cyclic-gmp and 20-hydroxy-5-8-11-14-eicosatetraenoic-acid

This study examined the response to nitric oxide (NO) in rat middle cerebral arteries (MCA). NO donors increased the activity of a 205-pS K(+) channel recorded from vascular smooth muscle (VSM) cells isolated from MCA 10-fold. Blockade of guanylyl cyclase activity with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10(-5) M) did not alter the effect of NO on this channel. In contrast, adding 20-hydroxyeicosatetraenoic acid (20-HETE) to the bath (10(-7) M) abolished the response to NO. NO donors also increased the diameter of serotonin-preconstricted MCA to 85% of control. Blockade of K(+) channels with iberiotoxin or a high-K(+) medium reduced this response by 50%. ODQ (10(-5) M) reduced this response by 47 +/- 3%, whereas preventing the fall of 20-HETE levels reduced the response by 59 +/- 2% (n = 5). Blockade of both pathways eliminated the response to NO donors. These results indicate that activation of K(+) channels contributes 50% to vasodilator response to NO in rat MCA. This is mediated by a fall in 20-HETE levels rather than a rise in cGMP levels or a direct effect of NO.

Topics: Animals; Cerebral Arteries; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitroprusside; Oxadiazoles; Patch-Clamp Techniques; Potassium Channels; Quinoxalines; Rats; Rats, Sprague-Dawley; Vasodilation; Vasodilator Agents

The present study examined the contributions of a rise in cGMP versus a fall in 20-HETE levels to the vasodilator response to nitric oxide (NO) in the cerebral circulation of the rat.. Intact rat middle cerebral and basilar arteries were bathed in physiological saline solution containing indomethacin (5 micromol/L) and baicalein (0.5 micromol/L) and pressurized at 90 mm Hg. Relaxations to sodium nitroprusside (SNP) were studied before and after addition of [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one] (ODQ, a guanylyl cyclase blocker), 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT5823, a protein kinase G blocker), and 20-hydroxyeicosatetraenoic acid (20-HETE). Cerebral blood flow was measured by using a laser Doppler flow probe over a thin cranial window in anesthetized rats, and the effects of intracerebroventricular infusion of 1-hexamine, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)N-methyl (MAHMA nonoate) and dibromododecenyl methylsulfimide (DDMS) were determined.. SNP-induced dilation of serotonin-preconstricted (0.2 micromol/L) middle cerebral arteries (10(-7) to 10(-3) mol/L) was attenuated in arteries treated with ODQ (10 micromol/L) or KT5823 (1 micromol/L) by 52% and 27%, respectively. Preventing the NO-induced fall in intracellular 20-HETE, by adding 20-HETE (100 nmol/L) to the bath, reduced the dilation to SNP by 62%. Simultaneous administration of ODQ and 20-HETE markedly attenuated the SNP-induced dilation by 90%. In basilar arteries, ODQ (10 micromol/L) alone completely blocked the response to SNP. Infusion of MAHMA nonoate (10 nmol/min ICV) in anesthetized rats increased cerebral blood flow by 52% before and 8% after blockade of the endogenous production of 20-HETE with DDMS (50 pmol/min).. These results suggest that NO dilates cerebral arteries through both cGMP-dependent and cGMP-independent pathways and that inhibition of 20-HETE formation contributes to the cerebral vasodilator response to NO both in vitro and in vivo.

Topics: Adenosine; Alkaloids; Amides; Animals; Basilar Artery; Carbazoles; Cerebrovascular Circulation; Cyclic GMP; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Hydrazines; Hydroxyeicosatetraenoic Acids; Indoles; Male; Microcirculation; Middle Cerebral Artery; Mixed Function Oxygenases; Nitric Oxide; Nitroprusside; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Sulfones; Vasodilation; Vasodilator Agents

The present study examined the contribution of elevations in cGMP versus inhibition of cytochrome P-4504A enzymes and the production of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) to the vasodilator actions of NO in renal arterioles. The NO donor sodium nitroprusside (SNP) at 10(-5), 10(-4), and 10(-3) M reduced the production of 20-HETE in microsomes prepared from renal arterioles to 80 +/- 2, 43 +/- 5, and 7 +/- 1% of control, respectively (n = 4). In other experiments, the vasodilator response to SNP (10(-7) to 10(-3) M) was examined in rat renal interlobular arteries (<90 micron ID), preconstricted with phenylephrine (1 microM) under control conditions and after blockade of the cGMP and P-4504A pathways. Inhibition of guanylyl cyclase with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (10 microM, n = 6) or of cGMP-dependent protein kinase with 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT-5823, 1 microM; n = 5) attenuated the vasodilator response to SNP by 26 and 30%, respectively. In contrast, inhibition of the endogenous production of 20-HETE with a suicide substrate, irreversible inhibitor [17-octadecynoic acid (17-ODYA), 1 microM, n = 5], or a selective, competitive inhibitor of 20-HETE formation (dibromo-dodecenyl-methylsulfimide, 25 microM, n = 5) markedly impaired the vasodilator response to SNP by 76 and 78%, respectively. Similarly, when 20-HETE levels were fixed at 100 nM (n = 6), the response to SNP was attenuated by 73%. Blockade of both pathways with ODQ and 17-ODYA completely abolished the response to SNP (n = 6). These results indicate that the vasodilator response to NO is largely cGMP independent and that inhibition of 20-HETE formation contributes to the cGMP-independent effects of NO in the renal microcirculation.

Topics: Alkaloids; Animals; Arterioles; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors; Guanylate Cyclase; Hydroxyeicosatetraenoic Acids; Indoles; Male; Nitric Oxide; Nitroprusside; Phenylephrine; Potassium Channels; Rats; Rats, Sprague-Dawley; Renal Artery; Vasoconstrictor Agents; Vasodilation

Recent studies from this laboratory demonstrated that bradykinin transiently elevates intracellular Ca2+ and inhibits Cl-reabsorption in the in vitro microperfused medullary thick ascending limb (mTAL) of the rat. The present study was designed to identify the intracellular signaling mechanism(s) that mediate this response. Preincubation with the intracellular calcium chelator BAPTA (10(-5) M) completely eliminated the bradykinin-dependent increase in intracellular Ca2+ and the suppression of Cl- transport. Preincubation with the cGMP-dependent protein kinase inhibitor H-89 (10(-5) M) had no effect on the transport response to bradykinin. In contrast, 17-octadecynoic acid (17-ODYA; 10(-5) M), a suicide-substrate inhibitor of renal cytochrome P450 omega-hydroxylase, completely blocked the transport response to bradykinin, while the cyclooxygenase inhibitor sodium meclofenamate (10(-5) M) had no effect. Finally, addition of the cytochrome P450 omega-hydroxylase metabolite 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M) to the bathing medium significantly inhibited Cl- transport in the mTAL (delta -39 +/- 6.0%; p < 0.05), while the epoxygenase metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET; 10(-8) M) had no effect. These data suggest that the bradykinin-dependent inhibition of Cl- transport in the mTAL of the rat is mediated by cytochrome P450 dependent metabolite(s) of arachidonic acid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Bradykinin; Calcium; Cyclic GMP; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Tubules, Distal; Male; Rats; Rats, Sprague-Dawley; Sodium Chloride