Inorganic nitrite bioactivation and role in physiological signaling and therapeutics.

The bioactivation of inorganic nitrite refers to the conversion of otherwise 'inert' nitrite to the diatomic signaling molecule nitric oxide (NO), which plays important roles in human physiology and disease, notably in the regulation of vascular tone and blood flow. While the most well-known sources of NO are the nitric oxide synthase (NOS) enzymes, another source of NO is the nitrate-nitrite-NO pathway, whereby nitrite (obtained from reduction of dietary nitrate) is further reduced to form NO. The past few decades have seen extensive study of the mechanisms of NO generation through nitrate and nitrite bioactivation, as well as growing appreciation of the contribution of this pathway to NO signaling in vivo. This review, prepared for the volume 400 celebration issue of Biological Chemistry, summarizes some of the key reactions of the nitrate-nitrite-NO pathway such as reduction, disproportionation, dehydration, and oxidative denitrosylation, as well as current evidence for the contribution of the pathway to human cardiovascular physiology. Finally, ongoing efforts to develop novel medical therapies for multifarious conditions, especially those related to pathologic vasoconstriction and ischemia/reperfusion injury, are also explored.

Methylene Blue: The Long and Winding Road From Stain to Brain: Part 2.

Methylene blue was the first synthetic drug ever used in medicine, having been used to treat clinical pain syndromes, malaria, and psychotic disorders more than one century ago. Methylene blue is a cationic thiazine dye with redox-cycling properties and a selective affinity for the nervous system. This drug also inhibits the activity of monoamine oxidase, nitric oxide synthase, and guanylyl cyclase, as well as tau protein aggregation; increases the release of neurotransmitters, such as serotonin and norepinephrine; reduces amyloid-beta levels; and increases cholinergic transmission. The action of methylene blue on multiple cellular and molecular targets justifies its investigation in various neuropsychiatric disorders. Investigations of methylene blue were instrumental in the serendipitous development of phenothiazine antipsychotic drugs. Although chlorpromazine is heralded as the first antipsychotic drug used in psychiatry, methylene blue is a phenothiazine drug that had been used to treat psychotic patients half a century earlier. It has also been studied in bipolar disorder and deserves further investigation for the treatment of unipolar and bipolar disorders. More recently, methylene blue has been the subject of preclinical and clinical investigations for cognitive dysfunction, dementia, and other neurodegenerative disorders. [Journal of Psychosocial Nursing and Mental Health Services, 54(10), 21-26.].

Estudio de la repetición del pentanucleótido CCTTT en el gen de la sintasa inducible del óxido nítrico en pacientes con hipertensión arterial pulmonar / CCTTT Pentanucleotide Repeats in Inducible Nitric Oxide Synthase Gene Expression in Patients With Pulmonary Arterial Hypertension

Introducción: La hipertensión arterial pulmonar (HAP) es una enfermedad en la que se implican, entre otras, la vía del óxido nítrico (NO). Se ha descrito un polimorfismo en el gen de la sintasa inducible del NO (NOS2) que consiste en la repetición del pentanucleótido CCTTT dando lugar a menor producción de NO. El objetivo del estudio fue conocer si este polimorfismo incrementa la susceptibilidad para desarrollar HAP. Métodos: Se compararon 64 pacientes diagnosticados de HAP grupos I y IV y 50 controles sanos. Mediante PCR se genotiparon las muestras de ADN para este polimorfismo. Se comparó la distribución en ambos grupos y se correlacionó con parámetros clínicos, hemodinámicos y respuesta terapéutica. Resultados: Se observó una distribución significativamente diferente en el número de repeticiones entre pacientes y controles (p < 0,0001). Agrupando las muestras en formas cortas (ambos alelos con menos de 12 repeticiones) y largas (≥ 12 repeticiones) se observó que los primeros tenían un riesgo casi 4 veces superior de desarrollar HAP (odds ratio: 3,83; IC 95%: 1,19-12,32; p = 0,024). No hubo diferencias entre los tipos más frecuentes de HAP ni en la respuesta terapéutica o supervivencia. No existió correlación entre parámetros hemodinámicos y el número de repeticiones en los pacientes, solo débil correlación con la presión arterial pulmonar sistólica. Conclusiones: Existen diferencias significativas en la distribución del polimorfismo CCTTT del gen NOS2 entre pacientes con HAP y población sana. Una menor repetición del pentanucleótido CCTTT en el gen de la NOS2 podría incrementar el riesgo de desarrollar HAP

Introduction: One of the pathways involved in pulmonary arterial hypertension (PAH) is the nitric oxide (NO) pathway. A polymorphism in the inducible NO synthase (NOS2) gene has been described, consisting of the CCTTT pentanucleotide repeat, which causes a reduction in NO production. The aim of this study was to determine if this polymorphism increases susceptibility to developing PAH. Methods: Sixty-four patients with a diagnosis of PAH groups I and IV and 50 healthy controls were compared. DNA genotyping of the samples for this polymorphism was performed using PCR. The distribution between both groups was compared and correlated with clinical and hemodynamic parameters and therapeutic response. Results: A significantly different distribution was observed in the number of repeats between patients and controls (P < 0.0001). When the samples were categorized by short forms (both alleles with less than 12 repeats) and long forms (≥12 repeats), it was observed that the former had an almost 4-fold risk of developing PAH (odds ratio: 3.83; 95% CI: 1.19-12.32, P = 0.024). There were no differences between the most common types of PAH, either in therapeutic response or survival. There was no correlation between hemodynamic parameters and the number of repeats in the patients, and only a weak correlation with systolic PAH. Conclusions: There are significant differences in the distribution of the NOS2 promotor CCTTT polymorphism between patients with PAH and the healthy population. A minor CCTTT pentanucleotide repeat in the NOS2 gene may increase the risk of developing PAH

Papel de la aldosterona en las alteraciones vasculares funcionales y en el proceso inflamatorio asociados a la hipertensión en ratas / Role of aldosterone in vascular functional alterations and the inflammatory process associated to hypertension in rats

Objetivos: Estudiar la participación de la aldosterona en la disfunción vascular, el proceso inflamatorio y estrés oxidativo vascular asociado a hipertensión. Material y método: Se utilizaron ratas (n = 16) espontáneamente hipertensas (SHR) de 22 semanas de edad. La mitad de las ratas fueron tratadas durante 10 semanas con eplerenona a una dosis de 30 mg/kg/día (E-30). Se utilizaron ratas (n = 8) normotensas (WKY) como grupo de referencia. La presión arterial se midió de manera indirecta en la arteria caudal de la cola de las ratas. Al final del tratamiento las ratas se sacrificaron y se pesaron los corazones. Se evaluó la función endotelial en anillos aórticos en respuesta a la acetilcolina. La expresión del ARN mensajero (ARNm) de las interleucinas 1 β y 6 (IL-1β e IL-6), del factor de necrosis tumoral α (TNF-α), de la enzima óxido nítrico endotelial (eNOS) y de la subunidad p22phox de la enzima NAD(P)H oxidasa se midió en la aorta de las ratas. Resultados: Las SHR presentaron unos valores de presión arterial sistólica mayores (p < 0,05) que las ratas controles WKY (199,8±4,2 frente a 125,3±2,0 mmHg). El tratamiento con eplerenona redujo (p < 0,05) ligeramente las cifras de presión arterial en las ratas hipertensas (E30; 181,0±2,0 mmHg). No hubo diferencias en el peso corporal de las ratas, sin embargo el peso relativo del corazón de las ratas hipertensas era significativamente mayor respecto a las ratas normotensas y se normalizó con el tratamiento con eplerenona. La relajación a acetilcolina estaba significativamente reducida en las ratas SHR así como la expresión vascular de la eNOS. Sin embargo, las ratas hipertensas presentaron una sobreexpresión vascular del ARNm de IL-1β, IL-6, TNF-α y p22phox respecto a las WKY (p < 0,05). El tratamiento con eplerenona normalizó la función endotelial en las ratas hipertensas; aumento la expresión del ARNm de eNOS y redujo la expresión vascular de las citocinas IL-1β, IL-6, TNF-α, así como de la p22phox. Conclusiones: La aldosterona participa en las alteraciones funcionales vasculares en las SHR reduciendo la biodisponibilidad de óxido nítrico, aumentando el estrés oxidativo y el proceso inflamatorio vascular(AU)

Objetives: To study the participation of aldosterone in the vascular dysfunction, inflammatory process and vascular oxidative stress associated to hypertension. Material and methods: Half of the group of 22 week-old spontaneously hypertensive rats (n=16) were treated with eplerenone (E-30; 30 mg/kg/day) for 10 weeks. Normotensive rats (WKY; n = 8) were used as reference group. Systolic arterial pressure (SAP) was measured by the tail-cuff method. Body weight and heart weight were measured at the end of the treatment. Endothelium-dependent relaxations, as well as vascular mRNA expression of interleukin 1 β and 6 (IL-1β and IL-6), tumor necrosis factor alpha (TNF-α), of endothelial nitric oxide synthase (eNOS), NAD(P)H oxidase subunit p22phox were studied in aorta from SHR untreated or treated with eplerenone. Results: SHR showed higher levels of systolic blood pressure (p < 0.05) as compare with control rats (199.8±4.2 vs. 125.33±2.0 mmHg). Although there were no differences in the body weight among the groups, hypertensive rats had a higher relative heart weight compare to normotensive rats and it was normalize with the treatment of eplerenone (p < 0.05). SHR showed higher vascular mRNA expression of IL-1β, IL-6, TNF-α and p22phox compared to WKY (p < 0.05). Treatment with eplerenone slightly reduced (p < 0.05) blood pressure in hypertensive rats (E30; 181.0±2.0 mmHg) and normalized acetylcholine relaxations. Eplerenone enhanced (p < 0.05) eNOS and reduced p22phox, IL-1β, IL-6, TNF-α of aortic mRNA expressions in SHR. Conclusions: In SHR, aldosterone participates in the functional vascular alterations through the diminution of nitric oxide availability and the enhancement of the inflammatory process and the increase of vascular oxidative stress(AU)

Effect of L-arginine on restraint stress induced modulation of immune responses in rats and mice.

The present study investigates the role of nitric oxide (NO) on restraint stress (RS)-induced modulation of humoral and cell-mediated immune responses in rats and mice. RS produced suppression of humoral immune response, i.e., anti-SRBC antibody titre ( 7.38 +/- 0.32 versus 4.13 +/- 0.30; mean +/- S.E.M., P < 0.001). In case of cell-mediated immunity, in delayed type hypersensitivity (DTH) response the change in paw volume decreased from 0.069 +/- 0.003 mm (mean +/- S.E.M.) in control non-stressed group to 0.038 +/- 0.002 mm in the stressed group (P < 0.001) while percentage leucocyte migration inhibition (% LMI) decreased from 39.7 +/- 1.95 in control non-stressed animals to 15.2 +/- 1.07 in animals subjected to stress (P < 0.01). Pretreating the animals with an NO precursor, L-arginine (1000 mg kg-1, i.p.) antagonized the effect of RS on humoral (anti-SRBC antibody titre 6.50 +/- 0.27 versus 4.13 +/- 0.30, P < 0.001 ) and cell-mediated (DTH response 0.066 +/- 0.002 mm versus 0.038 +/- 0.002 mm, P < 0.001; % LMI 41.5 +/- 1.46 versus 15.2 +/- 1.07, P < 0.01) immune responses. Administration of 7-nitroindazole (7-NI, 50 mg kg-1, i.p.), an inhibitor of neuronal NO synthase, alone further enhanced the immunosuppressive effect of RS (anti-SRBC antibody titre 2.75 +/- 0.25 versus 4.13 +/- 0.30, P < 0.001; DTH response 0.019 +/- 0.002 mm versus 0.038 +/- 0.002 mm, P < 0.001; % LMI 5.0 +/- 1.08 versus 15.2 +/- 1.07, P < 0.01). However, when given before L-arginine treatment, 7-NI reversed the effect of the latter drug on stress-induced immunomodulation (anti-SRBC antibody titre 3.00 +/- 0.27 versus 6.5 +/- 0.27, P < 0.001; DTH response 0.043 +/- 0.003 mm versus 0.066 +/- 0.002 mm, P < 0.001; % LMI 12.0 +/- 0.93 versus 41.5 +/- 1.46, P < 0.01). Unlike its effect on RS-induced immune responsiveness, L-arginine (250, 500, 1000 mg kg-1) when given for 5-7 days to naive non-stressed animals produced dose dependent suppression of both humoral (anti-SRBC antibody titre 6.4 +/- 0.32 versus 5.4 +/- 0.32, 4.0 +/- 0.27, 3.1 +/- 0.30, respectively) and cell-mediated (DTH 0.065 +/- 0.003 mm versus 0.064 +/- 0.004 mm, 0.039 +/- 0.003 mm, 0.020 +/- 0.002 mm, respectively and % LMI 37.52 +/- 1.58 versus 30.48 +/- 1.07, 28.18 +/- 1.22, 19.76 +/- 0.83, respectively) immune responses. 7-NI significantly blocked these immunosuppressive effects of L-arginine (anti-SRBC antibody titre 6.0 +/- 0.38 versus 3.1 +/- 0.030, P < 0.01; DTH response 0.056 +/- 0.004 mm versus 0.020 +/- 0.002 mm, P < 0.001; % LMI 34.76 +/- 1.31 versus 19.76 +/- 0.83, P < 0.01). However, 7-NI when given to non-stressed animals failed to modulate immune responsiveness. Thus, NO appears to play an important role in RS-induced immunomodulation and these effects are different from its effect on immune responsiveness in non-stressed animals.

Arginina, óxido nítrico y función endotelial / Arginine, nitric oxide and endothelial function

La arginina es un aminoácido semiesencial con importantes funciones fisiológicas. Entre ellas destaca su papel como precursora del óxido nítrico, una molécula producida a partir de la arginina por la enzima óxido nítrico sintasa en muchos tejidos y que en el endotelio vascular se comporta como vasodilatadora, antiaterogénica y antiagregante plaquetaria. El estudio detallado de esta reacción enzimática indica que la óxido nítrico sintasa tiene una gran afinidad por su sustrato, la arginina, que se encuentra en concentraciones altas en el endotelio. Por tanto, resultaba sorprendente que el funcionamiento de esta enzima estuviera condicionado por las variaciones en las concentraciones de arginina debidas al aporte nutricional. A esto se le llamó «paradoja de la arginina». Sin embargo, se ha demostrado recientemente la existencia de un inhibidor endógeno de la óxido nítrico sintasa denominado dimetilarginina asimétrica. Este compuesto disminuiría la formación del óxido nítrico por inhibición competitiva con el sustrato natural, la arginina. De ahí la importancia de la suplementación con arginina para contrarrestar este efecto. Además de la arginina, existen otros componentes de la dieta que pueden influir también en la síntesis de óxido nítrico por el endotelio vascular. el endotelio vascular (AU)

Arginine is a semi-essential amino acid with major physiological functions. One of the most outstanding of such is its role as an amino acid precursor of nitric oxide, a molecule produced, in many tissues, from arginine by the nitric oxide synthase enzyme. Within the vascular endothelium, nitric oxide behaves as a vasodilator, antiatherogenic, and anti-plaque aggregation agent. The detailed study of this enzymatic reaction indicates that nitric oxide synthase presents high affinity, aggregation agent. The detailed study of this enzymatic reaction indicates that nitric oxide synthase presents high affinity, for its substrate, arginine, which is found in high concentrations in the endothelium. Consequently, it is surprising that for its substrate, arginine, which is found in high concentrations in the endothelium. Consequently, it is surprising that the functionality of this enzyme is conditioned by variations in concentrations of arginine, produced by nutritional intake. the functionality of this enzyme is conditioned by variations in concentrations of arginine, produced by nutritional intake. This is known as «the arginine paradox». However, the existence of an endogenous nitric oxide synthase inhibitor, This is known as «the arginine paradox». However, the existence of an endogenous nitric oxide synthase inhibitor, known as asymmetric dimethylarginine, has been recently demonstrated. This compound would decrease the formation of nitric oxide through competitive inhibition with the natural substrate, arginine. It is for this reason that dietary supplementation with arginine would be important as a means to counteracting such an effect. In addition to arginine, supplementation with (..) (AU)

Indirect influences of radiation on unirradiated cells through irradiated cells.

There has been a recent upsurge of interest in radiation-induced bystander effects. Previously we reported that the accumulation of inducible nitric oxide (NO) synthase (iNOS) was induced only in human glioblastoma mutant (m) p53 cells by acute irradiation with X-rays, suggesting a suppression of iNOS induction after acute irradiation with X-rays in wtp53 cells. NO secreted from the irradiated mp53 cells induced the accumulation of p53 in unirradiated wtp53 cells. The radiosensitivity of wtp53 cells was reduced by exposure to the conditioned medium from irradiated mp53 cells, suggesting that NO is an initiator of radiation-induced bystander effects. In the present study, we found that the accumulation of iNOS in wtp53 cells was induced by chronic irradiation with gamma-rays followed by acute irradiation with X-rays, but not by each one. It is suggested that the accumulation of iNOS may be due to the depression of acute irradiation-induced p53 functions by pre-chronic irradiation. We found that chronic irradiation with gamma-rays did not inhibit the accumulation of p53 after exposure to the conditioned medium from the irradiated mp53 cells. However, the decay of accumulated p53 was stimulated by chronic irradiation with gamma-rays. At the same time, the accumulation of Hdm2 was observed; suggesting that chronic irradiation with gamma-rays may stimulate the degradation of p53 accumulated by NO-mediated bystander effects.

Real-time direct measurement of nitric oxide in bovine perfused eye trabecular meshwork using a clark-type electrode.

NO was detected in bovine trabecular meshwork (TM). Bovine eyes were perfused (posterior ciliary artery). In some eyes (operated eyes) a NO electrode was inserted adjacent to the TM (scleral flap). Vascular perfusion/intraocular pressures (VPP/IOP) were recorded. In operated eyes, epinephrine (1 nM-100 microM) increased NO (maximally 979.9 +/- 117.6 nM, mean +/- SDM). Timolol (1 mM) shifted the epinephrine-NO concentration-response curve rightward (2.94 log units) without significantly changing the maximal response (903.0 +/- 67.7 nM, mean +/- SDM). The non-selective NO synthase (NOS) inhibitor L-NMMA (100 microM) virtually abolished the NO response to epinephrine. L-NMMA alone (1 microM-100 microM) significantly reduced tonic NO generation (maximally 109.5 +/- 24.9 nM, mean +/- SDM), whereas timolol alone (1 microM-1 mM) had no effect. In unoperated eyes, epinephrine (1 nM-100 microM) reduced IOP (maximally 2.56 +/- 0.64 mmHg, mean +/- SDM). Epinephrine (100 microM) mildly increased VPP (4.6 +/- 1.3 mmHg, mean +/- SDM). Baseline aqueous humor formation rate (11.5 +/- 3.2 microl/min, mean +/- SDM) was unaffected. Effluent perfusate (effusate) total NO(2)(-) was determined by enzymatically reducing all NO(3)(-) to NO(2)(-), then assessing resultant NO(2)(-) (Griess assay). Epinephrine (1 nM-1 microM) increased effusate NO(2)(-) (maximally 15.8 +/- 4.9 microM, mean +/- SDM). Timolol (1 mM) reduced, and L-NMMA (100 microM) virtually abolished effusate NO(2)(-) response to epinephrine. L-NMMA alone (1 microM-100 microM) reduced tonic effusate NO(2)(-) (maximally from 5.8 +/- 1.6 microM to 1.1 +/- 0.9 microM, mean +/- SDM), whereas timolol alone (1 microM-1 mM) had no effect. NO is generated tonically in bovine TM and increases in response to epinephrine.

Effects of a selective nitric oxide synthase inhibitor on endotoxin-induced alteration in hypoxic pulmonary vasoconstriction in sheep.

It has been suggested that overproduction of nitric oxide (NO) by nitric oxide synthase (NOS) contributes to blunted hypoxic pulmonary vasoconstriction (HPV) during endotoxemia. We investigated the effect of a selective inducible NOS (iNOS) inhibitor, ONO-1714, on the loss of HPV during endotoxemia in awake sheep to clarify the role of iNOS. We prepared 11 intubated, awake sheep with hemodynamic monitoring. Hypoxic challenges (FiO2; 12%) were performed before, and 5, 24, 48, 72 hours after endotoxin (1 microg/kg) infusion for 15 minutes. Pulmonary artery (Ppa) and left atrial pressure (Pla) were continuously measured and cardiac output (CO) was measured by the thermodilution method. Pulmonary vascular resistance (PVR) was calculated by (Ppa - Pla)/CO. The percent change in PVR (%PVR) before (pre-PVR) and after (post-PVR) hypoxia was calculated as (post-PVR - pre-PVR)/pre-HPV x 100. ONO-1714 (0.1 mg/kg, n=5, Exp 1) or normal saline (n=6, Exp 2) was administered 5 hours before hypoxic challenge every day. ONO-1714 did not affect the baseline pulmonary hemodynamics before endotoxin administration. % PVR before and after hypoxic exposure was significantly decreased 5 hours after endotoxin administration and gradually improved to baseline at 72 hours. Treatment with iNOS inhibition significantly restored % HPV (24.7+/-5.5% in Exp1 versus -3.1+/-3.6% in Exp 2, 5 hours, 25.3+/-2.5% in Exp 1 versus 7.7+/-2.2% in Exp 2, 24 hours). It is suggested that inducible nitric oxide is related to pulmonary vascular hyporesponsiveness to hypoxia during endotoxemia in sheep.

Behavioral effects of NMDA receptor agonists and antagonists in combination with nitric oxide-related compounds.

Responding of rats was maintained under a 120-response fixed ratio (FR) schedule of food delivery, and animals received individual and combined injections of N-methyl-D-aspartic acid (NMDA), phencyclidine hydrochloride, (+)-MK-801 hydrogen maleate (MK-801), (+/-)-2-amino-5-phosphonopentanoic acid (AP5), 7-chlorokynurenic acid (7CK), ifenprodil tartrate, N(G)-nitro-L-arginine methyl ester hydorchloride (L-NAME), 7-nitroindazole, aminoguanidine hemisulfate, L-arginine, molsidomine, sodium nitroprusside, and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). Behavioral suppression after NMDA was completely and dose-dependently reversed by MK-801, phencyclidine, AP5, and aminoguanidine; partially and dose-dependently attenuated by molsidomine, ifenprodil, and 7CK; and not attenuated at all by L-NAME, 7-nitroindazole, or TMB-8. These findings suggested that behavioral suppression after NMDA was associated with nitric oxide from the inducible synthase. In a second series of experiments, comparable behavioral suppression by 0.1 mg/kg MK-801, but not 3 mg/kg phencyclidine, was attenuated by nitroprusside, molsidomine, and L-arginine, suggesting that suppressions from MK-801 and phencyclidine were mediated by different final common pathways, and that behavioral suppression from MK-801, but not phencyclidine, may be associated with Ca(2+)-dependent nitric oxide.

Óxido nítrico exhalado y asma / Exhaled nitric oxide and asthma

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