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Blood vessels have the vital responsibility to deliver nutrients and oxygen to the tissues apart from removing waste metabolites. There are many different types of blood vessels that constitute the vascular system namely the arteries, veins and capillaries. Although they may differ in functions, they share quite a number of basic features1. Smooth muscle cells are affected by the autonomic nervous system through neural mechanisms, also hormones, autocrine or paracrine agents and other locally acting chemicals2. Central to the function of blood vessels is the contractile activity of the smooth muscle.
Contraction of smooth muscle cells is initiated by an increase in the concentration of calcium ions, [Ca2+] which results in force generation from the interaction between the actin and myosin2, 3, 4, 28. This is depicted in Figure 1. Muscle contraction can be initiated by the change in the membrane potential, also sometimes caused by action potential firing or by the stretch-induced activation of plasma membrane ion channels as well as agonist-induced changes in ion channel activity2. Ca2+ entry into the cell cytosol is dependent on the electrochemical gradient arising from the membrane potential and concentration gradient4.
Figure 1: Regulators of intracellular Ca2+ concentration in the smooth muscle namely components of the plasma membrane and sarcoplasmic reticulum. It is clear that many mechanisms influence the intracellular Ca2+ concentration in smooth muscle. VOC = voltage operated channels, ROC = receptor operated channels, G = guanine nucleotide binding protein, PLC = phospholipase C, PIP2 = phosphatidylinositol 4-phosphate, DAG = diacylglycerol, RIP3 = IP3 receptors, NaK = Na+/K+ ATPase antiporter, NCX = Na+/Ca2+ exchanger28.
There are three major pathways in which muscle contraction may be initiated. In the first pathway, substances mainly neurotransmitters or hormones bind to their respective receptors triggering an increase in intracellular Ca2+ levels which initiate the contraction pathway. Different agonists activate the contraction pathway by various mechanisms. To emphasize different mechanisms, some examples will be explained. Binding proteins such as guanosine-5-triphosphate coupled to other ion channels and enzymes may be triggered to set up the contraction cascade. Another example would be enzymes (e.g. phospholipase C) causing the generations of IP3 and diacylglycerol (DAG). Alternatively, adenylate cyclase can be produced from the process which will then utilize adenosine triphosphate (ATP) to generate cyclic adenosine-3,5,-monophosphate (cAMP). IP3, DAG and cAMP are activators of smooth muscle contraction. Some ion channels on the other hand, for instance, the specific receptor for atrial natriuretic peptide, will directly activate guanylate cyclase which directly break down GTP to produce cyclic guanosine-3,5-monophosphate (cGMP) which also causes contraction in smooth muscle2, 6.
The second pathway in which contraction may be initiated is by directly increasing the levels of intracellular Ca2+. There are two pathways in which this can be achieved which are through Ca2+ influx from the external surroundings or through release from the internal stores (sarcoplasmic reticulum) which can be illustrated in Figure 2. Ca2+ influx involves a number of different types of ion channels namely the voltage-dependent Ca2+ channels (mainly L-type in smooth muscle cells), non-selective cation channels and the Na+/Ca+ exchanger in reverse mode. In addition to triggering Ca2+ release, receptor activation may initiate release from the internal Ca2+ stores (sarcoplasmic reticulum). Second messengers such as that produced in the first pathway (IP3, DAG, cAMP, cGMP) will affect these ion channels2, 3, 6. There are two types of calcium release channels located on the sarcoplasmic reticulum namely the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Although these two receptors act to achieve the same aim which is to increase cytosolic Ca2+ levels, they act via different pathways. Differences in these two pathways can be summarized by Figure 3. Ca2+ release via RyR is initiated by Ca2+ hence; it is termed the â€œcalcium-induced calcium releaseâ€Â mechanism where as Ca2+ release through IP3R is mediated through the production of IP3 (IP3-induced Ca2+ release)7.
Figure 2: The different Ca2+ channels which are responsible to increase Ca2+ levels in the cytosol5. Ca2+influx from exterior of the cell can be mediated through calcium channels and NCX. Contributing to the increase in the levels of intracellular Ca2+ is the release of the ion from the sarcoplasmic reticulum5.
Figure 3: The different mechanisms which are responsible for the increased intracellular Ca2+ levels. Ca2+ influx directly triggers RyR on the sarcoplasmic reticulum and indirectly triggers IP3R to release Ca2+. These activations then increase the levels of intracellular Ca2+ which brings about contraction7.
The third regulatory activator of muscle contraction involves the myosin light chain kinase (MLCK) activity. MLCK is activated by Ca2+-calmodulin complex. It phosphorylates myosin light chain (MLC) and produce cross bridging (contraction) in the presence of actin. The phosphorylated MLC will then be deactivated by MLC phosphatase. Therefore, the extent of contraction in this cascade is actually dependent on the equilibrium between the activity of MLC kinase and MLC phosphatase. However, to add to the complication, some agonists and second messengers have an effect on the activity of MLCK and MLC phosphatase which may have an effect on contractile activity. This process is termed the Ca2+ sensitivity of MLC phosphorylation. In this mechanism, cAMP and cGMP will interfere with the action of MLC kinase and phosphatase and cause relaxation2, 6. This whole process is showed in Figure 4.
Figure 4: Mechanism of contraction which involves MLCK and MLC phosphatase2. This diagram explains how activated MLC phosphatase influences the action of MLC kinase (which is one of the activator for muscle contraction).
When the stimuli for contraction are removed or there is inhibition of the contractile mechanism, smooth muscle will relax. Both processes cause relaxation by a reduction in cytosolic Ca2+ concentration or enhanced MLC phosphatase activity. There are a number of mechanisms assisting the removal of Ca2+ ions from the cytosol which is summarized in Figure 5.
In the first pathway, the uptake of Ca2+ into the SR depends on the hydrolysis of ATP2. The receptor responsible for this process is termed the sarcosplasmic/endoplasmic reticular Ca, Mg-ATPase (SERCA)2, 3. Phosphorylated SERCA binds two Ca2+ ions which then undergo translocation to the luminal part of the SR and the ions are released. SERCA may be inhibited by agents such as cyclopiazonic acid (used in present study), vanadate and thapsigargin2, 8. Details for the SERCA receptor will be explained in later section.
Another protein which helps to reduce the cytosolic Ca2+ concentration is the plasma membrane Ca, Mg-ATPases, which is different from SERCA in which they are located on the plasma membrane and are not regulated by phospholamban2, 9. The plasma membrane Ca, Mg-ATPase is an enzyme which contains an autoinhibitory domain where calmodulin binds, activating plasma membrane Ca2+ pump. The third mechanism that helps remove Ca2+ ions from the cytosol is the Na+/ Ca2+ exchanger2. Of these three Ca2+ removal process, SERCA is significant in causing Ca2+ uptake and relaxation in several smooth muscle types.
Figure 5: The three mechanisms involved in Ca2+ uptake in smooth muscle relaxation. The three receptors that are responsible for smooth relaxation is the SERCA, plasma membrane Ca, Mg-ATPase and the Na+/Ca2+ exchanger2. All three receptors aim to remove Ca2+ from the cytosol.
As described above, the levels of intracellular Ca2+ plays a crucial role in the vascular system. The major mechanism where Ca2+ uptake occurs is through SERCA receptor. SERCA is a receptor which is a membrane protein which has a molecular weight of 97-115 kDa found in the sarcoplasmic reticulum of all types of cells. There are four subtypes of SERCA identifies to date and each subtype can be found on different types of cell22.
Different subtypes of SERCA
Tissues in which they are found
Fast-twitch skeletal muscle
Heart and slow-twitch skeletal muscle
In all known cells including smooth muscle endothelium and platelets
Non-muscle cells, found in platelets and endothelial cells
Table 1 summarizes the different subtypes of SERCA and the tissues in which they are found22.
Since SERCA is an important regulator of Ca2+, an ion that is vital in smooth muscle contraction, this receptor is therefore important in regulating smooth muscle function. The function of SERCA is greatly influenced by the body regulatory mechanisms8.
Nitric Oxide as a regulator in smooth muscle function
The body has many regulatory functions which influences the mechanisms of contraction and relaxation. The endothelial cell coats the interior surface of blood vessels where its total mass is more than that of a liver 4, 10. The next layer surrounding the endothelial cells is the connective tissues which are then further coated with a single layer of mural cell (vascular cells in smooth muscle and pericytes) 1.
Endothelial cells are individually the chief endogenous component of the blood vessels which is capable of modulating the performance of the smooth muscle. Apart from being a barrier between tissues and blood, the endothelium is well known to play a pivotal role in cardiovascular system and is involved in autocrine and paracrine activities where vasodilating and vasoconstricting agents are secreted. This unique muscle regulates different function of the vascular system by contraction and relaxation processes. Both processes were controlled by altering the contractile status or modifying the signalling pathways in which contractile apparatus were stimulated11.
In response to external stimuli such as mechanical or biochemical stimuli, the endothelium will undergo changes in phenotype altering the shape of the cell, calcium entry, protein expression, mRNA expression, migration, cell differentiation, cell death, inflammatory response, leukocyte adhesion and migration4, 10, 12. If triggered, the endothelium secretes vasodilatory substances namely the nitric oxide (NO), prostacyclins, endothelium-derived hyperpolarizing factors as well as C-natriuretic peptide. Other than vasodilatory substances, the endothelium also releases vasoconstricting factors such as endothelin-1, angiotensin II, thromboxane A2, and reactive oxygen species (ROS)12. The endothelial cells generate vasoactive substances to produce a balance between vasodilatation and vasoconstriction, its initiation and inhibition of its functions, thrombogenesis and fibrinolysis. If there are shifts in the balance towards decreased vasorelaxation, a proinflammatory condition and prothrombic state brings about endothelial dysfunction4. The smooth muscle also plays an integral part in the regulation of blood pressure, flow of blood, microcirculation, and cardiovascular performance. Due to its importance, abnormalities in its function will result in disease states, hypertension being the most common example11.
One of the most crucial roles of the endothelial cell is to produce a free radical, nitric oxide 8, 10, 12. Nitric oxide works to induce relaxation to regulate arterial tone, suppresses growth and inflammation as well as preventing aggregation of platelets 13, 3, 14, 12. NO also plays a part in body defense as well as nerve transmission15. This vasodilating substance is produced in response to shear stress (force asserted by blood flow per surface unit of the blood vessel wall) and chemical substances15, 10. The generation of NO (Figure 6) occurs by NO synthases (eNOS) in the endothelium from L-arginine and molecular oxygen16, 3, 15, 10. The terminal nitrogen moiety from the guanidine component of L-arginine reacts with molecular oxygen and produces NO and L-citrulline as a byproduct. A number of cofactors are involved in this reaction namely calmodulin, tetrahydrobiopterin (THB4), NAPDH, flavin adenine dinucleotide and flavin mononucleotide17.
Figure 6: The reaction involved when NO is produced and the cofactors involved17. Sheer stress or chemical trigger may initiate this reaction.
After production, NO diffuses into the endothelium cells to cause relaxation. A small proportion of the produced NO affects platelets and leukocytes16. The diffusion of NO to the site of action can be reduced by the presence of reactive oxygen species, anions, and metal ions18. The half life of NO is short; NO is rapidly oxidised to nitrite and nitrate ions by haemoglobin found in blood or tissues. The rapid half life of NO explains the localised effect of NO5. The bioavailability of NO is also reduced by ROS which are scavengers of NO12.
The ability and extent of the NO-induced relaxation relies on the efficiency of reduced intracellular Ca2+. This efficiency varies between different types of blood vessels, the pathophysiology of the blood vessel and the different mechanisms of pre-activation3. The major signalling pathway for NO-induced relaxation is associated with the activation of soluble guanylyl cyclase followed by the pooling of cGMP and then, the activation of protein kinase G or potassium (K+) channels 8, 10, 14, 19.
The NO-induced vasodilation can be divided into two pathways; these pathways are either cyclic GMP-dependent or cyclic GMP-independent mechanisms. The cyclic GMP-dependent pathway is regulated by cGMP in which contractile proteins are desensitized via the MLC kinases/ phosphatases or Rho/Rho kinases relaxation pathway (refer to Figure 4)14. cGMP is a potential activator for many targets in mediating relaxation namely the L-type calcium channels, potassium channels, IP3R, phospholamban and MLC phosphatase18. In cyclic GMP-independent pathway on the other hand, NO triggers the activation of SERCA and increase Ca2+ sequestration into the SR, activation of potassium channels as well as Na+/K+ ATPase14, 20, 21. This is also followed by the inhibition of store-operated cation channels (SOC). Overall, this causes a decrease in cytosolic Ca2+ levels which causes dissociation of actin and myosin fibers and hence, relaxation14, 20. Cyclic GMP independent pathway is hugely affected by vascular diseases14. These two pathways is demonstrated in Figure 7.
Figure 7: The two pathways of authentic NO in mediating relaxation14, 22. In this diagram, it is suggested that sodium nitroprusside (SNP) and nitroglycerin (NTG) which are NO donors act by cGMP-dependent pathway while authentic NO may act by both mechanisms.
In normal endothelium cells, low levels of NO are produced and this low level of NO will stimulate SERCA thereby inducing Ca2+ uptake. However, if there is high amount of NO which is the case in endothelial dysfunction, they will initiate endothelial cells to produce superoxide anion. Superoxide anion and other types of oxidant will cumulatively cause increased oxidative stress and then, promote irreversible oxidation of proteins as well as SERCA22. This underlies the pathophysiology of several cardiovascular diseases namely hypertension, ischemic heart disease, pulmonary high blood pressure, diabetes, metabolic syndromes, chronic kidney failure and stroke 11, 10, 12.
Nitric oxide Donors
Nitric oxide donors are often used to mimic the action of authentic NO in experimental studies of the vascular function. Nitric oxide donors are pharmacologically active substances which release NO in vivo or in vitro where majority of them contains nitroso constituents19, 20. In sodium nitroprusside (SNP), a potent vasodilator used in the management of hypertension, an NO molecule bound to an iron metal forms a coordinated square pyramidal complex with five cyanide ions which leads to the NO formation in the presence of a reductant19. Toxicity to vascular cells is possible due to the release of toxic by-product, cyanide. It is also suggested that SNP will generate superoxide anions (O2-) which then interacts with NO to form peroxynitrite (ONOO-) which causes permanent tissue damage and apoptosis19, 14, 20.
SNP is commonly used as a nitric oxide donor in studying the relaxation in endothelial cells because it was believed to be acting in a similar manner to NO18. However, there is a slight difference in the relaxation effect of SNP and authentic NO14, 18, 21. A study showed that relaxation induced by SNP is less dependent on SERCA compared to authentic NO. The differences between the effects of SNP and authentic NO can also be explained by the fact that SNP is less susceptible to NO scavengers compared to the authentic NO18.
Nitric oxide and calcium handling via SERCA
It is suggested that the function of SERCA may be influenced by cGMP, mediated through protein kinases G- and A-dependent phosphorylation of phospholamban14, 23. It is believed that NO trigger the movement of cytosolic Ca2+ into the sarcoplasmic reticulum by activating SERCA. The mechanism involves the addition of a glutathione to the cysteine-674 of SERCA22. When the effect of NO gas was investigated in rat and mouse aorta, it was found that the vasodilatory effect of NO gas is mediated through two mechanisms. Initial effect of NO gas is mediated through Ca2+ uptake by SERCA followed by the inhibition of Ca2+ influx. Both of these mechanisms although they do not occur simultaneously help maintain the decreased levels of cytosolic Ca2+, hence, relaxation23. However, more studies should be carried out to support this.
The antagonist used for SERCA in the present study is cyclopiazonic acid. It is an indole tetramic acid mycotoxin produced by Aspergillus and Penicillium. It is regarded as a selective reversible antagonist of SERCA. It is used mainly in experiments investigating the effect of Ca2+ sequestration in muscle cells. It is found to inhibit SERCA in smooth muscles, skeletal muscles as well as cardiomyoctes24. It is found that CPA inhibits the conformation changes during ATP hydrolysis and Ca2+ transport affecting SERCA function. By doing this, the storage capacity for Ca2+ in SR is reduced and therefore, also inhibits contraction induced by drugs in which the contraction processes are reliant on the release of intracellular Ca2+. CPA promotes Ca2+ influx in endothelial cells through non-selective cation channel24. In a study where NO gas tested, CPA was found to inhibit SERCA by a time dependent mechanism where a substantial amount of time is needed before all the channels were completely blocked23.
There are many contradicting opinions found in the literature about the mechanism of action of SNP. All studies on SNP come to an agreement that the action of SNP is not mediated through Î±- or Î²-adrenoceptors. In a study, Kreye et al concluded that the mechanism of action of SNP is exerted beyond receptor level. In this article, it is emphasized that SNP produces relaxation by a mechanism which is independent to changes in membrane potential. A separate study emphasized on the importance of Ca2+ in the mechanism of relaxation25. In this study, it is hypothesized that smooth muscle relaxation is based on the inhibition of calcium entry, augmented calcium efflux and reduced sensitivity of contractile apparatus13, 3, 25. This reduced sensitivity is said to be due to the action of myosin phosphatase and dephosphorylation of myosin3. Another different source explains that the mechanism of action of SNP is said to be associated through activation of soluble guanylate cyclase followed by the escalating cGMP levels13. According to this source, in order for SNP to mediate relaxation, it requires both enzymatic and non-enzymatic bioactivation in tissues to produce NO21. Increased cGMP levels mediate relaxation via voltage independent processes through the activation of SERCA which extrude Ca2+ from the cytosolic into the lumen of SR, also through the action of Na+/K+-ATPase and various potassium channels or inhibition of L-type Ca2+ channels13, 26, 3, 14. There is also another view put forward in 1976 that SNP shifts the membrane potential away from the contraction threshold which causes hyperpolarisation and therefore, prevents the occurrence of contraction. At this point, smooth muscle relaxation proceeds27. Due to all these confusions about the action of SNP, there is need of a new approach to clarify the main mechanism of action in which SNP acts on.
The aim and objectives of this research is to deduce the mechanism of action of nitric oxide donor, sodium nitroprusside on rat aorta. Specifically, we are investigating whether the vasodilatory effect of sodium nitroprusside is mediated through the stimulation of SERCA. To do this, the effects of SERCA inhibitor (CPA) on SNP-induced relaxation will be studied in a traditional organ bath set-up.