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P. K.

Gupta

Cell signaling is such an important area of current research in biology, that the 1998
Nobel Prize in Medicine is the third Nobel Prize that has been awarded in this area of
research during the present decade. Earlier in 1992, the Nobel Prize for Physiology or
Medicine was awarded to Edwin G. Krebs and Edmond H. Fischer for their pioneering
work on reversible phosphorylation
as a biological regulatory mecha-nism; the 1994 Nobel Prize in Medicine was awarded
to Martin Rodbell and Arthur G. Gilman for their work on the role of G proteins in signal
transduction, and this year, the 1998 Nobel Prize for Medicine has been awarded to the
following three US scientists for their work concerning nitric oxide as a signaling
molecule in cardiovascular system: (1) Robert F. Furchgott of the State University of
New York in Brooklyn, (2) Louis J. Ignarro of the University of California-Los Angeles
and, (3) Ferid Murad of the University of Texas Medical School in Houston.

Cell signaling is a phenomenon that is ubiquitous and is an essential requirement in
multicellular organisms for intercellular and intracellular communications. Many
proteins and non-proteinaceous molecules have been identified, that take part in this
cell signaling. These molecules include receptor molecules and a variety of other
signaling molecules. The receptors are found on the cell surface (cell surface receptors)
or inside the cells (intracellular receptors) and recognize the extracellular and other
signaling molecules secreted by the cells. Hundreds of different kinds of cell signaling
molecules have been recognized which include proteins, small peptides, amino acids,
nucleotides, steroids, retinoids, fatty acid derivatives and even dissolved gases like
nitric oxide (NO) and carbon monoxide (CO). Most of these signaling molecules are
secreted by the signaling cell by exocytosis using vesicular transport and make use of
cell surface receptors for cell signaling. However, some of these signaling molecules like
NO diffuse through the plasma membrane and do not need either the vesicles for
secretion from the signaling cell or any cell surface receptors in the target cells for
triggering the signal. NO acts only locally, due to its short half life of about 5–10 seconds
in the extracellular space, where it is converted to nitrates by oxygen and water. As a
signaling molecule, NO is also a gaseous neuro-transmitter, which diffuses through the
plasma membrane at the presynaptic termini, into the synaptic cleft without the aid of
synaptic vesicles. NO has been found to play a key role in a variety of functions including
the following1: (i) dilation of blood vessels (vasodilation), correcting the inadequate
blood flow to the heart muscle, thus controlling the blood pressure; (ii) control of
bacterial and viral infection, through macrophages and neutrophils, as also evident from
the abnormality in the production of NO, reported in patients suffering with
atherosclerosis, diabetes and hypertension; (iii) neurotransmission involving biological
functions ranging from learning and memory to peristalsis and penile erection. When
produced in excess, it may also lead to neurotoxicity including septic shock.

NO as a paracrine signaling molecule for vasodilation

In late 1970s and 1980s F. Murad and L. J. Ignarro demonstrated for the first time the
role of NO in vasodilation (dilation of blood vessels), caused due to the formation of
cyclic GMP (cGMP) through activation of the enzyme, guanyl cyclase (GC)2–5.
Biologically, nitric oxide (NO) was first characterized as an ‘endothelial derived relaxing
factor (EDRF)’, which is released from the endothelial cells of blood vessels due to

Therefore. is lost in the blood vessels stripped of endothelium. NO mediates a large number of biological functions ranging from learning and memory to peristalsis and penile erection. This EDRF. brain extracts and slices. is converted to NO. where it can influence the formation of cyclic guanosine 3¢ . This change enhances the activity of guanyl cyclase (Figure 1). highest concentration of cGMP occurs in the cerebellum. bradykinin and ATP. Further. and corpus striatum). Vasodilation may also be influenced by the formation of NO in adventitial neurons in the walls of blood vessels or in the skeletal muscle (see later for some details). stimulates protein phosphorylation by cGMP dependent protein kinase. In brain also. Carbon monoxide may be one such other component involved in this relaxation. because in many vessels a substantial component of vessel relaxation is unaffected by NOS inhibitors. The formation of cGMP is achieved through activation of the enzyme. This is achieved by N-methyl-D aspartate (NMDA) receptor. etc. nitric oxide is not the only EDRF. increases the level of cGMP to ten times. bringing about a conformational change. suggesting the role of NO in this process.g.8. which relaxes the blood vessels in the heart. NO as a neurotransmitter NO has also been identified as a neurotransmitter (in addition to its role as a paracrine signaling molecule) in the brain and peripheral autonomic nervous system. is also responsible for NDP histochemical activity. Such a role of NO was also demonstrated with the help of cerebellar neuronal cultures. and the purified brain NOS has been shown to have NDP activity. As a neurotransmitter. where glutamate. NO acts as a messenger. while in other areas it is present only in 1–2% of the neurons (e.5¢ -monophosphate (cGMP).g. Possible functions of NO as neurotransmitter in the brain are also suggested by variations in the localization of NOS in the brain. Furchgott and his associates also demonstrated that the stimulatory effect of acetylcholine. basket and granule cells of cerebellum). distinct forms of NO synthases (NOSs) encoded in different genes are utilized. thus increasing the blood flow to the heart muscle. thus demonstrating the role of endothelium in release of nitric oxide (or EDRF)6. . it has been suggested that NOS catalytic activity requiring NADPH. later identified to be NO. In some areas of brain.stimulation by compounds like acetylcholine. which has been used for many years to treat patients with angina (pain due to inadequate blood flow to heart muscle). cerebral cortex. However. Cyclic GMP (cGMP) thus formed. which binds with very high affinity to iron in the heme group of guanyl cyclase. For instance. The nitroglycerine used for treatment. In the brain. hippocampus. guanyl cyclase (GC) by NO. the major neurotransmitter in the brain. which is a glutamate receptor7. leading to regulation of muscle relaxation. which were shown to release a factor with properties resembling NO. was found to function as a paracrine regulator causing vasodilation. For each of these sites of NO formation. it resides in all neurons (e. NO released by autonomic nerves in the penis. causes the local blood vessels to dilate resulting in enhanced blood supply leading to penile erection. the distribution of NOS neurons often matches that of the neurons staining for NADPH-diaphorase (NDP). The effect of NO in controlling blood pressure also provides an explanation for the mechanism of the action of nitroglycerine. Selective inhibition of NOS blocks NMDA-induced elevation of cGMP.

was found to block the tumoricidal and bactericidal action of macrophages1. such as septic shock (see later). When macrophages are activated by endotoxin in the form of lipopolysaccharides (LPS) in bacterial cell wall. Role of NO in checking infection NO serves as a messenger molecule. NBT. calmodulin. hours to weeks. LTP has attracted the attention of many. either due to removal of the substrate (arginine) or due to inhibitors of NOS. N-methyl-D-aspartate receptor. Hb. NO has been shown to work as a retrograde messenger for LTP. when macrophages and neutrophils exert their tumoricidal and bactericidal effects by killing the invading microorganisms and by blocking viral replication. inflammation response is elicited. thus functioning as a retrograde messenger in LTP induction9. This suggested that NO is produced in post- synaptic neurons and is diffused back to presynaptic terminals. CAM. suggesting that NO inhibits virus multiplication. superoxide free radical. This re-sponse involves activation of the enzyme NOS. Consequently. factor- a and interleukin-1. which transforms arginine into NO and citrulline. 8). NMDA-R. Any inhibition of the formation of NO. nitroblue tetrazolium. It is experimentally induced by synaptic stimulation and is implicated in memory formation in hippocampus in the brain. and the NOS inhibitors elevated such titers. spreading to the neighbouring synapses. hydroxide free radical. haemoglobin (redrawn from ref. . A model for the action of nitric oxide in neurons. macNOS also mediates pathogenic condition. as an aid to formation of new memories.NO as a retrograde messenger in long term potentiation Long term potentiation (LTP) is an enhancement of synaptic efficacy lasting Figure 1. since NOS inhibition when applied to post-synaptic neurons could inhibit induction of LTP. Addition of NOS in cultured cells also lowered viral titers. MacNOS (also called iNOS) activity is also induced by cytokines like interferon-g .

using either the DNA probes/primers representing the conserved sequences or through the use of antibodies1. FMN. including the bronchial tree and neurons in the brain.Excess levels of NO are neurotoxic Although NO participates in the normal synaptic transmission. themselves remain resistant to this neurotoxicity. although later each of these three NOSs were found to be located elsewhere also. Synthesis of NO using NOS Nitric oxide is synthesized by deamination of the amino acid arginine with the help of NOS. They also show close homology with cytochrome P450 reductase (CPR) having consensus sequences for the binding of NADPH. NOS also makes use of some cofactors including NADPH. chondrocytes. a model for learning and memory. since they suggest the location of the NOS. be misleading. thus mediating neurotoxicity. endothelial cells and fibroblasts. initially detected in macrophages and found to be inducible by substances like interferon g and lipopolysaccharide (LPS). NOS oxidizes the guanidine group of L-arginine in a process that consumes five electrons and results in the formation of NO with L-citrulline. but also occurs in the epithelium of several tissues. and (iii) endothelial NOS (eNOS or NOS-3). Structure of NOS and molecular cloning Molecular cloning of cDNA belonging to all the three forms of NOS has been successfully achieved in recent years. excess levels of NO are neurotoxic. It has been shown that DNA damage is central to NO neurotoxicity and a detailed mechanism for the same has been outlined1. where it is involved in long-term potentiation (LTP). Finally. which include the following10: (i) neuronal NOS (n-NOS or NOS-1). initially detected in brain. Arginine ® hydroxy-arginine ® L-citrulline + NO. Glutamate released in excess acts at NMDA receptors. FAD and FMN (Figure 2). although NOS neurons. Treatment with NOS inhibitors or the removal of arginine from the medium or scavenging NO with hemoglobin eliminates this neurotoxicity. but also in skeletal muscle. The close . nNOS occurs not only in the neurons. This activity of NOS resembles the activity of cytochrome P450 reductase (CPR). confirming the role of excess NO in this phenomenon. (ii) macrophage or inducible NOS (macNOS/iNOS or NOS-2). however. The names of NOSs can. All the three enzymes have also been shown to have binding sites for the cofactors FAD. iNOS occurs not only in macrophages. Like CPR. where it is complexed with dystrophin. eNOS is not only restricted to endothelium of blood vessels. Three forms of NOS There are three forms of NOS. its absence causes Duchenne’s muscular dystrophy. causing septic shock. Similarly. For instance. FAD and FMN (see later for some details). but in several other cell types including hepatocytes. which catalyses a series of detoxification reactions in the hepatocytes1. The enzyme varies in length from 133 kDa in iNOS or macNOS to 160 kDa in nNOS. This neurotoxicity is believed to be due to high levels of NO produced due to stimulation through NMDA receptor. NADPH and calmodulin.

acetylcholine acts at acetylcholine receptors on endothelial cells and activates phosphoinositide cycle to generate Ca2+. protein kinase A phosphorylation (P). Each of the three NOSs has ~36% homology to CPR in its C-terminal half containing the NOS reductase domain. FAD and FMN. The three NOSs also have considerable (> 50%) homology among themselves. alternate splicing and myristoylation (redrawn from ref. Regulation of nNOS and eNOS by Ca2+ NOS enzymes are regulated by calcium. In the brain. 1). Similarly. This homology reflects the oxidative mechanism of NO biosynthesis. L-Nw methylarginine or L-NMA and L-Nw aminoarginine or L-NAA) function as NOS inhibitors and their inhibitory effect can be reversed by the presence of excess arginine1. Thus the activity of NOS is regulated through Ca2+/calmo-dulin. and the structure of NOS reveals numerous regulatory mechanisms. This formation of NO is involved in neurotransmission. NADPH reduces FAD. L-Nw nitroarginine or L-NNA. both in neurotransmission and vasodilation. Regulation of inducible macNOS (iNOS) and immune response . Regulation of NOS NOS is one of the most regulated enzymes in biology. which activates NOS to form NO. which contains the binding sites for NADPH. stimulus such as glutamate (a neurotransmittor) acts at NMDA (N-methyl-D-aspartate) receptor and triggers Ca2+ influx. Cofactor recognition sites within NOS enzymes and cytochrome P450 reductase (CPR). which in turn reduces FMN. Figure 2. Predicted sites are shown for calmodulin binding (CaM). and is inhibited by calmo. L-Nw -substituted arginines (e.g. that is.dulin antagonists like trifluoperazine. which binds to calmodulin and activates nNOS to form NO.homology of NOS with CPR suggests that electrons follow the same path through NOS as they do through CPR. in blood vessels.

eNOS is predominantly localized in the plasma membrane. and against the proliferation of lymphoma tumour cells. but surprisingly they did breed and generally appeared to be normal. Other kinases including protein kinase C. where the NO system must have sufficed to repel the invading microorganisms without the aid of antibodies and/or T-cell receptors. It is known that unlike nNOS and iNOS.Calmodulin has been shown to be tightly bound to inducible macNOS (iNOS). have also been obtained to study the function of nitric oxide in different tissues/organs (Table 1)10. For instance. where it is rapidly phos-phorylated and then translocated from membrane to the cytosol and rendered inactive there. Regulation of NOS by phosphorylation Consensus sequences for phosphorylation by cAMP-dependent protein kinases have been shown to be present in nNOS and eNOS. after all. The ubiquitous distribution of this form of inducible NOS reflects a primitive form of immune response. where individual genes for different forms of NOS have been inactivated (knock out) by homologous recombination. where calmodulin cannot bind unless Ca2+ is present. which is active cannot be solubilized and is therefore presumably localized in the plasma membrane. But nNOS– mice are resistant to brain damage caused by vascular strokes. This would mean that nNOS is not so vital. neuronal NOS. In view of the ubiquitous vital role of NO. . suggesting that brain damage is mediated by NO. they should not be able to breed. Synthesis of inducible NOS is actually induced by stimuli such as interferon-g and LPS and this inducible synthesis is not restricted to macrophages (as was earlier believed) and has been reported in tissues lacking macrophages. active form of NOS may be unphosphorylated and restricted to the plasma membrane. which is released to the exterior. The nNOS– mice have also been shown to have markedly reduced defences against microorganisms like Listeria and Leishmania. In brain also. which are largely cytosolic. but this binding is unaffected by Ca2+ in contrast to neuronal NOS. cGMP-dependent protein kinase. have also been implicated in phosphorylation of neuronal NOS. Phosphorylation also regulates subcellular distribution of NOS. nNOS– mice also exhibited other abnormalities but did not die and were used as models for clinical tests. These animals were also resistant to carrageenan inflammation and hypotension elicited by endotoxin. but not in other types of NOS such as iNOS (Figure 2). Knockout mice for various forms of NOS Knockout mice. if nNOS (involved in penile erection) is absent in knockout nNOS– mice. Phosphorylation of NOS by these enzymes decreases its activity. Thus it is apparent that both in the neurons as well as in blood vessels. where it produces NO. viability of such knockout mice was doubtful. and Ca2+/calmodulin dependent protein kinase.

induction of NOS was observed in resistant. 3203–3207. F. 1989. Natl. H. Lippton. 288. and/or nNOS in skeletal muscles. 1980. Nature. 1977. These observations substantiated the role of eNOS earlier envisaged in vasodilation.. thus lowering the blood pressure. so that its activity is inhibited by NOS inhibitor. 373–376. Ignarro. where the role of NO in inducing defence response against pathogens in two plant species (tobacco and soybean) was demonstrated. J. K. Chem. In tobacco. H. through its vasodilating function. 739–749. USA. Katsuki. 3. Edwards. Furchgott. Ballot. Similarly. 1981.12 appeared. W. C. and Synder. USA. H. it is obvious that plants also produce NO in resistant genotypes using an enzymatic machinery similar to that found in animal cells. 3.. 2. Acad.. W. Acad. 63.. which contributes to increase in blood pressure. It is believed that NO collaborates with reactive oxygen intermediates (ROIs) to trigger transcriptional activation of plant defence genes and the hypersensitive response13. R. 86. Biol. 9030–9033. both the activity of NOS and release of NO were observed in response to either a bacterial pathogen or molecules that elicit defence responses. Pharmacol. L. J. Exp. 218. S. F. J. et al. V. 1984. Note added in proof: In August/September. Arnold. J. Bredt. Ther. Bredt.TABLE 1 The eNOS– mice were also produced and examined... but not in the susceptible plants11. FASEB J.. 1998. Ignarro. in cultured soybean cells. S. Baricos. Proc. B.. A. Annu. J. Sci. 4. S. These were viable. Therefore. D. Hyman. reproduced normally and were no different from the wild type in general appearance and behaviour. P.. which raises blood pressure in wild-type mice. L. and Synder. L-Nw -nitroarginine (L-NAA).. NO may also lead to contraction of skeletal muscles resulting in increase in blood pressure. H. confirming the role of endothelial NO in maintaining normal blood pressure. 175–195. This other form of NOS may be nNOS in the adventitial neurons in blood vessels.. two reports11. 74. The surprising result. Ignarro.. suggesting that there is a form of NOS other than eNOS.. 5. C. 1. however was that L-Nw - nitroarginine (NOS inhibitor).. S. 1989. L. K. D. 7. 1994. and Wood... Mittal.. . 259. S. L. and Zawadzki. Sci. The mean blood pressure in eNOS– mice was 35% higher than in control animals. S. 6201–6207. Natl. Biochem. 31–36. dramatically lowers blood pressure in eNOS– mice. Rev. and Murad. These mice displayed no relaxation in their aortic rings in response to acetylcholine and were unaffected by treatment with NOS inhibitor. Proc. 6.. J. J.

Dixon. 1998. Gupta is in the Department of Agricultural Botany. India Berdasarkan artikel di atas. F.. 1995. Nature. Delledonne. P. 394. Sci. 1994. H. Durner. Charan Singh University. 585–588. Ch. Science. A. Wendehenne. K. 1992. Synder. Barinaga. Y. D.. Nature.. Nature. Xia. Meerut. USA. C. maka dapat disimpulkan bahwa peran NO antara lain : Signaling oleh nitrat oksida 1) Sintesis NO akan menghasilkan NO dari deaminasi arginin 2) Berperan dalam merelaksasi otot a) Sequence of events b) Berkaitan dengan medis 3) Beberapa fungsi NO a) Memproduksi macrofag aktif dan neutrofil b) NO dilepaskankan/dihasilkan oleh banyak sel saraf 4) Mekanisme kerja nitrat oksida a) Berdifusi keluar sel melewati membran b) Bedifusi langsung ke sel tetangga (neighboring cells) c) Bekerja secara lokal d) Berikatan dengan enzim pada sel target e) Menghasilkan cGMP . Natl. 13. D. 257.. 394. 196–197. Dangl. 11. and Klessig. Synder. 263. 494–496. R. M. 12. 1998. 377. and Lamb. 9.. 250 005. H. Acad. Proc. J. 1998.... M. 525–526. Science. in press. J.8. 468. 10. S. S..