The hypothalamic-pituitary-adrenal axis response to stress in mice lacking functional vasopressin V1b receptors

Abstract

The role of arginine vasopressin (Avp) as an ACTH secretagogue is mediated by the Avp 1b receptor (Avpr1b) found on anterior pituitary corticotropes. Avp also potentiates the actions of CRH (Crh) and appears to be an important mediator of the hypothalamic-pituitary-adrenal axis response to chronic stress. To investigate the role of Avp in the hypothalamic-pituitary-adrenal axis response to stress, we measured plasma ACTH and corticosterone (CORT) levels in Avpr1b knockout (KO) mice and wild-type controls in response to two acute (restraint and insulin administration) and one form of chronic (daily restraint for 14 d) stress. No significant difference was found in the basal plasma levels of ACTH and CORT between the two genotypes. Acute restraint (30 min) increased plasma ACTH and CORT to a similar level in both the Avpr1b mutant and wild-type mice. In contrast, plasma ACTH and CORT levels induced by hypoglycemia were significantly decreased in the Avpr1b KO mice when compared with wild-type littermates. There was no difference in the ACTH response to acute and chronic restraint in wild-type mice. In the Avpr1b KO group subjected to 14 sessions of daily restraint, plasma ACTH was decreased when compared with wild-type mice. On the other hand, the CORT elevations induced by restraint did not adapt in the Avpr1b KO or wild-type mice. The data suggest that the Avpr1b is required for the normal pituitary and adrenal response to some acute stressful stimuli and is necessary only for a normal ACTH response during chronic stress.

Figure 1

Basal expression of neuropeptide mRNAs in PVN and pituitary and histology of the adrenal in Avpr1b KO mice. There was no difference in the expression (dark labelling on light background) of Crh (A,B), Avp (C,D) or Oxt (E,F) mRNAs in the PVN (arrowed) of wild-type (left panels) or Avpr1b KO (right panels) mice. A-F are images scanned directly from X-ray film and have been graphically adjusted in Adobe Photoshop CS to optimise brightness and contrast. The level of Pomc mRNA expression and relative numbers of Pomc mRNA-expressing cells (white labelling on dark background) were similar in the anterior pituitaries from wild-type (G) or Avpr1b KO (H) mice. There was no gross histological differences in the appearances of the adrenal glands between the two genotypes (I, wild-type; J, KO). IL, intermediate lobe of the pituitary; M, adrenal medulla; C, adrenal cortex.

Figure 2

Vasopressin does not stimulate ACTH secretion from anterior pituitary segments obtained from Avpr1b KO mice. Isolated anterior pituitary segments from wild-type or Avpr1b KO mice were incubated in vitro with buffer alone (basal), Crh (1nM), Avp (10nM), or a combination of both ACTH secretagogues (Avp/Crh). Values are mean total ACTH (ng)/well ± SEM, n = 4 separate pituitary preparations. a, P <0.001 Avp KO vs. Avp wild-type; Avp/Crh KO vs. Avp/Crh wild-type; Avp wild-type vs. basal wild-type; Avp/Crh wild-type vs. Crh wild-type. b, P <0.0001 Avp/Crh KO vs. Avp KO; Avp/Crh KO vs. basal KO; Crh KO vs. basal KO; Crh wild-type vs. Crh basal; Avp/Crh wild-type vs. Avp wild-type; Avp/Crh wild-type vs. basal wild-type. NS, not significant (P > 0.05) KO vs. wild-type basal; Crh wild-type vs. Crh KO; Crh KO vs. Avp/Crh KO; basal KO vs. Avp KO.

Figure 3

Diurnal variation in plasma CORT secretion in wild-type and Avpr1b KO mice. Values are mean ± SEM, n = 3-5 samples for each time-point. a, P < 0.05 female am vs. male am; female pm vs. male pm. b, P < 0.0001 male am vs. male pm; female am vs. female pm. There were no significant differences (P > 0.05) between am or pm values for male or female mice of both genotypes.

Figure 4

Effect of acute and repeated restraint stress on plasma ACTH (A) and CORT (B) levels in Avpr1b mutant and control mice. basal = control, naïve mice briefly handled 30 min prior to sacrifice; acute = mice subjected to 30 min acute restraint; 13d + 24h = 24 h after 13 daily 30 min restraint sessions; 14d = daily restraint fro 14 d, sacrifice immediately following the last 30 min restraint session. Values are mean ± SEM, n = 4-5 mice/group. Four out of five 14d restraint Avpr1b KO samples had detectable ACTH; in contrast, the vast majority (14/16) of samples in the Avpr1b KO and wild-type basal and 13d + 24h groups had no measurable ACTH (the assay limit of 10 pg/ml was recorded for these samples). a, P < 0.001 13d restraint + 24h wild-type vs. 14d restraint KO; b, P < 0.0001 acute restraint vs. basal; 14d restraint wild-type vs. 14d restraint KO (ACTH only). NS, not significant (P > 0.05) basal wild-type vs. basal KO; acute wild-type vs. acute KO; 13d + 24h wild-type vs. 13d + 24h KO; 14d wild-type vs. 14d KO (CORT only).

Figure 5

Levels of plasma glucose (A), ACTH (B) and CORT (C) following peripheral (ip) administration of insulin (Actrapid) to Avpr1b-deficient mice. Mice were sacrificed 1 h after treatment with saline vehicle, 0.75iu/kg or 3.0iu/kg insulin. Values are mean ± SEM, n = 3-4 mice/treatment. Plasma glucose = A: a, P < 0.05 0.75iu/kg insulin vs. 3.0iu/kg insulin. b, P < 0.01 saline vs. insulin-injected. b, P < 0.01 saline vs. insulin-injected. NS, no significant difference (P > 0.05) saline wild-type vs. saline KO; 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO; 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO. Plasma ACTH = B: a, P < 0.05 3.0iu/kg insulin KO vs. saline KO. b, P < 0.01 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO; 0.75iu/kg insulin wild-type vs. saline wild-type; 3.0iu/kg insulin wild-type vs. 0.75iu/kg insulin wild-type. c, P < 0.001 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO; 3.0iu/kg insulin wild-type vs. saline wild-type. NS, no significant difference (P > 0.05) saline wild-type vs. saline KO. Plasma CORT = C: b, P < 0.01 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO; 0.75iu/kg insulin KO vs. 3.0iu/kg insulin KO. c, P < 0.001 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO. NS, no significant difference (P >0.05) saline wild-type vs. saline KO.

Figure 5

Levels of plasma glucose (A), ACTH (B) and CORT (C) following peripheral (ip) administration of insulin (Actrapid) to Avpr1b-deficient mice. Mice were sacrificed 1 h after treatment with saline vehicle, 0.75iu/kg or 3.0iu/kg insulin. Values are mean ± SEM, n = 3-4 mice/treatment. Plasma glucose = A: a, P < 0.05 0.75iu/kg insulin vs. 3.0iu/kg insulin. b, P < 0.01 saline vs. insulin-injected. b, P < 0.01 saline vs. insulin-injected. NS, no significant difference (P > 0.05) saline wild-type vs. saline KO; 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO; 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO. Plasma ACTH = B: a, P < 0.05 3.0iu/kg insulin KO vs. saline KO. b, P < 0.01 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO; 0.75iu/kg insulin wild-type vs. saline wild-type; 3.0iu/kg insulin wild-type vs. 0.75iu/kg insulin wild-type. c, P < 0.001 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO; 3.0iu/kg insulin wild-type vs. saline wild-type. NS, no significant difference (P > 0.05) saline wild-type vs. saline KO. Plasma CORT = C: b, P < 0.01 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO; 0.75iu/kg insulin KO vs. 3.0iu/kg insulin KO. c, P < 0.001 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO. NS, no significant difference (P >0.05) saline wild-type vs. saline KO.

Figure 5

Levels of plasma glucose (A), ACTH (B) and CORT (C) following peripheral (ip) administration of insulin (Actrapid) to Avpr1b-deficient mice. Mice were sacrificed 1 h after treatment with saline vehicle, 0.75iu/kg or 3.0iu/kg insulin. Values are mean ± SEM, n = 3-4 mice/treatment. Plasma glucose = A: a, P < 0.05 0.75iu/kg insulin vs. 3.0iu/kg insulin. b, P < 0.01 saline vs. insulin-injected. b, P < 0.01 saline vs. insulin-injected. NS, no significant difference (P > 0.05) saline wild-type vs. saline KO; 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO; 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO. Plasma ACTH = B: a, P < 0.05 3.0iu/kg insulin KO vs. saline KO. b, P < 0.01 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO; 0.75iu/kg insulin wild-type vs. saline wild-type; 3.0iu/kg insulin wild-type vs. 0.75iu/kg insulin wild-type. c, P < 0.001 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO; 3.0iu/kg insulin wild-type vs. saline wild-type. NS, no significant difference (P > 0.05) saline wild-type vs. saline KO. Plasma CORT = C: b, P < 0.01 3.0iu/kg insulin wild-type vs. 3.0iu/kg insulin KO; 0.75iu/kg insulin KO vs. 3.0iu/kg insulin KO. c, P < 0.001 0.75iu/kg insulin wild-type vs. 0.75iu/kg insulin KO. NS, no significant difference (P >0.05) saline wild-type vs. saline KO.