Sexually diergic hypothalamic-pituitary-adrenal axis responses to selective and non-selective muscarinic antagonists prior to cholinergic stimulation by physostigmine in rats

Abstract

Central cholinergic systems regulate the hypothalamic-pituitary-adrenal (HPA) axis differentially in males and females (sexual diergism). We previously investigated the role of muscarinic receptors in this regulation by administering physostigmine (PHYSO), an acetylcholinesterase inhibitor, to male and female rats pretreated with scopolamine (SCOP), a nonselective muscarinic antagonist. SCOP pretreatment enhanced adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses in both sexes, but males had greater ACTH responses while females had greater CORT responses. In the present study, we further explored the role of muscarinic receptor subtypes in HPA axis regulation by administering PHYSO to male and female rats following SCOP or various doses of either the M1 or the M2 selective muscarinic receptor antagonists, pirenzepine (PIREN) or methoctramine (METHO). Blood was sampled before and at multiple times after PHYSO. ACTH and CORT were determined by highly specific immunoassays. M1 antagonism by PIREN prior to PHYSO resulted in sustained, dose-dependent increases in ACTH and CORT: ACTH responses were similar in both sexes, and CORT responses were greater in females. M2 antagonism by METHO prior to PHYSO resulted in overall decreases in ACTH and CORT: ACTH and CORT responses were higher in females but lower in both sexes than the hormone responses following PIREN or SCOP pretreatment. Area under the curve analyses supported these findings. These results suggest that specific muscarinic receptor subtypes differentially influence the HPA axis in a sexually diergic manner.

Keywords: Cholinergic; Methoctramine; Muscarinic; Pirenzepine; Scopolamine; Sexual diergism.

Figure 1. ACTH responses to PHYSO or SAL in Male and Female Rats

Response curves of absolute plasma ACTH (A, B) and corresponding percent changes from baseline (C, D). Baseline blood samples were collected at −25 min and −15 min to yield an average baseline at −20 min. Injection of SAL (1 ml/kg) occurred immediately after collection of the −25 min blood sample. Injection of either PHYSO (0.1 mg/kg) or SAL (1 ml/kg) occurred at 0 min. Four additional blood samples were collected at 10 min, 20 min, 40 min, and 60 min. Each bar represents the mean ± SEM. n = 23 to 24 rats for SAL + SAL groups; n = 7 to 8 rats for SAL + PHYSO groups. Females are represented by squares and males are represented by circles. a, sex difference at indicated dose and time point (p < 0.05); b, vs. baseline (p < 0.05); c, vs. SAL + SAL at indicated time point (p < 0.05).

Figure 2. CORT responses to PHYSO or SAL in Male and Female Rats

Response curves of absolute plasma CORT (A, B) and corresponding percent changes from baseline (C, D). See Fig. 1 for explanation.

Figure 3. Effects of SCOP Pretreatment on ACTH in PHYSO-Stimulated Male and Female Rats

Response curves of absolute plasma ACTH (A–D) and corresponding percent changes from baseline (E, F). Baseline blood samples were collected at −25 min and −15 min to yield an average baseline at −20 min. Injection of SCOP (0.3 mg/kg) or SAL (1 ml/kg) occurred immediately after collection of the −25 min blood sample. Injection of either PHYSO (0.1 mg/kg) or SAL (1 ml/kg) occurred at 0 min. Four additional blood samples were collected at 10 min, 20 min, 40 min, and 60 min. Each bar represents the mean ± SEM. n = 23 to 24 rats for SAL + SAL groups; n = 7 to 8 rats for SAL + PHYSO groups; n = 8 to 12 rats for SCOP pretreatment groups. Females are represented by squares and males are represented by circles. a, sex difference at indicated dose and time point (p < 0.05); b, vs. baseline (p < 0.05); c, vs. SAL + SAL at indicated time point (p < 0.05); d, vs. SAL + PHYSO at indicated time point (p < 0.05). * designates that letters apply to both sexes at indicated time point.

Figure 4. Effects of SCOP Pretreatment on CORT in PHYSO-Stimulated Male and Female Rats

Response curves of absolute plasma CORT (A–D) and corresponding percent changes from baseline (E, F). See Fig. 3 for explanation.

Figure 5. Dose-Dependent Effects of PIREN Pretreatment on ACTH in PHYSO-Stimulated Male and Female Rats

Response curves of absolute plasma ACTH (A–F) and corresponding percent changes from baseline (G, H). Baseline blood samples were collected at −25 min and −15 min to yield an average baseline at −20 min. Injection of PIREN (10, 30, or 70 mg/kg) or SAL (1 ml/kg) occurred immediately after collection of the −25 min blood sample. Injection of either PHYSO (0.1 mg/kg) or SAL (1 ml/kg) occurred at 0 min. Four additional blood samples were collected at 10 min, 20 min, 40 min, and 60 min. Each bar represents the mean ± SEM. n = 23 to 24 rats for SAL + SAL groups; n = 7 to 8 rats for SAL + PHYSO groups; n = 8 to 13 rats for PIREN pretreatment groups. Females are represented by squares and males are represented by circles. a, sex difference at indicated dose and time point (p < 0.05); b, vs. baseline (p < 0.05); c, vs. SAL + SAL at indicated time point (p < 0.05); d, vs. SAL + PHYSO at indicated time point (p < 0.05). * designates that letters apply to both sexes at indicated time point.

Figure 6. Dose-Dependent Effects of PIREN Pretreatment on CORT in PHYSO-Stimulated Male and Female Rats

Response curves of absolute plasma CORT (A–F) and corresponding percent changes from baseline (G, H). See Fig. 5 for explanation.

Figure 7. Dose-Dependent Effects of METHO Pretreatment on ACTH in PHYSO-Stimulated Male and Female Rats

Response curves of absolute plasma ACTH (A–F) and corresponding percent changes from baseline (G, H). Baseline blood samples were collected at −25 min and −15 min to yield an average baseline at −20 min. Injection of METHO (0.3, 1, or 3 mg/kg) or SAL (1 ml/kg) occurred immediately after collection of the −25 min blood sample. Injection of either PHYSO (0.1 mg/kg) or SAL (1 ml/kg) occurred at 0 min. Four additional blood samples were collected at 10 min, 20 min, 40 min, and 60 min. Each bar represents the mean ± SEM. n = 23 to 24 rats for SAL + SAL groups; n = 7 to 8 rats for SAL + PHYSO groups; n = 8 to 13 rats for METHO pretreatment groups. Females are represented by squares and males are represented by circles. a, sex difference at indicated dose and time point (p < 0.05); b, vs. baseline (p < 0.05); c, vs. SAL + SAL at indicated time point (p < 0.05); d, vs. SAL + PHYSO at indicated time point (p < 0.05).

Figure 8. Dose-Dependent Effects of METHO Pretreatment on CORT in PHYSO-Stimulated Male and Female Rats

Response curves of absolute plasma CORT (A–F) and corresponding percent changes from baseline (G, H). See Fig. 7 for explanation.