ABSTRACT
Alterations in phospholipase A(2) (PLA(2)) activity have been implicated in Alzheimer disease and other neurological disorders, although brain PLA(2) activity is currently measured using lengthy, non-continuous assays. We describe herein a rapid, continuous assay in which we measured PLA(2) activity in mouse brain cytosol (CB-57). Brains were homogenized in HEPES buffer (pH 7.5) and the cytosolic fraction was prepared by centrifugation at 25000xg for 20 min, followed by centrifugation of the supernatant at 100000xg for 60 min. Cytosolic protein content was determined using the Bradford assay. Pyrene labeled phosphatidylcholine was added to 50 microg of cytosolic protein in Tris buffer (pH 8.0) containing fatty acid free-bovine serum albumin for a final assay volume of 2 ml. Assay temperature was maintained at 30+/-1 degrees C. The excitation wavelength was 345 nm and emission was measured at 377 nm. Fluorescence intensity was converted to molar concentrations using a standard curve. Under these conditions, bromoenol lactone inhibited up to 58% of the PLA(2) activity with an IC(50) of 0.5 microM. In a separate experiment, lack of appreciable alternative acylhydrolase activity was verified chromatographically. Using this method, brain PLA(2) activity can be measured in a continuous, rapid, and sensitive manner.
Subject(s)
Brain Chemistry/physiology , Brain/enzymology , Cytosol/enzymology , Fluorometry/methods , Phospholipases A/analysis , Animals , Brain/cytology , Brain/drug effects , Brain Chemistry/drug effects , Cell Extracts/analysis , Cell Extracts/chemistry , Chromatography, Thin Layer/methods , Cold Temperature/adverse effects , Cytosol/drug effects , Female , Mice , Mice, Inbred C57BL , Naphthalenes/pharmacology , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/metabolism , Phospholipases A/metabolism , Pyrones/pharmacology , Temperature , Time FactorsABSTRACT
The differential uptake and targeting of intravenously infused [1-14C]palmitic ([1-14C]16:0) and [1-14C]arachidonic ([1-14C]20:4n-6) acids into heart lipid pools were determined in awake adult male rats. The fatty acid tracers were infused (170 microCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min postinfusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system, and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1-14C]20:4n-6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-14C]16:0. The unilateral incorporation coefficient, k*, which represents tissue radioactivity divided by the integrated plasma radioactivity for heart phospholipid, was sevenfold greater for [1-14C]20:4n-6 than for [1-14C]16:0. In contrast, [1-14C]16:0 was esterified mainly into heart neutral lipids, primarily triacylglycerols (TG), and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-14C]20:4n-6 was primarily targeted for esterification into phospholipids, while [1-14C]16:0 was targeted for esterification into TG or metabolized into nonvolatile aqueous components.
Subject(s)
Arachidonic Acid/pharmacokinetics , Myocardium/metabolism , Palmitic Acid/pharmacokinetics , Phospholipids/metabolism , Animals , Carbon Radioisotopes , Chromatography, Thin Layer , Femoral Vein/metabolism , Injections, Intravenous , Lipids/analysis , Male , Oxygen/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Triglycerides/metabolismABSTRACT
Because apolipoprotein E (ApoE) deficient mice have cognitive deficits (Neurosci. Lett., 199 (1995) 1-4; Neuroscience, 92 (1999) 1273-1286; Brain Res., 752 (1997) 189-196) that may involve decreased phospholipase A(2) (PLA(2)) activity (Neuroscience, 92 (1999) 1273-1286), striatal, hippocampal, and parieto-temporal PLA(2) activity was measured in cytosol from 3 and 20-month-old ApoE deficient and control mice. Samples were homogenized and cytosol prepared by ultracentrifugation. PLA(2) activity in each cytosolic fraction was measured in triplicate using a continuous fluorometric assay (J. Neurosci. Methods (2000) in press). In 3-month-old animals, there was a trend for decreased hippocampal PLA(2) activity between groups. In 20-month-old animals, hippocampal PLA(2) activity was significantly (P=0.0304) decreased nearly 20% in ApoE deficient mice as compared to age-matched control mice. No differences were found in other brain regions, although activity in the striatal samples were nearly 65% less than in the other two regions.
