AJP - Lung Cellular and Molecular Physiology, Vol 273, Issue 5 967-L979, Copyright © 1997 by American Physiological Society

ARTICLES

cAMP and purinergic P2y receptors upregulate and enhance inducible NO synthase mRNA and protein in vivo

S. S. Greenberg, X. Zhao, J. F. Wang, L. Hua and J. Ouyang
Department of Medicine, Louisiana State University Medical Center, New Orleans 70112, USA.

Adenosine 3',5'-cyclic monophosphate (cAMP) and purinergic P2y receptor agonists upregulate inducible nitric oxide (NO) synthase (iNOS) but inhibit Escherichia coli endotoxin lipopolysaccharide (LPS)- and cytokine-mediated upregulation of iNOS in cultured cells. We examined the effects of cAMP and P2y receptor agonists on the iNOS system in vivo. Intratracheal administration of dibutyryl-cAMP (DBcAMP, 0.1 and 1 mg/kg), a P2y receptor agonist [2-methylthioadenosine 5'-triphosphate (MeS-ATP), 5 mg/kg], or LPS (0.6 mg/kg) to rats 2 h before bronchoalveolar lavage (BAL) increased iNOS mRNA (competitor-equalized reverse transcription-polymerase chain reaction) and iNOS protein (Western blot) in rat alveolar macrophages compared with the effects of sterile phosphate-buffered saline (0.5 ml it). At equal levels of upregulation of iNOS mRNA, 1) LPS, but not DBcAMP or MeS-ATP, upregulated nuclear transcription factor-kappa B (NF-kappa B) and 2) iNOS protein and formation of NO were greater in alveolar macrophages from LPS- and MeS-ATP-treated rats than from DBcAMP-treated rats. Administration of DBcAMP or MeS-AMP 15 min before LPS did not inhibit LPS-induced alveolar macrophage-derived iNOS mRNA, iNOS protein, and NO. Diethyldithiocarbamate (DETC, 5 mg/kg it) inhibited LPS-induced iNOS mRNA but did not affect upregulation of iNOS mRNA produced by the other agonists. We conclude that an LPS-dependent and -independent pathway of iNOS mRNA induction exists in vivo. The former is activated by IPS and most cytokines, is associated with upregulation of NF-kappa B and inhibited by DETC, and elicits an inflammatory response. The latter, activated by DBcAMP and MeS-ATP, is not associated with upregulation of NF-kappa B, inhibition by DETC, or activation of inflammation. The two systems are additive in vivo rather than antagonistic. Speculatively, if the LPS-independent iNOS pathway exists in humans, the iNOS in tissues from patients taking drugs affecting cAMP or P2y receptors may be iatrogenic rather than pathogenetic in origin.

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