Corticotropin-releasing factor (CRF) receptor-1 is involved in cardiac noradrenergic activity observed during naloxone-precipitated morphine withdrawal

Abstract

Background and purpose: The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [noradrenergic activity, induction of the hypothalamic-pituitary-adrenocortical (HPA) axis and activation of heat shock proteins (Hsps)]. Corticotropin-releasing factor (CRF) pathways are important mediators in the negative symptoms of opioid withdrawal. We performed a series of experiments to characterize the role of the CRF₁ receptor in the response of stress systems to morphine withdrawal and its effect in the heart using genetically engineered mice lacking functional CRF₁ receptors.

Experimental approach: Wild-type and CRF₁ receptor-knockout mice were treated with increasing doses of morphine. Precipitated withdrawal was induced by naloxone. Plasma adrenocorticotropic hormone (ACTH) and corticosterone levels, the expression of myocardial Hsp27, Hsp27 phosphorylated at Ser⁸², membrane (MB)- COMT, soluble (S)-COMT protein and NA turnover were evaluated by RIA, immunoblotting and HPLC.

Key results: During morphine withdrawal we observed an enhancement of NA turnover in parallel with an increase in mean arterial blood pressure (MAP) and heart rate (HR) in wild-type mice. In addition, naloxone-precipitated morphine withdrawal induced an activation of HPA axis and Hsp27. The principal finding of the present study was that plasma ACTH and corticosterone levels, MB-COMT, S-COMT, NA turnover, and Hsp27 expression and activation observed during morphine withdrawal were significantly inhibited in the CRF₁ receptor-knockout mice.

Conclusion and implications: Our results demonstrate that CRF/CRF₁ receptor activation may contribute to stress-induced cardiovascular dysfunction after naloxone-precipitated morphine withdrawal and suggest that CRF/CRF₁ receptor pathways could contribute to cardiovascular disease associated with opioid addiction.

Keywords: CRF1 receptor knockout mice; Hsp 27; NA turnover; heart; naloxone-precipitated morphine withdrawal.

Figure 1

(A) Effect of saline or morphine injection on body weight gain in wild-type or CRF₁ receptor knockout (CRF/–) mice. The animals received increasing doses of morphine (10–60 mg·kg⁻¹, i.p.) or saline every 12 h for four days. (B) Effect of naloxone (1 mg·kg⁻¹, s.c.) injection on body weight loss in wild-type and CRF₁ receptor knockout mice treated with morphine or saline. Data are the mean ± SEM. ###P < 0.001 versus saline; ***P < 0.001 versus morphine + saline; +++P < 0.001 versus saline + naloxone; &&P < 0.01 versus wild-type mice.

Figure 2

Baseline MAP (mmHg) (A) and HR (beats·min⁻¹) (C) in wild-type and CRF₁ receptor knockout mice treated with saline or morphine (A, C). Effects of naloxone (1 mg·kg⁻¹s.c.) on changes in MAP and HR rate in wild-type and CRF₁ receptor-deficient mice (B, D). Naloxone was injected at time 0. Data are the mean ± SEM (n = 4–5). *P < 0.05, **P < 0.01, ***P < 0.001 versus saline + naloxone; + P < 0.05, +++P < 0.001 versus wild-type mice; &P < 0.05 versus saline.

Figure 3

NA (A), NMN (B) content and NMN/NA ratio (C) in left ventricle after naloxone or saline administration to placebo (saline) or morphine-treated wild-type and CRF₁ receptor knockout mice. Data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus morphine+saline; ++P < 0.01, +++P < 0.001 versus saline + naloxone; &&P < 0.01, &&&P < 0.001 versus wild-type mice.

Figure 4

Western-blotting analysis of membrane-COMT (MB-COMT) (A) and soluble-COMT (S-COMT) (B) in left ventricle after saline or naloxone (nx) administration to placebo (saline, s) or morphine (m)-treated wild-type and CRF₁ receptor knockout mice. The immunoreactivity corresponding to MB-COMT or S-COMT is expressed as a percentage of that in the control group defined as 100%. Data are the mean ± SEM. **P < 0.01, ***P < 0.001, versus morphine+saline; ++P < 0.01, +++P < 0.001 versus saline+naloxone; &&P < 0.01, &&&P < 0.001 versus wild-type mice.

Figure 5

Western-blotting analysis of Hsp27 (A) phospho (p)-Ser⁸² (B) and phospho(p)-Hsp27/Hsp27 ratio in left ventricle after saline or naloxone (nx) administration to placebo (saline, s) or morphine (m)- treated wild-type and CRF₁ receptor knockout mice. The immunoreactivity corresponding to Hsp27 and pHsp27 is expressed as a percentage of that in the control group defined as 100%. Data are the mean ± SEM **P < 0.01, ***P < 0.001 versus morphine + saline; + P < 0.05, ++P < 0.01 versus saline + naloxone; $P < 0.05 versus saline+saline; &P < 0.05, && P < 0.01 versus wild-type mice.

Figure 6

Plasma ACTH (A) and corticosterone (B) concentrations 1 and 24 h after saline or naloxone (nx) injection to placebo (saline) or morphine-treated wild-type and CRF₁ receptor knockout mice. Data are the mean ± SEM. ***P < 0.001, versus morphine + saline; ++P < 0.01, +++P < 0.001 versus saline + naloxone; &P < 0.05, &&&P < 0.001 versus wild-type mice.