Nitric Oxide, the Gas in the News

Transcription

1 GENERAL ARTCLE Nitric Oxide, the Gas in the News T Ramasarma T Ramasarma is an NS A Senior S~ientist at the Department of Bi~hemistry, ndian nstitute of Scien~e. Nitric Oxide(NO), a small molecule and a water-soluble gas, is formed in traces in several animal tissues. ts arrival on the biological scene was heralded by the magazine, Science as the 'molecule of 1992'. t is biolo.. gically active as a signal molecule in relaxing contracted smooth muscle. Furchgott, gnarro and Murad were awarded the Nobel Prize in Medicine or Physiology in 1998 for discovering this effect. Convergence of three independent lines of investigation led to recognition of vital roles of NO in cellular functionrelaxation of smooth muscle tissue, cytotoxic function in macrophages, and transmission of signals in brain cells. The performance of NO in these three major physiological roles will be briefly described here. t will become obvious that NO can be a saviour, a killer or a messenger. ntroduction Two oxides of nitrogen ~ NzO and NO - were discovered by Humphrey Davy. He inhaled N 2 0 and laughed and doing the same with NO almost killed himself. NO is a toxic molecule. t is long known that addition of salts, nitrate or nitrite, preserves meat, prevents growth of botulinism-producing organisms, and also enhances the red colour of the meat. This is essentially due to binding to biological iron of NO released under these conditions, a crucial property in the mechanism of action ofthis molecule. Dramatic relief from chest pain is obtained in a heart patient when a tablet of an organic nitrate is placed under the tongue. t is now found that this is due to NO formed which induces rapid relaxation of the contracted arterial smooth muscles and restores R -E-S-O-N-A-N-C-E---A-pr-il ~

2 GENERAL ARTCLE nterestingly one of the effective nitrates is trinitroglycerine, the explosive component of dynamite discovered by Alfred Nobel. the blood flow. nterestingly one of the effective nitrates is trinitroglycerine, the explosive component of dynamite discovered by Alfred Nobel. The fortune he made on this was eventually used to institute the Nobel Prizes. Some Characteristics of NO t is amazing that a mere gas NO can achieve a wide range of physiological actions. The high reactivity of NO is due to its unpaired electron. t is a radical with half-life less than 10 sec and can be readily oxidized by 02 to nitrate. t can complex with another radical, superoxide (02-:-)' to form a stable, potent oxidant, peroxynitrate. These two forms can modify proteins and their activities by their oxidation and nitrosylation of some amino acid residues. Water-soluble NO freely diffuses across membranes. As expected of a signal molecule, it can be generated in one cell and show effects in another cell. The basic reaction of NO is to bind to metals. ndeed the deep red colour imparted to meat by NO is due to its complexing with heme-iron of myoglobin, the oxygen-carrying protein of muscle. Basics of Relaxation of Smooth Muscle Smooth muscles are distributed widely and perform a variety of functions such as maintaining blood flow and pressure, moving contents of gastrointestinal tract, and elimination of matter as in labour, urination, defecation and sperm ejection. Contraction and relaxation of smooth muscle are the core of these activities. n general, muscle contraction depends on reversible formation of actin-myosin complex as a cross-bridge, consuming ATP. Excitation due to hormones and other initiator agonists increases calcium concentration, which, along with calmodulin, activates the kinase of myosin light chain (20 kda protein). The phosphorylated myosin light chain is the active form in contraction and its dephosphorylation is supposed to relax the muscle. Activation of guanylate cyclase and increase in cyclic GMP concentration occur during relaxation. Cyclic GMP is presently considered to have a role in relaxing contracted muscle, ~ R-ES-O-N-A-N-C-E--1 -A-p-rll

3 GENERAL ARTCLE although the mechanism is not known. The characteristic of smooth muscle contraction is the step response with successive additions in the medium of stimulus such as noradrenaline. On removing the stimulus this effect is reversed. Relaxation can be obtained on adding an effector such as acetyl choline or some organic nitro-compounds. The two processes are coupled, and an equilibrium between them determines the balance of the developed tension. 'The standard procedure of measuring this is as follows. The aortic tissue of animals was cut in transverse rings (about 5mm wide) and they were suspended with a resting tension (usually 2g weight) in temperature controlled organ bath in physiological salt solution. The rings are connected by silk suture to a transducer, an amplifier and a recorder. The isometric force displacement was recorded after addition of reagents to the medium. The contraction and relaxation processes of the aortic ring are illustrated in Figure 1. Figure 1. Stylized flow diagram of measurement of contraction and developed tension of aortic rings. On the left is a sketch of the arrangement of suspending the aortic ring with a weight and recording the change in its length. A cross-section of the ring is expanded showing the small inner lining of endothelial cells on the large section of muscle cells. On the right is shown the response of developed tension with increasing amounts of an agonist, phenylephrine, and its reversal on washing. ~nclotheljg,l ~ -~~-:~. t muse. cdl t nm Tim~,min pmnylephr~,aaaaaa RESONANCE April 1999 'V V V V V 'V 33

4 GENERAL ARTCLE Figure. 2. The classical experiment of Furchgott on the loss of acetylcholinedependent relaxation on rubbing of the endothelial layer. Notice the control aortic ring contracts with noradrenaline and relaxes with increasing concentration of acetylcholine (solid line). n the ring, which was rubbed with a wooden stick, the acetylcholine response was lost (broken line) and the contracted state can be reversed by washing away the effector. en s.. 0i! Acetylcholine 4 10nM 100nM 1000nM ~ + wash ----T-----~\ 1 2 Rubbed \ ~.s!,, ~ '---- \ \ O-t 1OnM '... Noradren aline Time, min Seminal Discoveries on Smooth Muscle Relaxation The seminal discovery on the process of relaxation of aortic smooth muscle was made by Robert Furchgott in the State University of New York, NY (USA) in Smooth muscle tissue has a top layer of endothelial cells over the inner layer of smooth muscle. n the rings where the endothelial cells were removed by gentle rubbing of the. inside of the ring with a 'wooden applicator stick',the noradrenaline-induced contraction was lost (Figure 2). This experiment showed that the endothelial layer is necessary for the relaxation process induced by acetylcholine. At the same time, the rubbing had no effect on the glycerol trinitrate-dependent aortic relaxation. Acting on the muscarinic receptors of endothelial cells, acetylcholine stimulates release of a factor that causes relaxation. t was called endothelium derived relaxing factor (EDRF). Louis gnarro found during , working at the Tulane University, New Orleans, LA (USA) that EDRF is in fact NO, that NO is generated from glycerol trinitrate in presence ofthiol, and that NO and nitrosothiols can relax contracted smooth muscle as well as activate guanylate cyclase in the arterial tissue. Salvador Moncada at Wellcome Research Laboratories (UK) showed that treatment of rabbit's aorta with bradykinin released NO and this accounted for the biological activity ofedrf that can fully be substituted by NO gas ~ RESONANCE April 1999

5 GENERAL ARTCLE n 1977, Ferid Murad at the University of Virginia, (USA) reported the activation effect of NO on soluble hemoprotein enzyme, guanylate cyclase, in several tissues of the rat and the cow. n the work of this group, Chandra Mittal from ndia participated. They also found high activities of this enzyme in smooth muscle tissues. t is now considered that the product of this enzyme, cyclic GMP, has a role to play in the relaxation process. NO Controls Smooth Muscle Functions ntensive efforts from many laboratories have documented the many facets ofn 0 actions since these early discoveries. t is now clear that the outer layer of endothelial cells produces NO by a complex enzyme, nitric oxide synthase, and this travels to the inner layer of smooth muscle where it activates guanylate cyclase and cyclic GMP produced causes relaxation (Figure 3). On reduction in the cells the organic nitrates produce NO which is then carried by the blood to arteries, and causes relaxation of Ac~tylcholint Figure 3. Schematic representation of synthesis of NO in endothelial cell and its action in muscle cell in reversing the actin-myosin complex during relaxation. -R-ES-O-N-A-N-C-E-----A-p-n-, ~~

6 GENERAL ARTCLE their contracted state, thereby lowering blood pressure. The balance of the two states maintains the blood pressure. ncreased NO gives a bonus benefit of suppressing aggregation of blood platelets. This assists in smooth flow of blood and preventing clots that are dangerous in heart patients. ncreased NO gives a bonus benefit of suppressing aggregation of blood platelets. This assists in smooth flow of blood and preventing clots that are dangerous in heart patients. With the entry of NO on the scene more attention is being given to relaxation process. The stages of synthesis of NO and NOdependent activation of guanylate cyclase, and also the cyclic GMP actions on the actin-myosin complex are soft spots amenable for modulation. Now it is recognized that NO forms the basis of controlling functions of most smooth muscles. An interesting example is provided by Solomon Snyder of Johns Hopkins University (USA) in 1992 on the implication of NO in promoting sexual activity. Relaxation of smooth muscle causes vasodilation and allows blood flow, an essential process in penile erection. t was found in the rat that nitric oxide synthase was localized to penile neurons that innervate corpora cavernosa and to neuronal plexuses in the adventitious layer of penile arteries. The stimulation of this enzyme provides NO that will relax the arterial smooth muscle, allows blood entry and erection of the penis. nhibitors of this enzyme (e.g. N-monomethylarginine, NMMA) prevent this erectile function. On the other hand conditions that retain cyclic GMP in the smooth muscle will keep the tissue in relaxed state. Preventing conversion of cyclic GMP to S'-GMP by inhibitors of the phosphodiesterase can achieve this. Sildenafil is such an inhibitor and is marketed as 'viagra', the blue pill for improved sexual vitality. Cytotoxic Function in Macrophages Another line ofinvestigation provided evidence for an immunoprotective role for NO. t is known that excreted nitrate is higher than is consumed in the food. The amounts excreted increased further during infection and on exposure to endotoxins in animals. t became apparent that the formation of this excess nitrate is related to the enhanced macrophage activity to combat the invading pathogens. Michael Marletta at the University of.aa~aa< 36 v V V V V v RESONANCE April 1999

7 GENERAL ARTCLE Michigan (USA) demonstrated in 1985 that murine macrophages, stimulated by bacterial lipopolysaccharide, produced high amount of nitrate. This occurred only when arginine, an essential amino acid, was present. t was obvious that arginine was the source of nitrogen and it must have gone through intermediate forms of oxidation. n another independent investigation, John Hibbs Jr. of University of Utah (USA) found that the ability of macrophages to kill tumor cells depended on the presence of arginine in the medium and provided evidence for the presence of an enzyme that produced NO from L-arginine by oxidation of its guanidino-nitrogen. Analogues of arginine with substitutions on the guanidino-nitrogen prevented the killing process. t then became known that macrophages use NO thus formed to destroy a variety of invading organisms such as mycobacteria, yeasts, protozoa and helminths. Previous work showed that reactive oxygen species, such as superoxide and H 2 0 2, are also formed and used by macrophages. Putting the two together, it appears that peroxynitrite formed from sup'eroxide and NO, is the active oxidant species. The cytotoxic effect of NO is illustrated in Figure 4. t became apparent that the formation of this excess nitrate is related to the enhanced macrophage activity to combat the invading pathogens. Transmission of Signals in Brain Cells The neurotransmitter potential of NO emerged in another line (lrgi-l~ + "-...\ i-no synthase... NO o~ macrophq~. y.. ONOO peoroxynltrite Figure 4. Schematic representation of formation of NO, 0/ and ONOO leading to killing of pathogen in a macrophage. R -E-S-O-N-A-N-C-E---A-p-rl ~~

8 GENERAL ARTCLE of research. Transmission of signals from one cell to another in the brain is effected through chemicals at a junction point called synapse. The amino acid, glutamate, acts as an excitatory molecule that specifically binds to the receptor of NMDA (Nmethyl-D-aspartate). John Garthwaite of the University of Liverpool (UK) noticed a parallel system in the brain tissue of formation of NO, and through that, activation of guanylate cyclase. The consequent increase in cyclic GMP mediates releases of glutamate in the pre-synaptic terminal. The cell that receives the glutamate signal at the N MDA receptor release calcium which in turn goes on to activate NO-synthase. NO thus produced diffuses back to the presynaptic terminal and acts as a 'retrograde messenger'. This cycle provides the basis for longterm potentiation and for learning and memory (Figure 5). The phenomenon of synthesis of NO in one type of cell and its use in another neighbouring cell is a special feature. Using the Figure 5. Schematic representation of nerve signal transmission. NO releases the neurotransmitter glutamate, in the presynaptic terminal. The NMDA receptoris activated by glutamate in the postsynaptic dendrite, and the released calcium activates NO synthase and NO formed diffuses back to the presynaptic terminal and provides for long- term potentiation ~~ R-E-S-O-N-A-N-C-E---A-pr-il

9 GENERAL ARTCLE specific antibody of NO-synthase it was found that NO synthesis occurs only in some cells in the brain, but not all (e.g. Purkinje cell). This finding helped understanding toxicity of excess monosodium glutamate in Chinese food syndrome. A resistant subpopulation of brain cells stained by tetrazolium salts, indicating the presence of activity of the enzyme, NAD(P)H diaphorase, produce NO. And this causes death to neighbouring cells in ischemic brain injury that also involves reactive oxygen species and therefore peroxynitrite. t is possible such a process of loss of brain cells is involved in Parkinson, Huntington and Alzheimer diseases. mplied in this is the possibility of control of damage under these conditions by regulating the synthesis of NO by NO-synthase. The Enzyme that Makes NO The enzyme NO-synthase, or more specifically 'L-arginine, N AD PH: oxidoreductase, NO forming', is a complex one for the apparent simple oxidation of 'guanidino-nitrogen of arginine yielding citrulline and NO. Besides arginine, NADPH and oxygen the reaction requires four cofactors - bound Fe, FAD, FMN and tetrahydrobiopterin. t has spectral properties similar to cytochrome P450 and is homologous to cytochrome P450 reductase. Calmodulin, the calcium-binding protein, is often associated as an integral component of NO synthase and is necessary for activation of the enzyme on arrival of calcium. Three isoforms of the enzyme are characterized and labelled cor n (cerebral, neuronal), e (endothelial, cardiovascular), i (macrophage, immune-responsive), with high molecular mass of 160, 133 and 130 KDa, respectively. The phenomenon of synthesis of NO in one type of cell and its use in another neighbouring cell is a special feature. The reaction occurs in two stages of oxidation of arginine to N hydroxyarginine and then to citrulline releasing NO. ts use of 1.5 moles each ofnadh and O 2 is unusual in itself in addition to undefined roles of many cofactors intrinsic in the protein, as well as others needed. The reaction sequence, shown in Figure 6, indicates the two dioxygen-d'ependent oxidations (reactions a and b) of = NH to NO and its release using the reducing power of -RE-S-O-N-A-N-c-e-- -A-pr-il ~

10 GENERAL ARTCLE L-Arginine l-cilruui,. Figure 6. The reactions nvolved in oxidation of L-arginine and generation of NO by NO-synthase.: (a) primary oxidation of guanidino-nltrogen by active-iron of the enzyme; (b) secondary oxidation of NG-hydroxy group releasing NO which needs tetrahydrobiopterin; (c) regeneration of reduced form of iron by NADPH; (e) reduction of quinonoid biopterin. FAD and FMN are involved in reactions band d in some unspecified manner. NADPH through active-iron species and biopterin (reactions c and d). Molecular Basis of NO Actions As a refrain it can be stated that the capability of NO to bind to iron in heme or Fe-S proteins forms the basis ofits many actions. The significant physiological action of NO is the activation of the soluble hemoprotein, guanylate cyclase that acts on GTP and produces the active cyclic GMP. The same binding of NO to heme-iron of myoglobin happens in curing meat and enhancing its colour. This remarkably high binding capacity of NO with hemoproteins also gives toxic effects. ts presence as an atmospheric pollutant leads to some of the respiratory problems. Nitrosylation of protein-thiols yields modified proteins, while providing' long-lived reservoirs ofbioactive NO'. n the process some activities of enzymes can be modulated. The enzyme glyceraldehyde-3-phosphate dehydrogenase, the first step of oxidation leading to A TP formation in glycolysis, is inactivated by NO-generating compounds. As a secondary response a small ~ RESONANCE April 1999

11 GENERAL ARTCLE fraction of the modified protein self-adp-ribosylates its own active thiol group. This effect of NO represents a more significant action in creating conformational change in proteins conferring a 'gain in function'. A more general action of NO is to deplete protein-bound nonheme iron. This effect explains loss of essential activities in the cell such as NADH- and succinate-dependent ubiquinone reductases, cis-aconitase and ribonucleotide reductase. Moreover NO releases ferritin-bound iron and thereby promotes lipid peroxidation and consequent damage to cell membranes. As NO hits so many reactions, its overproduction can be toxic. A convergence of multiple approaches in the research on NO helped in explaining the age-old precepts and practices in terms of new concepts and understanding of control of smooth muscle action, toxicity to cells and information transfer between cells. Amazingly these are achieved by NO, a small molecule and a gas, but elaborated with a combination of multiple cofactors and complex set of proteins. Suggested Reading [1] R F Furchgott and P M Van Boutte, Endothelium derived relaxing and contracting factors,faseb J.,3, ,1989. [2] S Moncada, R M J Palmer and E A Biggs, Biosynthesis of nitric oxide from L-arginine, Biochem. Pharmacol., 38, , [3] J Garthwaite, Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends in Neurosciences, 14,60-67,1991. [4] S H Snyder and D S Bredt, Biological roles of nitric oxide, Sci. Amer ,1992. [5] C Nathan, Nitric oxide as a secretory product of mam,malian cells, FASEB J. 6, ,1992. Address for Correspondence T Ramasarma Dept. of Biochemistry ndian nstitute of Science Bangalore , ndia. trsobiochem.lisc.emet.in Arnold Sommerfeld about Wolfgang Pauli: " can't teach him anything; at my suggestion he is writing a summary of Einstein's relativity theory". R -E-S-O-N-A-N-C-E---A-p-rl ~ '