OBJECTIVE: The first aim of the present study was to determine if morphine, a prototypic mu-opioid agonist drug, affects pituitary-adrenocortical activity in developing rat pups (first and second weeks of postnatal life). The second aim of this study was to explore, in vivo, if nitric oxide (NO) could be involved in the neurohormonal response to morphine in the early stages of postnatal life. METHODS: Plasma ACTH and corticosterone concentrations were determined by RIA in rat pups (n=5-14 rats/experimental group) after they had been killed by decapitation. In a first experiment, 1-day and 1- and 2-week-old rats were treated s.c. with morphine (20 mg/kg) or with vehicle (0.9% NaCl) and killed 5-90 min later. In a second experiment, 2-week-old pups were pretreated s.c. with naltrexone (NAL; 0.4 mg/kg or 10 mg/kg), and injected 1 h later with either morphine (20 mg/kg) or vehicle, and killed 30 min later. Some pups injected with only NAL were killed 60 or 90 min later. On the other hand, pups injected with NAL (10 mg/kg) or NAL and morphine were killed 30 min later. In a third experiment, 2-week-old pups were pretreated s.c. with N-omega-nitro l arginine methyl-ester (L-NAME; 30 mg/kg or 100 mg/kg), and injected 1 h later with either morphine (20 mg/kg) or vehicle, and killed 30 min later. Moreover, some pups injected with L-NAME (100 mg/kg) or L-NAME with morphine were killed 30 min later. In a final experiment, pups were injected s.c. with either S-nitroso-N-acetylpenicillamine (SNAP; 5 mg/kg) or vehicle, and killed 60 or 90 min later. RESULTS: Morphine administered to rat pups elicited marked rises in both ACTH and corticosterone secretion. Moreover, these responses increased with advancing postnatal age. In 2-week-old rat pups, NAL, a competitive antagonist at mu-opioid receptors, administered alone increased both plasma ACTH and corticosterone concentrations 30 min later. L-NAME, a specific NO synthase inhibitor, did not affect plasma ACTH and corticosterone concentrations 30 min later when administered alone. NAL, when concomitantly administered with morphine, was unable to block morphine responses. In contrast, morphine responses were blocked by pretreatment (60 min before) with NAL or with L-NAME. Acute injection of SNAP increased both ACTH and corticosterone release. CONCLUSION: Our results suggest that opioids have controversial effects on pituitary-adrenocortical activity in the early postnatal period in the rat, and that endogenous NO is one of the major factors in the response of the pituitary-adrenocortical axis to morphine.
J Lesage, F Bernet, V Montel and JP Dupouy
N Sebaai, J Lesage, A Alaoui, JP Dupouy and S Deloof
OBJECTIVE: The first aim of this work was to investigate, under basal conditions in adult male rats, the long-term consequences of perinatal maternal food restriction on the plasma concentrations of vasopressin (VP), aldosterone and atrial natriuretic peptide (ANP) and on plasma renin activity (PRA). Furthermore, under these same conditions, the hypothalamic VP gene expression as well as the density (B(max)), affinity (K(d)) and gene expression of ANP receptors were determined in kidneys and adrenals. The second aim of this work was to examine the responsiveness to dehydration in perinatally malnourished rats. Thus, the latter parameters were studied in these rats after 72 h water deprivation. METHODS: This study was conducted on 4-Month-old male rats from mothers exposed to 50% food restriction (FR50) during the last week of gestation and lactation and on age-matched control animals (C). At this stage, both C and FR50 rats were killed by decapitation between 0900 h and 1000 h in order to determine parameters under basal conditions or after 72 h water deprivation. Plasma VP, ANP and aldosterone levels and PRA were determined by radioimmunoassay. Hypothalamic VP gene expression was determined in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization. The B(max) and K(d) values of ANP receptors were evaluated from Scatchard plots. ANP receptor gene expression was determined by Northern blot analysis. RESULTS: Under basal conditions, perinatal malnutrition reduced body weight, absolute weight of kidneys and adrenals, and haematocrit. Compared with control rats, FR50 rats had significantly greater plasma VP and aldosterone levels but PRA, plasma ANP levels, plasma osmolality and hypothalamic VP gene expression were not significantly different. Perinatal malnutrition did not significantly affect glomerular ANP receptor density, but in adrenals it decreased both B(max) and K(d) values of ANP-B receptors (biological receptors) and increased B(max) of ANP-C receptors (clearance receptors). ANP-B(A) (receptor subtype A of ANP-B receptors) receptor gene expression was not significantly affected, whereas ANP-C receptor gene expression was enhanced in both adrenals and kidneys in FR50 rats. After 72 h dehydration, control rats showed a significant rise in haematocrit, plasma osmolality, PRA, circulating levels of VP and aldosterone and VP gene expression in both PVN and SON but showed a decrease in plasma ANP levels. B(max) of ANP-B receptors was decreased whereas B(max) of ANP-C receptors was enhanced in both adrenals and kidneys. ANP-B(A) receptor gene expression was not significantly affected in either kidneys or adrenals in dehydrated control rats. Similarly, ANP-C receptor gene expression was unaffected in kidneys whereas it was significantly enhanced in adrenals. In FR50 rats, the effects of water deprivation were qualitatively similar to those reported in controls concerning plasma parameters except for plasma VP levels which tended to rise (not significant) but this increase was only very slight compared with controls. Moreover, unlike controls, VP gene expression in both PVN and SON was not enhanced after dehydration in FR50 rats. In kidneys, dehydrated FR50 rats, like controls, upregulated ANP-C receptors, but they were unable to downregulate ANP-B receptors. In adrenals, unlike controls, FR50 rats enhanced ANP-B receptor density whereas they decreased both ANP-C receptor density and expression after 72 h dehydration. Similar to controls, the expression of ANP-B(A) receptors in both kidneys and adrenals as well as the expression of ANP-C receptors in kidneys, were unaffected in dehydrated FR50 rats. CONCLUSION: Perinatal malnutrition had long-lasting effects on regulation of the fluid and electrolyte balance under basal conditions. The main effects were a significant rise in circulating levels of VP and aldosterone, and changes in density of adrenal ANP-binding sites and ANP-C receptor gene expression in both adrenals and kidneys. Perinatal malnutrition also affects the responsiveness to water deprivation with alterations in both hypothalamic VP gene expression and regulation of ANP-binding sites.