RESUMO
The activation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and other ligands has been extensively investigated for many years. In the present study we considered the effect of molecular oxygen (O2) on sGC both as a direct ligand and its affect on other ligands by measuring cyclic guanosine monophosphate (cGMP) production, as an index of activity, as well as investigating smooth muscle relaxation under hypoxic conditions. Our isolated enzyme studies confirm the function of sGC is impaired under hypoxic conditions and produces cGMP in the presence of O2, importantly in the absence of NO. We also show that while O2 could partially affect the magnitude of sGC stimulation by NO when the latter was present in excess, activation by the NO independent, haem-dependent sGC stimulator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was unaffected. Our in vitro investigation of smooth muscle relaxation confirmed that O2 alone in the form of a buffer bolus (equilibrated at 95% O2/5% CO2) had the ability to dilate vessels under hypoxic conditions and that this was dependent upon sGC and independent of eNOS. Our studies confirm that O2 can be a direct and important mediator of vasodilation through an increase in cGMP production. In the wider context, these observations are key to understanding the relative roles of O2 versus NO-induced sGC activation.
Assuntos
Músculo Liso Vascular/fisiologia , Oxigênio/fisiologia , Animais , Peróxido de Hidrogênio/metabolismo , Masculino , Relaxamento Muscular , Óxido Nítrico/metabolismo , Coelhos , Superóxidos/metabolismoRESUMO
Thienopyridines (ticlopidine, clopidogrel and prasugrel) are pro-drugs that require metabolism to exhibit a critical thiol group in the active form that binds to the P2Y12 receptor to inhibit platelet activation and prevent thrombus formation in vivo. We investigated whether these thienopyridines participate in S-nitrosation (SNO) reactions that might exhibit direct anti-platelet behaviour. Optimum conditions for in vitro formation of thienopyridine-SNO formation were studied by crushing ticlopidine, clopidogrel or prasugrel into aqueous solution and adding sodium nitrite, or albumin-SNO. Ozone-based chemiluminescence techniques were utilised to specifically detect NO release from the SNO produced. Effect on agonist-induced platelet aggregation was monitored using light transmittance in a 96 well microplate assay. Pharmaceutical grade preparations of ticlopidine, clopidogrel and prasugrel were found to exhibit significant free thiol and formed SNO derivatives directly from anionic nitrite in water under laboratory conditions without the need for prior metabolism. Thienopyridine-SNO formation was dependent on pH, duration of mixing and nitrite concentration, with prasugrel-SNO being more favourably formed. The SNO moiety readily participated in trans-nitrosation reactions with albumin and plasma. Prasugrel-SNO showed significantly better inhibition of platelet aggregation compared with clopidogrel-SNO, however when compared on the basis of SNO concentration these were equally effective (IC50=7.91 ± 1.03 v/s 10.56 ± 1.43 µM, ns). Thienopyridine-derived SNO is formed directly from the respective base drug without the need for prior in vivo metabolism and therefore may be an important additional contributor to the pharmacological effectiveness of thienopyridines not previously considered.
Assuntos
Nitritos/metabolismo , Compostos de Sulfidrila/metabolismo , Tienopiridinas/metabolismo , Animais , Bovinos , Estabilidade de Medicamentos , Humanos , Nitrosação , Agregação Plaquetária/efeitos dos fármacos , Compostos de Sulfidrila/farmacologiaRESUMO
Inorganic nitrite has recently been recognized to possess vascular activity that is enhanced in hypoxia. This has been demonstrated in humans in the forearm vascular bed. In animal models nitrite reduces pulmonary vascular resistance, but its effects upon the pulmonary circulation of humans have not yet been demonstrated. This paradigm is of particular interest mechanistically since the pulmonary vasculature is known to behave differently to the systemic. To investigate, 18 healthy volunteers were studied in a hypoxic chamber (inspired oxygen, 12%) or while breathing room air. Each received an infusion of sodium nitrite (1 micromol/min) or 0.9% saline. Three protocols were performed: nitrite/hypoxia (n = 12), saline/hypoxia (n = 6), and nitrite/normoxia (n = 6). Venous blood was sampled for plasma nitrite, forearm blood flow was measured by strain-gauge plethysmography, and pulmonary arterial pressure was measured by transthoracic echocardiography. Plasma nitrite doubled and clearance kinetics were similar whether nitrite was infused in hypoxia or normoxia. During hypoxia, nitrite increased forearm blood flow (+36%, P < 0.001) and reduced three separate indirect indexes of pulmonary arterial pressure by 16%, 12%, and 17% (P < 0.01). Pulmonary, but not systemic, arterial effects persisted 1 h after stopping the infusion, at a time when plasma nitrite had returned to baseline. No effects were observed during normoxia. Therefore, in hypoxic but not normoxic subjects, sodium nitrite causes arterial and pulmonary vasodilatation. In addition, hypoxia-induced pulmonary vasoconstriction was attenuated for a prolonged period and not dependent on a simultaneous elevation of plasma nitrite. This finding is consistent with the direct extravascular metabolism of nitrite to nitric oxide to effect hypoxia-associated bioactivity.
Assuntos
Hipóxia/fisiopatologia , Nitratos/farmacologia , Nitritos/sangue , Artéria Pulmonar/fisiopatologia , Vasodilatação/efeitos dos fármacos , Adulto , Pressão Sanguínea/fisiologia , Débito Cardíaco/fisiologia , Relação Dose-Resposta a Droga , Ecocardiografia , Humanos , Hipertensão Pulmonar/fisiopatologia , Pulmão/irrigação sanguínea , Masculino , Nitratos/farmacocinética , Artéria Pulmonar/efeitos dos fármacos , Método Simples-Cego , Vasodilatação/fisiologiaRESUMO
The vasorelaxant properties of red blood cells (RBCs) have been implicated in both the control of normal vascular tone and the protection of tissues from ischemic events. The identity of the vasorelaxant released from RBCs has yet to be elucidated, however growing evidence suggests that nitric oxide bound to the beta 93 cysteine residue of haemoglobin (SNO-Hb) may be responsible. The vasorelaxant moiety is released during the transition of haemoglobin from its R (oxygenated) to T (deoxygenated) state. We subsequently chose to assess the significance of haemoglobin saturation on the capacity of RBCs to mediate hypoxic vasorelaxation. Human RBC samples suspended in saline were manipulated in a thin film rotating tonometer, designed to rapidly change haemoglobin saturation within the time frame of circulatory transit. Various cycles of oxygenation and deoxygenation were performed. The vasorelaxant properties of the RBCs were analysed using an aortic ring bioactivity assay, wherein changes in isometric tension were recorded to study vessel relaxation. The rabbit aortic rings were preconstricted with phenylephrine under hypoxic conditions (approximately 1% O2) prior to RBC addition. Highly saturated RBCs (98.22% +/- 0.45 HbO2) elicited significantly (P<0.001) more relaxation of hypoxic blood vessels compared to those partially saturated (20.40% +/- 5.28 HbO2). Upon re-oxygenation, previously de-oxygenated RBCs were also capable of eliciting vessel relaxation, which was not significantly different from that observed with the original oxygenated RBC relaxation response. Interestingly, the relaxant capability was not simply returned from extracellular milieu upon re-oxygenation. This data provides further evidence that the conformational switch of haemoglobin from the R-state (oxygenated) to the T-state (deoxygenated) is essential for the release of the vasoactive moiety contained within red blood cells.
Assuntos
Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Vasodilatação , Hipóxia Celular , Feminino , Humanos , MasculinoRESUMO
This study uses an organ chamber bioactivity assay to characterise the direct effect of sodium nitrite upon rabbit blood vessels (aorta (Ao), inferior vena cava (IVC) and pulmonary artery (PA)) in a haemoglobin independent/variable oxygen environment. In 95% oxygen constriction to 8g (Ao), 6g (PA) and 4g (IVC) was achieved using 1 microM phenylephrine. The same constriction in 1% oxygen required 3 microM. During 95% oxygen constriction was consistent and sustained for all vessels. However under 1% oxygen PA was quick to constrict but rapidly gave up this tension whereas Ao was slower to constrict but exhibited a more sustained response. Relaxation of each vessel was assessed post constriction using 10 microM sodium nitrite. Results were expressed as a percentage loss in tension compared to the maximum achieved and corrected by controls which received no nitrite. At 95% oxygen PA relaxed greater than Ao (10.04% +/- 2.28% vs. 5.25% +/- 1.51%). IVC response was varied (2.26% +/- 9.43%). At 1% oxygen all vessels relaxed more. However the pattern was reversed with both IVC (14.20% +/- 3.63%) and PA (16.55% +/- 0.93%) relaxing less than Ao (42.20% +/- 5.21%). These results suggest that relatively low concentrations of sodium nitrite can vasodilate blood vessels. This effect is independent of haemoglobin and tissue specific.
Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Hipóxia/metabolismo , Nitritos/farmacologia , Oxigênio/metabolismo , Animais , Masculino , CoelhosRESUMO
A plethora of publications on techniques and methodologies for measuring nitric oxide (NO) or reaction products of NO (NO metabolites) has served in recent years to complicate and confuse the majority of researchers interested in this field. Here, we provide a practical approach and summarize the key issues and corresponding solutions regarding quantification with the use of ozone-based chemiluminescence, which is the most accurate, sensitive, and widely used NO detection method. We have drawn on the vast experience of leaders in the field to produce this consensus, but the views and implications presented herein represent our own, and we limit our advice to those techniques with which we have direct experience. Hopefully, this guide will allow authors to make more informed decisions regarding NO metabolite measurement methodology, without the need for each subsequent group to rediscover previously observed advantages and pitfalls.