Purinergic Receptor Modulation of Lipopolysaccharide Signaling and Inducible Nitric-oxide Synthase Expression in RAW 264.7 Macrophages

doi:10.1074/jbc.273.42.27170

Purinergic Receptor Modulation of Lipopolysaccharide Signaling and Inducible Nitric-oxide Synthase Expression in RAW 264.7 Macrophages*

From the Departments of ^tOaBiomolecular Chemistry,^tOcCellular and Molecular Biology, ^tOdMedical Microbiology and Immunology, ^tOfMolecular and Cellular Pharmacology, and ^tOhMedicine, University of Wisconsin Medical School, Madison, Wisconsin 53706

Abstract

Previous studies have suggested that the P2Z/P2X₇ purinergic receptor can participate in nucleotide-induced modulation of lipopolysaccharide (LPS) stimulated inflammatory mediator production. To test this hypothesis, we evaluated whether antagonism of the P2Z/P2X₇ receptor can influence LPS signaling and expression of the inducible form of nitric-oxide synthase (iNOS) in RAW 264.7 macrophages. In the present study, we demonstrate that pretreatment of RAW 264.7 macrophages with a P2Z/P2X₇ receptor antagonist, periodate oxidized adenosine 5′-triphosphate (o-ATP), substantially inhibits LPS-stimulated NO production and iNOS expression without altering cell viability. This effect on LPS-induced iNOS expression is mimicked by a pyridoxal-phosphate-based antagonist (pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid) of the P2Z/P2X₇ purinergic receptor, indicating that these results are not unique to o-ATP. Additionally, o-ATP prevents cell death induced by P2Z/P2X₇ receptor agonists. To ascertain how P2Z/P2X₇ receptor antagonists influence LPS signaling, we evaluated the capacity of o-ATP to regulate LPS-mediated activation of the transcription factor, nuclear factor-κB, and the mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2. These experiments reveal that pretreatment of RAW 264.7 cells with o-ATP attenuates the LPS stimulation of a nuclear factor-κB-like binding activity. Moreover, the activation of ERK1 and ERK2 by LPS, but not by the phorbol ester, phorbol 12-myristate 13-acetate, is also blocked in RAW 264.7 cells by o-ATP pretreatment. In summary, these data suggest that the P2Z/P2X₇ receptor modulates LPS-induced macrophage activation as assessed by iNOS expression and NO production. This report implicates the P2Z/P2X₇ receptor in the control of protein kinase cascades and transcriptional processes, and these observations are likely to be important for the development of selective purinergic receptor antagonists for the treatment of septic shock.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵FNb Supported by National Institutes of Health Training Grant 5 T32 HD07259.
↵FNe Supported by the University of Wisconsin Molecular Biosciences Training Grant, National Institutes of Health Grant T32-GM07215, and a University-Industry Research (UIR) grant from the University of Wisconsin.
↵FNg Supported by National Institutes of Health, Biotechnology Training Program No. T32-GM08349, University of Wisconsin.
↵i Supported by funds awarded from Gensia, Inc., the University of Wisconsin Medical School, the Department of Medicine, and the Graduate School.
↵FNj Supported by awards from Gensia, Inc., the Milwaukee Foundation (Shaw Scholar Award), National Institutes of Health Grant CA47881/GM53271, and the Draper Fund (University-Industry Research (UIR) Fund). To whom all correspondence should be addressed: Dept. of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-8667; Fax: 608-262-5253; E-mail: pbertics{at}macc.wisc.edu.
Abbreviations:

LPS

lipopolysaccharide

iNOS

inducible nitric-oxide synthase

NO

nitric oxide

2-MeS-ATP

2-methylthio-ATP

o-ATP

periodate oxidized ATP

Bz-ATP

2′- and 3′-O-(4-benzoylbenzoyl)-ATP

ATPγS

adenosine 5′-O-(3-thiotriphosphate)

PMA

phorbol 12-myristate-13-acetate

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PPADS

pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid

MAP

mitogen-activated protein kinase

ERK

extracellullar signal-regulated kinase

NF-κB

nuclear factor-κB.
- Received April 7, 1998.
- Revision received July 16, 1998.