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PMID: 3092807 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.

The Biochemical journal ·Vol. 235 ·No. 3 ·1986-05-01 ·Pages 869-77

Pollock WK, Rink TJ, Irvine RF

Abstract

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

MeSH Terms
Aminoquinolines Arachidonic Acid Arachidonic Acids/blood Benzofurans Blood Platelets/drug effects,metabolism Calcium/blood Collagen/pharmacology Cytosol/metabolism Ethers/pharmacology Fluorescent Dyes Fura-2 Humans In Vitro Techniques Ionomycin Lipids/blood Stimulation, Chemical Tetradecanoylphorbol Acetate/pharmacology
Chemicals
Aminoquinolines Arachidonic Acids Benzofurans Ethers Fluorescent Dyes Lipids Arachidonic Acid Ionomycin Collagen Tetradecanoylphorbol Acetate Quin2 Calcium Fura-2
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Pollock W K
Rink T J
Irvine R F
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41 references, click to expand
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2350869
Published
1986-05-01
Pages
869-77
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1146767
Subset
IM
External Links
PubMed source DOI: 10.1042/bj2350869 PMC Full Text View journal details
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