11-12-epoxy-5-8-14-eicosatrienoic-acid and 17-octadecynoic-acid

P-450-dependent epoxygenase pathway of arachidonic acid and the products of epoxyeicosatrienoic acids (EETs) have been demonstrated to be involved in angiogenesis and tumor progression. This study examined the expression of EETs and the role of the pathway in the angiogenesis of multiple myeloma (MM). MM cell lines of U266 and RPMI8226 were cultured, and the EETs levels (11, 12-EET and 14, 15-EET) in the supernatant were determined by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured and used for analysis of the angiogenesis activity of the two MM cell lines, which was examined both in vitro and in vivo by employing MTT, chemotaxis, tube formation and matrigel plug assays. 11, 12-EET and 14, 15-EET were found in the supernatant of the cultured MM cells. The levels of the two EETs in the supernatant of U266 cells were significantly higher than those in the RPMI8226 cell supernatant (P<0.05), and the levels paralleled the respective angiogenesis activity of the two different MM cell lines. 17-octadecynoic acid (17-ODYA), as a specific inhibitor of P450 enzyme, suppressed HUVECs proliferation and tube formation induced by MM cells. Furthermore, 17-ODYA decreased the EET levels in the supernatant of MM cells. These results suggest that EETs may play an important role in the angiogenesis of MM, and the inhibitor 17-ODYA suppresses this effect.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Bone Marrow Neoplasms; Cell Line; Cell Line, Tumor; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Endothelial Cells; Fatty Acids, Unsaturated; Humans; Mice; Mice, Inbred C57BL; Multiple Myeloma; Neovascularization, Pathologic

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome p-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential (Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, congruent with 630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aryl Hydrocarbon Hydroxylases; Blotting, Western; Calcium Channel Blockers; Cell Membrane; Chronic Disease; Cyclooxygenase Inhibitors; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Fatty Acids, Unsaturated; Hypoxia; In Vitro Techniques; Male; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Oxygenases; Potassium Channels, Calcium-Activated; Proadifen; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Vasoconstriction

The present study evaluated the effects of cytochrome P-450 inhibitors on the response of the renal microvasculature to changes in renal perfusion pressure and on autoregulation of glomerular capillary pressure using the rat juxtamedullary nephron microvascular preparation perfused in vitro with a cell-free perfusate containing 5% albumin. The basal diameters of the proximal and distal afferent arterioles averaged 28 +/- 1 (n = 32) and 18 +/- 1 micron (n = 23), respectively, at a control perfusion pressure of 80 mmHg. The diameters of these vessels decreased by 8% when perfusion pressure was elevated from 80 to 160 mmHg. After addition of cytochrome P-450 inhibitors (either 17-octadecynoic acid, 20 microM; 7-ethoxyresorufin, 10 microM; or miconazole, 20 microM) to the perfusate, the diameters of the proximal and distal afferent arterioles increased by 6% in response to the same elevation in perfusion pressure. Control glomerular capillary pressure averaged 43 +/- 1 mmHg (n = 32) at a renal perfusion pressure of 80 mmHg and increased by only 9 +/- 1 mmHg when perfusion pressure was elevated to 160 mmHg. Autoregulation of glomerular capillary pressure was impaired after addition of the cytochrome P-450 inhibitors, and it increased by 18 +/- 2 mmHg when perfusion pressure was varied over the same range. These results indicate that cytochrome P-450 inhibitors attenuate the vasoconstrictor response of afferent arterioles to elevations in renal perfusion pressure and impair autoregulation of glomerular capillary pressure, suggesting a possible role for cytochrome P-450 metabolites of arachidonic acid in these responses.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arterioles; Cytochrome P-450 Enzyme Inhibitors; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney; Kidney Cortex; Miconazole; Microsomes; Muscle, Smooth, Vascular; Oxazines; Perfusion; Rats; Rats, Sprague-Dawley; Renal Circulation