Pyrimidinoceptor‐mediated activation of phospholipase C and phospholipase A₂ in RAW 264.7 macrophages

Abstract

Taiwan1 As well as the presence of P2Z purinoceptors previously found in macrophages, we identified pyrimidinoceptors in RAW 264.7 cells, which activate phospholipase C (PLC) and phospholipase A2 (PLA2).2 The relative potency of agonists to stimulate inositol phosphate (IP) formation and arachidonic acid (AA) release was UTP = UDP > > ATP, ATPyS, 2MeSATP. For both signalling pathways, the EC50 values for UTP and UDP (3 uiM) were significantly lower than that for ATP and all other analogues tested (> 100 yM).3 UTP and UDP displayed no additivity in terms of IP formation and AA release at maximally effective concentrations. 4 UTP-, but not ATP-, evoked AA release was 60% inhibited by pertussis toxin (PTX), while stimulation of IP formation by both agonists was unaffected. Short-term treatment with phorbol 12-myristate 13-acetate (PMA) led to a dose-dependent inhibition of IP responses to UTP and UDP, but failed to affect the AA responses. Removal of extracellular Ca2+ inhibited the PI response to UTP, but abolished its AA response. 5 ATP-induction of these two transmembrane signal pathways was decreased in high Mg2+-containing medium but potentiated by the removal of extracellular Mg2'. 6 Suramin and reactive blue displayed equal potency to inhibit the IP responses of UTP and ATP.7 Both UTP and UDP (0.1-100 pM) induced a sustained increase in [Ca21], which lasted for more than 10 min. 8 Taken together, these results indicate that in mouse RAW 264.7 macrophages, pyrimidinoceptors with specificity for UTP and UDP mediate the activation of PLC and cytosolic (c) PLA2. The activation of PLC is via a PTX-insensitive G protein, whereas that of cPLA2 is via a PTX-sensitive G proteindependent pathway. The sustained Ca2+ influx caused by UTP contributes to the activation of cPLA2.RAW 264.7 cells also possess P2z purinoceptors which mediate ATP4--induced PLC and PLA2 activation.

Amlodipine Inhibits Pro-inflammatory Cytokines and Free Radical Production and Inducible Nitric Oxide Synthase Expression in Lipopolysaccharide / Interferon-γ-Stimulated Cultured Vascular Smooth Muscle Cells

“…Previous studies have demonstrated that UTP, acting via pyrimidinoceptors, can stimulate phosphoinositide breakdown and increase intracellular Ca 2+ levels (20). Although UTP alone had no effect, UTP potentiated the LPS-induced NO production, activation of transcription factor NF-kB and iNOS expression in mouse J774 macrophages (21).…”

Lipoxygenase Metabolites as Mediators of UTP-Induced Intracellular Acidification in Mouse RAW 264.7 Macrophages

“…Because we previously characterized the UTP-triggered PI signaling cascades in RAW 264.7 cells (Lin and Lee, 1996), we explored the possible involvement of PI/PLC-triggered second messengers in the pH i decrease. With respect to PKC pathways, we found that 1 M phorbol-12-myristate-13-acetate, a PKC-activating phorbol ester, had no effect on pH i .…”

“…AA release was measured as described previously (Lin and Lee, 1996). In brief, cells were prelabeled with 0.3 Ci/ml [ 3 H]AA in DMEM for 24 hr at 37°and then washed twice with HEPES-buffered solution and incubated in HEPES solution containing 0.5% fatty acid-free bovine serum albumin before stimulation with UTP, thapsigargin, or ionomycin (1 M) at 37°for 30 min.…”

“…Our previous studies first demonstrated the presence in the murine macrophage RAW 264.7 cell line of pyrimidinoceptors that are more selectively activated by UTP and UDP than by ATP and are coupled to the stimulation of PI breakdown and activation of cPLA 2 (Lin and Lee, 1996;Lin, 1997), the key enzyme in the release of AA from phospholipids and in the biosynthesis of eicosanoids via the cyclooxygenase or lipoxygenase pathways (Mayer and Marshall, 1993). These findings have stimulated interest in the physiological and pathological roles of endogenous nucleotides, particularly UTP, in macrophage function.…”

“…After our previous study, which demonstrated the activation of PI/PLC and cPLA 2 by pyrimidinoceptors in RAW 264.7 macrophages (Lin and Lee, 1996), we became interested in understanding the cellular signal transduction of UTP and its role in macrophage function. In the current study, we found that cellular acidification can be induced by UTP, thapsigargin, or ionomycin.…”