Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid

https://doi.org/10.1016/j.freeradbiomed.2003.10.026Get rights and content

Abstract

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.

Introduction

Nitric oxide (NO), an important messenger molecule involved in various physiological processes [1], [2], [3], is synthesized by three distinct isoforms of NO synthase (NOS) [4], [5]. Two isoforms are constitutively expressed, neuronal NOS (nNOS or type I) and endothelial NOS (eNOS or type III), and the third member of the family is the inducible NOS (type II). nNOS and eNOS are classified as Ca2+/calmodulin-dependent enzymes [6] and their enzymatic activities are regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i).

In the nervous system, activation of cell surface receptors triggers the Ca2+-dependent nNOS activation and subsequent synthesis of NO, which regulates neurotransmitter release [7], plays a crucial role in synaptic plasticity [8], and affords cytoprotection in various toxicity paradigms [9], [10], [11]. One of the most relevant mechanisms operative in the inhibition of nNOS activity is enzyme phosphorylation. In particular, in vitro studies indicate that phosphorylation of nNOS by protein kinase A, cAMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II (CaMK-II) inhibits its catalytic activity [12], [13], [14], whereas the opposite effect is observed upon its dephosphorylation [15], [16]. In intact cells, rapid inhibition of nNOS activity was associated with enzyme phosphorylation induced by either protein-tyrosine kinases [17] or CaMK-II [18]. To our best knowledge, however, the upstream events leading to kinase activation remain unknown.

Cytosolic phospholipase A2 (cPLA2) is a high-molecular-mass member of the family of PLA2 enzymes, and its activity is regulated by increases in [Ca2+]i. cPLA2 plays a central role in the release of arachidonic acid (AA) that occurs in many types of cells in response to a variety of stimuli [19]. AA and/or its metabolites regulates the activity of numerous kinases, including protein-tyrosine kinases [20] or CaMK-II [21], both kinases being able to elicit nNOS phosphorylation, as previously described [17], [18]. Hence, cPLA2 may represent a good candidate for triggering a downregulation of NOS activity and may be an important component of the “cross talk” between Ca2+- and NO-regulated signal transduction pathways in neuronal cells. In our opinion, it is reasonable to hypothesize that Ca2+ has the potential of triggering nNOS activation as well as parallel pathways (i.e., PLA2) leading to activation of kinases which eventually phosphorylate and inactivate nNOS.

In the present study, we investigated the above hypothesis using nNOS-expressing rat PC12 pheochromocytoma cells [22], [23], as a cellular model. Our results clearly indicate that increasing the [Ca2+]i promotes a progressive activation of cPLA2 and NOS, paralleled by an accumulation of the respective products AA and NO. The stimulation of cPLA2 may in turn produce critical levels of an AA-triggering signaling pathway(s), which then causes inhibition of NOS activity.

Section snippets

Materials

AA, A23187, 5,8,11,14-eicosatetraynoic acid (ETYA), melittin, Nω-nitro-l-arginine methyl ester (l-NAME), 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), thapsigargin (Tg), S-nitroso-N-acetylpenicillamine (SNAP), and other reagent-grade biochemicals were from Sigma–Aldrich (Milan, Italy). AACOCF3, cyclosporin A (CsA), and FK506 were purchased from Calbiochem (Milan, Italy). [3H]AA and l-[3H]arginine were from Amersham Italia (Milan, Italy). 4,5-Diaminofluorescein diacetate

Results and discussion

PC12 cells were loaded with 10 μM DAF-2DA for 20 min, treated as detailed below, and examined by confocal microscopy. Fig. 1 shows that a 15 min exposure to the calcium ionophore A23187 (2.5 μM) promoted a remarkable fluorescence indicative of NO formation because it was sensitive to both the NOS inhibitor l-NAME (1 mM) and the NO scavenger PTIO (50 μM). Additional evidence is provided by the observation that two different calcineurin inhibitors, CsA (1 μM) and FK506 (1 μM), also blunted

Acknowledgements

This work was supported by a grant from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Progetti di Ricerca di Interesse Nazionale.

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