Kohei Sawaki

Inhibitory effect of diazepam on muscarinic receptor-stimulated inositol 1,4,5-trisphosphate production in rat parotid acinar cells

This study examined the effect of diazepam (DZP) on phosphoinositide turnover, which plays an important role in the regulation of salivary secretion, in rat parotid acinar cells. DZP (10−9 M to 10−5 M), a potent agonist of both central‐ and peripheral‐type benzodiazepine receptors, dose‐dependently decreased inositol 1,4,5‐trisphosphate (IP3) production stimulated by carbachol, a muscarinic receptor agonist, in the cells. DZP produced a maximum inhibitory response at a concentration of 10−5 M, with IP3 production decreased to 63% of maximal levels. The concentration inducing half maximal inhibition of IP3 production was approximately 3.5×10−8 M. An inhibitory response to DZP was produced by a short‐term pretreatment (<3 min) of the cells and prevented by antagonist and competing ligand for the central‐ and peripheral‐type benzodiazepine receptors, flumazenil and PK 11195, respectively. DZP showed a non‐competitive inhibition of carbachol‐stimulated IP3 production. It did not directly inhibit the activities of GTP‐binding regulatory proteins and phosphatidylinositol 4,5‐bisphosphate‐specific phospholipase C (PLC) in the parotid gland membranes, though choline chloride inhibited PLC activity. DZP (10−5 M) attenuated the increase in the intracellular Ca2+ concentration ([Ca2+]i) in the cells following stimulation of the muscarinic and α1‐adrenoceptors. These results suggest that in the parotid acinar cells, DZP inhibits muscarinic receptor‐stimulated IP3 production through benzodiazepine receptors and that PLC activity which produces IP3 is inhibited by chloride. The decreases in IP3 and [Ca2+]i in the cells may be connected with the suppression of salivary secretion induced by DZP.

Effects of Anticonvulsants on Local Anaesthetic‐Induced Neurotoxicity in Rats

The effects of various anticonvulsants on local anaesthetics procaine- and lidocaine-induced convulsions were investigated in rats. Pretreatment with diazepam (2.5-5 mg/kg, intraperitoneally) and clonazepam (5-10 mg/kg, intraperitoneally) completely protected the rats against both local anaesthetic-induced convulsions. Phenobarbital (12.5-50 mg/kg, subcutaneously) also significantly decreased the incidence of both convulsions and prolonged their latencies. Carbabazepine (10-40 mg/kg, intraperitoneally) did not completely repress both convulsions, but it prolonged their latencies. Phenytoin (5-20 mg/kg, intraperitoneally) and primidone (30-60 mg/kg, intraperitoneally) markedly enhanced both local anaesthetic-induced convulsions, as shown by shortening of latency and increase in mortality. Valproate (100-200 mg/kg, intraperitoneally) produced a protective effect against procaine-induced convulsions, while it strongly enhanced lidocaine-induced convulsions. These results suggest that the benzodiazepines are effective drugs to prevent neurotoxicity induced by local anaesthetics, while phenytoin and primidone potentiate them.

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.

Expression of BMP7 is associated with resistance to diabetic stress: Comparison among mouse salivary glands

We determined mRNA levels of bone morphogenetic protein 7 (BMP7), a growth and differentiation factor belonging to the transforming growth factor-beta superfamily, in the salivary glands of mice with streptozotocin (200 mg/kg, i.p.)-induced diabetes. We also examined the effects of BMP7 on secretion of saliva and degenerative change in salivary glands in diabetic mice. In normal mice, BMP7 mRNA levels were high in the submandibular gland and low in the parotid gland, while in diabetic mice, levels were significantly decreased in the parotid gland, but not in the submandibular gland. No significant difference was observed in mRNA levels of BMP receptors between normal and diabetic mice. In diabetic mice, pilocarpine (4 mg/kg, i.p.)-stimulated salivary secretion showed a remarkable decrease in both parotid and submandibular gland, although degree of reduction was smaller in the latter. Notable degeneration with vacuolation and atrophy was also found in parotid gland, whereas degeneration of submandibular gland was slight. Administration of BMP7 (50 and 100 microg/kg, i.v.) in diabetic mice induced a significant increase in salivary secretion, with rate of recovery higher in parotid gland than in submandibular gland. In diabetic mice, BMP7 also exhibited a powerful protective effect in degenerated salivary gland, especially in parotid gland. These results suggest that BMP7 acts to prevent diabetic damage in salivary gland, and that its cytoprotective effect is closely correlated with mRNA levels in tissue.

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.

Modulation of benzodiazepine receptor, adrenoceptor and muscarinic receptor by diazepam in rat parotid gland.

This study investigated the influence of diazepam on the binding characteristics of adrenoceptor, muscarinic and benzodiazepine receptors in rat parotid gland membrane using a radioligand binding assay. At a concentration of >10(-6)M, diazepam competed with [(3)H]dihydroalprenolol for β-adrenoceptor, but not [(3)H]prazosin for α-adrenoceptor or [(3)H]quinuclidinyl benzilate for muscarinic receptor. Continuous administration of diazepam at doses of 0.4mg/kg/day, i.p. for 7days in rat significantly decreased pilocarpine (4.0mg/kg, i.p.)-induced parotid salivary flow. Diazepam also produced a significant increase in the dissociation constant (Kd) value for [(3)H]dihydroalprenolol binding, but no change in the maximal binding capacity (Bmax) value, and a decrease in the Kd value for [(3)H]diazepam binding to benzodiazepine receptors, but no change in the Kd or Bmax values for [(3)H]prazosin or [(3)H]quinuclidinyl benzilate binding. These results suggest that continuous administration of diazepam modifies affinity for β-adrenoceptor and benzodiazepine receptor binding sites in parotid gland membrane and that changes in these binding sites may be closely related to diazepam-induced suppression of salivary secretion.