Eicosanoid profiling in cerebral vessels and brain of WKY, SHR, and SHR‐SP rats

Abstract

A previous F2 linkage analysis identified a QTL for sensitivity to cerebral ischemia on rat chromosome 5 in the region of the cytochrome P-450 (CYP) 4A genes. CYP4A is a potential candidate gene because it metabolizes arachidonic acid to form 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor of cerebral arteries. The goal of this study was to use liquid chromatography mass spectrometry (LC/MS) to characterize CYP eicosanoid formation in cerebral vessels and surrounding brain parenchyma of Wistar-Kyoto (WKY), spontaneously hypertensive (SHR), and spontaneously hypertensive stroke prone (SHR-SP) rats. Microdissected cerebral vessels and microsomes prepared from the remaining brain tissue were incubated in 0.1M potassium phosphate buffer in the presence of 1mM NADPH, 40μM arachidonic acid, 2μM indomethacin and oxygen at 37°C for 90 minutes. Samples were homogenized and extracted with ethyl acetate. Intact cerebral microvessels and brain microsomes generated 5-, 9-, 11-, 12-, 15-, and 20-HETE, as well as 14,15- and 11,12-EET when incubated with arachidonic acid. The formation of 20-HETE by cerebral vessels of SHR and SHR-SP rats averaged 0.2814±0.050 and 0.2719±0.033pmol/min/mg protein, respectively (n=5). This was significantly greater than that seen in WKY rats (0.1509±0.012 pmol/min/mg protein, n=5). These studies support the potential for differences in the formation of 20-HETE by cerebral arteries to contribute to genetic differences in sensitivity to ischemic stroke. This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-59996 and HL-29587.