Paul H. Figard

Brain microvessels produce 12-hydroxyeicosatetraenoic acid.

Abstract: Cerebral microvessels isolated from perfused, adult murine brain produce a compound with the chromatographic properties of a monohydroxyeicosatetraenoic acid when incubated with arachidonic acid or stimulated with calcium ionophore A23187. The formation of this arachidonic acid metabolite is not reduced in the presence of the cyclooxy‐genase inhibitor ibuprofen, but it is abolished by the lipoxygenase inhibitor nordihydroguaiaretic acid. Analysis by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonre‐duced derivatives of the metabolite indicate that the compound is 12‐hydroxyeicosatetraenoic acid. Fractions of isolated microvessels enriched with capillaries produce 2.1 times more 12‐hydroxyeicosatetraenoic acid per microgram of protein than do fractions of microvessels enriched with arterioles. These studies confirm that brain microvessels can produce 12‐hydroxyeicosatetraenoic acid and strongly suggest that cerebral endothelia are the primary source of microvessel‐derived 12‐hydroxyeicosatetraenoic acid. They further suggest that in brain injury, the liberation and accumulation of arachidonic acid in cerebral tissues may lead to the production of 12‐hydroxyeicosatetraenoic acid within microvessels. The 12‐hydroxyeicosatetraenoic acid formed in this way may mediate some of the blood‐brain barrier and cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.

Formation of 8-hydroxyhexadecatrienoic acid by vascular smooth muscle cells.

Smooth muscle cells derived from the human umbilical vein produce four radioactive metabolites when they are incubated in culture with [3H]-12-hydroxyeicosatetraenoic acid. This conversion does not require the addition of an agonist for eicosanoid formation. The main product, which accounts for 60% of the radioactivity converted to these metabolites, has been identified as 8-hydroxyhexadecatrienoic acid.

Docosatetraenoic acid in endothelial cells: Formation, retroconversion to arachidonic acid, and effect on prostacyclin production☆

Cultured bovine aortic endothelial cells convert arachidonic acid to docosatetraenoic acid and also take up docosatetraenoic acid from the extracellular fluid. After a 24-h incubation with biosynthetically prepared [3H]docosatetraenoic acid, about 20% of the cellular fatty acid radioactivity was converted to arachidonic acid. Furthermore, in pulse-chase experiments, the decrease in phospholipid docosatetraenoic acid content was accompanied by an increase in arachidonic acid, providing additional evidence for retroconversion. These findings suggest that one possible function of docosatetraenoic acid in endothelial cells is to serve as a source of arachidonic acid. The endothelial cells can release docosatetraenoic acid when they are stimulated with ionophore A23187, but they do not form appreciable amounts of eicosanoids from docosatetraenoic acid. Enrichment of the endothelial cells with docosatetraenoic acid reduced their capacity to produce prostacyclin (PGI2) in response to ionophore A23187. This may be related to the fact that docosatetraenoic acid enrichment caused a 40% reduction in the arachidonic acid content of the inositol phosphoglycerides. In addition, less prostacyclin was formed when the enriched cells were incubated with arachidonic acid, suggesting that docosatetraenoic acid also may act as an inhibitor of prostaglandin synthesis in endothelial cells.

Eicosanoid Production by Isolated Cerebral Microvessels and Cultured Cerebral Endothelium

Many studies indicate that eicosanoids have an important role in normal cerebrovascular physiology and in the cerebrovascular response to brain injury.’.’ However, little is known about which eicosanoids are contributed by the cells of the cerebral vessels themselves. To examine this contribution more closely, we have been studying the metabolism of arachidonic acid in isolated murine cerebral microvessels and in cultured murine cerebromicrovascular endothelium (CME). Prostaglandins (PG) Iz, E2. and Fh are produced by cultured CME in response to exogenous arachidonic acid, calcium ionophore A23 1 87, or physiologic agonists including thrombin, bradykinin, serotonin, and acetylcholine. Arachidonic acid and ionophore A23 187 stimulated PG production by as much as 30and 40-fold, respectively, above the background level, while the physiologic agonists increased PG production by 3to 12-fold above background. Analysis of CME eicosanoids by reverse-phase high-performance liquid chromatography demonstrates production of primarily PGI, and E2 by one line of endothelium (FIG. lA), while another also produces PGF,, HHT, and a compound with chromatographic properties of 12-hydroxyeicosatetraenoic acid (12-HETE) (FIG. 1 B).3 Two other minor compounds have chromatographic properties of 12-HETE metabolites (FIG. lB).3 CME grown on micropore filters and incubated 30 min with 7.5 pM arachidonic acid release PG from both apical and basal surfaces, but primarily from their basal surfaces in a ratio as high as 4:1, basal:apicaL3 Although PG are the major arachidonic acid metabolites of cultured CME, microvessels isolated from perfused adult mouse brains additionally produce a compound from arachidonic acid with chromatographic properties of a monohydroxyeicosatetraenoic acid (FIG. 1 C). Analysis of the trimethylsilyl ether, methyl ester derivative of this metabolite by gas chromatography-mass spectrometry revealed a base peak at m / e 295 and alpha cleavage ions at m / e 215 and 301 from its reduced derivative. Chemical ionization of the reduced derivative in NH, revealed a molecular ion at m / e 41 5. Together these spectra prove that the primary cerebral microvessel metabolite of arachidonic acid is 12-HETE.4 While little or no 12-HETE is produced by cultured CME, IZHETE is rapidly incorporated into CME phospholipids, primarily phosphatidylcholine. 12-HETE is also rapidly metabolized by cultured CME to more polar compounds with chromato,graphic properties of two of the minor CME arachidonic acid metabolites, (FIG. 1). Furthermore, 12-HETE inhibits PG production by cultured CME. When stimulated