Chronic administration of methamphetamine (MAP) up-regulated the mRNA expression of diazepam binding inhibitor (DBI) in rat brain, possibly leading to anxiety. Acute effects of MAP on anxiety associated with DBI, however, are not clear. In this study, we examined the effects of acute administration of MAP on behavior related to anxiety and the expression level of DBI mRNA and pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA, calibrated with the glyceraldehydes 3-phosphate dehydrogenase mRNA as the internal control in rat brain. The elevated plus-maze test was applied to the analysis of the possible anxiety-related profile of MAP. Acute administration of MAP (5 mg/kg, intraperitoneal administration) significantly increased spent time in the open-space arms at 4 h after the administration compared with a saline-treated group. The expression of DBI mRNA in a large number of regions of rat brain significantly decreased 2, 4, 8 and 16 h after acute administration of MAP. In contrast, the expression of PACAP mRNA in a large number of regions of rat brain significantly increased 4 and 8 h after the administration of MAP. These results suggest that MAP, at this dose, has an anxiolytic effect, based on the reduction of the putative anxiogenic peptides, DBI.

Long-term treatment with morphine increases the D-serine content in the rat brain by regulating the mRNA and protein expressions of serine racemase and D-amino acid oxidase.

Recent studies indicate that an endogenous co-agonist for an N-methyl-D-aspartate (NMDA) receptor-related glycine site, D-serine, is synthesized by serine racemase and is metabolized by D-amino acid oxidase (DAO) and that acute treatment with morphine augments the gene expression of serine racemase and DAO in rat brain. To further elucidate the mechanism underlying the activation of NMDA receptors following chronic opioid administration, we have evaluated the effects of the chronic administration of morphine on the mRNA and protein expressions of serine racemase and DAO and on the contents of D-serine in several areas of the rat brain. Repeated administration of morphine for 30 days produced a significant augmentation of both the mRNA and protein expressions of serine racemase in all the brain regions, whereas no significant change in the protein expression of DAO was observed in all the brain regions. Furthermore, the chronic administration caused a slight but significant elevation in the concentration of D-serine in the cortex, striatum, and hippocampus. These results indicate the elevated D-serine level following the chronic morphine treatment could at least in part be involved in the activation of NMDA receptors via the glycine site.

Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma.

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.

Activation of pro-MMP-2 mediated by MT1-MMP in human salivary gland carcinomas: possible regulation of pro-MMP-2 activation by TIMP-2.

Matrix metalloproteinases (MMPs), a family of extracellular matrix-degrading enzymes, are considered to play important roles in cancer invasion and metastasis. The present study examined the production levels of eight different MMPs (MMP-1, 2, 3, 7, 8, 9 and 13, and MT1-MMP) and two tissue inhibitors of metalloproteinases (TIMP-1 and 2) in homogenates of human salivary gland carcinomas [mucoepidermoid carcinomas (MECs), adenoid cystic carcinomas (ACCs), and adenocarcinomas (ADEs)] and non-neoplastic control salivary glands using sandwich enzyme immunoassay systems. The levels of MMP-1, MMP-2, MMP-13, MT1-MMP, and TIMP-1 were significantly higher in the carcinoma samples than in the controls (p < 0.05). Gelatin zymography demonstrated that the activation ratio of the MMP-2 zymogen (pro-MMP-2) was significantly higher in the carcinomas than in the controls (p < 0.05). In addition, the activation ratio in MECs was significantly higher than that in ACCs or ADEs (p < 0.01) and also correlated with histological grade and lymph node metastasis in MECs (p < 0.05), whereas the ratio showed no such correlation in ACCs or ADEs. Although the production levels of pro-MMP-2 and MT1-MMP were similar among these carcinoma groups, TIMP-2 levels were significantly higher in ACCs and ADEs than in MECs (p < 0.01). In carcinoma samples, the pro-MMP-2 activation ratio correlated directly with the MT1-MMP/TIMP-2 ratio (r = 0.736, n = 23; p < 0.01). Immunohistochemistry and in situ zymography demonstrated localization of MMP-2, MT1-MMP, and TIMP-2 to carcinoma cells, but only in MECs did carcinoma cell nests exhibit gelatinolytic activity, which was inhibited by 1,10-phenanthroline. These results suggest that enhanced activation of pro-MMP-2 mediated by MT1-MMP is implicated in the invasion and metastasis of MECs and that TIMP-2 may regulate pro-MMP-2 activation in salivary gland carcinomas.