Migiwa Okubo

Inhibitory regulation of amylase release in rat parotid acinar cells by benzodiazepine receptors

This study examined the influence of benzodiazepine receptors on amylase release from rat parotid acinar cells. Diazepam (10(-8)-10(-6) M), which is a potent agonist of both central- and peripheral-type benzodiazepine receptors, dose dependently decreased amylase release induced by isoprenaline and carbachol, which are beta-adrenoceptor and muscarinic receptor agonists, respectively. The maximum inhibitory response was obtained with 10(-6) M diazepam: amylase release was decreased to 57% (isoprenaline) and 39% (carbachol) of maximal levels, while these responses were completely inhibited by propranolol and atropine, respectively. Clonazepam and 7-chloro-1,3-dihydro-1-methyl-5-p-chlorophenyl)-2H-1,4-benzodiazepine-2- one (Ro 5-4864), which are selective agonists of central- and peripheral-type benzodiazepine receptors, respectively, also produced a significant and dose-dependent decrease in isoprenaline-induced amylase release. The inhibitory potency was diazepam > clonazepam > Ro 5-4864. Flumazenil and 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK 11195), which are selective antagonists of central- and peripheral-type benzodiazepine receptors, respectively, dose dependently blocked the inhibition of isoprenaline-induced amylase release by diazepam. At a concentration of 10(-5) M, flumazenil and PK 11195 restored amylase release to approximately 75% of that in the presence of isoprenaline alone. The combination of both antagonists completely prevented the inhibition by diazepam. Similarly, the inhibitory responses of clonazepam and Ro 5-4864 were completely blocked by flumazenil and PK 11195, respectively. These results suggest that, in rat parotid acinar cells, benzodiazepines inhibit beta-adrenoceptor and muscarinic receptor-stimulated amylase release and that both central- and peripheral-type benzodiazepine receptors contribute to this inhibitory regulation.

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.

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, EP3β, 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 EP3β 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 EP3β 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.

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.

The effects of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and meso-2,3- dimercaptosuccinic acid (DMSA) on the nephrotoxicity in the mouse during repeated cisplatin (CDDP) treatments

Previously, we reported that specific lower dose of sodium 2,3-dimercapto-1-propanesulfonic acid (DMPS) which is an antidote to heavy metal intoxication, inversely enhanced cisplatin (CDDP)-induced antitumor activity to S-180 cell-bearing mouse. This activity was only weak with meso-2,3-dimercaptosuccinic acid (DMSA), however. This study investigated the effects of lower doses of DMPS or DMSA on the nephrotoxicity and kinetics of CDDP. Kidney and blood isolated from female mice which received CDDP with or without DMPS or DMSA once daily for 4 days were provided for measuring levels of blood urea nitrogen (BUN) and transporter proteins (OCT2: organic cation transporter; MATE1: multidrug and toxin extrusion) mRNA, and CDDP-originated platinum, and TUNEL staining of renal tubular cells. DMPS or DMSA reduced effectively CDDP-induced BUN, and caused a moderate reduction of platinum in kidney. Additionally, both dimercapto-compounds restored the CDDP-reduced mRNA levels of transporter proteins (OCT2 and MATE1), and apparently suppressed the CDDP-induced apoptosis. These results suggest that DMPS, as well as DMSA, at approximate 17-fold dose (μmol/kg) of CDDP, has an enough potential to reverse the CDDP nephrotoxicity, and concomitant use of DMPS considering both dose and timing for administration is potentially useful for preventing nephrotoxicity and enhancing antitumor activity during CDDP chemotherapy.