ENDOTOXIN INDUCES INTERLEUKIN-1-BETA AND NITRIC-OXIDE SYNTHASE MESSENGER-RNA IN RAT HYPOTHALAMUS AND PITUITARY

The gases nitric oxide (NO) and carbon monoxide (CO) may be involved i n hypothalamopituitary-adrenal axis (HPA) modulation. In the brain, NO is synthesized by two forms of NO synthase (NOS), a constitutive neur onal form (nNOS) and an inducible form (iNOS). There are also a consti tutive heme oxygenase (HO2) and an inducible form (HO1) which generate CO. We have therefore investigated the effect of peripheral lipopolys accharide (LPS) administration on the gene expression of these enzymes along with interleukin-1 beta (IL-1 beta) gene expression in the hypo thalamus, pituitary and liver. Male Wistar rats (200-250 g body weight ) were injected intraperitoneally with endotoxin (Escherichia coli, 05 5 B5) dissolved in sterile normal saline [250 mu g/kg first group, 2.5 mg/kg (second group) and 6.25 mg/kg (third group)] in a final volume of 0.5 mi, or saline alone in the control group. The first and the sec ond groups were studied 1, 3, 8 and 24 h after LPS (n = 4 per group); the third group was studied at 3 h. Total RNA was extracted from the h ypothalamus, pituitary and liver, and cDNA was made using standard rev erse transcriptase methods. Duplex polymerase chain reaction (PCR) was standardised in order to quantify the expression of a specific gene i n relation to the 'house-keeping' gene beta-actin. The specific genes studied were iNOS, nNOS, HO1, HO2 and IL-1 beta. The PCR products were separated on agarose gel and densitometric analysis of the bands allo wed semi-quantification. In the second group, iNOS and IL-1 beta were induced in hypothalamus, pituitary and liver, showing a peak at 3 h (p < 0.001), returning to baseline levels at 24 h. Neuronal NOS was not expressed in the liver under basal conditions or after LPS; in the hyp othalamus and pituitary, nNOS was expressed basally but there was no c hange after LPS. In the first group, iNOS and IL-1 beta were again ind uced in all three tissues studied, but with a delayed time course comp ared to the second and third groups; the peak change for IL-1 beta occ urred at 8 h (p < 0.05), again returning to baseline levels at 24 h. T he peak for iNOS occurred at 24 h. HO1 and HO2 were expressed in all t hree tissues under basal conditions; HO1 was increased at 1 h in the l iver in the second group, and at 3 h in the pituitary in the third gro up, There was no change in either HO1 or HO2 in the hypothalamus at an y dose at any time point. We conclude that IL-1 beta and iNOS are indu ced in rat hypothalamus and pituitary following various doses of endot oxin. We speculate that while IL-1 beta may mediate stimulation of the HPA by endotoxin, NO generation may be involved in the counter-regula tion of this response.