CELLULAR STUDY OF INHIBITORY EFFECT OF BENZODIAZEPINES ON SALIVARY SECRETION

2000 Fiscal Year Final Research Report Summary

• Project/Area Number: 10671752
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional basic dentistry
• Research Institution: TOKYO DENTAL COLLEGE
• Principal Investigator: SAWAKI Kohei TOKYO DENTAL COLLEGE, DEPARTMENT OF DENTISTRY, LECTURER, 歯学部, 講師 (50178828)
• Co-Investigator(Kenkyū-buntansha): KAWAGUCHI Mitsuru TOKYO DENTAL COLLEGE, DEPARTMENT OF DENTISTRY, PROFESSOR, 歯学部, 教授 (20096473)
• Project Period (FY): 1998 – 2000
• Keywords: Salivary secretion / Salivary gland / Phospholipase C / Benzodiazepine / Benzodiazepine receptor / Chloride ion / Calcium ion / Intracellular pH

Research Abstract

Benzodiazepines (BDZs) suppress salivary secretion as a side effect. The fluid secretion is caused through an intracellular phosphoinositide pathway by muscarinic and α-adrenoceptor stimulations. We have previously reported that BDZs modified Cl^- transport and Ca^<2+> flux in the parotid acinar cells. BDZs also inhibited inositol 1,4,5-trisphosphate (IP_3) generation and decreased an intracellular pH in this cell. These findings led us to the suggestion that an intracellular changes in these factors may affect membrane-bound phospholipase C (PLC) activity related to IP_3 generation. In this study, we investigated the effects of pH, Cl^-, and C^<2+> ion on PLC activity in further detail.
Rat parotid gland membranes were prepared by centrifugation of the tissue homogenates. Cholate extract was prepared by treating the membranes with 2M KCl and 1% sodium cholate. PLC activity was measured by monitoring the hydrophilic products from the hydrolysis of exogenously added [^3H]PIP_2.
At concent rations from 10^<-8> M to 10^<-5> M, diazepam had no effect on AlF_4^--stimulated PLC activity in the membranes and the cholate extracted PLC activity. PLC activities stimulated by carbachol plus GTPγS in the membranes and the cholate extracts were activated at an acidic pH in the assay buffer and decreased at an alkaline pH : the optimum pH for PLC activity was about 6.4-6.6. Their activities were also maximally activated by Ca^<2+> at concentrations of 5×10^<-7> M to 10^<-7> M, but were significantly decreased by choline chloride (>80 mM) : they decreased to about 70% of maximum levels. The decrease rate was similar to that of carbachol-stimulated IP_3 generation by diazepam, namely about 65%.
These results demonstrate that : (1) BDZs do not inhibit directly the activities of GTP-binding protein and PLC in the parotid gland membranes ; (2) a decrease in intracellular pH and an increase in Ca^<2+> concentration in the parotid cells do not necessarily modify PLC activity ; (3) Cl^- inhibits PLC activity in the parotid membranes ; and (4) in the parotid cells, an increased intracellular Cl^- produced by BDZs inhibits membrane-bound PLC activity and IP_3 generation, and an intracellular Ca^<2+> concentration is decreased and the fluid secretion is suppressed.