SCLA مجلة التشخيص المخبري - هيئة مخابر التحاليل الطبية

المجلد 5 , العدد 3 , ربيع الثاني 1430 - نيسان (أبريل) 2009

The Importance of Nitric Oxide Signaling in the Vascular System

أهمية إشارة أكسيد النتريك في الجهاز الوعائي

Dr. Zaher Raslan and Prof Khalid Naseem
Hull York medical school, UK

الملخص Abstract

The discovery of nitric oxide about 28 years ago has had a great impact on the understanding of a variety of biological functions, due to its effect on the nervous system, body defense and the cardiovascular system. In this review, we will shed the light on the effects of NO on the cardiovascular system as a vasodilator and platelets anti aggregator. However, not every thing is fully understood regarding the NO signaling in the cardiovascular system as there are a lot of questions still need to be answered, but the fact that the cardiovascular diseases are the main killer in the world and the great link between the occurrence of those diseases and NO deficiencies make the effort to dig deep in this field indispensable.

إن اكتشاف أكسيد النتريك منذ حوالي 28 سنة كان له أثر كبير على فهم الكثير من الوظائف الحيوية، نظراً لأثره على كل من الجهاز العصبي، الدفاع عن الجسم، إضافة إلى الجهاز القلبي الوعائي. في هذه المراجعة العلمية سوف نسلط الضوء على آثار أكسيد النتريك على الجهاز القلبي الوعائي كموسع وعائي بالإضافة إلى دوره كمضاد لتكدس الصفيحات. من جهة أخرى، إن دور إشارة أكسيد النتريك في الجهاز القلبي الوعائي لا يزال غير مفهوم بشكل كامل، والكثير من الأسئلة ما تزال بحاجة إلى الإجابة، لكن نظراً لكون الأمراض القلبية الوعائية هي من أسباب الوفاة الرئيسية في العالم، بالإضافة إلى أن العلاقة الكبيرة بين حدوث تلك الأمراض وأعواز أكسيد النتريك تجعل من البحث العميق في هذا المجال ضرورة ملحة.

Abbreviations:

EDRF: endothelial-derived relaxing factor, NOS: nitric oxide synthase, HSP: heat shock protein, GTP: Guanosine triphosphate, cGMP: cyclic guanosine monophosphate, MLCP: myosin light chain phosphatase, MLC: myosin light chain, IRAG: IP3 R-associated cGMP kinase, RGS: regulated G-protein signaling, LASP: LIM and SH3 protein, PLC: phospho lipase C.

Introduction:

NO is a very important chemical messenger that mediates a lot of essential functions in the body. It was first known as the endothelial-
derived relaxing factor (EDRF), which was discovered by Furchgott in 1980. Then in 1987 Ignarro et al and Moncada et al; came up with the fact that EDRF is in fact NO. NO stimulates vasodilation, inhibits inflammation, inhibits platelets aggregation and limits vascular smooth muscle proliferation. All these functions make NO a very important chemical to study. It is a complicated area though. In health, it is in charge of regulating the vascular tone and inhibiting platelets aggregation. "Now, it is hard to find a disease that is not associated with altered NO homeostasis"

The synthesis of NO:

NO synthase (NOS):
First, (Mayer et al, 1989) demonstrated that NO is synthesized by an enzymatic reaction mediated by an enzyme called NO synthase, which is regulated directly and indirectly by Ca+2 cations and converts the amino acid L-arginine into NO. After that, NO activates soluble guanylyl cyclase sGC (Mayer et al., 1989).
Three separate isoforms of NOS have been identified so far. First, nNOS or NOS1, which is found in both neurons and endothelial cells and was the first isoform to be isolated (Bredt and Snyder, 1990). The second isoform to be isolated was the iNOS or NOS2, which was isolated from macrophages. It is induced by inflammatory response and after exposing to cytokines and microbial products (Hevel et al., 1991). eNOS or NOS3 was the last to be isolated by Pollok's group in 1991 from bovine aortic endothelial cells (Pollock et al., 1991). All those isozymes are Ca+2-depandent except for iNOS-derived NO and its activity is only induced by cytokines and endotoxin (Cho et al., 1992).
NOS in general is a homodimeric enzyme, which has a reductase domain, in the C-terminus, into which necotinamide adinine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN) and flavin adinine dinucleotide (FAD) bind. However, the N-terminus has the oxygenase domain, into which (6R-) 5,6,7,8 tetrahydrobiopterine BH4, L-arginine, a prosthetic heme group and a molecular oxygen bind (Forstermann et al., 1994). What is more, a structural zinc ion forms a zinc-thiolate cluster in the homodimeric NOS. The zinc ion binds to a CXXXXC motifs from each monomer. This zinc ion has no catalytic effect (Raman et al., 1998) figure (1).
This review will only focus on eNOS since it is the most important isozyme for the regulation of the vascular system. The mechanism, by which eNOS is activated is extremely complicated and is not fully understood. We can divide the process of eNOS activation into two different stages. Those two stages are linked by hsp90. The first stage is Ca+2-dependent, which requires additional calcium influx in order to form Ca+2-calmodulin complex. The other stage is phosphorylation-dependent stage, which with some agonists requires calcium influx, whereas with others it does not. So, in short, the activation of eNOS involves different phosphorylation sites and calcium mobilization mechanisms depending on the type of stimuli. It is not possible to cover all the mechanisms in detail. To find out more about eNOS activation the reader is advised to refer to this review (Shaul, 2002).

Figure 1: The electron transfer pathway and the structure of NOS: found as a homodimer. NADPH, FAD, FMN and CAM are all located in the C-terminus, whereas BH4, the heme group and L-argininee are located in the N-terminus. (Stuehr et al., 2001)

NO: L-arginine reaction:
NO: L-arginine reaction is a two-step oxygenation reaction that is applied on the L-arginine substrate. The first oxygenation step is a conventional one. One mol of NADPH, which is the electron donor in this reaction, and one mol of O₂ contribute to convert L-arginine to a compound called guanido nitrogen-hydroxy-L-arginine. In this step the molecular oxygen binds to the iron atom in the heme. Consequently, the heme group is activated and then it hydroxylates L-arginine. The second step is a non-conventional oxygenation of the guanido nitrogen-hydroxy-L-arginine, which needs 3 electrons from one mol of O₂ and 0.5 mol of NADPH and terminates with the formation of NO and L-citrolline. In other words, the first step is a subsequent electron transfer that starts from the NADPH, then to the FAD, then to the FMN, which delivers it to the NOS heme (go back to figure 1). Calmudolin plays a significant role at this stage as it facilitates the electron transfer by repressing the effect of negative controls that are located in the reductase domain (Abu-Soud et al., 2000). On the other hand, the donor of the electron in the second step is either BH4 or the outcome of the first step, which is NG-hydroxy-L-arginine. It is theoretically thought that BH4 is preferred to be in charge of this donation but this is not proved practically (Stuehr et al., 2001)

NO:cGMP Pathway:
After the synthesis of NO, it moves from the endothelial cell to either the vascular smooth muscle cells (VSMC) or to the platelets, where it activates soluble Guanylyl Cyclase (sGC), which in its turn leads to elevated levels of cGMP. cGMP also stimulates a cGMP-dependent protein kinase (cGKI), which induces the effects of NO in either VSMCs or the platelets (Munzel et al., 2003). This cascade is illustrated in figure 2.
The only definitely proved receptor of NO is soluble Guanylyl cyclase (sGC) which is a heterdimeric enzyme that has two subunits, namely α and β, with a prosthetic heme group. The activation of this enzyme leads to the conversion of GTP into cGMP (Gerzer et al., 1981). The activation of sGC is both sophisticated and debatable as there is more than one opinion regarding the mechanism by which it is activated. Generally it is activated via the binding of NO to a prosthetic heme group in the enzyme, which leads to conformational changes that leads to its activation (Friebe and Koesling, 2003). For more information about Guanylyl Cyclases the reader is advised to go back to this review (Lucas et al., 2000).

cGMP signaling in the cardiovascular system:

After activating guanyly cyclase in the VSMC membrane, the outcome would be the conversion of guanosine 5'-triphosphate (GTP) into cGMP.
cGMP signaling is cell-specific and depends on the type of receptors that are presented. cGMP has three types of receptor proteins namely: Cyclic nucleotide-gated (CNG) cation channels, cGMP-dependent protein kinases and cGMP-regulated phosphodiesterases (PDEs). In addition, cAMP-dependent protein kinase (cAK) could be, to some extent, regulated by cGMP (Munzel et al., 2003).
The first cGMP receptor is a group of enzymes called 3',5'-cyclic nucleotide phosphodiesterases (PDEs). These enzymes play a very important role in regulating the cardiovascular system, by degrading both cAMP and/or cGMP. They are encoded by 21 genes and comprise 11 families and each family has its own subfamilies. Some of them are cGMP-specific, whereas the others are both cGMP and cAMP-specific. They are allocated in different organs and different tissues. The common isoforms in the vasculature are PDE1, PDE3 and PDE5; Reviewed by (Omori and Kotera, 2007).
The second protein receptor downstream cGMP is (CNG). Although there is not too much information available about these channels, as the knock-out mice showed no specific cardiovascular phenotype (Biel et al., 1999), we are sure that those channels are up-regulated by NO (Zhang et al., 2002).
The last and the most important protein downstream cGMP is the cGMP-dependent protein kinases cGKs (PKG) as it mediates the effects of NO in the vascular system. Two genes encode two isozymes, cGKI and cGKII. In addition, cGKI is existed in two isoforms, cGKIα and cGKIβ, as the N-terminus of it is encoded by two spliced exons. cGKs are serine/threonin protein kinases, which comprise three domains, namely the catalytic domain (C), the regulatory domain (R) and the an N-terminus domain (A), reviewed by (Hofmann et al., 2000). As we are discussing NO signaling in vascular system, the light will only be shed on the role of cGKs in vascular smooth muscle cells and platelets in terms of normal physiological effects, which are vasorelaxation and platelets anti-aggregation. This involves only cGKI, because cGKII's effects are solely demonstrated in the intestinal secretion, bone growth and renine release (Lohmann et al., 1997).

1- Regulation of the vascular tone by NO:

The involvement of cGKs in the vasorelaxation process was investigated by Pfeifer et al in 1998, using cGKI-knock out mice, which resulted in hypertensive mice in week 4-6 (Pfeifer et al., 1998).
Ca+2 concentration in VSMCs is the major factor that controls the contractility in vasculature. On the other hand, vasorelaxation is conducted by cGKI through many targets in VSMCs. This is usually fulfilled by inhibiting both hormone-dependent constriction, which is the triggering stage, in addition to the depolarization of Ca+2 channels by different mechanisms. Those depolarizing mechanisms include decreasing [Ca+2] levels, inhibiting phospholipase C and negatively affecting the Ca+2 sensitivity. However, there are other mechanisms that still under investigation. To date, four pathways have been discovered in the VSMCs, through which cGKI exerts its effects. All these pathways are presented in figure 3.
The first pathway is the MLCP pathway, through which cGKI reduces Ca+2 sensitivity. cGKI activates MLCP indirectly by phosphorylating MYPT1, which in turn activates MLCP. MLCP inhibits the phosphorylation of MLC under constant [Ca+2] levels, which leads to vasorelaxation (Surks et al., 1999).
The second pathway is the cGKI/IRAG pathway (figure 3). In this pathway cGKI induces vasorelaxation in VSMCs by decreasing the hormone-induced release of intracellular Ca+2 through the phosphorylation of IRAG, which results in the inactivation of IP3-induced release of internal Ca+2 and as a result inhibits smooth muscles contraction (Schlossmann et al., 2000).
Another pathway, through which cGKI regulates the vascular tone is by causing Ca+2 depolarization and reducing Ca+2 influx. The target in this situation is the large-conductance Ca+2-activated K+ (BKca) channels (figure 3). The phosphorylation and the open of those channels decrease the levels of external Ca+2, because voltage-dependent Ca+2 channels will be closed as a result. This is fulfilled through either, a direct mechanism by which a direct phosphorylation is applied on BKca channels (Alioua et al., 1998) or through a protein phosphatase (Zhou et al., 1996).
The last pathway, to be discussed, is the cGKI/RGS pathway (figure 3). Regulator G protein signaling (RGS-2) is another target for cGKI. This protein is responsible for the regulation of Gq proteins in pathways that activate vasoconstriction. Not only does cGKI activate RGS-2, through phosphorylation, but also it inhibits those Gq proteins by increasing GTPase activity (Tang et al., 2003).

Figure 3: (Hofmann et al., 2006) cGKI-mediated vasorelaxation pathways in the VSMCs.

2- Platelet inhibition:

This is the second substantial physiological function of NO. Platelets are the other destination, where NO heads to, after being synthesized in the endothelium and the location of eNOS in the caveolae facilitates that, as it is adjacent to both VSMCs and the blood lumen. The evidence of NO effect on platelets, as an anti aggregator, was established long time ago when the role of NO and NPs were first discovered, since high concentrations of cGKI were noticed in deactivated platelets (Walter, 1989). The signaling pathway of NO in platelets is very similar to that in VSMCs.
Platelets activation involves several steps beginning with the adhesion, shape change, granule secretion, cytoskeleton reorganization and is triggered by the increase in Ca+2 concentrations. cGKI inhibits platelets by activating several substrates that regulate those mechanisms (the most important mechanisms are illustrated in figure 4). To begin with, an inactivation of polyphosphoinositide-specific phospholipase C (PLC) was found to be associated with the high levels of cGMP and cAMP in platelets. PLC is responsible for the conversion of PIP2 into IP3 which is responsible for the release of intracellular Ca+2. It is thought that cGKI inhibits the re-synthesis of PIP2 (Ryningen et al., 1998). It is also believed that IP3, itself, is a cGKI target and the consequent phosphorylation of it results in the inhibition of intracellular Ca+2 release (Cavallini et al., 1996). Another outcome of PLC deactivation is the inactivation of PKC, which is thought to have an important role in platelets granule secretion through phosphorylating pleckstrin (Yang et al., 1996) and the phosphorylation of another protein called myristoylated alanine-rich C kinase substrate (MARCKS) (Elzagallaai et al., 2000).
One of the most important substrate, which is phosphorylated by cGKI and has a significant role in platelets as a negative regulator through cytoskeleton reorganization, is the vasodilator-stimulated phosphorprotein (VASP). This protein exists in platelets in high concentration (Eigenthaler et al., 1992) and is in charge of regulating actin polymerization and its filament formation (Reinhard et al., 2001). Heat shock protein 27 (Hsp27) also participates in actin polymerization. It is also phosphorylated by cGKI and the outcome of this phosphorylation is the inhibition of its effect in mediating actin polymerization, which proves its role in inhibiting platelets aggregation (Butt et al., 2001). LIM and SH3 protein (LASP) is another novel substrate that binds to actin filaments and is inhibited by cGKI (Butt et al., 2003).
MLCK is another target for cGKI in platelets as well and it activates MLC through phosphorylation. MLC plays a role in platelet shape change by activating myosin ATPase (Daniel et al., 1984). It was found in vitro that the phosphorylation of MLCK by cGKI reduces its sensitivity to Ca+2/calmudolin complex and inhibits the subsequent phosphor-rylation of MLC (Nishikawa et al., 1984).
Rap1b is another protein that is found in platelets in high concentrations. It is a "small GTPase of the Ras family". However the exact role of Rap1b has not been investigated fully, but it is thought that it has a role in cell adhesion and it is rapidly activated during platelets aggregation. It is proved that the presence of NO leads to the phosphorylation of it by the downstream-activated cGKI and as a result it is inactivated (Reep and Lapetina, 1996). However, this inactivation by NO needs to be studied deeper. Finally, cGKI is known to inhibit Gq/Gi-coupled receptor signaling especially P2Y12 which is an ADP receptor which in its turn is a platelet agonist (Aktas et al., 2002).

Figure 4: PKG I (cGK I) and cAK substrates and signaling pathways in platelets; Taken from (Munzel et al., 2003).

Conclusion:

Having discussed NO signaling pathway in the cardiovascular system, we can conclude that the basic steps to produce NO and exert its functions in the vascular system have been reasonably established. However, we still do not know how exactly the production of NO is actually triggered. In addition, some of cGMP and cGKI possible substrate as well as other suggested pathways need to be studied more profoundly. However, in order to benefit from those findings clinically, more work is needed to clarify every detail in the NO signaling pathway.

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المجلد 5 , العدد 3 , ربيع الثاني 1430 - نيسان (أبريل) 2009

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