Abstract
Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different brain cells in response to various cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. This study underlines the importance of cAMP in inhibiting the induction of NO production by lipopolysaccharide (LPS) and cytokines in rat primary astrocytes. Compounds (forskolin, 8-bromo-cAMP, and (Sp)-cAMP) that increase cAMP and activate protein kinase A (PKA) were found to inhibit LPS- and cytokine-mediated production of NO as well as the expression of iNOS, whereas compounds (H-89 and (Rp)-cAMP) that decrease cAMP and PKA activity stimulated the production of NO and the expression of iNOS in rat primary astrocytes. Forskolin, but not the inactive analogue 1,9-dideoxyforskolin, inhibited NO production and iNOS expression in a dose-dependent manner in astrocytes. The inhibition of LPS- and/or cytokine-induced NO production in rat C6 glial cells by forskolin suggest that similar to astrocytes, iNOS expression in C6 cells is also regulated by similar mechanisms. In contrast, in rat peritoneal macrophages the cAMP analogues stimulated the LPS- and cytokine-induced production of NO. In vitro, the PKA had no effect on iNOS activity in LPS-treated astrocytes or macrophages, suggesting that PKA modulates the intracellular signaling events associated with the induction of iNOS biogenesis rather than the post-translational modification of iNOS. The compounds which activate PKA activity, blocked the activation of NF-kappabeta in astrocytes but stimulated the activation of NF-kappabeta in macrophages. This differential regulation of NF-kappabeta activation in two different cell types (astrocytes and macrophages) by the same second messenger (cAMP) indicates that intracellular events or pathways in the activation of NF-kappabeta may be different. Moreover, this inhibition of iNOS expression in LPS- and cytokine-treated astrocytes by cAMP may be of therapeutic potential in NO-mediated cytotoxicity in neurodegenerative diseases.
Highlights
Modulation of LPS-induced Nitric oxide (NO) Production and Expression of inducible nitric-oxide synthase (iNOS) in Rat Primary Astrocytes by Compounds That Modulate Intracellular Levels of cAMP—Primary astrocytes in serum-free DMEM/F-12 were treated with different activators and inhibitors of protein kinase (PKA) 15 min before the addition of 1 g/ml LPS
Recent studies have provided evidence that cAMP induces the expression of iNOS in LPS- and cytokine-stimulated glomerular mesangial cells [33], smooth muscle cells [34], cardiac myocytes [35], murine 3T3 fibroblasts [28], and peritoneal macrophages [26]
The increase in cAMP as a result of inhibition of phosphodiesterase, or an exogenous supply of cAMP derivatives or compounds that enhance intracellular levels of cAMP cause induction of iNOS. Contrary to these results we have observed that intracellular levels of cAMP negatively regulate the expression of iNOS in LPS or cytokine-stimulated primary astrocytes from rat brain
Summary
At low concentration NO has been shown to play a role in neurotransmission and vasodilation and NO secreted at higher concentrations is implicated in having a role in the pathogenesis of stroke and other neurodegenerative diseases [2] This is of particular importance in conditions associated with infiltrating macrophages and production of proinflammatory cytokines such as demyelinating conditions (e.g. multiple sclerosis, experimental allergic encephalopathy, and X-adrenoleukodystrophy) and in ischemic and traumatic injuries [3,4,5,6]. The differential effect of Rolipram, an inhibitor of type IV phosphodiesterase on the production of TNF-␣ and NO in a murine macrophage cell line suggests a role for cAMP in LPS-induced immune response [12]. We report here that cAMP-dependent protein kinase (PKA) significantly inhibits LPS- and cytokine-mediated activation of NF- and the expression of iNOS in rat primary astrocytes. This is the first example where cAMP regulates the activation of NF- and induction of iNOS differentially in two cell types (astrocytes and macrophages) of the same animal species (rat)
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