ABSTRACT
Nitric oxide (NO) along with Carbon monoxide (CO) and Hydrogen Sulphide (H2S) are biologically significant gaseous molecules generally called as “gasotransmitters”. At a concentration higher or lower than optimum value may result in toxicity or malfunctioning of mammalian tissues. Soon after the acknowledgment of NO as multifunctional bio-signalling molecule in 1987, many interesting implications of this field emerged out. Meanwhile, several studies have proven the NO-biosynthetic pathway responsible for normal functioning of eye. High intraocular pressure (IOP) has been suggested as the main risk factor in this context and collaborative approach with nitric oxide releasers is said to control IOP and hence the relation with glaucoma. Similar miracles were reflected from several other naturally produced gaseous molecules,viz ., CO and H2S after year 1990. The biological roles of both these molecules are now widely accepted and in the current era investigations focused mainly with development of efficient CO and H2S releasing compounds. CO and H2S donors are also said to help in normalising IOP like NO. Therefore the trio-gasotransmitters have collective relation with the ophthalmic homeostasis in association with nervous control. On one hand, the antimicrobial efficiency of these three molecules is widely known and on the other hand, their collaborative key-role in ocular nerve functioning makes it remarkable to state here that their donors are supposed to act as a shield for both the infectious as well as the non-infectious eye defects.
Keywords: Gasotransmitters; Ophthalmic diseases; NO; CO; H2S, NORMs, CORMs, H2S-donors.
Introduction
The scientific recognition of carbon monoxide (CO) and hydrogen sulphide (H2S) as bio-conjugated molecules sharing similar functional role as nitric oxide (NO) resultedin coining the term “gasotransmitters” for these molecules based on size, lipophilic character,half-life and several other features [1, 2].Even though these gases share a number of common features, they also possess dissimilar characteristics and display noteworthy interactions, which complicate the interpretation of their physiological activities.
In the late 1990’s, the scientific community saw a very unusual phenomenon, the conversion of nitric oxide from harmful gases into an important chemical messenger. The remarkable role of this molecule in signal transduction and cytotoxicity is considered to be one of the greatest marvels of biological chemistry in recent times. The biological diversity of this molecule is well documented in neuroscience, physiology and immunology [3,4]. Recommendations obtained from the chemical stress of nitric oxide included the ”Molecule of the Year” vote in 1992 by the journal ”Science”, published by the American Association for the Advancement of Science (AAAS) [5]. At present, NO has been accepted to be linked to many physiological mechanisms including platelet aggregation and adhesion, neurotransmission, synaptic plasticity, vascular permeability, hepatic metabolism, senescence, and renal function [6-9]. In high concentration (μM), NO also plays a strategic role in the immune system [10] and in suppression of carcinogenic state [11, 12]. The prominence in the absence of biology and medicine was emphasized in 1998 when the Nobel Prize for Physiology and Medicine was awarded to Robert Furchgott, Louis Ignarro and Ferid Murad for their role in transforming the role of NO in the nervous, cardiovascular and physiological systems. In microbial world NO plays mediator role in denitrification [13-15] (Figure 1 ). In addition, the molecule is tailored in various ways due to its important therapeutic potential [16,17].
NO2 NO N2O N2
Figure 1 . Denitrification involving NO as intermediate
Carbon monoxide (CO) has long been known as a dangerous gas for mammals and is called as a “silent killer” [18]. Carbon monoxide, when inhaled enters the bloodstream, formscarboxyhaemoglobin (COHb) at a rate 240 times greater than oxygen [19]. This reduces the oxygen transport ability and results in hypoxia [20]. Biologically, CO is considered as a by-product of heme oxygenase (HO) metabolism [21], and in the early stage of its biological exploration, CO was found as a chronic neurotransmitting agent [22]. Therefore, the further studies have altered the general perception of CO as a harmful molecule[23]. CO has now become an important molecule in the physical monitoring of many organ systems. In the last few decades, investigations related to CO have shown this gaseous molecule as a major chemical messenger.
In the seventeenth century, Carl Wilhelm Scheele recognized H2S through chemical analysis. However, it has long been speculated that the gas is derived from the sewage system, and is linked to a series of special eye diseases that occur in sewage workers. The disease is associated with severe inflammation, secondary bacterial attacks and even blindness [24].Similar to NO and CO, internally generated H2S is now considered a significant gasotransmitter [25] and within a neuromodulator, which draws a lot of attention in literature. Traditional neurotransmitters bind to and activate membrane receptors, whereas gasotransmitters are able to freely distribute to adjacent cells and directly bind to their target proteins to supplement biological functions by contributing to their short-term mutations [26].
Like NO which S-nitrosylates a variety of proteins, H2S physically regulates the different protein functions by S-sulfhydration. However, S-nitrosylation inhibits enzymes, while S-sulfhydration stimulates them. Therefore, H2S is an important physiologic gasotransmitter such as NO and CO [27-30].
The eye is one of the most sensitive parts of the brain. Any impairment in eye function requires high quality care. Among eye health problems intraocular pressure (IOP), cataract and retinal hypertension continue to remain as potential risk factors in treatment. Due to our growing interest in the synthesis of various chemicals tagged with NO, CO and H2S [31-39] and the many biological actions of H2S and the more precise delivery of this flexible gas to target tissue in the form of H2S sponsors [40], the current chapter focuses on in the applied interest of NO, CO and H2S-compounds on eye-physiology. A historical view of the emergence of the term ”gasotransmitter”, within the production of NO, CO and H2Sin mammals, and to sought strong sponsors of NORMS, CORMS and H2S-releasers (in the event of chronic biosynthesis and digestion) applicable in the most common eye-defects are the main objectives of thisliterature update.
A Meaningful Introduction Towards “Gasotransmitters”
In general, gasotransmitters refer to the distinctive class of molecules like NO, CO and H2S, responsible for communication amongstbody cells for a particular biological action. Albeit, these molecules exist in solvated form while in biological medium, the respectivedifferences in size, action, shape and bio-membrane interactions stems their multitude biological roles reported so far. The signal transduction pathway among such carriers may range from short to long distances to transmit the required information[41]. The properties and functional diversity found in these bioessential signalling molecules, therefore gave rise to coin a new term in reference to their biological relevanceas “gasotransmitters”.
Several parameters may be found differentiating neurotransmitters from gasotransmitters. From the cellular biology it is clear that neurotransmitters stored in vesicles get released by the intervention of a suitable stimulus (Figure 2 , top). These responses are receptor-specific in nature and depend on the molecular signalling to bring forth a physiological move. Hence, synaptic vesicles behave as a reservoir of information required at the time of safety or normal physiological functioning. Whereas, gasotransmittersare endogenously availedsmall molecules of signalling potentiality (“gaso” refers to their gaseous nature under normal conditions) [41, 42]. Gasotransmitters have the main characteristic feature of diffusing through cellular membrane without the aid of any receptor (Figure 2 , bottom). No reservoir is required (like vesicles in neurotransmitters), but are rapidly produced in response to a stimulus when needed[1, 2]. Moreover, here in gasotransmitters cell exocytosis fashion followed in neurotransmitters is not pronounced at al.Therefore a separate term ‘gasotransmitter’ coined by Wang in 2002 is suitable to distinguish them from neurotransmitters [25].The vasorelaxant and gasotransmitter labelling of NO enhanced scientific vigour to an extraordinary fashion and activated the seek for other molecules of this class [43,44, 25]. [44]. Gases other than these are also under interrogation to add further possible members to this group.
Figure 2. Diagram showing the mechanism of neurotransmitter (2A, top) and gasotransmitter (2B, bottom)action.
Biosynthesisand target of NO, CO and H2S
Biological synthesis and target of NO
NOis biologically synthesized by the catalytic action (oxidation of the nitrogen coloured red in Figure 3 ) ofnitric oxide synthase (NOS) over L-arginine as substrate and resulting in the formation of L-citrulline [45]as shown in Figure 3 . Since, the NO production is involved in every system of a human body, there are three distinctive gene products and isoforms of NOS, producing NO in the presence of oxygen, flavins and NADPH [46]. Figure 4(i) andFigure 4(ii) represent these three members as NOS-I, NOS-II and NOS-II. These are also called as eNOS (endothelial), iNOS (inducible) and nNOS (neural), respectively, because of the specific target/production locus during their biosynthesis.NOS-I is central and peripheral nervous system linked [47], NOS-III is vaso-relaxationconnected [46] and NOS-II is immunological directed. From the literature it is found that NOS-II functions independent of the presence of calcium, and is a source of considerable amount of NO produced for a longer duration as compared to NOS-I and NOS-III [48].Several examples L-arginine structural analogues have been found to prevent NOS from producing NO,viz ., NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine (LNA), NG-nitro-L-arginine methyl ester (L-NAME), etc. [49]. However, by making instant and sufficient arginine availability, the action could get reversed initially.
Figure3. Reaction showing thebiosynthesis of nitric oxide
Figure4. Interaction of three NOS isoforms
Biological Production and Target of COAs per the metabolic pathways concerned with the CO-biosynthesis, almost 14% of 500 μmol/day is obtained from lipid peroxidation and from photooxidation plus self-activation of cytochrome p-450. Bacteria and Xenobiotics also contribute the same minor percentage [67,68]. Major contribution(almost 86%) is generated by the erythrocyte-breakdown, wherein, the haem-oxygenase (HO) catalyzes this oxidation. Like NOS, HO also exists in two isoforms,viz, HO-1 and HO-2. These are alsocalled as inducible and constitutive,respectively. Both the isoforms show same rate-limiting step while catabolizing heme, the difference lies with the regulation, amino acid sequence, and distribution in the tissues. Another HO has been recently identified and named as HO-3. This form of HO was detected in the several organs of rats. Till date no haem-degradation study has been reported for this newly detected HO-member [69].The metabolic pathwayof HO-catalyzed haem oxidation involves several important stages as has been illustrated in Figure 5 . In addition to CO other intermediatory products like of α-meso-hydroxyheme, verdoheme, biliverdin (converts to bilirubin as excretory product conjugated by glucoronic acid shown inFigure 6 ) are also involved. [70,71].The bioaction of HO-1 under stressful situation gets enhanced and the CO-production gets increased than the optimal value [72]. Therefore, suchan elevation in the concentration can be used as a sign convention medically to read the associated behaviour. The similar correlation has been found in several diseases wherein a patient is expected to suffer from stress and strain conditions.For instance in bronchiectasis,asthma, cystic fibrosis, hyperglycemia and other diseases CO level appears higher than the normal [73]. Hence, the detection level of CO because of inducible HO-1 can help in diagnosis of pathophysiological state.
Figure 5. Oxidation of heme by heme oxygenase (HO)forming CO as a by-product.
Figure 6. Chemical structure ofGlucuronic acid
CO in mammals has been found to show target specific action in two ways,viz. ,soluble guanylyl cyclase (sGC) pathway and Non-cGMP pathways. It is well established fact that NO binds with heme by replacing one histidine unit to result in the activation of soluble guanylyl cyclase (sGC) [74]. Generally NO binds to heme b to formaunstable short lived six-coordinate systemto finally cleave the heme-His105 bond, and forms a five coordinated heme complex with NO (Figure 7 ). Therefore, it is conformational change in other words that leadsto sGC activation [75].The resulting sGC catalyzes the conversion of GTP to cGMP in the fashion as given inScheme-1. The cGMP formation is related to a number of sequential pathways entailed with several clinical implications asillustrated inScheme-2 (red coloured).
Figure.7 . Diagram showing the activation of guanylyl cyclase by NO and CO/YC-1.[Taken from Ref. E. Martin, K. Czarnecki, V. Jayaraman, F. Murad and
J. Kincaid, J. Am. Chem. Soc., 127(2005) 4625-4631]
Scheme-1: sGC catalyzing the Conversion of GTP tocGMP
Scheme-2: Cyclic guanosine monophosphate (cGMP)routes ameliorating medical conditions shown as red.cGKs = cGMP-dependent protein kinases, Iα and Iβ; IRAG =Inositol 1,4,5-triphosphate(IP3 )receptor-associated cGKIβ substrate; VASP = vasodilator-stimulated phosphoprotein; PDEs =phosphodiesterases; cAMP = cyclic adenosine monophosphate [Adopted from Ref. Brian E. Mann and Roberto Motterlini, Chem. Commun., (2007) 4197-4208]
Therefore, in an analogous way like NO, this molecule (CO) alsoyields cGMP by activating guanylyl cyclase. However, it may be mentioned here that this activation is only 1/80theffective as NO. Some trials have been found to use synthetic compounds like YC-1 (Figure 8 ), when used in combination with CO can increase this effectiveness up to the level of NO. This indicates that there should be some naturally existing signalling molecule like YC-1 that shows the guanyl cyclase activation similar to NO.There are contrary observations reported for CO-activity in the same activation study. CO while coordinating withguanylyl cyclase results in six-coordinated iron complex and central metal ion continues to remain as FeII. The retention of five-coordinated inability of CO with iron is due to the increased radii of the ion anddecrease in the electronegativity. This shows CO-based vasorelaxation goes through a no similar pathway as in NO. On the other hand, considering YC-1 interaction with the displacement of His-105 produces a 5-coordinate complex with retaining CO as co-ligand [79](Figure8 ),there is another interpretation for the CO-dependent stimulation of sGC [80].
Figure 8. Structure of YC-1:1-Benzyl-3-(5′-hydroxymethyl-2furyl)indazole
Non-cGMP CO-pathway is remarkably in other targets to lead vasorelaxation, incorporating the involvement of potassium channels (BKCa). The conductance BKCachannels are found distributed almost in everytissue, and get affected byseveral modulators. Among contributing factors towards such channels, protein kinases, endogenous NO and CO along with heme are mentionable. Many physiological processes including neuronal excitability, contractility of muscles, and vascular tone maintenance are because of BKCa activity by involving the suitable yield of action potential [81].
Biosynthesis and target sites of H2S
H2S is considered as the recently recognized third member among gasotransmitters. This gas is also produced endogenously through enzymatic reactions (Figure 9 ) and is responsible for maintaining physiological balance.Recently, non-enzymatic pathways have also been reported to be responsible for biosynthesis of hydrogen sulphide. The optimal concentration of H2S under normal conditions is said to be present in micromoles (µM) [82].L-cysteine is the important bioessential source of this gas involving the role of cystathionine β-synthase (CBS), cytosolic/pyridoxal-5′-phosphate (P5P)-dependent enzymes, and cystathionineγ-lyase (CSE)[83,84] or the tandem enzymes, cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulphur transferase (3-MST), mainly confining the activity in mitochondria. Absolute H2S is produced from CSE and CBS, whereas 3-mercaptopyruvate transfers sulphur to cysteine through the catalytic application of 3-MST resulting in the formation of persulfide [85, 86]. Thereafter, persulfide acts as a H2S-releaser endogenouslycausing the reduction of disulfides,e.g, reduction of dihydrolipoic acid (DHLA) or thioredoxin(Trx) under physiological terms [87]. Strictly, therefore it is thioredoxin reductase (TR) acting sequence wise in combination with glutathione (GSH) and sulfur oxidase (SO) in this phenomenon. The significance of H2S (oxidized form) in respiratory chain is well documented acting as electron donor in Q,III and IV steps of the chain that results in the generation of energy currency (ATP) and cellular oxygenconsumption. Hence, biosynthesis and H2S-mediated sensing of oxygen represent essential role of H2S-biochemistry.
Production of H2S in tissues via cystosolic enzymes CBS and CSE
(ii) (a) Generation of H2S via tandem enzymes, CAT and (3-MST) and(b)
H2S oxidation in the mitochondria
Figure 9. Biosynthesis of H2S and its oxidation [Adopted from Ref. K. R. Olsona, J. A. Donald, R. A. Dombkowski and S. F. Perry, Respiratory Physiology & Neurobiology ,184 (2012) 117– 129]
The biological synthesis of H2S as in the case of NO and CO, must be followed by the consumption or target phenomenon. This gas has no colour, flammable and smellslike that of rotten eggs.Itsacid strengthis weak (pKa value of 6.98 at 25 °C and 6.76 at 37 °C). The dissociation of this gas in water may be represented as shown in Scheme-3 (Ka1=1.3×10−7 M, Ka2 =1×10-19) [39]. The H2S that remains undissociated is volatile, while the dissociated form HS is not volatile. In physiological medium these dissociation patterns are pH-dependant.At the 7.4 pH, one-third of H2S remains udissociated. Physiological pH does not support the substantial presence of S2−(because high pH is required for it). As per another scientific observation H2Smainly persists in HS-form(82%) because of having weak acid strength (pKa1: 6.76; pKa2: 19.6)[97].
Scheme-3. Reactions showing the dissociation of H2S in aqueous solution
Therefore, both the H2S as well as SHare contributoryunder biological activity of hydrogen sulfide, despite the fact that SH, represents more nucleophilic potential than cysteine (Cys) or reduced form of glutathione (GSH), thatswiftly coordinates with bio-metallic centres or interacts with othercompounds[98].In mitochondrial chain reactions, H2Sdisplays sequential oxidation trend. Initially, it gets oxidized to thiosulfate, followedby conversion to sulfite and subsequently to sulfate. The first oxidation stage isnon-enzymatic in nature, while the rest steps are carried out enzymatically using thiosulfate cyanide sulfurtransferase (TST). Despite the observation showing sulfate as major end product of the metabolic pathways followed by H2S, urinary thiosulfate represents a non-specific indicator to sense thequantity of H2S production within a body [99]. Overall, the target of H2S is highly influenced by several factors especially the rapid oxidation disfavouring the long-distance transport. Therefore the development of efficient storage system endogenously applicable is suggested.For instance,in NO-association,H2S gets stored as nitrosothiols(RS-NO) implying the dual gas-combinatory fashion. This opens several areas of interest to seek answer for the unexplored queries regarding the isolated and combined gasotransmitters research.Meanwhile, the solvated fashion of H2S reveals different solubility trend based on the nature of the solvent. Due to lipophilic feature of this gas, H2Seasily crosses cell membrane. The tested solubility experiments have shown itto be five times moresoluble in lipophilic solvent as compared to water. The data furnished from its solubility behaviour in various solvents other than water have been keenly recorded [100, 101].The solution chemistry on further exploration depicts that concentration H2S under varied physiological abnormalities within mammalian blood. Harmful implications of H2S at the level of <100 ppm gets expressed in the formsore throat, eye irritation, dizziness, etc [102–104].The exposure at greater than 1000 ppm affects CNS, respiratory chain and may more even cause death[104].
Gasotransmitters in the Mission of Vision (Eye-Health Contribution)Eye is one of the most important sense organs performing the function of vision through interacting with light, involving several physicochemical phenomena to memorize the surroundings, and therefore acts as a natural perception mediator to translate the observations to the brain. So, ultimately light-phenomenon to nerve actions, so many tissues collaborate to let such a complex process to happen. The physiology of eye is not restricted to a simple conduction process only, but is subjected to the role of the gasotransmitters introducedvide supra . This section details the role of NO, CO and H2S in maintaining a healthy eye, so is the title established as “the aim of gasotransmitters in the mission of vision”.
NO News is Good News for Eyes: NO Donors for the Treatment of Eye Diseases
Since the dawn of NO-recognition as a key signalling molecule, the diversified biological role of this free radical met with its extended role in so many areas of physiological investigations. Keeping in view the combinatory functional status of NO and cGMP are entailed with a range of biological actions, there are numerous evidencessupporting the fact that the NO-metabolic pathways are also involved in the normal functioning of an eye.The respective neurological role served as a motivational move for the researchers to find the possible responsiveness while studyingeye functioning. These responsible roles include dynamics of aqueous humour (AqH) dynamics, retinal neurotransmission and other light induced pathways. Any malfunctioning that results in the respective NO-generation can cause eye abnormality [48]. As of now, the normal tissue functioning of eye in concern with eNOS and nNOSbasedrolehas got wide acceptance [105]. This is because neural and immunologic expressive forms of NOS have been found in retina. Several reports are evidential in supporting nNOSresponsible in photoreception via NO-generation, and similar effect in bipo­lar cells. This in turn leads to stimulus for guanylate cyclase photosensitive rod cells and thereby increasing the calcium channel activation. By stopping NOS action in the retina of cats, results indicate impairment in photo-transduction [106]. Moreover, iNOS has also been found responsible for keeping normal phagocytosis in the outer section of retina. Also, the role that NO plays in maintaining circulation of retina, links the molecule with ophthalmologic role.
The optimal concentration as discussed in the biosynthesis is always mainly eyed to confirm the normal functioning of any tissue. At the concentrations other than optimum value results in so many eye diseases [105]. In case of low NO-concentration (eNOS or nNOS abnormal functioning) substances that could act as NO-donors could be administered. On the other side, iNOS as pointed in the above sections isonly intervening in pathological conditions expressive in terms of several cytokines (interleukin-1, interleukin-6, etc), inflammation and endotoxins. Once initiated, iNOS continues to produce sufficient NO followed by conversion phenomenon free radicals, nitrogen dioxide or nitrites as defensive way against pathogens.In hyperactivity of iNOS in several disorders like cataracts, age-related macular degeneration (AMD), myopia and uveitis, iNOS inhibitioncould be suggested.
As is widely known that blood pressure plays important role in keeping normal eye-sight, and on the other hand NO is also considered as blood pressure regulator. Under such a stemming fact, some recent studies have foundeffectivemutual relationof NO with hypertension based cataracts. The increase in lens nitrite (as NO metabolite) is suggested as one of the key regulator for toincrease oxidative stress of lenticular part and hypertensive cataract formation [107]. Thus, NOinvolvement in retinal action continues to be of significant interest [108]. Let us specify this role by illustrating the role of NO in eye defects as elaborated below figures (Figure 10−figure 13 ): [Adopted from L. K. Wareham1, E. S. Buys and R. M. Sappington, The Nitric Oxide-Guanylate Cyclase Pathway and Glaucoma, Nitric Oxide , 77(2018) 75–87, Ref. 111].