nitroarginine and tetrabutylammonium

We have performed a nonlinear analysis of fluctuations in red cell velocity and arteriolar calibre in the mesenteric bed of the anaesthetized rat. Measurements were obtained under control conditions and during local superfusion with NG-nitro-L-arginine (L-NNA, 30 microM) and tetrabutylammonium (TBA, 0.1 mM), which suppress NO synthesis and block Ca2+ activated K+ channels (KCa), respectively. Time series were analysed by calculating correlation dimensions and largest Lyapunov exponents. Both statistics were higher for red cell velocity than diameter fluctuations, thereby potentially differentiating between global and local mechanisms that regulate microvascular flow. Evidence for underlying nonlinear structure was provided by analysis of surrogate time series generated from the experimental data following randomization of Fourier phase. Complexity indices characterizing time series under control conditions were in general higher than those derived from data obtained during superfusion with L-NNA and TBA.

Topics: Animals; Arterioles; Blood Flow Velocity; Blood Vessels; Cardiovascular Physiological Phenomena; Mathematics; Nitroarginine; Nonlinear Dynamics; Quaternary Ammonium Compounds; Rats; Time Factors

1. To elucidate the mechanism of the relaxation mediated by endothelium-derived hyperpolarizing factors (EDHFs), the effect of brefeldin A, a membrane transport blocker, on cytosolic Ca(2+) concentration ([Ca(2+)]i) and tension was determined in the porcine coronary arterial strips. We also examined the effect of brefeldin A on [Ca(2+)]i in the endothelial cells of the porcine aortic valve. 2. In the presence of 10 microM indomethacin and 30 microM N(G)-nitro-L-arginine (L-NOARG), both bradykinin and substance P induced a transient decrease in [Ca(2+)]i and tension in arterial strips contracted with 100 nM U46619 (thromboxane A2 analogue). A 6 h pre-treatment with 20 microg ml(-1) brefeldin A abolished the bradykinin-induced relaxation, while it had no effect on the substance P-induced relaxation. 3. In the absence of indomethacin and L-NOARG, brefeldin A had no effect on the bradykinin-induced relaxation during the contraction induced by U46619 or 118 mM K(+). 4. The indomethacin/L-NOARG-resistant relaxation induced by bradykinin was completely inhibited by 3 mM tetrabutylammonium (non-specific Ca(2+)-activated K(+) channel blocker), while that induced by substance P was not inhibited by 3 mM tetrabutylammonium or 1 mM 4-aminopyridine (voltage-dependent K(+) channels blocker) alone, but completely inhibited by their combination. 5. Brefeldin A had no effect on the [Ca(2+)]i elevation in endothelial cells induced by bradykinin or substance P. 6. In conclusion, bradykinin produce EDHF in a brefeldin A-sensitive mechanism in the porcine coronary artery. However, this mechanism is not active in a substance P-induced production of EDHF, which thus suggests EDHF to be more than a single entity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Animals; Apamin; Biological Factors; Bradykinin; Brefeldin A; Calcium; Coronary Vessels; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; In Vitro Techniques; Indomethacin; Male; Nitroarginine; Peptides; Potassium; Potassium Channel Blockers; Quaternary Ammonium Compounds; Substance P; Swine; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

In isolated, perfused mesenteric vascular beds from female rats, it was assessed whether the constrictor response to cirazoline, an alpha(1)-adrenergic agonist, or acetylcholine (ACh)-induced relaxation was altered by oestrous cycle or pregnancy and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. Mesenteries, removed from female rats on each oestrous cycle day and gestation day 16, were perfused with physiological salt solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to ACh generated. Responsiveness to ACh was tested in the presence of N(omega)-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit nitric oxide synthase (NOS), cyclo-oxygenase and K(+) channels respectively. Cirazoline-induced tone was smaller in pro-oestrous and pregnant groups, but the increase in tone to L-NA was larger in pregnant compared with oestrous and dioestrous groups. Control responses to ACh were not different, but L-NA attenuated the response in virgin groups only. IBU did not affect the ACh response, but TBA attenuated it in all groups. When TBA was introduced first, ACh-induced dilatation was significantly reduced and not altered by L-NA addition. These results suggest that in the mesenteric vascular bed from cycling and pregnant rats, EDHF is the major mediator of ACh-induced dilatation and NOS may be up-regulated in pregnant and pro-oestrous rats.

Topics: Acetylcholine; Adrenergic alpha-Agonists; Animals; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Estrus; Female; Ibuprofen; Imidazoles; Nitric Oxide Synthase; Nitroarginine; Potassium Channel Blockers; Pregnancy; Pregnancy, Animal; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Splanchnic Circulation; Vasoconstriction; Vasodilation

Isolated uterine vascular beds from virgin and pregnant rats were used to assess vascular reactivity and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. One uterine horn from female rats in each oestrous cycle day and gestation day 17 was removed and perfused with physiological saline solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to acetylcholine (ACh) generated. Responsiveness to ACh was tested in the presence of N-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit NO synthase, cyclo-oxygenase and K+ channels respectively. Cirazoline-induced tone was smaller in the pregnant compared with the proestrous group. Sensitivity to ACh was cycle day and pregnancy dependent with pregnant > dioestrous day-1 > dioestrous day-2 > proestrous and oestrous. L-NA shifted the curve to the right in all groups except dioestrous day-1. IBU inhibited the ACh response in the pregnant group only. TBA virtually abolished the response in all groups. These results suggest that in the uterine vascular bed from pregnant rats, EDHF, along with NO and a dilator prostanoid mediate ACh-induced dilatation. In contrast, in the dioestrous day-1 group, only EDHF seems to be released by ACh in this vascular bed. In the oestrous, dioestrous day-2 and proestrous groups, ACh releases both EDHF and NO.

Topics: Acetylcholine; Adrenergic Agonists; Animals; Biological Factors; Cyclooxygenase Inhibitors; Diestrus; Enzyme Inhibitors; Estrus; Female; Gestational Age; Ibuprofen; Imidazoles; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Potassium Channel Blockers; Pregnancy; Proestrus; Prostaglandins; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Uterus

Studies were designed to compare the N(G)-nitro-L-arginine- and indomethacin-resistant, endothelium-dependent relaxation to acetylcholine in isolated renal artery rings from normal and cholesterol-fed rabbits. It was assumed that the resistant part in response to acetylcholine is mediated by the endothelial-derived hyperpolarizing factor (EDHF). Rabbits were fed normal (n = 15) or cholesterol enriched chow (n = 13, 1% cholesterol for 4 weeks, 0.5% for 12 weeks). In organ chamber experiments, renal artery rings were precontracted with 0.1-1 microM phenylephrine or 35 mM KCl, and relaxed with acetylcholine (0.001-10 microM) in the presence of 10 microM indomethacin. Studies were performed in the presence or absence of: 100 microM N(G)-nitro-L-arginine (L-NOARG) to inhibit the nitric oxide pathway, 100 nM charybdotoxin (CTX) or 1 mM tetrabutylammonium (TBA) to inhibit Ca2+-activated K+ channels, and 100 microM SKF 525a to inhibit cytochrome P450 monoxygenase pathway. In normal arteries, L-NOARG partially inhibited acetylcholine-induced relaxation. The resistant part was almost abolished when the arteries were depolarized with KCl, or when L-NOARG was combined with either CTX, TBA or SKF 525a. In arteries from hypercholesterolemic animals, the relaxation to acetylcholine was only slightly impaired as compared to normal animals. However, in comparison to arteries from normal animals, the L-NOARG-resistant part of acetylcholine-induced endothelium-dependent relaxation was enhanced. It is speculated that differences in the balance between nitric oxide (NO)- and EDHF-mediated control of vascular tone may maintain acetylcholine-induced vasodilatation of the renal artery in hypercholesterolemia.

Topics: Animals; Aorta, Thoracic; Cholesterol, Dietary; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypercholesterolemia; In Vitro Techniques; Indomethacin; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroarginine; Proadifen; Quaternary Ammonium Compounds; Rabbits; Renal Artery

We examined the role of K+ channels in the endothelium-dependent relaxation which is resistant to nitric oxide (NO) synthase inhibition in porcine coronary artery. In the presence of 0.2 mM NG-nitro-L-arginine (L-NNA), a potent inhibitor of NO synthase, 10 nM substance P (SP) added to 9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F2alpha (U46619) contractures elicited a relaxation. The L-NNA-resistant relaxation induced by SP was strongly inhibited by 5 mM tetrabutylammonium chloride (TBA), a non-specific inhibitor of K+ channels. Interestingly, 4-aminopyridine (4-AP, 1 mM), a relatively specific inhibitor of voltage-sensitive K+ channels, shortened the duration of SP response, but it had no effect on the peak of SP response. Although 4-AP has also been shown to inhibit Ca2+-activated K+ channels, the shortening effect of 4-AP in SP response was observed in the presence of 1 microM apamin, an inhibitor of small conductance Ca2+-activated K+ channels, or 100 nM charybdotoxin, and inhibitor of large conductance Ca2+-activated K+ channels. Moreover, although SP stimulates both L-NNA-resistant relaxation and endothelium-derived NO-dependent relaxation (EDNO) in porcine coronary arteries, a low concentration of 4-AP (1 mM) affected only the L-NNA-resistant response, but not the EDNO response. These are the first results to show that the L-NNA-resistant relaxation induced by SP, probably, endothelium-derived hyperpolarizing factor(s) (EDHF) response, is dependent on voltage-dependent K+ channels in porcine coronary artery.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Animals; Apamin; Calcium; Charybdotoxin; Coronary Vessels; Endothelium, Vascular; Enzyme Inhibitors; Glyburide; Ion Transport; Isometric Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Potassium Channels; Quaternary Ammonium Compounds; Substance P; Swine

1. In the presence of indomethacin (IM, 10 microM) and N omega-nitro-L- arginine (L-NOARG, 0.3 mM), acetylcholine (ACh) induces an endothelium-dependent smooth muscle hyperpolarization and relaxation in the rat isolated hepatic artery. The potassium (K) channel inhibitors, tetrabutylammonium (TBA, 1 mM) and to a lesser extent 4-aminopyridine (4-AP, 1 mM) inhibited the L-NOARG/IM-resistant relaxation induced by ACh, whereas apamin (0.1-0.3 microM), charybdotoxin (0.1-0.3 microM), iberiotoxin (0.1 microM) and dendrotoxin (0.1 microM) each had no effect. TBA also inhibited the relaxation induced by the receptor-independent endothelial cell activator, A23187. 2. When combined, apamin (0.1 microM) + charybdotoxin (0.1 microM), but not apamin (0.1 microM) + iberiotoxin (0.1 microM) or a triple combination of 4-AP (1 mM) + apamin (0.1 microM) + iberiotoxin (0.1 microM), inhibited the L-NOARG/IM-resistant relaxation induced by ACh. At a concentration of 0.3 microM, apamin + charybdotoxin completely inhibited the relaxation. This toxin combination also abolished the L-NOARG/ IM-resistant relaxation induced by A23187. 3. In the absence of L-NOARG, TBA (1 mM) inhibited the ACh-induced relaxation, whereas charybdotoxin (0.3 microM) + apamin (0.3 microM) had no effect, indicating that the toxin combination did not interfere with the L-arginine/NO pathway. 4. The gap junction inhibitors halothane (2 mM) and 1-heptanol (2 mM), or replacement of NaCl with sodium propionate did not affect the L-NOARG/IM-resistant relaxation induced by ACh. 5. Inhibition of Na+/K(+)-ATPase by ouabain (1 mM) had no effect on the L-NOARG/IM-resistant relaxation induced by ACh. Exposure to a K(+)-free Krebs solution, however, reduced the maximal relaxation by 13% without affecting the sensitivity to ACh. 6. The results suggest that the L-NOARG/IM-resistant relaxation induced by ACh in the rat hepatic artery is mediated by activation of K-channels sensitive to TBA and a combination of apamin + charybdotoxin. Chloride channels, Na+/K(+)-ATPase and gap junctions are probably not involved in the response. It is proposed that endothelial cell activation induces secretion of an endothelium-derived hyperpolarizing factor(s) (EDHF), distinct from NO and cyclo-oxygenase products, which activates more than one type of K-channel on the smooth muscle cells. Alternatively, a single type of K-channel, to which both apamin and charybdotoxin must bind for inhibition to occur, may be the target for EDHF.

Topics: Acetylcholine; Animals; Arginine; Biological Factors; Chloride Channels; Endothelium, Vascular; Enzyme Inhibitors; Female; Gap Junctions; Hepatic Artery; In Vitro Techniques; Indomethacin; Membrane Potentials; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Potassium Channel Blockers; Potassium Channels; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase

We investigated whether formation of endothelium-derived relaxing factor (EDRF) and endothelium-derived hyperpolarizing factor (EDHF) in porcine and bovine endothelial cells (PAECs) was stimulated by different kinin receptors and studied pharmacologic differences and similarities between the two types of bradykinin-induced relaxation of bovine or porcine coronary arteries. Cultured PAECs were used for [3H]bradykinin binding assay and for measurement of the endothelial free [Ca2+]i by the fura-2/AM method. In organ bath studies with strips of bovine and porcine coronary arteries (endothelium intact), changes in length were recorded and cyclic GMP was measured by radioimmunoassay (RIA). Two bradykinin binding sites were detected, suggesting the presence of two subtypes of B2 kinin receptors. Bradykinin increased [Ca2+]i, and this action was antagonized by the B2 kinin receptor antagonist Hoe 140 and the K channel inhibitor tetrabutylammonium (TBA). Hoe 140 competitively antagonized the relaxing effects of bradykinin, whereas a B1 antagonist was inactive. L-omega N-nitro-arginine (L-NNA) diminished one part of bradykinin-induced relaxation and abolished the increases in cyclic GMP; TBA inhibited another part of the relaxing effect and attenuated (but not significantly) increases in cyclic GMP, and Hoe 140 completely inhibited relaxation and increases in cyclic GMP. The results indicate that the bradykinin response is mediated by biosynthesis of EDRF, which is sensitive to L-NNA, and of EDHF, which is sensitive to TBA.

Topics: Animals; Arginine; Biological Factors; Bradykinin; Bradykinin Receptor Antagonists; Cattle; Cells, Cultured; Coronary Vessels; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Quaternary Ammonium Compounds; Receptors, Bradykinin; Swine