Faculty Collaboration Database

Kinetic evidence that arachidonate-induced calcium efflux from platelet microsomes involves a carrier-type ionophoric mechanism. Biochim Biophys Acta 1990 Feb 28;1022(2):215-28

Date

02/28/1990

Pubmed ID

2137712

DOI

10.1016/0005-2736(90)90117-7

Scopus ID

2-s2.0-0025257084 (requires institutional sign-in at Scopus site)   16 Citations

Abstract

Arachidonate, at concentrations up to 50 microM, induced dose-dependent calcium efflux from preloaded microsomes prepared from human platelets, but not from unilamellar egg phosphatidylcholine vesicles. Arachidonate-induced efflux from microsomes was not inhibited by indomethacin, 13-azaprostanoic acid, or catalase and superoxide dismutase, indicating that the release was due to arachidonate and not a metabolite. Linolenate (18:3, cis) and linoleate (18:2, cis) induced calcium efflux in a manner similar to arachidonate (20:4, cis), while arachidate (20:0), linolelaidate (18:2, trans), elaidate (18:1, trans), oleate (18:1, cis), stearate (18:0) and palmitate (16:0) had no effect. An experimental method was developed for distinguishing between carrier ionophore, small aqueous pore (i.e., calcium channel), or large aqueous pore (i.e., detergent effect) mechanisms in vesicular efflux systems in which calcium efflux occurs over a period of minutes. This development predicted that with a carrier ionophore mechanism, an increase in either internal or external calcium should competitively inhibit 45Ca efflux. In contrast, 45Ca efflux by diffusion through a small aqueous pore or a large aqueous pore should be measurably insensitive to variations in internal or external calcium. These predictions were experimentally verified in the platelet microsomal system using efflux agents with known mechanisms. Efflux of 45Ca by A23187, a calcium ion carrier ionophore, was sensitive to internal or external calcium competition, while alamethicin, a small aqueous pore channel model, and Triton X-100, a detergent which forms large aqueous pores, mediated 45Ca efflux which was measurably insensitive to variations in internal or external calcium concentration. Arachidonate-induced 45Ca efflux was inhibited by increasing either internal and external calcium concentration, suggesting that the fatty acid functions as a carrier ionophore. Arachidonate-induced 45Ca efflux was also inhibited with extravesicular Sr2+, but not Mn2+ or Ba2+. The dependence of the initial arachidonate efflux rate on arachidonate concentration showed that at least two arachidonates were contained in the calcium-carrier complex. These results are consistent with a model in which arachidonate (A) and an endogenous microsomal component (B) translocate calcium across the membrane through a carrier ionophore mechanism as part of a complex with a stoichiometry of A2B.Ca.

Author List

Fischer TH, Griffin AM, Barton DW, White GC 2nd

Author

Gilbert C. White MD Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Adenosine Triphosphate
Arachidonic Acid
Arachidonic Acids
Biological Transport
Blood Platelets
Calcimycin
Calcium
Calcium-Transporting ATPases
Carrier Proteins
Fatty Acids
Humans
Indomethacin
Ionophores
Kinetics
Liposomes
Microsomes
Thromboxane B2