8-11-14-eicosatrienoic-acid and ethoxyresorufin

The metabolic pathways of arachidonic acid (AA) have been shown to be important in the regulation of cellular function. Several studies have demonstrated both direct and indirect effects of products of these pathways in the regulation of vascular actions, and in particular on signaling pathways. Because intracellular calcium concentration is a significant mediator of stimulus-coupled signal transduction, we investigated the effects of AA pathway inhibitors on angiotensin II (Ang II)-induced calcium mobilization in cultured rat vascular smooth muscle cells (VSMC). Thus, specific calcium pools were examined for differential effects resulting from inhibitors of the three major pathways of AA metabolism. Inhibition of lipoxygenase (LO) with 2.5 micromol/L of 5,8,11 eicosatriynoic acid (ETI), cyclooxygenase (CO) with 2 micromol/L of ibuprofen (IBU), and cytochrome P-450 (P450) with 1 micromol/L of 7-ethoxyresorufin (7ER) all reduced total Ang II-induced intracellular calcium transients ([Ca2+]i) in fura 2-loaded cultured rat VSMC. However, the sites of action affected were unique for each inhibitor. Pretreatment of VSMC with either ETI or IBU inhibited thapsigargin (TG) (1 micromol/L)-sensitive calcium increments (control; 118.0 +/- 33.1 nmol/L, n = 9, ETI; 34.7 +/- 4.8 nmol/L, n = 9, IBU; 40.3 +/- 8.8 nmol/L, n = 8, P < .05 v control). Both caffeine (CAF) and ryanodine (RY) treatment attenuated Ang II-induced [Ca2+]i; however, pretreatment with ETI, IBU, or 7ER did not alter this effect. In other studies, the LO inhibitor ETI attenuated Ang II-induced Ca2+ influx, whereas inhibitors of CO and P450 pathways had no effect. These data show that 1) E

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Caffeine; Calcium; Calcium Signaling; Cells, Cultured; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Ibuprofen; Lipoxygenase Inhibitors; Male; Muscle, Smooth, Vascular; Oxazines; Rats; Ryanodine

The cytochrome P450-dependent monoxygenase pathway represents a major route for the metabolism of arachidonic acid (AA) in the kidney. In turn, AA metabolites have been shown to affect renal electrolyte metabolism, including sodium transport. Specifically AA, 20-HETE and 12-HETE inhibit sodium-dependent (Na+-Pi) uptake into renal culture cells, and both 12-HETE and 14,15 EET have been shown to reduce renin release from renal cortical slices. Since the bulk of Pi transport occurs in the proximal tubule (PT), and the PT is a major site of AA metabolism, we studied the effect of AA and several of its metabolites on Na+-Pi uptake into PT-like opossum kidney (OK) cells. Incubation of OK cells in AA (10(-8) M) resulted in 17% inhibition of Pi uptake. Three metabolites of omega-hydroxylation of AA induced significant decreases in Pi uptake: 19R-HETE (10(-8) M) by 36% (P=0.008), 19S-HETE (10(-8) M) by 24% (P=0.002) and 20-COOH-AA (10(-8) M), a metabolite of 20-HETE, by 25% (P<0.0001). 14,15 EET (10(-8) M), a breakdown product of AA by the epoxygenase pathway, had the greatest effect on Pi uptake in OK cells. It decreased Pi uptake by 47% (P < 0.0001). Addition of the P450 inhibitor, 7-ER (10(-8) M), to OK cells resulted in a significant stimulation (28%) of Pi uptake (P=0.016). These results indicate that these AA metabolites have a significant inhibitory effect on Na+-Pi uptake in OK cells.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Biological Transport; Cell Line; Electrolytes; Hydroxyeicosatetraenoic Acids; Ion Transport; Kidney Cortex; Kidney Tubules, Proximal; Opossums; Oxazines; Phosphates; Renin; Sodium

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