IMMOBILIZATION-INDUCED STRESS ACTIVATES NEURONAL NITRIC-OXIDE SYNTHASE (NNOS) MESSENGER-RNA AND PROTEIN IN HYPOTHALAMIC-PITUITARY-ADRENAL AXIS IN RATS

The purpose of this study was to determine whether immobilization stre ss can cause changes in the enzyme activity and gene expression of neu ronal nitric oxide synthase (nNOS) in the hypothalamus, pituitary, and adrenal gland in rats. NOS enzyme activity was measured as the rate o f [H-3]arginine conversion to citrulline, and the level of nNOS mRNA s ignal was determined using in situ hybridization and image analysis. N OS-positive cells were also visualized using nicotinamide adenine dinu cleotide phosphate-diaphorase (NADPH-diaphorase) histochemistry and by immunohistochemistry using an anti-nNOS antibody. A significant incre ase 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 str essed animals (immobilization of 6 h) as compared to non-stressed cont rol 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 hypotha lamic paraventricular nucleus (PVN) significantly increased after the stress for 6 h. This increase in NOS enzyme activity was confirmed usi ng NADPH-diaphorase staining and immunostaining in the PVN and adrenal cortex. An increase of NOS enzyme activity in adrenal medulla after i mmobilization for 6 h posited by far longer than in the adrenal cortex and anterior pituitary. The present findings suggest that psychologic al and/or physiological stress causes NO release in hypothalamic-pitui tary-adrenal (HPA) axis and in sympatho-adrenal system. It is suggeste d that NO may modulate a stress-induced activation of the HPA axis and the sympatho-adrenal medullary system. The different duration of stre ss-induced NOS activity in HPA axis and the adrenal medulla may sugges t NO synthesis is controlled by separate mechanism in the two HPA and the sympatho-adrenal systems.