Vol. 53, Issue 2, 313-321, February 1998

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

Wan-Wan Lin, Sheng-Ho Chang, and Mei-Lin Wu

Departments of Pharmacology (W.-W.L., S.-H.C.) and Physiology (M.-L.W.), College of Medicine, National Taiwan University, Taipei, Taiwan

In previous studies, we have shown that mouse RAW 264.7 macrophages possess pyrimidinoceptors, coupled to a phosphoinositide-specific phospholipase C, with a higher specificity for UTP than for ATP. In the current study, we explored the mechanism involved in the UTP-induced intracellular acidification seen in this cell line. UTP (30 µM) caused a reversible pH_i decrease of 0.16 ± 0.01 unit; this effect was not influenced by the removal of extracellular Cl or Na⁺ ions or by pretreatment with 5-(N-ethyl-N-isopropyl)-amiloride (10 µM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 µM), staurosporine (1 µM), or Ro 31-8220 (1 µM) but was completely abolished by the removal of extracellular Ca²⁺. UTP (30 µM), thapsigargin (1 µM), and ionomycin (1 µM) each induced a similar extent of external Ca²⁺-dependent acidification with a similar time-dependency, but the effects were nonadditive. To further investigate the Ca²⁺-dependent mechanism, we studied the involvement of arachidonic acid (AA) and eicosanoid metabolites. The addition of AA (10 µM) but not arachidic acid (100 µM) produced a reduction in pH_i. UTP, thapsigargin, and ionomycin induced Ca²⁺-dependent AA release. Furthermore, 4-bromo-phenacyl bromide [30 µM, a phospholipase A₂ (PLA₂) inhibitor], nordihydroguaiaretic acid (50 µM, a lipoxygenase inhibitor), and MK-886 (10 µM, a 5-lipoxygenase-activating protein inhibitor) abolished the UTP- or ionomycin-induced responses, whereas indomethacin (30 µM, a cyclooxygenase inhibitor) and baicalein (10 µM, a selective 12-lipoxygenase inhibitor) had no effect. MAFP (a cPLA₂ inhibitor) and REV 5901 (a 5-lipoxygenase inhibitor as well as a competitive antagonist of peptide leukotrienes), but not RHC 80267 (a diacylglycerol lipase inhibitor), also inhibited the UTP-induced response. In contrast, the pH_i response to AA was unaffected by the presence of 4-bromo-phenacyl bromide or the removal of extracellular Ca²⁺ ions but abolished by addition of NDGA. Exogenous 5-hydroperoxyeicosatetraenoic acid (2 µM) also produced marked acidification, and UTP and ionomycin both induced peptide leukotriene formation. In conclusion, this is the first report indicating that lipoxygenase metabolites act as mediators of the Ca²⁺-dependent acidification seen in macrophages in response to UTP or ionomycin via activation of cPLA₂ and AA release.