HIV-1 gp120 protein modulates corticotropin releasing factor synthesis andrelease via the stimulation of its mRNA from the rat hypothalamus in vitro: involvement of inducible nitric oxide synthase

In the present study, we examined whether the human immunodeficiency virus type I (HIV-I) gp120 coat protein can modulate corticotropin releasing fact or (CRF) secretion by using the incubation of rat hypothalamic explants as an in vitro model. Treatment of the hypothalamic fragments with recombinant gp120 resulted in a time- and concentration-dependent increase in CRF rele ase. The maximal dose of 10 nM gp120 increased CRF release by 56.4% after 1 h, and 78.4% after 3 h, as compared with their respective controls. The in tra-hypothalamic amount of CRF was also increased by 54.7% and 77.3% vs. co ntrols after I and 3 h. respectively. Moreover. the action of gp120 was blo cked by pretreatment with cycloheximide, suggesting that the viral protein modulates CRF secretion via an increase in its synthesis. We also investiga ted the effects of gp120 on CRF gene expression. RNase protection analyses of total RNA isolated from the explants indicated that 10 nM gp120 signific antly increases CRF mRNA in a time-dependent manner. Furthermore, gp120 did not modify CRF mRNA stability, suggesting that the viral protein modulates CRF gene expression at the transcriptional level. Analysis of the mechanis ms that mediate gp120-induced CRF synthesis was conducted. The incubation o f the explants with recombinant interleukin-1 (IL-1) type I receptor antago nist (hrIL-1 ra)did not antagonize the actions of gp120 at 1 and 3 h. indic ating that the effect of the latter is independent of IL-I mediated mechani sms. The involvement of some second messenger pathways was also investigate d. Specific inhibitors of cAMP-PKA, cyclo-oxygenase or heme oxygenase pathw ays failed to antagonize the gp120-induced increase in CRF production. By c ontrast. incubation with nonselective inhibitors of nitric oxide synthase ( NOS), L-NAME and L-NNA. or aminoguanidine (AG), a selective inhibitor of in ducible NOS (iNOS), blocked CRF release and, AG. its mRNA accumulation, sti mulated by gp120, whereas selective inhibitors of endothelial and neuronal NOS had no effect. In addition, only L-NAME, L-NNA and AG were able to inhi bit the gp120-stimulated production of nitrites. These results indicate tha t gp120 directly stimulates CRF gene expression and peptide synthesis from the rat hypothalamus in vitro via the activation of iNOS. Therefore, the ac tions of this viral protein on the HPA axis may. in part, reflect its abili ty to modulate CRF synthesis. (C) 2001 Elsevier Science B.V. All rights res erved.