Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

W. W. Lin, B. C. Chen

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+](i)), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 μM) and thapsigargin (1 μM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 μM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 μM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml-1) or 4-bromophenacyl bromide (100 μM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 μM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+](i) rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 μM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 μM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 μM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+](i), but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 μM), Ro 31-8220 (10 μM), Go 6976 (1 μM) and the down-regulation of PKC. 7. Following treatment of cells with SK and F 96365 (30 μM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 μM), MEK a inhibitor, nor genistein (100 μM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+](i)-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+](i) was also shown to be involved in AA release.

Original languageEnglish
Pages (from-to)1173-1181
Number of pages9
JournalBritish Journal of Pharmacology
Volume123
Issue number6
DOIs
Publication statusPublished - 1998
Externally publishedYes

Fingerprint

Uridine Triphosphate
Thapsigargin
Arachidonic Acid
Macrophages
Pharmacology
Inositol Phosphates
Phosphatidylinositols
Protein Kinase C
1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole
Pertussis Toxin
Buffers
Phosphoinositide Phospholipase C
Staurosporine
Neomycin
Lipoprotein Lipase
Genistein
Mitogen-Activated Protein Kinase Kinases

Keywords

  • [Ca](i)
  • Cytosolic phospholipase A
  • Phospholipase C
  • Protein kinase C
  • Raw 264.7 macrophages
  • Thapsigargin
  • Uridine 5'-triphosphate (UTP)

ASJC Scopus subject areas

  • Pharmacology

Cite this

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages. / Lin, W. W.; Chen, B. C.

In: British Journal of Pharmacology, Vol. 123, No. 6, 1998, p. 1173-1181.

Research output: Contribution to journalArticle

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AU - Chen, B. C.

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KW - [Ca](i)

KW - Cytosolic phospholipase A

KW - Phospholipase C

KW - Protein kinase C

KW - Raw 264.7 macrophages

KW - Thapsigargin

KW - Uridine 5'-triphosphate (UTP)

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