Regulation of Inducible Nitric Oxide Synthase Expression by Macrophage Purinoreceptors and Calcium

Abstract

Macrophage activation is central to the progression of multiple diseases via the release of inflammatory mediators such as cytokines and nitric oxide. Despite the recognized overlap in the regulatory mechanisms involved in mediator production, little formation exists regarding receptor-initiated signaling pathways that coordinately control multiple end points, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide production. In this study, the expression of inducible nitric oxide synthase (iNOS) in macrophages is shown to be regulated by calcium and by a purinoreceptor signaling system. The P2Y purinoreceptor partial agonist, 2-methylthio-ATP (2-MeS-ATP), inhibits the expression of iNOS induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) in primary macrophages. Additionally, 2-MeS-ATP attenuates the expression of iNOS in macrophages isolated from CD-1 mice challenged with LPS, and it inhibits LPS-induced TNF-alpha and interleukin-1 alpha (IL-1 alpha) release, thereby preventing endotoxic death. Thus, purinoreceptors and calcium are likely to be critical for macrophage activation and the production of inflammatory mediators stimulated by LPS.

Endotoxin Activation of Macrophages Does Not Induce ATP Release and Autocrine Stimulation of P2 Nucleotide Receptors

“…Activation of the c-Jun N-terminal kinase cascade and production of IL-1␤ in response to endotoxin have also been observed in this particular macrophage line (33) (R. D. Beigi, S. B. Kertesy, and G. R. Dubyak, unpublished observations). Most experiments and results described below were replicated using RAW 264.7 macrophages, another murine cell line widely used for studies of endotoxin signaling (6,7,(17)(18)(19)34).…”

“…In further support of this model, macrophages are known to express both G protein-coupled (P2Y) and ionotropic (P2X) nucleotide receptors, including the P2X7 pore-forming receptor (12)(13)(14). Additionally, a subset of macrophage responses to endotoxin is modulated by costimulation with exogenous nucleotides (5,(15)(16)(17)(18)(19)(20)(21).…”

Lipopolysaccharide-induced Apoptosis of Macrophages Determines the Up-regulation of Concentrative Nucleoside Transporters Cnt1 and Cnt2 through Tumor Necrosis Factor-α-dependent and -independent Mechanisms

“…Nucleosides, and particularly adenosine, can inhibit TNF-␣ and NO synthesis in LPS-stimulated and IFN-␥-induced macrophages (7)(8)(9)(10)(11)(12)19) by mechanisms that rely, at least partially, upon the interaction of adenosine with membrane receptors. The long term control of these regulatory interactions appears to involve receptor synthesis and insertion into the plasma membrane of the activated macrophage, as suggested by evidence that IFN-␥ can up-regulate the synthesis of adenosine receptors of the A2B type (20).…”

“…Macrophage activation and apoptosis appear to be modulated by extracellular nucleosides and nucleotides (7)(8)(9)(10)(11)(12). These effects are partially explained by their interaction with adenosine and P2 receptors, respectively.…”

ATP Stimulates Human Macrophages to Kill Intracellular Virulent Mycobacterium tuberculosis Via Calcium-Dependent Phagosome-Lysosome Fusion

“…Because Ca 2ϩ regulates several phagocyte antimicrobial responses including generation of reactive oxygen species (33) and reactive nitrogen intermediates (34,35), granule secretion (36), synthesis of cytokines (37), and, in certain cases, phagosome-lysosome (P-L) fusion (9), the Ca 2ϩ -dependence of ATP e -induced killing of M. tuberculosis may be due to multiple effects. We tested the hypothesis that increases in [Ca 2ϩ ] c promote ATP e -induced killing of M. tuberculosis via enhancement of P-L fusion based on the following rationale: 1) During initial infection of naive human M by M. tuberculosis, mycobacterial inhibition of M Ca 2ϩ signaling is tightly coupled to inhibition of P-L fusion and promotion of intracellular bacterial viability (9); 2) pharmacologic reversal of M. tuberculosis-induced inhibition of M Ca 2ϩ signaling results in increased levels of both P-L fusion and mycobacterial killing (9); and 3) ATP e -stimulated killing of BCG does not involve reactive oxygen or reactive nitrogen intermediates species and has not been linked to synthesis or secretion of inflammatory mediators by infected M (8).…”