Septic Shock Case Study: Causes and Effects

Septic Shock Case Study: Causes and Effects Introduction Septic Shock is a serious circulatory disorder often characterised by a whole- body inflammatory state and the systemic response to infection (Titheradge, 1998), with the most common cause being the contamination of blood with bacteria. Septic shock is defined as sepsis with hypotension which develops in almost half of all septic patients as a complication with a mortality rate of 40-60% (Titheradge, 1998). Septic shock often results in a progressive failure of the circulation to provide blood and oxygen to vital organs of the body resulting in impaired tissue perfusion and oxygen extraction (Thiemermann, 1997). The key symptoms include a severe fall in blood pressure (hypotension) with hypo-reactivity to vasoconstrictor agents (vasoplegia) which may lead to the dysfunction or failure of major organs including lungs, liver, kidneys and brain (multiple organ dysfunction, MODS) and ultimately death (Goligorski et al., 1997). Presently it is widely assumed that septic shock rarely shows similar symptoms in affected individuals and therefore it is difficult to detect and then consequently treat it (Groeneveld and Thijs, 1986). It is, however, also agreed that most of the therapeutic interventions invariably focus on the primary aim of fighting the refractory hypotension by the use of aggressive fluid infusions, glucocorticoids, large doses of vasoconstrictors (Baumgartner and Calandra, 1999) and occasionally renal replacement therapy (Wheeler Bernard, 1999). Yet these interventions do not offer consistent success (Parratt, 1997). In recent times, our understanding of the pathophysiology of septic shock has developed significantly through experimental and clinical trials, though the discovery of a suitable treatment with therapeutic efficacy is proving elusive (Baumgartner and Calandra, 1999). This is probably because of the heterogeneity of the clinical situations and the differences in host response to i dentical pathogens. Moreover evidence suggests that different pathogens which cause septic shock respond differently to the conventional treatments. Still, no effort has been made to treat patients according to the nature of the infecting organism (Gao, Anonymous, 1992). During the early 1990s nitric oxide (NO) emerged as a potentially substantial step towards the treatment of septic shock. This finding directed scientists to carry out numerous clinical trials and animal experiments with the objective of finding out more about the interrelation of NO and Sepsis (Cobb, 1999; Kilbourn, 1999). Later, the discovery of nitric oxide synthase (NOS) made this enzyme the primary target of therapeutic agents (Rosselet et al., 1998). However, recently a substantial amount of literature has been published with evidence contradicting the discoveries of earlier studies. Today more than a decade has elapsed without any resolution to the matter and even in this 21st century Septic Shock is invading the developing countries rapidly (Cobb, 1999; Kilbourn, 1999). This problem of a lack of significant advances in this field can be highlighted by the fact that 17 years have passed since the final publication of the Consensus Conference on sepsis and sepsis related syndromes (Rangel-Frausto, 2005). This paper conclusively defined sepsis and its symptoms with the hope of finding a suitable treatment in order to eradicate the disease (Rangel-Frausto, 2005). Today, however, the situation has not changed significantly with up to 750,000 new sepsis cases every year and 215,000 annual deaths in the United States (Trzeciak et al., 2008, Mitchell M. Levy, 2007). Additionally, in the population that survives such attacks, there is considerable morbidity with many scoring low in health related quality of life assessments (Kaarlola et al., 2003, Perl et al., 1995). Hence the 5 million lives campaign instigated by the US Institute of Healthcare Improvements, aims to minimize the prevalence of nosocomial sepsis (sepsis originating in a hospital) (Gao et al., 2008). This campaign aimed specifically at increasing safety and transforming the quality of hospital care (McCannon et al., 2007). Moreover, the Surviving Sepsis Campaign aims to improve the quality of life of septic patients using the best evidence available currently (Dellinger et al., 2004). Though the world mortality rates have declined in recent times, sepsis is gradually becoming more prevalent in the elderly in the developing countries (Gao et al., 2008).This is mainly due to the extended longevity of patients with chronic illnesses, the increased occurrence of immunosuppression, and the more frequent use of invasive procedures (Bone, 1991; Parrillo, 1993). Sepsis, severe sepsis, septic shock and multiple organ failure still dominate the mass cases of non coronary intensive care units (ICUs). This essay will attempt to explore the fundamental mechanisms leading to tissue and organ damage in septic shock through the investigation of a case study. Following this, the discovery and general biology of nitric oxide (NO) shall be discussed, and the experimental evidence implicating NO as an effector in sepsis, will be examined in detail. The results obtained from various pharmacologic interventions directed at NO in animal studies will also be considered. This report will also give an account of conventional and innovative treatments for the management of septic shock. Finally, data from the few available relevant clinical trials will be reviewed and possible future avenues of interest will be discussed. Systemic Inflammatory response syndrome and Septic Shock (Clinical Manifestation) Sepsis, severe sepsis and septic shock were inaccurately defined until a recently reviewed consensus conference in 1992 (Bone et al., 1992, Dellinger et al., 2004). In this meeting the term systemic inflammatory response syndrome (SIRS) was invented insinuating a clinical response arising from a nonspecific tissue damaging event (insult) (Rangel-Frausto, 2005). Sepsis as mentioned earlier is the result of substantial release of inflammatory mediators in response to an infection. However, often the same mediators are released in the absence of a documented infected in several medical conditions such as cardiopulmonary bypass (Wan S. et al., 1997), pancreatitis and trauma (Shanley TP. et al., 2006). Due to this, the early definitions of sepsis or septic shock were found to be misleading hence a North American consensus conference proposed a new terminology. In this conference sepsis was defined as a condition in which critically ill patients meet criteria for SIRS, in the context of infection. SIRS was defined as two or more of the following: 1) heart rate >90/min; 2) temperature >38Â °C or 12,000 or 20/min or a PCO2 3.5 L min-1 M-23; 2) hyperglycaemia (plasma glucose >120 mg/dL) in the absence of diabetes; 3) significant oedema or positive fluid balance (20 mL/kg over 20 h); 4) inflammatory variables: plasma C-reactive protein >2 SD above t he normal value or plasma procalcitonin >2 SD above the normal value and 5) mixed venous oxygen saturation (SVO2) >70% . (Dellinger RP et al., 2004). SIRS is generally not considered as a disease and its recognition does not provide any clinical conclusion. However, since it has proven to be a very useful for the identification of sepsis and its sequelae namely severe sepsis and septic shock it is regarded as a very important tool (Shanley TP. et al., 2006). Case Study: A 3-year-old boy was admitted to the intensive care unit because of fever, hypotension, and lethargy. A purpuric rash was noted on his arms and legs. Arterial blood gas analysis demonstrated hypoxemia and metabolic acidosis. The arterial lactate level was 10 mmol/L. He was intubated, resuscitated with crystalloid solution, started on broad-spectrum antibiotic therapy, and given dopamine to maintain a MAP above 55 mmHg. His chest radiograph revealed bilateral interstitial-alveolar infiltrates with a left sided predominance. Gram stain of the cerebrospinal fluid showed gram-negative cocci. The dopamine infusion was increased to 18Â µg kg-1min-1 for persistent hypotension and oliguria. Pulmonary and radial arterial catheter data yielded the following blood pressure, 85/30 mm Hg; MAP, 48 mm Hg; heart rate, 140 beats per minute; CVP, 10 mm Hg; pulmonary artery pressure, 22/14 mm Hg; PCWP, 12 mm Hg; cardiac index, 2.5L min-1m-2; and SVRI, 1226 dyne-sec-cm-5m-2. The boy was treated with ad ditional saline and an epinephrine infusion, which increased the MAP, cardiac index, and urine output. Arterial lactate levels decreased over the next 12 hours. Subsequent cultures of blood and cerebrospinal fluid grew Neisseria meningitidis. In this case the patient with acute organ failure and hypotension was diagnosed with septic shock. The young boy with meningococcal septic shock had a borderline-low cardiac index. Due to this a decrease in the cardiac index in this patient by the administration of an NOS inhibitor might not be desirable. This is suggestive of the fact that NO or its synthesising means cannot always be targeted in order to battle septic shock. I will attempt to go into further details with regards to this statement as we go along the essay. Nitric Oxide discovery: Joseph Priestly first identified gaseous NO in 1772. NO also named Nitrogen Monoxide is a very simple and tiny molecule consisting of one nitrogen and one oxygen molecule. Unknown of its fundamental physiological roles in the mammals, until the 1980s NO was widely considered as a mere toxic atmospheric pollutant (Konstantin J. Ovodov et al., 2000). In the 1980s researchers were examining how blood vessels expand (dilate) hence regulate the mean arterial blood pressure. Dilation of blood vessels, also termed vasodilation is a very important physiological response which partly regulates the blood pressure. By increasing the diameter of blood vessels, vasodilation causes the blood to travel more freely due to lower resistance (RF Furchgott, 1980). Since the blood vessel lumen widens during vasodilation, the blood imposes less outward pressure on the vessel wall hence reducing the blood pressure. In opposition, vasoconstriction reduces the diameter of the lumen increasing the BP. These physiological responses occur all the time in the human body regulating the BP and therefore are one of the most fundamental mechanisms of the human body. Dr Robert Furchgott and his group, later in 1980s investigated the role of acetylcholine in the smooth muscle relaxation and found that relaxation only occurred if a special class of cells called endothelial cells were present (RF Furchgott, 1980). These cells line the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. Behind the endothelial cells are the smooth muscle cells which either relax or contract thus regulating the vascular tone (RF Furchgott, 1991). The same research group also discovered that smooth muscle were only able to vasodilate the blood vessels in the presence of endothelial cells. This indicated that there was some kind of factor that was being released by the endothelial cells which was involved in the dilation of the blood vessel. This factor was named Endothelium Derived Relaxing Factor (EDRF) and subsequently specified as NO (S Moncada et al., 1997). In 1977, Ferid Murad independantly investigated the mechanism of action of nitroglycerin and found that it worked by inducing the release of NO which in turn was able to cause relaxation of smooth muscle cells (F Murad et al., 1977). Louis Ignarro in 1986 finally resolved the whole perplex of EDRF and NO by declaring that EDRF was in fact NO. It was stated that both molecules showed identical properties when he compared gas Nitric Oxide and EDRF (Ignarro, L. J. Et al. 1987). In 1998, Nobel Prize in Physiology and Medicine was awarded to Drs. Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries that vascular endothelial cells make nitric oxide (NO) and that such endothelium-derived NO stimulates cyclic guanosine monophosphate (cGMP) synthesis in the underlying vascular smooth muscle, causing relaxation (Kilbourn, 1999). Since the discovery of NO, vast number of its physiological roles in normal conditions have been reported including in the immune system, nervous system, reproductive system and other cellular functions. It has also been found to play important roles in variety of species ranging from mammals, to insects and plants. Role of Nitric Oxide in Biology Since it was first discovered to play a role in the dilation of blood vessels many new roles for Nitric Oxide (NO) have been discovered. In human body, NO is metabolised by its diffusion into red blood cells where it oxidizes the ferrous iron of oxyhemoglobin yielding methemoglobin and nitrate ions (NO3-) (J.M. Hevel et al., 1994 and Konstantin J. Ovodov and Ronald G. Pearl, 2000). This meachanism limits local NO build up and is particularly important in keeping NO concentration in naomolar range, at least in nonhydrophobic compartments (i.e. outside cell membranes) (Beckman Koppenol, 1996). Nitric oxide has been found to be produced by effectively every cell type in vivo and plays an important role in both controlling the normal function of cells as well as in regulating larger scale processes such as the reproductive, immune and nervous systems. A few of these biological functions for NO are described in more detail below. The Immune System Nitric oxide plays many important roles in the immune system although it is expressed in many cell types following endotoxin or pro-inflammatory cytokine treatment (C. Nathan et al., 1991 and R.G. Knowles et al., 1994). NO, in immune system, is produced in high amounts from specific cells called macrophages. Proceedingly to an infection, chemicals known as cytokines are release in vivo which activate the cells of the immune system such as macrophages, and help guide them to the site of infection (S. Moncada et al., 1991 and C. Nathan et al., 1991). NO produced by the macrophages is toxic to the bacteria and play an important role in their destruction (Fig 1). The production of nitric oxide in this way also helps protect against other types of infection including parasites and viruses (S. Moncada et al., 1991). However, too much nitric oxide production can not only lead to septic shock but has also been implicated in conditions where the immune system is too active autoimmune diseases like arthritis (Jang D and Murrell G A, 1998). The Nervous System Nitric oxide has been shown to be involved in both the peripheral and central nervous system. The three nitric oxide producing isotypes of enzyme (iNOS, eNOS, nNOS) (see section) in humans, one isotype (neuronal NOS (nNOS)) is found almost exclusively in the nervous system (Forstermann et al., 1995). nNOS is thought to be involved in promoting the transfer of interneuronal nerve signals. This is thought to take place by the stimulation of exocytosis (release) of endogenous chemicals called neurotransmitters of one neurone (Moncada et al., 1991; Forstermann and Kleinert, 1995). These NT then diffuse across the synaptic cleft (gap between neuronal terminals) and stimulate the neighbouring nerve cell terminal to transmit the signal (Otto Loewi, 1961). NO has shown to play a substantial role in diseases of the nervous system like Alzheimers and Parkinsons. In both diseases, the inhibition of NO has shown to slow down the progression of the disease in mice (Weill Cornell Team, 2005 and Jo hns Hopkins et al., 2004). Reproductive Biology Nitric oxide is one of the most important molecule in the course of reproduction and is involved in many aspects of it. As well as dilating the blood vessels and thereby helping to regulate maternal blood pressure, NO is also involved in implanting the early embryo in the uterus (Rogers, 1995). During pregnancy, nitric oxide is also suggested promote angiogenesis (a process in which new blood vessels are formed) (RayChaudhury et al., 1996). It is also known to play a role in the survival of trophoblasts (form placenta) (Enders et al., 1978). Furthermore deficiency of NO has been found in patients with preeclampsia (a medical condition in pregnancy) suggesting its partial role in growth of embryo (Yallampalli et al., 1994; Liao et al., 1996). In addition, drugs (Viagra) used to treat erectile dysfunction also affect nitric oxide signalling. Cellular function A notable number of cellular activities can be controlled by nitric oxide including cell division, cell movement and cell survival. The majority of cells in human body have the ability to undergo programmed cellular death. This is a self-destructive mechanism usually called apoptosis which often occurs when a cell is damaged and beyond repair, infected with a virus, or undergoing stressful conditions such as starvation (Kerr et al., 1972). Cells in these conditions go through apoptosis so that they dont hinder the proper functioning of the rest of the tissue. During apoptosis, the structures of the cell break down in an organised manner, forming a packaged cell that is smaller in size so that it can be easily removed by the cells of the immune system (Kerr et al., 1972). Nitric oxide was first shown to inhibit apoptosis hence promoting cell survival in human B lymphocytes (Mannick, J. B. Et al., 1994). Subsequently similar finding were reported in an ample number of other cells. However, high doses of nitric oxide also have deleterious effects. They can be toxic to many cell types and can lead to septic shock and multiple organ dysfunction syndrome (MODS) in which case NO causes cell death instead of promoting cell survival (Beal Cerra, 1994). Nitric Oxide and Inflammation (Pathophysiology of septic shock) This section will deal with mechanism by which wall fragments of Gram-negative or Gram-positive bacteria and other inflammatory agents induce nitric oxide synthase (iNOS) in cells and tissues. Exogenous toxins which enter the circulation stimulate the synthesis and release of a number of endogenous cytokines. During a gram-negative infection which can lead to septic shock, lipopolysaccharide (LPS) and endotoxins present on bacterial wall and many other inflammatory agents bind to a co-receptor (CD14) on the surface of specific immune cells like macrophages, resulting in their activation (J.C. Lee et al., 1996). LPS also bind to LPS-binding proteins which are produced by the liver. These proteins facilitate LPS binding to the CD14 co-receptor of the macrophages. The CD14 co-receptor is activated through the binding of LPS to a toll-like molecule (TLR4), (Re F, Strominger J. Et al., 2001) which is responsible for initiating the transmembrane signaling. TLR2 molecules act in the same way as TLR4, (Leppper PM et al., 2002) though; these are activated by gram-positive bacteria, mycobacteria and yeast. Gram-positive bacteria such as Staphylococcus aureus have further additional wall fragments such as peptidoglycan (PepG) and lipoteichoic acid (LTA). Both PepG and LTA have been shown to synergise to produce the characteristic features of septic shock, MODS and ultimately death in rodent models (S.J. De Kimpe et al., 1995 and G.M. Millar et al., 1997). These effects were not observed with either LTA or peptidoglycan alone, although high doses of LTA can cause circulatory failure but not MODS (S.J. De Kimpe et al., 1995). Gram-positive bacteria may also release other enterotoxins and exotoxins, for example toxic shock syndrome toxin 1, which are involved in the pathogenesis of sepsis (reviewed in (R.C. Bone et al., 1994)). The additional fragments released by Gram-positive bacteria bind to unknown receptors however, like LPS binding, cause the release of proinflammatory cytokines TNF-a, IL-1ß, and IFN-? (Thiemermann, 1997, Titheradge, 1999). These cytokines as well as IL-6 are often produced in response to immune stimulation of macrophages and monocytes hence also in septic shock patients. IL-1 and TNF each occur in two forms, a and ß. TNF- a and both forms of IL-1 are made by activated monocytes and macrophages, whereas TNF- ß is made by activated T lymphocytes (Review by J. Saklatvala et al., 1996). There are two receptors for both IL-1 and TNF and the two forms of each cytokine interact with the same receptors. IL-1 a and ß interact with the type 1 IL-1 receptor for signal transduction, whereas type II does not appear to transmit any signal and functions as an inhibitor of IL-1 action (J. Saklatvala et al., 1996). The two types of TNF receptor, p55 (type I) and p75 (type II) have different end effects; p75 mediates the proliferative actions of TNF- a while p55 receptor signals the inflammatory response and apoptosis (J. Saklatvala et al., 1996). Complex interactions between these different mediators produce intense pathophysiological modification, which eventually lead to diffuse tissue injury and ultimately sequential system failure (multiple organ dysfunc tion syndrome), which accounts for the majority of deaths among patients with sepsis, severe sepsis and septic shock (Beal Cerra, 1994). IL-1ß and TNF-a have a very short half life compared to IL-6 and therefore IL-6 is a very good indicator of cytokinemia. The initial studies of septic shock, showed a very strong positive correlation between IL-6 levels and fatal outcome (Casey L. Et al., 1993). NO is equally a very short lived molecule with an estimated in vivo half life of only 0.1 seconds hence again it is hard to measure its levels in order to detect the severity of cytokinemia hence the severity of sepsis or septic shock. There are several molecules that contribute to the pro and anti-inflammatory responses in septic shock (Table 4); however I shall only focus on a few due to the limited word allowance. In response to inflammatory agents in septic shock, the released cytokines (TNF-a, IL-1ß, and IFN- ?), bind to their specific receptors activating a protein kinase called tyrosine kinase leading to both the activation of the nuclear factor-kB (NF-?B) (a transcription factor) and the phosphorylation of intracellular protein (Gao et al., 2008). A precise mechanism by which these cytokines act was proposed by J. Saklatvala et al. in 1996 however this has yet to be confirmed. Nitric oxide producing cells contain I-?B which is an inhibitor of NF-?B. For the activation of NF-?B, proteolytic cleavage of I-?B from NF-?B is required which forms NF-?B. This biological change allows the activated NF-?B to translocate to the nucleus, where it binds to the promoter region of the iNOS gene inducing transcription. It has also been reported to induce other inflammatory agents, such as cytokines and leukocyte-endothelial adhesion molecules (Janssen-Heininger et al., 2000). Tyrosine kinase present inside the cell acts as a messenger molecule involved in the proteolytic cleavage of I-?B/NF-?B and hence in the activation of NF-?B and iNOS expression (Hecker M, et al., 1996). In septic shock, the translated products of iNOS mRNA subsequently assemble forming the iNOS protein which in turn causes local NO proliferation (Thiemermann, 1997). Fig 2 shows the signal transduction pathway of iNOS expression in response to inflammatory agents. The physiological role of iNOS is to enhance the formation of NO (due to iNOS activity), which in turn may contribute to either the pathophysiology of septic shock (clinbical symptoms) or the host defence (Reviewed in Gao et al., 2008). Fig 3 shows a simplified schematic of the Anti Inflammatory cascade in the context of septic shock. Excess NO produced by iNOS has been reported to both induce and inhibit NF-?B (Kalra et al., 2000; Umansky et al., 1998). In year 2000, it was proposed that low levels of NO may induce further NO production while high concentrations do the opposite exhibiting a feedback mechanisms that would oppose the over expression of genes regulated by NF-?B (Janssen-Heininger et al., 2000). In addition NO at high concentrations competes with O2 at the active site of NOS, thus providing a feedback mechanism of its own synthesis (Griscavage et al., 1995; Rengasamy Johns, 1993). Biological Synthesis of Nitric Oxide (Nitric Oxide Synthases) NOS structure and substrates for NO production: In mammals, NO is exclusively formed from the enzymatic oxidation of one terminal guanidino nitrogen of the amino acid L-arginine. When expressed in moles, this reaction utilizes 1 mol each of arginine and O2, and 1.5 mol of NADPH, yielding 1 mol of NO, 1 mol of L-citrulline and 1.5 mol of NADP (R.G. Knowles et al., 1994). The reaction sequence involves the generation of an Ng-hydroxy-L-arginine intermediate, followed by the oxidation of Ng-hydroxy-L-arginine in presence of molecular oxygen to form L-citrulline and NO (Dennis J. Stuehr et al., 1991 and R.G. Knowles et al., 1994). The enzymes that accelerate the reaction above are a family of relatively large heme proteins known as NO synthase (NOS) which resemble cytochrome P450 structurally (M. M. Chan et al., 2001 and Francois Feihl, 2001) (The general mechanism of NO production from NOS is illustrated in Fig 4). All members of this family share a similar homodimeric structure, where each monomer consists of a an oxygenase domain and a reductase domain, separated by a short amino acids (30aa) sequence for the attachment of the Ca2+-binding protein calmodulin. In addition to calmodulin attachment, enzymatic activity requires the presence of four cofactors: FAD, flavin mononucleotide (FMN), tetrahydrobiopterin (BH4), and heme (Francois Feihl, 2001). Fig 5 shows the general structure of the NOS enzymes. Nitric Oxide Synthase isoforms and their locus in the Human Body: There are three known isoforms of NOS, each the product of a different gene: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). nNOS and eNOS were first described in rat cerebellum and bovine aortic endothelial cells, respectively, but their tissue distribution is far wider than suggested by their names. eNOS and nNOS are involved in regulating vascular tone (R.G. Knowles et al.,1992 and S. Moncada et al., 1991) and neurotransmission respectively, whereas iNOS is involved in the immune defence although it is expressed in many cell types following endotoxin or pro inflammatory cytokine treatment (R.G. Knowles et al., 1994). nNOS is typically expressed in skeletal muscle and myenteric plexuses. eNOS is ubiquitous in vascular endothelium, but may also be found in the kidney tubular epithelial cells, placenta (Forstermann et al., 1995), and neurons (Nathan Xie, 1994). In these tissues, the expression of nNOS and eNOS is constitutive, although it may be regulated (Michel Feron, 1997). For instance, the levels of transcript for eNOS in vascular endothelial cells is increased by shear stress (Topper et al., 1996; Uematsu et al., 1995) and exercise (Wang et al., 1997), reduced by inflammatory stimuli such as TNF-a (Nathan Xie, 1994) and variably affected by hypoxia (Le Cras et al., 1998; Toporsian et al., 2000). In the physiological state, the iNOS isoform is only present at a few locations, notably the respiratory epithelium, the gravid uterus (Nathan Xie, 1994), and perhaps the ileal mucosa (Hoffman et al., 1997). iNOS expression has been demonstrated in numerous cell types including macrophages, neutrop hils, vascular endothelial (Hoffmann et al., 1999), smooth muscle cells, endocardium, myocardium, fibroblast, mesangial cells, renal tubular epithelium (Kunz et al., 1994), neurons, hepatocytes, pancreatic islet cells and astrocytes (Nathan Xie, 1994). iNOS can be induced by a number factors including UV light; cyclic AMP-elevating agents; trauma; ozone and bacterial products described earlier (see section). On the other hand, many endogenous agents may oppose cytokine induction of iNOS. These include anti-inflammatory cytokines such as IL-10, chemokines such as monocyte chemoattractant protein-1 and growth factors such as tumor growth factor-ß (Forstermann et al., 1995). In all NOS isoforms, calmodulin binding is an absolutely vital for enzymatic activity. In the cases of eNOS and nNOS, this binding necessitates relatively high concentrations of Ca2+ (see Fig 7), in the range of 0.1-1 mM (Forstermann et al., 1995). In contrast, iNOS is able to bind calmodulin virtually independ ant of Ca2+ (Table 2). Therefore, once iNOS is expressed, NO synthesis may only be limited by the availability of substrates and cofactors (J. Stuehr, 1990). As NO produced from iNOS predominantly depends only on iNOS expression, it lasts much longer than NO formed from the other isoforms of NOS. In addition iNOS produces much higher concentrations of Ca2+ compared to the consecutive forms of NOS (Cobb et al., 1996). The production of NO by eNOS and nNOS, compared to iNOS, can be controlled relatively easily by decreasing or increasing intracellular Ca2+, whereas iNOS can only be controlled through transcription (Cobb et al., 1996). In most cell types iNOS protein levels are either very low or undetectable. However, stimulation of these cells by cytokines or growth factors, can lead to increased transcription of the iNOS gene, with subsequent production of NO. On the other hand, for the prevention of iNOS expression through endotoxins, TGF-ß (Szabo, 1995) and anti inflammatory gl ucocorticoids can be administered which lower the magnitude of vascular hyporeactivity. Glucocorticoids such as Dexamethasone inhibit iNOS activity by blocking arginine transport and inhibiting tetrahydrobiopterin biosynthesis (A.J.B. Brady et al., 1992 and Thiemermann C et al., 1993). Regulation of NO production In the normal as well as in extreme physiological states (e.g. during infection), nitric oxide is considered as one of the most important signalling molecules in vivo. It is however also highly reactive and highly diffusible due to it being a free radical (one unpaired electron) (see fig 8). It is therefore important that there is strict control and regulation of nitric oxide production. The synthesis of NO within cells can be regulated in several ways such as the cellular distribution of NOS, changes in NOS gene expression, enzymatic activation by phosphorylation and the presence of cellular inhibitors NOS activity. Intracellular distribution of NOS Nitric oxide is principally regulated through strict control over the location of NO production. The NOS isoforms can be targeted to different regions of the cell, where NO will be produced in close contact with its target proteins. The image below shows the distribution of iNOS (shown in green in image 1) and eNOS (shown in red) in a trophoblast cell. The nucleus is shown in blue. Co-localisation between iNOS and eNOS will show up as a yellow colour (Phil Dash, University of Reading). The image shows that eNOS and iNOS are fairly variably distributed inside the cell, with hardly any yellow colour suggesting very little overlap in their cellular distribution (Phil Dash, University of Reading).Although both iNOS and eNOS produce NO it is likely that their different cellular distribution will lead to NO interacting with different targets and therefore having different effects (Phil Dash, University of Reading). It is very likely that the distribution of NOS isoforms is an important mechanism for regulating when and where NO is produced. Therefore the current research on NOS mainly focuses on how endogenous signals trigger NOS transport and redistribution (Rahul S. Koti et al., 2005). Activation of NOS activity NOS enzyme synthesis is principally regulated by changes in intracellular calcium levels. The constitutive isoforms of NOS, (eNOS and nNOS) have shown to proliferate following increases in Ca2+, and therefore calmodulin levels, in the cell (Rameau et al., 2003). Additionally both nNOS and