When human blood platelets are exposed to hypotonic medium they swell frwst but, shortly thereafter, revert toward their original volume in a process termed regulatory volume decrease (RVD). RVD is the result of an enhanced efflux of K+ and Cl-ions and associated water. Platelet RVD is controlled by a short-lived lipoxygenasederived product (LP). By using a combination of highperformance liquid chromatography, gas chromatographymass spectrometry, and RVD reconstitution bioassay, we show that LP is identical with hepoxilin A3. In addition we demonstrate that authentic hepoxilin A3 possesses the same biological properties on RVD reconstitution as LP and that the activity of both compounds is amplified through epoxide hydrolase inhibition with 3,3,3-trichloropropene-1,2-oxide. Therefore, we report here that volume expansion causes the formation and release of hepoxilin A3 from intact human platelets and that this hepoxilin plays a major role in volume regulation.Regulation of cell volume is an important physiological process expressed by many cell types (for review, see ref. 1). Platelets respond to hypotonic-induced swelling by an increase in K+ conductance which occurs simultaneously with an increase in an independent conductive Cl-transport. The outward movement of KCl, driven by the K+ gradient, results in an osmotically obliged water efflux causing a volume loss designated as regulatory volume decrease (RVD) (2). Margalit and Livne (3) have shown that volume retraction of human platelets after hypotonic swelling is controlled by an unidentified lipoxygenase-derived product (LP). LP is released into the medium in response to hypotonic shock, and when added to volume expanded cells whose retraction has been inhibited by the presence of a lipoxygenase inhibitor, it initiates volume retraction by promoting exclusively K+ permeability. The identity of this metabolite with hepoxilin A3 [8-hydroxy-11,12-epoxyeicosa-5 (CIBA-Geigy), and N-(3-phenoxycinnamyl)acetohydroxamic acid (BW A4C) was provided by L. G. Garland (Wellcome). Hepoxilin A3 was kindly provided by E. J.Corey (Harvard University, Cambridge, MA). [1-14C]Hepoxilin A3 was prepared in our laboratories as previously described (4).Solutions. Acid citrate/dextrose solution was composed of 65 mM citric acid, 11 mM glucose, and 85 mM trisodium citrate. The standard isotonic medium contained 137 mM NaCl, 1 mM KCl, 0.42 mM NaH2PO4, 0.5 mM MgCl2, 5.5 mM glucose, and 20 mM Hepes, pH 7.4, adjusted to 285 mOsm. Hypotonic solutions were prepared by 1:1 dilution of the standard isotonic medium with distilled water. Media were filtered through a 1.2-,um membrane filter (Schleicher & Schuell, AE 9S) to remove particles that would interfere with the cell sizing measurement. Stock solution of NDGA (20 mM) was made in ethanol, BW A4C (20 mM) was made in dimethyl sulfoxide, and CGS 8515 was made in dimethylformamide. Fresh stock solutions (0.5 and 0.1 mM) of hepoxilins were made daily in dimethyl sulfoxide.Platelet Preparation. Venous blood was obtained from healthy v...
the activation of 15-LOX in the heart tissue of rabbits by Abstract Arachidonic acid (AA) metabolism via the lipoxygenase (LOX) pathway in rat hearts and in cultured rat hypercholesterolemia [7]. cardiomyocytes was investigated using 1-[14C]AA. LOX activityWe describe here the presence of 12-LOX activity in rat was detected in the mierosomal fraction, in the high speed heart muscle and in cultured rat cardiomyocytes. supernatant prepared from rat hearts and in rat eardiomyocyte supernatant. LOX products from all fractions comigrated in thin 2. Materials and methods layer chromatography as 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE. Enzyme linked immunosorbent assay for 2.l. Preparation of enzyme from rat heart 12-HETE showed its formation by the microsomal fraction, theThe microsomal fraction and the cytosolic fraction were prepared ammonium sulfate (AS) pellet, and by rat cardiomyocyte as described by Shahin et al. [8]. Fresh hearts taken from Charles supernatant, while radioimmunoassay for 15-HETE showed itsRiver female rats (200-300 g) were homogenized in 0.25 M sucrose formation only by the AS pellet. The properties of LOX in each and 0.01 M phosphate buffer (pH 7.2-7.4) containing 2.5 mM EGTA (buffer A), at 4°C (3 ml/g tissue), using a Virtis homogenizer. The fraction are reported here.microsomal and high speed supernatant (S-100) were prepared by differential centrifugation, as described by Shahin et al. [8]. The SKey words: Lipoxygenase; 12-HETE; 15-HETE; Cardiac 100 was fractionated by 0-55% saturation of ammonium sulfate (AS) muscle; Cardiomyocyte at 4°C, and the pellet was collected by centrifugation at 12 000 ×g for 10 min (AS pellet) [9]. The microsomal fraction was washed with buffer A, containing 0.05 mg/ml fatty acid free bovine serum albumin [10].
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