Published Online on 21 February 2018 Proc Indian Natn Sci Acad 84 No. 1 March 2018 pp. 153-168  Printed in India. DOI: 10.16943/ptinsa/2018/49300 Review Article Recent Advancement in Nitric Oxide Research in India ANIL N GAIKWAD, SACHIN KUMAR and MADHU DIKSHIT* Pharmacology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow 226 031, India (Received on 30 September 2017; Accepted on 30 November 2017) Discovery of nitric oxide (NO), as endothelial derived relaxing factor that has been awarded with Nobel Prize, highlights its importance as master signalling molecule in diverse systems. Recent research has unfolded several unknown facets of this intriguing biomolecule in cardiovascular system, immunomodulation neurotransmission and plant physiology. NO produced by nitric oxide synthases (NOSs), is a short lived radical that reacts with superoxide radicals to generate reactive nitrogen species and potent oxidant, peroxynitrite implying damage in various pathological conditions. This adds another level of complexity in the understanding of pathological conditions associated with NO paucity or due to increased reactive oxygen nitrogen species (RONS). Researchers from India have been instrumental in unfolding various important and novel functions of this molecule and associated signalling by using multipronged approaches in different systems. Association of NO signaling with increased burden of life style diseases in recent years provide sufficient rationale to investigate NO in diverse pathophysiological conditions in Indian perspective. Here we review recent and important contributions of Indian science during last five years in understanding of NO signaling fundamentals in human, animals and also plants, its association with diverse pathological conditions and therapeutic targeting with possible ameliorative strategies. Keywords: Nitric Oxide; Nitric Oxide Intermediate; Oxidative Stress; Cardiovascular System; Central Nervous System; Immunomodulation Introduction of these associated pathologies. Recent research work on this intriguing molecule has identified more facets Several systems and their functions are regulated at of its contribution in various disease physiology and local levels though autacoid signaling, where auto pathophysiology. Together, recent upshot in life style means “self” and acos means “relief”. An imbalance diseases, cardiovascular diseases burden since last of such autocoids at the level of synthesis, release or decade and association of NO signaling has been an signal tranductions may contribute to pathological active rationale to investigate nitric oxide biology and conditions. Nitric oxide (NO), an important autacoid, its implications in Indian perspective. Furthermore, is released from the cell and act as local environment role of NO has been implicated in plant physiology with short activity duration to regulate physiological including plant-pathogen interaction, stress responses functioning based on its chemically diffusible property. and food biotechnology field that is an national priority NOis generated by reactive oxidation of nitrogen by as agriculture based country and thus provide many cell types in vertebrates and non-vertebrates sufficient reasons to better understand nitric oxide following enzymatic catalysis (Fig. 1). Imbalance in research in diverse systems. In this current review, NO signaling causes development of patho- we overview major research work performed by physiological conditions such as allergy, Indian authors along-with their collaborators abroad hypersensitivity, inflammation, neurotransmission, in the field of nitric oxide biology. We have covered cardiovascular disorders and ischemia-reperfusion publications from year 2012-2017 (five years) for injury. Anti-oxidants and anti-inflammatory agents are purpose of this review. Though there are significant being suggested as the interventive agents for many amount of publications that have used NO and its *Author for Correspondence: E-mail: [email protected] 154 Anil N. Gaikwad et al. byproducts as inflammatory markers in association with other inflammatory molecules and similarly high number of publications have reported modulations of NO pathways using natural products and other approaches, we sincerely apologize that we have not accomodated all of such publications to this review. Nitric Oxide Synthase isoforms and Generation of NO Fig. 1: NO generation by Nitric Oxide Synthase (NOS) NO is produced enzymatically by the action of nitric and its association with oxidants oxide synthase (NOS) isoforms. NOS enzyme oxidizes the guanidine group of the enzyme substrate conditions (Fig. 1). This cause scavenging of NO and L-arginine. Arginine is converted into Nw- superoxide to cause reduction in their functions, while hydroxyarginine and then to citrulline and NO. The adds pathological insults through peroxynitrite and enzyme activity that required dimerization of NOS, is other reactive oxygen -nitrogen species (RONS) regulated by the number of co-factors such as tetra- mediated damage. hydrobiopterine (BH4), FAD, FMN and NADPH (Fig. 1). This process also requires acceleration by Research on fundamental of NOS isoforms calcium calmodulin as an activator. The endothelial regulation is highly important and has provided NOS isoform was discovered for the first time in different isoforms specific inhibitors. Recent research vascular endothelium and now being reported to be in this area has identified interesting phenomenon like present in many other cell types too. Eventually NOS uncoupling that provides superoxide instead of induced NOS (iNOS) and neuronal NOS (nNOS) NO. In India, laboratory of Dr. Koustubh Panda at were also identified. These nNOS, iNOS and eNOS Kolkata is focusing on basic regulation of NOS are also defined as NOS1, NOS2 and NOS3 system. Pyrimidine imidazoles inhibits NOS respectively. These three isoforms differ in their dimerization, required for activity but precise genetic locations. NOS1, NOS2 and NOS are located mechanism of their action has remained unclear. A on chromosome 12, 17 and 7 respectively with single recent study using pyrimidine imidazole and its copy of the gene in the haploid human genome. All derivative (PID) identified mechanism of iNOS these three different isoforms have different catalytic inhibition using rapid stopped-flow kinetic, gel filtration, properties, inhibitor sensitivity and have around >50% and spectrophotometric analysis (Nagpal et al., 2013). homology between human isoforms. Interestingly, two Precisely, PID bound to iNOSheme generated an of these i.e., nNOS and eNOS are constitutive in irreversible PID-iNOS monomer complex that could nature and depend on calcium levels for activation, not be converted to active dimers by while iNOS is inducible as suggested by its name and tetrahydrobiopterin and L-arginine. PID also caused trigerred by various cytokines and other factors in a irreversible monomerization of active iNOS dimers calcium independent manner. Interestingly, various L- (Nagpal et al., 2013). This study established PID as arginine analogues can inhibit NO production and a versatile iNOS inhibitor for complete physiological enzyme activity, though these NOS isoforms differ in inhibition of iNOS in inflammatory, immunological, and terms of their sensitivity to these analogues. Many of neurodegenerative diseases. Interestingly, NO plays the single gene level variants are also found out and a regulatory role as signaling molecule at low getting many more cannot be negated. NO is highly concentration, while high NO and associated nitrogen unstable molecule and gets transformed into nitrite species cause insult to cardiovascular, central nervous and nitrate that are commonly used as indicator of system and other tissues and implicated in various nitric oxide signaling in various systems. NO also diseases (Fig. 2). Following sections will discuss NO reacts with superoxide radicals to generate reactive signaling in these systems in details. nitrogen species (RNS) and potent oxidant, peroxynitrite implying damage in various pathological Recent Advancement in Nitric Oxide Research in India 155 functions of several proteinsimportant in cardiovascular and diabetes biology. Dr. Srinivas Gopala group’s at Thiruvananthapuram has recently reviewed the functional importance of NO signaling in many mitochondrial and cytosolic proteins in diabetic heart using nitrated proteome elucidation studies (Jayakumari et al., 2014). Laboratory of Dr. Madhu Khullar at Chandigarh has also significantly contributed in understanding of NO and eNOS pathway in diabetic cardiomyopathy, hypertension nephropathy, preterm labour and rheumatoid arthritis. In addition, epigenetic regulation of myocardial NOS has also been suggestedin diabetic cardiomyopathy (Khanna et al., 2014) and eNOS gene polymorphism Fig. 2: Major functions of NO link was revealed in type 2 diabetic Asian Indians (Cheema et al., 2013). Dr. Madhulika Dixit at IIT Madras has been investigating role of NO Signaling Studies on Cardiovascular System and Related in endothelial dysfunction, atherosclerosis and edema. Pathological Conditions Catestatin (CST), an endogenous antihypertensive/ In cardiovascular system, NO is mainly produced by antiadrenergic peptide regulates cardiovascular eNOS constitutively expressed in the endothelial cells. physiology. A recent study has revealed association In addition, diverse blood cells also contribute to NO of the naturally-occurring human CST-Gly364Ser availability in the vicinity to endothelium. Together, variant with increased risk of systemic blood pressure NO plays key role in maintenance of vascular function and hypertension in human populations, possibly via and vasodilation. The endothelial cells derived NO diminished endothelial derived NO production due to prominently from eNOS plays a central role in altered interactions of CST-364Ser peptide with beta- vascular tone regulation via acting as endogenous adrenergic receptors (Kiranmayi et al., 2016). vasodilator. Usually, cytosolic eNOS is not catalytically Dr. Suvro Chatterjee’s group at Anna University, active, while active enzyme is localized at the plasma Chennai has been investigating NO-cGMP signaling membrane where NO generation takes place that in endothelial permeability, nonalcoholic fatty liver subsequently released into extracellular environment disease (NAFLD), atherosclerosis and during and the abdominal side of the blood vessels. mechanical stresses in RBCs(Balaguru et al., 2016, Continuous generation of NO is favourable towards Nagarajan et al., 2016, Saran et al., 2017, Seth et al., maintenance of integrity of cardiovascular system. 2017) and are summarizedhere. Rho GTPases Further NO in association with other free radicals downstream effecter, Rho-associated protein kinase and their balance with anti-oxidant system is vital for (ROCK) isapotential target for cardiovascular normal cellular functioning and imbalance of which diseases.Interestingly, ROCK inhibitorY-27631 was causes risk for cardiovascular health. Consistently, found to modulate NO production in endothelial cells deficiency in production and/or bioavailability of NO in a biphasic manner suggesting caution for its use in have long been associated with endothelial dysfunction cardiovascular diseases (Kolluru et al., 2014) and and cardiovascular diseases [summarized in a recent advocated a combination therapy of chemotherapeutic review (Charles et al., 2017)]. At equimolar ratio, drugs and cGMP analogs, which would confer better NO and superoxide form speroxynitrite that decides protection against chemotherapy mediated vascular cellular fate towards necrosis or apoptosis depending dysfunctions in cancer patients (Gajalakshmi et al., on its concentration in various cardiovascular 2013). RBCs-eNOS contributes to intravascular NO disorders(Islam et al., 2015). NO and reactive oxygen pool and regulates physiological functions. Nagarajan intermediates (ROS) also mediatepost translational et al., have shown that mechanical stimuli perturb modifications such as tyrosine nitration, cysteine S- RBC membrane that in turn triggered a signaling nitrosylation and S-glutathionylation that regulate cascade to activate the eNOS via phosphorylation of 156 Anil N. Gaikwad et al. the serine-1177 moiety of RBC-eNOS and promoted (Jacob et al., 2017). Mercury exposure led to loss of important endothelial functions such as migration and endothelium-dependent vaso-relaxation due to reduce vascular sprouting (Nagarajan et al., 2016). This study NO bioavailability via enhanced oxidative stressand implicated mild mechanical/physical perturbations (like can function as an early trigger to consequent excersize) to sensitize RBC-eNOS for NO production cardiovascular complications (Omanwar and Fahim, in vivo and during storage to improve viability of 2015). Organochlorine, endosulfan pesticides that are RBCs in blood banks (Nagarajan et al., 2016). Tip associated with cardiovascular disease (CVD) and cell formation from single leader endothelial cell is an atherosclerosis cause endothelial dysfunction by essential process in angiogenesis, studies have decreasing eNOS activity(Ghosh et al., 2017). performed on the role of eNOS-NO-cGMP signalling Decreased NO production or bioavailability was also during this process that confirmed loss of eNOS found in chronic kidney disease (CKD) patients suppressed tip cell formation (Priya et al., 2015). (Reddy et al., 2015). Further adiponectin specially Further, dissection of NO downstream signaling using IL-6 was negatively correlating with circulating NO pharmacological inhibitors and inducers indicated that levels in CKD patients (Ambarkar et al., 2016). Many NO via sGC/cGMP pathway in the tip cells led of amino acid such as arginine, lysine, glycine and angiogenesis (Priya et al., 2015). A comparative study methionine intake has significant impact on the NO of NONOate based NO donors and linking NO levels and cardiovascular parameters. Glycine release dynamics with physiological functions supplementation in hypercholesterolemic rats suggested spermine NONOate applicability for significantly increased total NO concentration angiogenesis (Majumder et al., 2014). Thalidomide (Venkatesh et al., 2017). L-Arginine supplementation treatment to pregnant women, causes limb deformities. a well tolerated safe amino acid that improved Interestingly, NO was found to prevent limb endothelial dysfunction, ameliorates arterial stiffness deformities in thalidomide affected chick and zebrafish and oxidative stress in chronic kidney disease mediated embryos by promoting angiogenesis, reducing corroborating NO levels (Reddy et al., 2015). Organic oxidative stress and inactivating caspase-3 dependent “nitro” compounds like nitroglycerine are useful in apoptosis (Siamwala et al., 2012). Similarly donating acute coronary syndrome, but the mechanism remains NO can be a preventive measure for cadmium speculative. Using different anti-anginal agents, Bank mediated teratogenicity (Nagarajan et al., 2013, et al., found that organic nitro compounds, acetyl Veeriah et al., 2015). Secreted Frizzled-Related salicylic acid, insulin and glucose activate NOS in the Protein 4 (sFRP4), a secreted glycoprotein caused arterial endothelial cells to generate continuous NO endothelial dysfunction followed by suppression of that seems to control the chest pain in acute coronary angiogenesis. Saran et al dissected the mechanism syndrome(Bank et al., 2017). In yet another study, of sFRP4 mediated inhibition of angiogenesis that NO donors sodium nitroprusside (SNP), S-nitroso- envisaged NO-cGMP signaling and elevated N-acetylpenicillamine (SNAP) and S- corresponding ROS levels and apoptosis for the nitrosoglutathione (GSNO) were found to inhibit ion induction of endothelial dysfunctions (Saran et al., channel pannexin 1 (Panx1) mediated currents in 2017). HEK-293 cells (Poornima et al., 2015). Role of NO in ischemic preconditioning (IPC)-induced During physiological conditions, NO levels can cardioprotection is suggested. Ovariectomyalso be regulated at multiple levels. Constent to this, reduces atrial natriuretic peptide (ANP) with endogenous asymmetric dimethyl arginine (ADMA) subsequent reduction in the level of NO. IPC-mediated in serum competitively inhibits NO synthase. cardioprotection was thus significantly attenuated in Interestingly, serum ADMA/NO ratio has been shown ovariectomized rat (Vishwakarma et al., 2017). to be better predictive marker for the severity of the Further perfusion with ANP protected that was coronary artery disease in patients at the risk of angina attenuated by perfusion with N(ω)-nitro-l-arginine pectoris or myocardial infarction(Shivkar and Abhang, methyl ester hydrochloride (L-NAME) suggesting 2014). NO augmentation by sports and supplements ANP mediated availability of NO as central in are currently used for physical fitness of the sport cardioprotection (Vishwakarma et al., 2017). persons. These products exhibited increase in Administration of recombinant erythropoietin (rEPO) circulating levels of nitrite and nitrate and in saliva Recent Advancement in Nitric Oxide Research in India 157 is often associated with systemic and pulmonary while NO donor (DETA-NO) increased BDNF arterial hypertension in animals and human. A recent release (Datta et al., 2017). In other in-vitro study, study showed that even a short-term exposure of 6-OHDA or lipopolysaccharides (LPS) has erythropoietin impaired endothelial function through significantly decreased the viability of astrocytes, by inhibition of NO production (Sultan et al., 2017). inducing iNOS, nitrite level, ROS and decrease in During pregnancy, hypertension is the most common mitochondrial membrane potential (Gupta et al., medical problem, that is associated with maternal 2015). Role of iNOS was further confirmed by using endothelium. Recently Gaikwad et al., observed a iNOS inhibitor aminoguanidine that significantly decrease in NOx and Thiol levels in pregnancy induced attenuated the 6-OHDA/LPS induced cell death hypertension (PIH) (Gaikwad et al., 2017). including mitochondrial activity, ROS levels (Gupta et Interstingly S-nitrothiols were increased in PIH, al., 2015). Another view of NO role in Parkinson suggesting nitrosative stress a potential factor for this disease has also been suggested that NO from nNOS clinical manifestations. While in another study causes neurodegeneration, while NO produce from fractional exhaled nitric oxide (FENO) was failed to iNOS in proliferating microglia mediates the disease differentiate controlled and uncontrolled asthma with progression. In addition antagonistic pleiotropic effects FENO value of 16(11-23) ppb and 13 (11-25) ppb, of NO has been suggested in the pathophysiology of respectively (P=0.26) in children (Meena et al., 2016). Parkinson’s disease (Tripathy et al., 2015). Intra- A study aimed to investigate effect of stored RBC cerebroventriculars trepto zotocin (STZ) model of transfusions that increase cell-free Hb on NO cognitive impairment in rats exhibited increased NO, availability in postoperative surgical patients, results oxidative stress, inflammatory cytokines, increased obtained suggested decrease in NO metabolites expression of Rho kinase in cortex and hippocampus. irrespective of stored RBC transfusions, but most Taurine, the essential amino acid exerted neuro- likely due to hemodilution (Nagababu et al., 2016). protective and beneficial effects for cognitive impairment of Alzheimer’s type by suppressing above- Studies on Central Nervous system and Related mentioned parameters (Reeta et al., 2017). In mouse Pathological Conditions model of STZ induced chronic hyperglycemia, NO (using SNP) caused oxidative stress in addition to Nitric oxide is prominently produced by nNOS in brain molecular alteration in the neurons and glial cells and playsa keyrole in the central nervous system through neuroinflammation via NF-κB signaling pathophysiology including regulation of stress(Gulati (Richa et al., 2017). Consistently in a rat model of et al., 2015). It is important to mention significant ischemia, NOS Inhibitor, L-NAME exhibited neuro- contribution made by Dr. Kavita Gulati and Dr. protective effects by mitigating glutamate Arunabha Ray, New Delhi and others in NO regulation excitotoxicity, inflammation and oxidative stress in CNS physiology and dysfunction. NO and its stable mediated by decreased nitrate/nitrite content (Pramila metabolites (nitrite, peroxynitriteetc) cumulatively et al., 2015). Hyper-ammonemia found in many cause nitrosative stress in brain especially in neurological disorders is associated with urea cycle nigrostriatal systemthat is implicated in Parkinson’s dysfunction and altered brain energy metabolism. disease. Consistently, NO Sinhibitors have moderately Glutamate-NO-cGMP pathway on modulation of rescued Parkinson’s disease (Gupta et al., 2014), glutamate receptors and transporters altered important which was also suggested by the earlier findings from cerebral processes causing cerbraledema and cell Dr Madhu Dikshit’s lab. Datta et al. revealed that death (Natesan et al., 2016). In another study, Zinc- localization and number of astrocytes decided induced nigrostriatal dopaminergic neurodegeneration dopaminergic neuron survival and function under 6- was found to be dependent on reduction in nitrite hydroxydopamine (6-OHDA) stress, as astrocytes content and total/nNOS activity/expression. NO from midbrain provided better dopaminergic neuronal donors discernibly alleviated Zn-induced (TH) cell survival in comparison to forebrain and neurobehavioral impairments, neurodegeneration, and hindbrain through BDNF secretion (Datta et al., other associated changes (Singh et al., 2017). 2017). Further BDNF released from astrocytes is Curcumin have significantly attenuated vincristine mediated through autocrine and paracrine signaling induced neuropathic pain in a mice model owing to its of NO, as NOS inhibitor mitigated this BDNF release, 158 Anil N. Gaikwad et al. anti-nociceptive, calcium inhibitory and anti-oxidant synthase inhibitor L-NAME pre-treatment increased effects (Babu et al., 2015). The hyper-angelic pain it significantly (Gulati et al., 2015). Together suggests induced by CNS stimulant Modfinil was reversed by that estrogen and its interactions with oxidative stress NOS inhibitors indicating role of NO pathways (Gupta including NO are central to gender based differences et al., 2014). In addition, NO precursors have in cold restraint stress induced gastric ulceration exacerbated and NOS inhibitor attenuated low (Gulati et al., 2015). In a study, hypobaric hypoxia frequency magnetic field induced OCD like using high altitude simulation chamber (294.4 mmHg) behaviours by modulating levels of NO (Salunke et for 24 h resulted in elevation of arterial blood pressure, al., 2014). Chronic alcohol administration altered the renal sympathetic nerve activity, right ventricular functioning of CNS with increased ROS and NO levels systolic pressure, lung wet to dry weight ratio and and decrease in mitochondrial complex I, III and IV Evans blue dye leakage (Sharma et al., 2015). These activities (Reddy et al., 2013). Opioid agonist, responses were significantly attenuated after lesioning morphine protected from restraint stress induced posterior hypothalamus or after chronic infusion of anxiogenesis and neurobehavioral suppression in rats GABA receptor agonist muscimol into posterior that was associated with reductions in oxidation hypothalamus. Interestingly, chronic infusion of the products (NOx) of NO in the brain (Anand et al., NO donor SNAP into the posterior hypothalamus 2012). Importantly, NO levels were rescued with mitigated such attenuation (Sharma et al., 2015). morphine. Further, L-arginine synergized with sub- Together during hypobaric hypoxia over-activity of effective doses of morphine to protect stress-induced posterior hypothalamic neurons via local decrease in anxiety, whereas L-NAME blocked morphine GABA-ergic inhibition increased the sympathetic mediated protection (Anand et al., 2012). In another drive and thus pulmonary hypertension and edema. study, morphine and L-arginine pre-treatment Nitric Oxide in Dyslipidaemia, Insulin ameliorated stress mediated effects via decrease Resistance, Sepsis and Diseases inHSP-70 levels and demonstrated involvement of NO in brain (Joshi et al., 2015).Chronic predictable and Here we would like to mention that studies from our unpredictable stresses also modulated immunological group have been instrumental to demonstrate responses by decreasing IgG type antibody and significant alterations in the NO signaling in delayed type hypersensitivity. Stable metabolite of NO experimental models of thrombosis, hypoxia- including peroxinitrite formation through 3-nitrotyrosine reoxygenation, sepsis, and in CNS disorder patients. (3-NT) formation impacted immuno-modulation. Pre- NO has been suggested to play an important role in treatment with iNOS inhibitor amino guanidine the initiation of dyslipidaemia induced insulin resistance attenuated effects of stress in decreasing NOx and (IR) with contrary reports. Recently by using iNOS 3-NT levels indicating involvement of iNOS during KO mice, our group has reveal an altered glucose modulation of adaptive immunity to stress (Thakur et and lipid homeostasis in liver and adipose tissue that al., 2017). Acute and chronic restraint stress causes pre-dispose to insulin resistance (Kanuri et al., 2017). anxiety, while both acute and chronic restraint stress The respiratory exchange ratio (RER), volume of correlated with increased HSP-70 levels, only acute carbon dioxide (VCO ), and heat production were restraint stress led to decrease in NOx level. Acute 2 lower as compared to WT mice. Significant reduction restrained stress induced anxiogenesis is more in male in eNOS and nNOS gene expression, hepatic and rats than female rats, that can be associated with adipose tissue nitrite content, circulatory nitrite suggest increased level of ADMA and reduced level of NOx a link between the NO status with systemic and tissue in brain homogenates (Chakraborti et al., 2014). specific IR. Furthermore, a potential link between Markedly higher level of gastric ulceration was NO, leptin and adipocyte insulin responsiveness has observed in male rats than female rats upon cold been suggested (Gupta et al., 2017). Recently chronic restraint stress (Gulati et al., 2015). These effects hyper-leptinemia was found to induce insulin signaling were associated with the reduced brain and plasma disruption in adipocytes through increased expression NOx and GSH levels while MDA levels were of iNOS. Further, leptin effects on insulin signaling elevated. L-Arginine pre-treatment prior to cold were mitigated by pharmacological depletion of iNOS restraint stress prevented ulceration while NO and were absent in iNOS knockout animals (Gupta et Recent Advancement in Nitric Oxide Research in India 159 al., 2017). Reduced NO generation in the kidney is progression of autoimmune diseases (Khan et al., associated with hypertension in insulin resistance. 2017). Interestingly insulin was found to increase NO Nitric Oxide and Host-pathogen Interaction production in mouse renal inner medullary collecting duct cells via increased p-eNOS (Ser1177) levels Role of nitric oxide in host and pathogen interplay has (Pandey et al., 2015). Other experiments suggested also been a point of focus. Recently, high NO levels contribution of reduced insulin receptor signaling in were found in the samples with high mononuclear renal inner medullary collecting duct cells towards cell counts and chronic tuberculous meningitis thus hypertension in the insulin-resistant state (Pandey et suggesting important role of NO (Kumar et al., 2017). al., 2015). Active nitrogen molecules have been Autophagy is important innate immune defense suggested to play an important role in vascular mechanism though lysosomal degradation. Sustained instability of septic shock. Plasma levels of nitrite and activation of Raw264.7 macrophages by IFN-γ and nitrate in systemic inflammatory response syndrome LPS has limited autophagy in NO dependent activation (SIRS), sepsis and septic shock has revealed the of AKT-mTOR signalling. Using Si-RNA approaches association of active nitrogen molecules in the authors have demonstrated AKT was responsible for progression of septic shock. Plasma nitrite and nitrate glycolytic shift, decreased mitochondrial potential and were high in patients with sepsis and septic shock, autophagy inhibition in activated macrophages (Matta which increases with severity of sepsis (Kothari et and Kumar, 2015). Interestingly, Plasmodium al., 2012). Endogenous ADMA inhibits NOS and thus falciparum parasite drive cerebral malaria exhibited regulates vascular tone. A recent study revealed the persistent debilitating neurological deficit due to blood association of ADMA and diabetes induced kidney brain barrier disruption, endothelial cell activation, NO injury. Significant elevation in plasma ADMA levels bioavailability, apoptosis and neuro-inflammation (Hora was observed in T2DM micro and macroalbuminuric et al., 2016). In Northeast India, the Jaintia tribes patients, suggesting a causative role of ADMA in the utilize aqueous extract of the medicinal plant development of kidney injury in terms of renal fibrosis Carexbaccans to control helminthiasis. A recent study (Jayachandran et al., 2017). This study also suggested identified that phytochemicals resveratrol- and alpha- 0.66muM/l of plasma ADMA level as a predictive viniferin decrease acetylcholinesterase and NOS in risk threshold for diabetic nephropathy in south Asian helminth parasite Raillietina echinobothrida (Giri Indian population (Jayachandran et al., 2017). and Roy, 2015). This study highlights NO signaling in Tobacco smoke induced oxidative damage to lung helminth intracellular communications through proteins, activated pro-inflammatory Rtp801 that neuromuscular system and potential for anthelmintic triggers nuclear factor kappa B and consequent iNOS potential purpose (Giri and Roy, 2015). In another mediated overproduction of NO to induce oxido- study, rabies virus induced pathologies in mouse model nitrosative stress and lung protein nitration (Gupta et were reduced with U0126 (inhibitor of MEK1/2) al., 2016). Interestingly, lung protein nitration was treatment (Manjunatha et al., 2017). The better inhibited with lung-specific inhibition of iNOS using survival was positively correlated with reduced viral N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) load and reduced viral spread in the brain. RV-infected but fails to inhibit/reverse the oxidative lung injury mice were present with higher levels of serum NO, (Gupta et al., 2016). Ascorbate or vitamin C, a dietary iNOS, and TNF-α. CD4+, CD8+ T lymphocytes and antioxidant substantially prevented tobacco smoke- NK cells were increased in blood and spleen of U0126- induced lung protein oxidation as well as Rtp801 treated group (Manjunatha et al., 2017). Furthermore, activation and iNOS/NO-induced nitration and thus intra-macrophage survival of L. donovani depended provided holistic prevention to pulmonary on the availability of extracellular L-arginine (Mandal emphysema.A recent review article advocated role et al., 2017). Leishmania, resulted in upregulation of of oxidatively nitrated histones in the initiation/ L-arginine transport while Leishmania survival was progression of autoimmune inflammatory diseases. greatly impaired when the L-arginine transporters Interestingly, systemic lupus erythematosus and CAT-2 were blocked either using inhibitor or siRNA- rheumatoid arthritis sera shows oxidatively and mediated downregulation (Mandal et al., 2017). NO nitrated modified histones involve in the initiation and also plays an indispensable role in killing of invading 160 Anil N. Gaikwad et al. pathogens by enhancing RONS in immune cells. A group has investigated an direct effect on study using novel and alternative approach for hematopoietic potential, NO donor (SNP) treatment intracellular delivery of NO using inhalable has led to high expression of CD34+ cells in murine microparticles (MP) containing NO donors has bone marrow Lin-cells or sorted LSK-CD34-cells, induced phagosome maturation and kill suggesting upregulation of CD34, that has contrasting Mycobacterium tuberculosis Mtb H37Rv (Verma et age-dependent effects on the functionality of murine al., 2013). Importantly, inhalable MP were able to hematopoietic stem cells (Jalnapurkar et al., 2016). target NO donors to the macrophage and NO release Another interesting study has revealed the role of NO in cytosol to reduce Mtb (Verma et al., 2013). It would in migration and/or invasion of colon cancer cells by be interesting to further investigation these MP as an up-regulating cGMP-PKG-ERK1/2-AP1 pathway adjunct to standard anti-tuberculosis chemotherapy. leading to increase expression of MMP-2/9 (Babykutty et al., 2012). DEPTOR endogenously Nitric Oxide in Immune Cells, Hematopoie- inhibit mTOR complexes and are often deregulated sisand Leukemia in carcinogenesis. DEPTOR overexpression and silencing studies concluded that it promotes survival Nitric oxide has been shown to have contributory role of cervical squamous cell carcinoma cells by reducing in hematopoietic cell growth and differentiation. To apoptosis mediated by differential effects of iNOS/ validate this presumption, our group recently has eNOS expression, PI3K-AKT and P38-MAPK assessed the alterations in nitrite level in control and pathway (Srinivas et al., 2016). leukemic cell growth by using myeloid leukemias including AML and CML patients. The significant Recent focus of our lab research has been decrease in nitrite levels in the blood plasma, marrow investigation of NO signaling and its effect on fluid and cellular fractions in BM and blood of myeloid neutrophil function and death (Fig. 3). Recent study leukemia suggests towards decrease in NOS activity has revealed a crucial role of NO/iNOS in neutrophil (Jain et al., 2017). Further current work is focused to apoptosis via enhanced ROS generation and caspase- unfold molecular targets for therapeutic role of NO 8 mediated activation of mitochondrial death pathway modulators. Another study from Dr. Vaijayanti Kale’s (Dubey et al., 2016). Prolonged treatment of human Fig. 3: NO signaling and its effect on neutrophils functions Recent Advancement in Nitric Oxide Research in India 161 PMNs or mice neutrophils with NO led to enhanced Studies targeting NO in Health and Diseases ROS generation, caspase-8/caspase-3 cleavage, and Several natural products have been evaluated for NO reduced mitochondrial membrane potential and finally mediated cardiovascular diseases, neurodegeneration cellular apoptosis (Dubey et al., 2016). Involvement and inflammatory syndromes. Various flavonoids, of NOX2 in NO-induced apoptosis of PMNs was carotenoids, phytoestrogen, phytosterols contribute to further suggested by inhibition of caspase-8 and BID improve endothelium dependent vaso-relaxation by cleavage in BMDN from neutrophil cytosolic factor- modulating availability of NO and has been reviewed 1 (NCF-1) knockout mice. Furthermore, ROS, NO recently (Upadhyay and Dixit, 2015). To discuss all generation and iNOS expression were enhanced in a of such studies in this review was not feasible. We time-dependent manner in PMNs undergoing recommend another review that has described the spontaneous apoptosis. Pharmacological and genetic herbal plants and phytochemicals with ablation of iNOS in human PMNs and mice BMDN hepatoprotective and Immunomodulatory via targting significantly reduced the levels of apoptosis (Dubey chemokines, and cytokines, and release of the et al., 2016). Furthermore, nitric oxide induced inflammatory mediator (Ilyas et al., 2016). oxidative stress-related posttranslational modifications (PTMs) of cytoskeleton proteins were investigated Cardiovascular Associated Diseases in human PMNs by using in vitro and genetic approaches. Importantly S-glutathionylation of L- Traditional essential oil rich in curzerene, phenone, plastin (LPL) and β-actin promotes reduced germacrone and other sesquiterpenes caused chemotaxis, polarization, bactericidal activity, and significant vaso-relaxant effects in ex vivo model phagocytosis. S-Thiolation diminished binding as well system through NO dependent pathway (Shiva as the bundling activity of LPL (Dubey et al., 2015). Kumar et al., 2017). Amaranth extract increases NO Enhanced nitroxidative stress with LPL S- levels in circulation suggesting improvement of overall glutathionylation identified disease relevance in performance of sport persons (Subramanian and diabetic patients and db/db mice with impaired PMN Gupta, 2016). A novel class of ‘1-(nitro-oxy) ethyl functions (Dubey et al., 2015). In diabetes-associated ester’ group-containing NSAIDS as efficient NO vascular complications, lower levels of glutathione and releasing ‘true’ prodrugs of aspirin and naproxenwas increased oxidative stress have been reported. Thus reported recently with parallel bioactivity and exhibited provide a mechanistic basis for their impaired protective effects in rats from gastric damage (Gund functions in diabetes mellitus(Sanchez-Gomez et al., et al., 2014). Atorvastatin has ameliorated arsenic 2013). Another study has identified interaction of iNOS induced hypertension by improving lipid profile and with rac2 that has regulated ROS and RNS generation aortic NO signalling that restored vascular redox in the human neutrophil phagosomes to mediate homeostasis (Kesavan et al., 2014, Sarath et al., microbial killing. During phagocytosis neutrophils 2014). In another study, Gentianalutea (GL) and its showed significant elevation in NO and RONS, these component isovitexinexerted anti-atherosclerotic responses were inhibited in iNOS, Nox2 and Rac2 effects (Kesavan et al., 2016). GL aqueous root silenced human or iNOS-knockout mice neutrophils. extract and isovitexin prevented endothelial Interestingly iNOS-Rac2 complex formed translocate inflammation and smooth muscle cell migration to block to phagosomes after phagocytosis accompanied by the onset and progression of atherosclerosis in STZ superoxide, NO, ROS/RNS. Rac inhibitor, NSC23766 induced diabetic rats. Interestingly, GL treatment led that significantly abrogated NO release and microbial to reduction of iNOS expression in aortic segments killing in vivo suggests its importance in inflammatory of diabetic rats (Kesavan et al., 2016). Sinapinic acid conditions (Jyoti et al., 2014). In a study focused to increased level of plasma NO metabolites in L-NAME explore estrogen mediated regulation of immune induced hypertension model and protect from high responses, Estrogen through ER-α was found to blood pressure, cardiac fibrosis, cardiac dysfunction, differentially modulates β2-AR-induced immune kidney fibrosis and lipid metabolic (Silambarasan et response pathways, and NO that seems to be al., 2014, Silambarasan et al., 2016). Widely used responsible for estrogen-induced immunosenescence chemotherapeutic breast cancer drugs has been and development of female-specific diseases shown to dampen vascular functions by interfering (Priyanka et al., 2014). with NO signaling in endothelium and these effects 162 Anil N. Gaikwad et al. could be recovered using pharmaceutical agonists of (CMCE) was found to exert potent anti-inflammatory NO signaling pathway (Gajalakshmi et al., 2013). L- activity and cytotoxic effect. Interestingly, CMCE theanine, a non-protein amino-acid found in tea induced a significantly decrease in LPS-induced (Camellia sinensis), promotes NO production in nitrite, aortic iNOS expression, and thus rescue endothelial cells (Siamwala et al., 2013) to improve vascular dysfunction and thus suggests a therapeutic vascular function and is linked to lowering the risk of potential for its use in sepsis and leukemia (Rana et cardiovascular disease. al., 2016). Interestingly, Curcuma oil was also found to ameliorate insulin resistance and associated CNS Associated Diseases thrombotic complications in hamster and rat models (Singh et al., 2015). Curcuma oil also reduces Sesame which is reported lipid lowering agent of endothelial cell-mediated inflammation in post Sesame indium Linn (Pedaliaceae) corrected the myocardial ischemia/reperfusion in rats (Manhas et aluminium chloride (AlCl3) induced cognitive al., 2014). dysfunction and memory impairment in rodents and also reversed NO and inflammatory cytokines in NO and Plant Physiology hippocampus and frontal cortex of these rodents (John et al., 2015). In another study, fisetin, a naturally NO also plays role in plant stress responses including occurring flavonoid, exhibited therapeutic benefits by infection resistance and tolerence. Dr. Sanjay Ghosh modulating urea cycle enzymes in ammonium chloride and others are understanding the plant-pathogen induced hyper-ammonaemic rats. This effect was interaction and nitrosative stress participation in plant derived from decrease in iNOS and NF-κB in hyper- responses. In a recent study, salicylic acid (SA) and ammonaemic rats (Subramanian et al., 2014). NO using SNP were found to mitigate injury Naringenin another flavonoid has reduced focal symptoms of saline stress in Pisumsativum L. through cerebral ischemia reperfusion injury by supressing substantial decline in reactive oxygen species neuro-inflammation and NF-kB mediated accumulation and inducing effects on activities of inflammatory pathway, thus improved functional superoxide dismutase, catalase, guaiacol peroxidase outcome (Raza et al., 2013). Naringenin has also and ascorbate peroxidase (Yadu et al., 2017). Thus decreased oxidative stress by reducing increased lipid may be efficiently utilized to overcome the adverse peroxide and NO in type-2 diabetes induced memory signatures of salinity stress (Yadu et al., 2017). dysfunction in rats and improved cholinergic function Furthermore, NO is an important signaling molecule (Rahigude et al., 2012). Bacopa moneri extract has in plants under physiological and stress conditions. A been shown to reverse cognitive dysfunction in many recent review has described the influence of NO on neurodegenerative disorders/diseases. Brahmi has chloroplasts possibly by influencing also increased the reduced age related NO production photophosphorylation, electron transport activity and in lymphocytes in rats (Priyanka et al., 2013). Fish oil oxido-reduction state. In addition, NO can change the enrichment with quercetin has provided higher degree gene expression to influence the photosynthetic of neuro-protection in 3-nitropropionic acid rat model apparatus and its functions (Misra et al., 2014).A of neuro-degeneration by attenuating oxidative stress recent finding demonstrates that sunflower seedling in brain regions (striatum/cerebellum) as observed roots exhibit high sensitivity to salt stress in terms of reduction in reactive species, hydropreoxides and NO nitrite accumulation. Salt stress cause reduction in S- levels (Denny Joseph and Muralidhara, 2013, K and nitrosoglutathione reductase (GSNOR) activity that Muralidhara, 2014). Pre-treatment of NO mimetic, was restored with dithioerythritol (Jain et al., 2017). L-arginine as well as melatonin reduced aminophylline Opposite patterns of S-nitrosylation in seedling induced anxiogeneic response including anxiety and cotyledons and roots was observed using LC-MS/ seizures while NO synthase inhibitors L-NAME and MS analysis, suggesting S-nitrosylation as a key 7-NI aggravated it (Gulati and Ray, 2014). mechanism of salt stress sensing in sunflower seedling (Jain et al., 2017). Another study has Inflammatory Syndromes revealed ROS/NO regulation of phenolic metabolism under water stress and abscisic acid (ABA) by using Recently, a novel chemically modified, non-carbonyl tolerant and sensitive wheat cultivar (Kaur and compound enriched Curcuma longa L. extract