Abstract 410 Poster Session III, Monday, 5/3 (poster 159)

Background: Animals chronically addicted to morphine have enhanced NOS activity in the brain following naloxone-induced withdrawal. NO levels also increase following discontinuation of MS infusion with a concomitant increase in brain NOS (bNOS) activity, indicating withdrawal. The objectives of this study were (1) to investigate the effect of MS infusion on NO levels in systemic and sagittal sinus vein plasma during and following infusion, and in brain homogenates at 2 hours post MS infusion; (2) to determine the mRNA expression of NOS in the brain at 2 hours post MS infusion.

Methods: Six newborn piglets received a loading dose of MS (100 µg/kg) over 5 minutes followed by continuous intravenous infusion of MS (100 µg/kg/hr) in 5% dextrose for 4 hours. The control animals (n=6) received equivalent volumes of 5% dextrose. Systemic and sagittal sinus vein blood samples were collected at baseline (pre-bolus), 0.5 hr post-bolus, 1, 2, 3, & 4 hours during continuous infusion, and at 1 & 2 hours following cessation of infusion. Total nitrite(NO2-) and nitrate (NO3-) levels, as indicators of NO production, were determined using the Griess method. The cerebral cortex, thalamus, brainstem, and cerebellum were sectioned for analysis of total NO2- and NO3- levels and for extraction of total RNA.

Results: MS infusion resulted in a transient increase in mean systemic total NO2- and NO3- levels at 0.5 hr post-bolus dose (18.0±1.7 µM, p<0.05) compared to baseline (14.4±1.2 µM). However, the values declined at 1 and 2 hours during continuous infusion (9.8±0.6 µM and 6.5±1.0 µM, respectively, p<0.001) and at 1 and 2 hours post infusion (6.4±2.0 µM and 8.3±0.8 µM, respectively, p<0.01) compared to baseline values. Mean sagittal sinus vein total nitrite and nitrate levels decreased at 30 minutes post-bolus (9.6±0.4 µM, p<0.05) and at 2, 3, & 4 hours during continuous infusion (8.2±0.8 µM, p<0.05, 8.4±1.5 µM, p<0.05, and 7.6±1.1 µM, p<0.01, respectively) compared to baseline(12.8±0.4 µM). Brain total nitrite and nitrate levels increased in the brainstem and cerebral cortex of MS-treated animals (15.3±3.4 and 20.0±3.2 µM/mg protein, respectively, p<0.05) compared to control levels (7.0±1.1 and 10.1±2.1 µM/mg protein, respectively). RT-PCR assays revealed >90%, (p<0.01) increases in NOS mRNA expression in the brain sections taken from the animals in the MS-treated group at 2 hours post infusion compared to the control group.

Conclusions: MS appears to inhibit NO production during infusion. NO levels increase in brain stem and cerebral cortex by 2 hours of discontinuation of MS with concomitant increase in NOS mRNA expression in all regions of the brain studied. These data may imply that the molecular evidence of withdrawal starts at least by 2 hours following discontinuation of MS infusion in newborn piglets.