Castration decreases amylase release associated with muscarinic acetylcholine receptor downregulation in rat parotid gland

Abstract

1 The mechanism and receptor subtypes involved in carbachol-stimulated amylase release and its changes after castration were studied in parotid slices from male rats. 2 Carbachol induced both amylase release and inositol phosphate (IP) accumulation in parotid slices from control and castrated rats, but castration induced a decrease of carbachol maximal effect. The effect of castration was reverted by testosterone replacement. 3 The selective M(1) and M(3) muscarinic receptor antagonists, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, respectively, inhibited carbachol-stimulated amylase release and IP accumulation in a dose-dependent manner in parotid slices from control and castrated rats. 4 A diminution of binding sites of muscarinic receptor in parotid membrane from castrated rats was observed. Competition binding assays showed that both, M(1) and M(3) muscarinic receptor subtypes are expressed in membranes of parotid glands from control and castrated rats, M(3) being the greater population. 5 These results suggest that amylase release induced by carbachol in parotid slices is mediated by phosphoinositide accumulation. This mechanism appears to be triggered by the activation of M(1) and M(3) muscarinic receptor subtypes. Castration induced a decrease of the maximal effect of carbachol evoked amylase release and IP accumulation followed by a diminution in the number of parotid gland muscarinic acetylcholine receptors.

Figure 1

Panel (a) Concentration-dependent effect of carbachol on amylase release in parotid slices from control, castrated and castrated testosterone-treated male rat. Results are expressed as the mean±s.e.m. of six experiments in each group. Panel (b) Effect of atropine (1 × 10⁻⁷ M), U-73122 (5 × 10⁻⁶ M), TFP (5 × 10⁻⁶ M), staurosporine (1 × 10⁻⁹ M) and L-NMMA (1 × 10⁻⁴ M) on carbachol-induced amylase release on parotid slices from control and castrated rat, represented as the percentage of carbachol maximal effect. A: carbachol (1 × 10⁻³ M); B: atropine; C: U-73122; D: TFP; E: staurosporine; F: L-NMMA. Results are expressed as the mean±s.e.m. of six experiments in each group.

Figure 2

Panel (a) Concentration-dependent effect of carbachol on IP accumulation in parotid slices from control, castrated and castrated testosterone-treated male rat. Results are expressed as the mean±s.e.m. of six experiments in each group. Panel (b) Effect of atropine (1 × 10⁻⁷ M) and U-73122 (5 × 10⁻⁶ M) on carbachol-induced IP accumulation on parotid slices from control and castrated rats, represented as the percentage of carbachol maximal effect. A: carbachol (1 × 10⁻³ M); B: atropine; C: U-73122. Results are expressed as the mean±s.e.m. of six experiments in each group.

Figure 3

Antagonism of muscarinic receptor-stimulated amylase release by 4-DAMP and pirenzepine in parotid slices from control (upper panel) and castrated (lower panel) male rat. Parotid slices were preincubated in the absence or presence of indicated concentrations of 4-DAMP (a) or pirenzepine (b) added 20 min before the agonist, followed by determination of carbachol dose-dependent stimulation of amylase release as described in Methods. (c) Schild plots of 4-DAMP and pirenzepine antagonism of carbachol-mediated amylase release by parotid slices from control and castrated male rats. Logs of dose ratios minus 1 are from data shown in (a) and (b), and are plotted as a function of the pAx of the antagonist. Results shown are one of four experiments with similar results.

Figure 4

Antagonism of muscarinic receptor-stimulated IP accumulation by 4-DAMP and pirenzepine in parotid slices from control (upper panel) and castrated (lower panel) male rat. Parotid slices were preincubated in the absence or presence of indicated concentrations of 4-DAMP (a) or pirenzepine (b) added 20 min before the agonist, followed by determination of carbachol dose-dependent stimulation of IP accumulation as described in Methods. (c) Schild plots of 4-DAMP and pirenzepine antagonism of carbachol-mediated IP accumulation by parotid slices from control and castrated male rats. Logs of dose ratios minus 1 are from data shown in (a) and (b), and are plotted as a function of the pAx of the antagonist. Results shown are one of four experiments with similar results.

Figure 5

Correlation between IP accumulation and amylase release in parotid slices from control and castrated male rat. The data were obtained from Figure 1 and Figure 2, and amylase release was plotted as a function of the IP accumulation. Correlation values: 0.9747 and 0.9992 for control and castrated animals, respectively.

Figure 6

Saturation curves (a) and Scatchard plots (b) on rat parotid gland membranes from control, castrated and castrated testosterone-treated rats, incubated with different concentrations of [³H]-QNB. The results shown are the mean of four experiments performed in duplicate in each group.

Figure 7

Competition binding assays of muscarinic antagonists with [³H]-QNB in parotid membranes from control (a), castrated (b) and castrated testosterone-treated (c) rats. Parotid gland membranes were obtained and competition binding assays were performed as indicated in the text with the muscarinic antagonists atropine, 4-DAMP and pirenzepine. Results shown are representative of three separate experiments performed in duplicate with similar results. % B means per cent of [³H]-QNB bound.