RESUMEN
BACKGROUND AND PURPOSE: Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. EXPERIMENTAL APPROACH: Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. KEY RESULTS: Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K(+) channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, L-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with L-NAME, N(omega)-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO(*)) with hydroxocobalamin (HXC) or HNO with L-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and L-cysteine reduced ACh-mediated relaxation, as did L-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with L-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO(*), as HXC and L-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. CONCLUSIONS AND IMPLICATIONS: The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO(*) = HNO > EDHF and EDHF > HNO = NO(*) respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels.
Asunto(s)
Factores Biológicos/fisiología , Factores Relajantes Endotelio-Dependientes/fisiología , Arterias Mesentéricas/fisiología , Óxidos de Nitrógeno/metabolismo , Resistencia Vascular/fisiología , Vasodilatación/fisiología , 4-Aminopiridina/farmacología , Animales , Cisteína/farmacología , Hidroxocobalamina/farmacología , Técnicas In Vitro , Masculino , Arterias Mesentéricas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/fisiología , Ratas , Ratas Endogámicas WKY , Resistencia Vascular/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
The free radical form of nitric oxide (NO(.)) is a well-known mediator of vascular tone. What is not so well recognized is that NO(.) exists in several different redox forms. There is considerable evidence that NO(.) and its one-electron reduction product, nitroxyl (HNO), have pharmacologically distinct actions that extend into the regulation of the vasculature. The aim of this study was to compare the vasorelaxation mechanisms of HNO and NO(.), including an examination of the ability of these redox variants to hyperpolarize and repolarize vascular smooth muscle cells from rat mesenteric arteries. The HNO donor Angeli's salt (0.1 nM-10 microM) caused a concentration-dependent hyperpolarization of vessels at resting tone and a simultaneous, concentration-dependent vasorelaxation and repolarization of vessels precontracted and depolarized with methoxamine. Both vasorelaxation and repolarization responses to Angeli's salt were significantly attenuated by both the HNO scavenger l-cysteine (3 mM) and the voltage-dependent K(+) (K(v)) channel inhibitor 4-aminopyridine (4-AP; 1 mM) and virtually abolished by the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM) or 30 mM K(+). In contrast, NO(.) (0.01-1 microM) repolarized arteries to a lesser extent than HNO, and these responses were resistant to inhibition by ODQ (10 microM) and 4-AP (1 mM). Blockade of K(v) channels (1 mM 4-AP) also significantly inhibited the repolarization response to YC-1 (0.1-10 microM), confirming a role for sGC/cGMP in the activation of K(v) channels in this preparation. We conclude that HNO causes vasorelaxation via a cGMP-dependent activation of K(v) channels and that there are different profiles of vasorelaxant activity for the redox siblings HNO and NO(.).
Asunto(s)
Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/metabolismo , Resistencia Vascular , Vasodilatación , 4-Aminopiridina/farmacología , Animales , GMP Cíclico/metabolismo , Cisteína/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Técnicas In Vitro , Masculino , Potenciales de la Membrana , Arteria Mesentérica Superior/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Nitritos/farmacología , Oxadiazoles/farmacología , Oxidación-Reducción , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Canales de Potasio con Entrada de Voltaje/metabolismo , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa Soluble , Resistencia Vascular/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Until recently, most of the biological effects of nitric oxide (NO) have been attributed to its uncharged state (NO*), yet NO can also exist in the reduced state as nitroxyl (HNO or NO(-)). Putatively generated from both NO synthase (NOS)-dependent and -independent sources, HNO is rapidly emerging as a novel entity with distinct pharmacology and therapeutic advantages over its redox sibling, NO*. Thus, unlike NO*, HNO can target cardiac sarcoplasmic ryanodine receptors to increase myocardial contractility, can interact directly with thiols and is resistant to both scavenging by superoxide (*O2-) and tolerance development. HNO donors are protective in the setting of heart failure in which NO donors have minimal impact. Here, we discuss the unique pharmacology of HNO versus NO* and highlight the therapeutic potential of HNO donors in the treatment of cardiovascular disease.
Asunto(s)
Antioxidantes/farmacología , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/metabolismo , Alcoholismo/tratamiento farmacológico , Animales , Antioxidantes/química , Enfermedades Cardiovasculares/tratamiento farmacológico , Humanos , Donantes de Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/uso terapéutico , Óxidos de Nitrógeno/químicaRESUMEN
The nitroxyl anion (HNO) is emerging as a novel regulator of cardiovascular function with therapeutic potential in the treatment of diseases such as heart failure. It remains unknown whether tolerance develops to HNO donors, a limitation of currently used nitrovasodilators. The susceptibility of the HNO donor, Angeli's salt (AS), to the development of vascular tolerance was compared with the NO donors, glyceryl trinitrate (GTN) and diethylamine/NONOate (DEA/NO) in rat isolated aortae. Vasorelaxation to AS was attenuated (P<0.01) by the HNO scavenger l-cysteine, whereas the sensitivity to GTN and DEA/NO was decreased (P<0.01) by the NO. scavenger carboxy-[2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidozoline-1-oxy-3-oxide]. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one impaired responses to GTN>or=AS>>DEA/NO. Pretreatment with 10, 30, and 100 micromol/L of GTN for 60 minutes induced a 4- (P<0.05), 13- (P<0.01), and 48-fold (P<0.01) decrease in sensitivity to GTN, demonstrating tolerance development. In contrast, pretreatment with AS or DEA/NO (10, 30, and 100 micromol/L) did not alter their subsequent vasorelaxation. All of the nitrovasodilators (30 micromol/L) displayed a similar time course of vasorelaxation and cGMP accumulation over a 60-minute period. Unlike vasorelaxation, the magnitude of peak cGMP accumulation differed substantially: DEA/NO>>AS>GTN. GTN did not induce cross-tolerance to either AS or DEA/NO. In contrast, pre-exposure to DEA/NO, but not AS, caused a concentration-dependent attenuation (P<0.01) of GTN-mediated relaxation, which was negated by the protein kinase G inhibitor guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-,Rp-isomer, triethylammonium salt. In conclusion, vascular tolerance does not develop to HNO, nor does cross-tolerance between HNO and GTN occur. Thus, HNO donors may have therapeutic advantages over traditional nitrovasodilators.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Nitritos/farmacología , Óxidos de Nitrógeno/administración & dosificación , Animales , Aorta Torácica/metabolismo , Benzoatos/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Cisteína/farmacología , Tolerancia a Medicamentos , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Hidrazinas/farmacología , Imidazoles/farmacología , Técnicas In Vitro , Masculino , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxidos de Nitrógeno/metabolismo , Nitroglicerina/farmacología , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Endogámicas WKY , Factores de Tiempo , Vasodilatación/efectos de los fármacosRESUMEN
OBJECTIVE: The nitroxyl anion (HNO) is the one-electron reduction product of NO(). This redox variant has been shown to be endogenously produced and to have effects that are pharmacologically distinct from NO(). This study investigates the vasodilator and chronotropic effects of HNO in the rat isolated coronary vasculature. METHODS: Sprague-Dawley rat hearts were retrogradely perfused with Krebs' solution (8 ml/min) using the Langendorff technique. Perfusion pressure was raised using a combination of infusion of phenylephrine and bolus additions of the thromboxane mimetic U46619 to attain a baseline perfusion pressure of 100-120 mm Hg. The vasodilator effects of a nitroxyl anion donor, Angeli's salt, were examined in the absence and presence of HNO and NO* scavengers, K+ channel inhibition, and soluble guanylate cyclase (sGC) inhibition. In addition, the inotropic and chronotropic effects of Angeli's salt were examined in hearts at resting perfusion pressure (50-60 mm Hg) and compared to responses evoked by acetylcholine and isoprenaline. RESULTS: Angeli's salt causes a potent and reproducible vasodilatation in isolated perfused rat hearts. This response is unaffected by the NO* scavenger hydroxocobalamin (0.1 mM) but is significantly inhibited by the HNO scavenger N-acetyl-L-cysteine (4 mM), suggesting that HNO is the mediator of the observed responses. Vasodilatation responses to Angeli's salt were virtually abolished in the presence of the sGC inhibitor ODQ (10 microM). The magnitude of the vasodilatation response to Angeli's salt was significantly reduced in the presence of 30 mM K+, 10 microM glibenclamide and in the presence of the calcitonin gene-related peptide (CGRP) antagonist CGRP((8-37)) (0.1 microM). Angeli's salt had little effect on heart rate or force of contraction, whilst isoprenaline and acetylcholine elicited significant positive and negative cardiotropic effects, respectively. CONCLUSIONS: The HNO donor Angeli's salt elicits a potent and reproducible vasodilatation response. The results suggest that the response is elicited by HNO through sGC-mediated CGRP release and K(ATP) channel activation.
Asunto(s)
Antioxidantes/farmacología , Vasos Coronarios/fisiología , Nitritos/farmacología , Vasodilatación , Acetilcolina , Animales , Aniones/farmacología , Péptido Relacionado con Gen de Calcitonina/farmacología , Vasos Coronarios/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Isoproterenol/farmacología , Masculino , Contracción Miocárdica/efectos de los fármacos , Nifedipino/farmacología , Óxidos de Nitrógeno , Oxadiazoles/farmacología , Oxidación-Reducción , Fragmentos de Péptidos/farmacología , Perfusión , Potasio/metabolismo , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Nitrito de Sodio/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
This study characterises the vasorelaxation and hyperpolarisation effects of the negatively charged quaternary compound tetraphenylboron (TPB) in the rat small mesenteric artery. Segments of rat small mesenteric artery were mounted in a myograph and vessel tone and membrane potential were measured simultaneously. In vessels pre-contracted with vasopressin (0.3-0.6 nM), U46619 (30-90 nM) or methoxamine (0.3-3 microM), TPB (0.1-100 microM) produced a marked endothelium-independent relaxation. However, vasorelaxation responses to TPB were abolished in tissues pre-contracted with K(+) (50 mM), and significantly inhibited by glibenclamide (glib, 10 microM). In the absence of tone, TPB (1-30 microM) caused a concentration-dependent membrane hyperpolarisation of rat mesenteric artery smooth muscle cells, which was not dependent on the endothelium, but sensitive to glibenclamide (10 microM). In methoxamine (0.3-3 microM) pre-contracted vessels, the relaxation response was associated with a marked hyperpolarisation, which was also sensitive to glibenclamide (10 microM), further inhibited by a combination of K(+) channel blockers (glib [10 microM], charybdotoxin [100 nM], apamin [100 nM], 4-aminopyridine [1 mM] and Ba(2+) [30 microM]) and abolished by 50 mM K(+). The results of this study show that TPB causes a vasorelaxation and hyperpolarisation response in the rat small mesenteric artery through a direct action on the vascular smooth muscle. TPB exerts its effects partially via the activation of K(ATP) channels, but also by another mechanism involving K(+)-dependent hyperpolarisation.
Asunto(s)
Arteria Mesentérica Inferior/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Tetrafenilborato/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Membrana Celular/fisiología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Masculino , Potenciales de la Membrana , Arteria Mesentérica Inferior/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Potasio/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Tetrafenilborato/administración & dosificaciónRESUMEN
Nitric oxide (NO) plays an important role in the control of vascular tone. Traditionally, its vasorelaxant activity has been attributed to the free radical form of NO (NO*), yet the reduced form of NO (NO-) is also produced endogenously and is a potent vasodilator of large conduit arteries. The effects of NO- in the resistance vasculature remain unknown. This study examines the activity of NO- in rat small isolated mesenteric resistance-like arteries and characterizes its mechanism(s) of action. With the use of standard myographic techniques, the vasorelaxant properties of NO* (NO gas solution), NO- (Angeli's salt), and the NO donor sodium nitroprusside were compared. Relaxation responses to Angeli's salt (pEC50=7.51+/-0.13, Rmax=95.5+/-1.5%) were unchanged in the presence of carboxy-PTIO (NO* scavenger) but those to NO* and sodium nitroprusside were inhibited. l-Cysteine (NO- scavenger) decreased the sensitivity to Angeli's salt (P<0.01) and sodium nitroprusside (P<0.01) but not to NO*. The soluble guanylate cyclase inhibitor ODQ (3 and 10 micromol/L) concentration-dependently inhibited relaxation responses to Angeli's salt (41.0+/-6.0% versus control 93.4+/-1.9% at 10 micromol/L). The voltage-dependent K+ channel inhibitor 4-aminopyridine (1 mmol/L) caused a 9-fold (P<0.01) decrease in sensitivity to Angeli's salt, whereas glibenclamide, iberiotoxin, charybdotoxin, and apamin were without effect. In combination, ODQ and 4-aminopyridine abolished the response to Angeli's salt. In conclusion, NO- functions as a potent vasodilator of resistance arteries, mediating its response independently of NO* and through the activation of soluble guanylate cyclase and voltage-dependent K+ channels. NO- donors may represent a novel class of nitrovasodilator relevant for the treatment of cardiovascular disorders such as angina.
Asunto(s)
Arterias/fisiología , Guanilato Ciclasa/metabolismo , Óxidos de Nitrógeno/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Vasodilatación , Animales , Arterias/efectos de los fármacos , Arterias/enzimología , Arterias/metabolismo , Benzoatos/farmacología , Técnicas de Cultivo , Cisteína/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Imidazoles/farmacología , Masculino , Donantes de Óxido Nítrico/farmacología , Nitritos/farmacología , Nitroprusiato/farmacología , Oxadiazoles/farmacología , Potasio/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Quinoxalinas/farmacología , Ratas , Ratas Endogámicas WKY , Resistencia Vascular , Vasodilatación/efectos de los fármacosRESUMEN
The ability of a series of novel imidazoline (IMID) compounds (fluoryl-, methoxy- and methyl-phenyl derivatives of clonidine) to inhibit the vasorelaxation and hyperpolarisation response to exogenous K+ (1-10 mM) was assessed in the rat middle cerebral artery (MCA) using the small vessel myograph. In this preparation, K+ -induced relaxation was inhibited by low concentrations of Ba2+ (30 microM) but not affected by the Na+/K+ ATPase inhibitor ouabain (10 microM), or a combination of tetraethylammonium (TEA; 1 mM), 4-aminopyridine (1 mM) and glibenclamide (10 microM). These results are consistent with K+ eliciting a vasorelaxation response through the activation of inwardly rectifying K+ channels (Kir channels) in this tissue. K+ -mediated vasorelaxation was assessed in the absence and in the presence of two concentrations of the IMID compounds (1 and 10 microM). The majority of the compounds investigated caused marked inhibition of K+ -mediated relaxation at these concentrations. In electrophysiological studies the fluoryl-derivative (IMID-4F; 10 microM) potently inhibited the hyperpolarisation response that accompanies the relaxation response to exogenous K+. In conclusion, we have identified a number of IMID compounds that inhibit relaxation and hyperpolarisation responses mediated via Kir channels in the rat MCA. Many of these compounds have a greater potency as inhibitors of Kir channels than Ba2+, and may be a useful tool in studying Kir channel function.