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

Abstract

1. 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. 2. 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. 3. 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 x 10 (-8) M. 4. 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. 5. 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. 6. DZP (10(-5) M) attenuated the increase in the intracellular Ca2+ concentration ([Ca(2+)](i)) in the cells following stimulation of the muscarinic and alpha(1)-adrenoceptors. 7. 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 [Ca(2+)](i) in the cells may be connected with the suppression of salivary secretion induced by DZP.

Figure 1

Time response effect of CCh (a) and effect of doses of CCh (b) on IP₃ production in rat parotid cells. (a) Parotid cells were incubated with or without CCh (10⁻⁴ M) for the periods indicated at 37°C after which IP₃ levels were measured. (b) Parotid cells were incubated with the indicated concentrations of CCh for 5 s at 37°C after which IP₃ contents were measured. The IP₃ level is shown as a percentage of the basal value taken as 100%. Basal IP₃ values of (a) and (b) experiments were 8.92±0.76 and 8.66±0.51 pmol mg protein⁻¹, respectively. The data are expressed as the mean±s.e.mean of three experiments.

Figure 2

Effect of DZP on CCh-stimulated IP₃ production in rat parotid cells. After preincubation in the absence or presence of the indicated concentrations of DZP for 5 min at 37°C, parotid cells were further incubated with or without CCh (10⁻⁴ M) for 5 s at 37°C. The basal IP₃ value was 8.56±0.50 pmol mg protein⁻¹. The data are expressed as the mean±s.e.mean of three to four experiments. *P<0.05, compared to the values obtained with CCh alone in the absence of DZP.

Figure 3

Effect of DZP on the concentration-response curves for CCh-stimulated IP₃ production in rat parotid cells. (a) After preincubation in the absence or presence of DZP (10⁻⁷ and 10⁻⁵ M) for 5 min at 37°C, parotid cells were further incubated with the indicated concentrations of CCh for 5 s at 37°C. The basal IP₃ value was 8.09±0.44 pmol mg protein⁻¹. The data are expressed as the mean±s.e.mean of four experiments. (b) Double reciprocal plots are taken from the data of experiment (a). The ordinate and abscissa represent the reciprocals of IP₃ production and CCh concentration, respectively.

Figure 4

Effects of PK 11195 (a) and flumazenil (b) on the inhibitory effect of DZP on CCh-stimulated IP₃ production in rat parotid cells. After preincubation in the absence or presence of DZP, atropine (ATR), PK 11195, and flumazenil (FLZ) for 5 min at 37°C, parotid cells were further incubated with or without CCh (10⁻⁴ M) for 5 s at 37°C. Basal IP₃ levels of (a) and (b) experiments were 9.03±0.65 and 9.22±0.74 pmol mg protein⁻¹, respectively. The data are expressed as the mean±s.e.mean of three to five experiments. *P<0.05 and #P<0.05, compared to the values obtained with CCh alone or DZP plus CCh, respectively.

Figure 5

Effect of DZP on the CCh- and phenylephrine-stimulated Ca²⁺ response in rat parotid cells. Parotid cells loaded with fura-2/AM were preincubated with or without DZP (10⁻⁵ M) for 5 min and then [Ca²⁺]_i following stimulation of CCh (10⁻⁴ M) or phenylephrine (Phe; 10⁻⁴ M) was measured as described under ‘Methods'. CCh and Phe were added at the arrow. Cells were incubated in HBSS-H (a, c) or Ca²⁺-free HBSS-H+3 mM EGTA (b, d). The traces are presented as the average of results obtained with six or seven cells.