Immobilization-induced stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamic-pituitary-adrenal axis in rats

The purpose of this study was to determine whether immobilization stress can cause changes in the enzyme activity and gene expression of neuronal nitric oxide synthase (nNOS) in the hypothalamus, pituitary, and adrenal gland in rats. NOS enzyme activity was measured as the rate of [H-3]arginine conversion to citrulline, and the level of nNOS mRNA signal was determined using in situ hybridization and image analysis. NOS-positive cells were also visualized using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) histochemistry and by immunohistochemistry using an anti-nNOS antibody. A significant increase of NOS enzyme activity in the anterior pituitary, adrenal cortex, and adrenal medulla (1.5-, 3.5-, and 2.5-fold) was observed in the stressed animals (immobilization of 6 h) as compared to non-stressed control rats. Up-regulation of nNOS mRNA expression in anterior pituitary and adrenal cortex was already detectable after stress for 2 h with 1.5- and 2-fold increase, respectively. The nNOS mRNA signals in hypothalamic paraventricular nucleus (PVN) significantly increased after the stress for 6 h. This increase in NOS enzyme activity was confirmed using NADPH-diaphorase staining and immunostaining in the PVN and adrenal cortex. An increase of NOS enzyme activity in adrenal medulla after immobilization for 6 h posited by far longer than in the adrenal cortex and anterior pituitary. The present findings suggest that psychological and/or physiological stress causes NO release in hypothalamic-pituitary-adrenal (HPA) axis and in sympatho-adrenal system. It is suggested that NO may modulate a stress-induced activation of the HPA axis and the sympatho-adrenal medullary system. The different duration of stress-induced NOS activity in HPA axis and the adrenal medulla may suggest NO synthesis is controlled by separate mechanism in the two HPA and the sympatho-adrenal systems.