nitroarginine and 17-octadecynoic-acid

Hyperthyroidism was induced by subcutaneous injections of L-thyroxine (T4) (0.5 mg/kg/day) for 3 days in order to investigate the effects of acute hyperthyroidism on the vasorelaxing responses to isoprenaline and acetylcholine in isolated rat aortae. In the aortae, there was no significant difference in isoprenaline-induced relaxation between hyperthyroid and control rats, however acetylcholine-induced relaxation was significantly greater in hyperthyroid rats than in control rats. N(G)-nitro-L-arginine (L-NOARG), an inhibitor of nitric oxide (NO) synthase, reduced isoprenaline- and acetylcholine-induced relaxations in both hyperthyroid and control rats and in the presence of L-NOARG no significant difference in the acetylcholine-induced relaxation was seen between the two groups of rats. Indomethacin, a cyclo-oxygenase inhibitor, had no significant influence on both isoprenaline- and acetylcholine-induced relaxations in both control and hyperthyroid rats. 17-Octadecynoic acid (17-ODYA), a cytochrome P-450 mono-oxygenase inhibitor, reduced the both isoprenaline- and acetylcholine-induced relaxation in both hyperthyroid and control rats, and acetylcholine-induced relaxation was still greater in hyperthyroid rats than in control rats. These results indicate that an acute hyperthyroidism significantly enhances muscarinic receptor- but not adrenoceptor-mediated relaxations of the aortae and L-NOARG abolished an enhancement by acute hyperthyroidism of muscarinic receptor-mediated relaxation, suggesting that the effects may be due to an alteration in muscarinic receptor-mediated NO systems of the aortae at early stage of hyperthyroidism.

Topics: Acetylcholine; Adrenergic beta-Agonists; Animals; Aorta, Thoracic; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fatty Acids, Unsaturated; Hyperthyroidism; Isoproterenol; Male; Nitroarginine; Rats; Rats, Wistar; Receptors, Cholinergic; Vasodilation; Vasodilator Agents

The effects of acute hyperthyroidism on the vasorelaxing responses to isoprenaline and acetylcholine were investigated in isolated rat renal and femoral arteries. In the renal artery, isoprenaline- and acetylcholine-induced relaxations were significantly greater in hyperthyroid rats than in control rats. In the femoral artery, only the acetylcholine-induced relaxation was significantly greater in hyperthyroid rats than in control rats. In the renal artery, NG-nitro-L-arginine (L-NOARG), an inhibitor of nitric oxide (NO) synthase, reduced isoprenaline- and acetylcholine-induced relaxations in both hyperthyroid and control rats and the isoprenaline-induced relaxation was still greater in hyperthyroid rats than in control rats, but no difference in the acetylcholine-induced relaxation was seen between the two groups of rats since L-NOARG almost abolished the acetylcholine-induced relaxation. In the femoral artery, L-NOAGR reduced the isoprenaline-induced relaxation in control rats but not in hyperthyroid rats, while it almost abolished the acetylcholine-induced relaxation in both groups of rats. 17-Octadecynoic acid (17-ODYA), a cytochrome P-450 monooxygenase inhibitor, reduced the isoprenaline-induced relaxation in renal and femoral arteries from hyperthyroid and control rats, but it did not change the acetylcholine-induced relaxation in both arteries. These results indicate that acute hyperthyroidism significantly enhances beta-adrenoceptor-mediated relaxation of the renal artery and muscarinic receptor-mediated relaxation of both renal and femoral arteries, suggesting that these effects may be due to an alteration in the NO and cytochrome P-450 systems of the artery.

Topics: Acetylcholine; Acute Disease; Animals; Cyclooxygenase Inhibitors; Fatty Acids, Unsaturated; Femoral Artery; Heart Rate; Hyperthyroidism; Indomethacin; Isoproterenol; Japan; Male; Muscle, Smooth, Vascular; Nitroarginine; Nitroprusside; Organ Size; Rats; Rats, Wistar; Receptors, Adrenergic; Receptors, Muscarinic; Renal Artery; Thyroxine; Vasodilation

Recent advances in transgenic mouse technology provide novel models to study cardiovascular physiology and pathophysiology. In light of these developments, there is an increasing need for understanding cardiovascular function and blood flow control in normal mice. To this end we have used intravital microscopy to investigate vasomotor control in arterioles of the superfused cremaster muscle preparation of anesthetized C57Bl6 mice. Spontaneous resting tone increased with branch order and was enhanced by oxygen. Norepinephrine and acetylcholine (ACh) caused concentration-dependent vasoconstriction and vasodilation, respectively. Microiontophoresis of ACh evoked vasodilation that conducted along arterioles; the local (direct) response was inhibited by N(omega)-nitro-L-arginine (LNA), and both local and conducted responses were inhibited by 17-octadecynoic acid (17-ODYA). Microejection of KCl evoked a biphasic response: a transient conducted vasoconstriction (inhibited by nifedipine), followed by a conducted vasodilation that was insensitive to LNA, indomethacin, and 17-ODYA. Phenylephrine evoked focal vasoconstriction that did not conduct. Perivascular sympathetic nerve stimulation evoked constriction along arterioles that was inhibited by tetrodotoxin. These findings indicate that for arterioles in the mouse cremaster muscle, nitric oxide and endothelial-derived hyperpolarizing factor (as shown by LNA and 17-ODYA interventions, respectively) mediate vasodilatory responses to ACh but not to KCl, and that vasomotor responses spread along arterioles by multiple pathways of cell-to-cell communication.

Topics: Acetylcholine; Animals; Arterioles; Cell Communication; Electric Stimulation; Fatty Acids, Unsaturated; Indomethacin; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Nitroarginine; Norepinephrine; Phenylephrine; Vasoconstriction; Vasodilation

We tested whether local and conducted responses to ACh depend on factors released from endothelial cells (EC) in cheek pouch arterioles of anesthetized hamsters. ACh was delivered from a micropipette (1 s, 500 nA), while arteriolar diameter (rest, approximately 40 microm) was monitored at the site of application (local) and at 520 and 1,040 microm upstream (conducted). Under control conditions, ACh elicited local (22-65 microm) and conducted (14-44 microm) vasodilation. Indomethacin (10 microM) had no effect, whereas N(omega)-nitro-L-arginine (100 microM) reduced local and conducted vasodilation by 5-8% (P < 0.05). Miconazole (10 microM) or 17-octadecynoic acid (17-ODYA; 10 microM) diminished local vasodilation by 15-20% and conducted responses by 50-70% (P < 0.05), suggesting a role for cytochrome P-450 (CYP) metabolites in arteriolar responses to ACh. Membrane potential (E(m)) was recorded in smooth muscle cells (SMC) and in EC identified with dye labeling. At rest (control E(m), typically -30 mV), ACh evoked local (15-32 mV) and conducted (6-31 mV) hyperpolarizations in SMC and EC. Miconazole inhibited SMC and EC hyperpolarization, whereas 17-ODYA inhibited hyperpolarization of SMC but not of EC. Findings indicate that ACh-induced release of CYP metabolites from arteriolar EC evoke SMC hyperpolarization that contributes substantively to conducted vasodilation.

Topics: Animals; Arterioles; Biological Factors; Cheek; Cricetinae; Electrophysiology; Endothelium, Vascular; Enzyme Inhibitors; Fatty Acids, Unsaturated; Indomethacin; Male; Mesocricetus; Miconazole; Muscle, Smooth, Vascular; Nitroarginine; Vasodilation

It is unclear to what extent the endothelium-derived hyperpolarizing factor (EDHF) contributes to the control of microcirculatory blood flow in vivo. We analyzed, by intravital microscopy in hamster muscles, the potential role of EDHF along the vascular tree under stimulated (ACh) or basal conditions. Experiments were performed in conscious as well as anesthetized (pentobarbital, urethan) animals. Additionally, cellular effects of the potential EDHF were studied in isolated small arteries. In pentobarbital-anesthetized animals, treatment with Nomega-nitro-L-arginine (L-NNA; 30 micromol/l) and indomethacin (3 micromol/l) reduced the dilation in response to 10 micromol/l ACh from 60 +/- 6 to 20 +/- 4%. This nitric oxide/prostaglandin-independent dilation (NPID), which was of a similar magnitude in large and small arterioles, was abolished by potassium depolarization or charybdotoxin (ChTX, 1 micromol/l) but not by glibenclamide. In conscious animals, NPID amounted to 33 +/- 3%. The inhibitor of the P-450 monooxygenase 17-octadecynoic acid (ODYA) reduced NPID further to 9 +/- 4%. ChTX abolished the NPID and also reduced basal diameters (by -11 +/- 3%). The induction of anesthesia with pentobarbital reduced NPID (to 12 +/- 6%), whereas urethan anesthesia was without effect. Pentobarbital also reduced the ACh-induced hyperpolarization of vascular smooth muscle in isolated arteries, whereas ChTX abolished it. This study suggests that a considerable part of the ACh dilation in the microcirculation is mediated by EDHF, which also contributes to the control of basal tone in conscious animals. The direct inhibitory effect of pentobarbital and ODYA supports the idea that "microcirculatory" EDHF is a product of the cytochrome P-450 pathway. The role of EDHF might be underestimated in pentobarbital-anesthetized animals.

Topics: Acetylcholine; Adjuvants, Anesthesia; Animals; Arterioles; Biological Factors; Charybdotoxin; Cricetinae; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Fatty Acids, Unsaturated; Indomethacin; Mesocricetus; Microcirculation; Muscle, Skeletal; Muscle, Smooth, Vascular; Nitroarginine; Penicillamine; Pentobarbital; Potassium; Potassium Channels; Skin; Vasodilator Agents

In the canine coronary microcirculation, acetylcholine (ACh)-induced vasodilation of large (> or = 100 microns) epicardial arterioles (LgA), but not small (< 100 microns) epicardial arterioles (SmA), is blocked by nitric oxide (NO) synthase inhibitors in vivo. We hypothesized that the ACh-induced vasodilation of SmA is mediated by a cytochrome P-450 metabolite of arachidonic acid (AA). Epicardial coronary microvascular diameters in dogs were measured at baseline and after treatment with topically applied ACh (1, 10, and 100 microM), AA (1, 5, and 10 microM), or sodium nitroprusside (SNP; 10-100 microM). Coronary microvascular diameters were compared among control dogs (group OO); dogs pretreated with N omega-nitro-L-arginine (L-NNA; 70 microM topically) (group NO); dogs pretreated with L-NNA plus clotrimazole (Clo; 1.6 microM topically) or 17-octadecynoic acid (ODYA; 2 microM topically), cytochrome P-450 monooxygenase inhibitors (groups NC and NY, respectively); dogs pretreated with Clo alone (group OC); and dogs pretreated with L-NNA plus Clo with AA as the agonist (group AA). ACh-induced vasodilation of LgA was abolished by L-NNA alone, whereas in SmA, L-NNA was without effect. Clo alone did not inhibit ACh-induced dilation in either SmA or LgA. However, the combinations of L-NNA plus either Clo or ODYA abolished ACh- and AA-induced dilation of SmA (100 microM ACh: NC, 3 +/- 5%; NY, 8 +/- 2%; 10 microM AA: 6 +/- 3%) but did not affect responses to SNP. These results suggest that the ACh-induced vasodilation of SmA is mediated in part by cytochrome P-450 metabolites of AA and provide the first evidence that the cytochrome P-450 pathway contributes to the regulation of coronary resistance vessels in vivo.

Topics: Acetylcholine; Animals; Arachidonic Acid; Arterioles; Clotrimazole; Coronary Circulation; Cytochrome P-450 Enzyme System; Dogs; Enzyme Inhibitors; Fatty Acids, Unsaturated; Female; Male; Muscle, Smooth, Vascular; Nitroarginine; Nitroprusside; Vasodilation; Ventricular Function, Left

To date, the release of the endothelium-derived hyperpolarizing factor (EDHF) has been demonstrated only in response to receptor-dependent Ca2+-elevating agonists. Since endothelial cells in situ are continuously subjected to rhythmic distension, we investigated the effect of rhythmic stretch on the release of EDHF from isolated porcine coronary arteries. In the combined presence of diclofenac and N(G)-nitro-L-arginine (L-NNA), sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) led to simultaneous oscillations in the external diameter of coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. In order to directly demonstrate the release of EDHF, the intraluminal solution from endothelium-intact coronary segments exposed to pulsatile stretch was applied to detector rat aortic smooth muscle cells, the membrane potential of which was continuously measured using the patch-clamp technique. The hyperpolarization of detector cells induced by the intraluminal solution was proportional to the amplitude of the pressure oscillations applied to the donor artery and was attenuated by either preincubation of donor arteries with 17-octadecynoic acid or application of either tetrabutylammonium or iberiotoxin to detector cells. In contrast to the bradykinin-induced release of EDHF, the EDHF synthesized in response to pulsatile stretch did not exhibit any tachyphylaxis. These findings demonstrate for the first time that the synthesis of EDHF in coronary arteries can be mechanically stimulated by rhythmic vessel wall distension and suggest that the continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Apamin; Biological Factors; Cells, Cultured; Coronary Vessels; Diclofenac; Electric Stimulation; Endothelium, Vascular; Fatty Acids, Unsaturated; In Vitro Techniques; Muscle, Smooth, Vascular; Nitroarginine; Oscillometry; Patch-Clamp Techniques; Peptides; Rats; Stress, Mechanical; Swine; Vasodilation

In the presence of N omega-nitro-L-arginine and indomethacin, acetylcholine (ACh) induced endothelium-dependent relaxation in guinea pig coronary artery preconstricted with 9,11-dideoxy-9 alpha, 11 alpha-epoxymethano prostaglandin F2 alpha. Dexamethasone and arachidonyltrifluoromethyl ketone, inhibitors of phospholipase A2, and 17-octadecynoic acid, an inhibitor of cytochrome P450 epoxygenase, had no effect on the response to ACh. Although proadifen, which is used widely as an inhibitor of cytochrome P450-dependent enzymes, suppressed the ACh-induced relaxation, the drug also inhibited the relaxation induced by cromakalim, a K+ channel opener. In isolated smooth muscle cells of guinea pig coronary artery, proadifen, but not 17-octadecynoic acid, almost abolished delayed rectifier K+ current. Epoxyeicosatrienoic acids failed to relax the artery. Apamin and iberiotoxin, inhibitors of small- and large-conductance Ca(++)-activated K+ channels, respectively, did not affect the relaxation induced by ACh. A combination of charybdotoxin plus apamin, but not iberiotoxin plus apamin, abolished the response. However, the combination of charybdotoxin plus apamin had no effect on ACh-induced increase in intracellular free Ca++ concentration in endothelial cells. These results suggest that epoxyeicosatrienoic acids do not contribute to N omega-nitro-L-arginine/indomethacin-resistant relaxation induced by ACh in the guinea pig coronary artery. The present study also proposes that K+ channels on vascular smooth muscle cells, which both charybdotoxin and apamin must affect for inhibition to occur, are the target for endothelium-derived hyperpolarizing factor.

Topics: Acetylcholine; Animals; Biological Factors; Calcium; Coronary Vessels; Endothelium, Vascular; Fatty Acids, Unsaturated; Guinea Pigs; In Vitro Techniques; Indomethacin; Male; Nitric Oxide; Nitroarginine; Phospholipases A; Phospholipases A2; Potassium Channels; Prostaglandins; Vasodilation

In several blood vessels, endothelium-dependent vasorelaxation is in part mediated by an endothelium-derived hyperpolarizing factor (EDHF), the nature of which is as yet unknown. However, some evidence suggests that EDHF might be a cytochrome P-450-dependent monooxygenase metabolite of arachidonic acid. By using isometric tension measurements on rat main mesenteric arteries, the influence of four structurally and mechanistically different cytochrome P-450 inhibitors (proadifen, miconazole, 1-amino-benzotriazole, and 17-octadecynoic acid) was investigated on relaxations elicited by EDHF, assessed as the nitro-L-arginine-resistant component of acetylcholine-induced relaxation, and on relaxations provoked by the endothelium-independent potassium channel opener cromakalim. Proadifen (30 microM) inhibited the EDHF- as well as the cromakalim-induced relaxation, but not that elicited by nitroprusside. Also miconazole (30 microM) inhibited both the EDHF and the cromakalim-induced relaxation. On the other hand, 17-octadecynoic acid (5 microM) had no influence, and 1-aminobenzotriazole (1 mM) even potentiated EDHF- and cromakalim-induced relaxations. We conclude that the EDHF, released from the rat mesenteric artery by acetylcholine, is unlikely to be a cytochrome P-450-dependent monooxygenase metabolite of arachidonic acid and that proadifen and miconazole interfere with the action of cromakalim.

Topics: Acetylcholine; Animals; Benzopyrans; Biological Factors; Cromakalim; Cytochrome P-450 Enzyme Inhibitors; Fatty Acids, Unsaturated; Female; In Vitro Techniques; Mesenteric Arteries; Miconazole; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroarginine; Norepinephrine; Proadifen; Pyrroles; Rats; Rats, Wistar; Triazoles; Vasodilator Agents

1. The possibility that the endothelium-derived hyperpolarising factor (EDHF) in the rat hepatic artery is a cytochrome P450 mono-oxygenase metabolite of arachidonic acid was examined in the present study. In this preparation, acetylcholine elicits EDHF-mediated relaxations in the presence of the nitric oxide (NO) synthase and cyclo-oxygenase inhibitors N omega-nitro-L-arginine (L-NOARG) and indomethacin, respectively. 2. 17-Octadecynoic acid (17-ODYA, 50 microM), a suicide-substrate inhibitor of the cytochrome P450 mono-oxygenases responsible for the production of 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs), had no effect on acetylcholine-induced relaxations in the presence of L-NOARG (0.3 mM) plus indomethacin (10 microM). Furthermore, 5,6-, 8,9-, 11,12- and 14,15- EETs failed to relax arteries without endothelium in the presence of L-NOARG plus indomethacin. 3. Proadifen and clotrimazole, which are inhibitors of several isoforms of cytochrome P450 mono-oxygenases, inhibited acetylcholine-induced relaxations in the presence of L-NOARG plus indomethacin. The concentration of acetylcholine which caused half-maximal relaxation was about 3 and 30 times higher in the presence than in the absence of clotrimazole (3 microM) and proadifen (10 microM), respectively. The maximal relaxation was reduced by proadifen but not by clotrimazole. Proadifen (10 microM) also inhibited acetylcholine-induced hyperpolarization in the presence of L-NOARG plus indomethacin. 4. In the presence of 30 mM K+ plus indomethacin (10 microM), acetylcholine induced an L-NOARG-sensitive relaxation mediated via release of NO. Under these conditions, proadifen (10 microM) shifted the acetylcholine concentration-response curve 6 fold to the right without affecting the maximal relaxation. Clotrimazole (3 microM) was without effect on these responses. The relaxant actions of the NO donor, 3-morpholino-sydnonimine, were unaffected by proadifen (10 microM). 5. The relaxant effects of the opener of ATP-sensitive potassium channels, levcromakalim, were abolished by proadifen (10 microM) and strongly attenuated by clotrimazole (3 microM). Proadifen (10 microM) also abolished the hyperpolarization induced by levcromakalim (1 microM). 6. The lack of effect of 17-ODYA on relaxations mediated by EDHF, together with the failure of extracellularly-applied EETs to produce relaxation, collectively suggest that EDHF is not an EET in the rat hepatic artery. It seems likely that inhibition

Topics: Acetylcholine; Animals; Benzopyrans; Biological Factors; Clotrimazole; Cromakalim; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fatty Acids, Unsaturated; Female; Hepatic Artery; Indomethacin; Membrane Potentials; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Potassium Channels; Proadifen; Pyrroles; Rats; Rats, Sprague-Dawley; Vasodilator Agents