Abstract count: 289 Abbreviations Bile acid (BA), Estrone-3-sulfate



Bile acids (BA) are
known to facilitate the absorption of dietary fatty acids, fat soluble vitamins
and maintain cholesterol homeostasis. Recently it has been shown that BA also
interact with other receptors to stimulate various responses for example
affecting vascular tone. This project explores the mechanism of how
taurocholate (a primary BA conjugated with taurine) can induce
vasoconstriction. This can help us understand how conditions like cholestasis
(abnormal BA flow) encourages vascular constriction.



To investigate the
mechanisms of vascular constriction induced by taurocholate.



Isolated thoracic
aorta segments from male Wistar rats were mounted onto a wire myograph, bathed
in physiological salt solution and gassed (95% O2, 5% CO2).
Responses to the cumulative addition of a vasoconstriction agent phenylephrine
(1 nM – 100 ?M) and taurocholate (1 nM – 100 ?M) were measured. To determine
the mechanism behind any taurocholate-induced vasoconstriction, responses to
taurocholate were also performed in the presence of inhibitors for: L-type Ca2+
channel (LTCC); endothelin, muscarinic and thromboxane receptors;
cyclooxygenase (COX) enzyme; organic anion transporter (OAT) and/or OAT
polypeptides; and finally endothelial nitric oxide synthase (eNOS). Responses
were analysed by two-way ANOVA and significance accepted at p<0.05.         Results Taurocholate produced a concentration-dependent constriction that was significantly less potent than phenylephrine (n=15, p<0.001). The inhibition of LTCC, endothelin and muscarinic receptors, COX and OAT/OATP significantly reduced taurocholate-induced vasoconstriction. The drug inhibiting thromboxane receptor alternatively had no effect on taurocholate responses, while inhibiting eNOS significantly amplified taurocholate-induced constriction.   Discussion This data indicates taurocholate induces vasoconstriction. The mechanisms appear to involve: LTCC, endothelin and muscarinic receptors, but not thromboxane receptors. Equally important appear to be the OAT/OATP family of transporters. This provides insight into developing treatments to prevent complications from constriction in cholestasis, by inhibiting vasoactive pathways stimulated by BA.   Abstract word count: 289                           Abbreviations   Bile acid (BA), Estrone-3-sulfate (E3S), Intracellular calcium concentration (Ca2+i), L-type Ca2+ channel (LTCC), Organic anion transporter (OAT), Organic anion transport polypeptide (OATP), Sulfobromophthalein sodium (BSP), Taurocholate (TC), Ursodeoxycholic acid (UDCA), Vascular smooth muscle (VSM).       Acknowledgments I would like to express my appreciation to Dr. Christopher Torrens, who provided patient guidance while continuously teaching, supporting me and providing useful critiques throughout all aspects of the project. Furthermore, I would also like to thank Dr. Emily Lofthouse for the ongoing encouragement and helping me in the laboratory.     My contribution The design for the study had already been created including the type of vessel, bile acid and inhibitors to use and investigate. This experiment was a new project for looking at the mechanisms of taurocholate contraction in the presence of various drug inhibitors.  My roles involved conducting the experiment after being taught and carrying out the following: ·         Dissection of the thoracic aorta fat and connective tissue; after the aorta had been removed from the rat by Dr. Christopher Torrens. ·         Setting up and using the wire myograph to conduct the study ·         Creating some of the solutions needed e.g. physiological salt solution (PSS) and K+ in PSS (KPSS) ·         Diluting the bile acid and phenylephrine into the concentrations required for the experiments ·         Carrying out the experiment procedure of adding taurocholate and inhibitor to the vessels, and measuring the effect taurocholate had. ·         Inserting and translating the data results into usable form I had received help on creating the solutions from the raw powder ingredient. The drugs used were pre-made at the concentrations required.     Table of contents Abstract 2 Abbreviations. 4 Acknowledgments. 5 My contribution. 5 Chapter 1 – Introduction. 8 1.1 Bile Acids. 8 1.2. Pathologies due to bile acid accumulation. 9 1.3. Mechanism of smooth muscle contraction. 10 1.4 Channels, receptors and mediators involved in VSM and BA interaction. 12 Chapter 2 – Aim and hypothesis. 17 Chapter 3 – Methods. 18 3.1 Animals. 18 3.2 Myography. 18 3.3 Measuring Taurocholate Contractility. 19 3.4 Measuring Taurocholate Relaxation. 20 3.5 Statistical Analysis. 20 Chapter 4 – Results. 21 4.1 Taurocholate-induced constriction. 21 4.2 Receptors & channels involved in taurocholate-induced constriction. 21 4.3 Endothelial mediators and taurocholate-induced constriction. 23 4.4 Organic anion transporter and organic anion polypeptide transporter inhibitor 23 4.5 eNOS inhibition with other inhibitors and taurocholate-induced constriction. 24 4.6 eNOS inhibition and phenylephrine-induced constriction response. 25 4.7 Endothelial mediators in pre-constricted aorta and taurocholate-induced relaxation. 26 Chapter 5 – Discussion. 28 Chapter 6 – Summary. 36 Chapter 7 – References. 37             Chapter 1 – Introduction   1.1 Bile Acids Abnormal bile acid (BA) concentration can lead to the development of several conditions including: liver disease and intrahepatic cholestasis of pregnancy (ICP). At present the mechanisms of how BA negatively contribute to these pathologies is unknown.   Bile acids are synthesized within hepatocytes and transported into the gallbladder for storage. From the gallbladder BA are released on stimulation and enter the duodenum to aid digestion and absorption of dietary lipids, whilst maintaining cholesterol homeostasis1. Primary BA (cholic acid and chenodeoxycholic acid) are conjugated with glycine or taurine within the hepatocyte. It is this process that leads to the formation of taurocholate; which is a taurine conjugated to primary BA cholic acid. These primary BA and their conjugated complex enter the small intestine; intestinal bacteria modify them into secondary BA.   There is increasing evidence that BA functions expand beyond the digestive system and can act as signalling molecules. They interact with nuclear receptors and G-protein-coupled receptors (GPCRs) which can influence cardiovascular functions, glucose homeostasis, thyroid functions and more2-4.   BA are transported across cell membranes via various transporters including Na+?taurocholate co-transporting polypeptide (NTCP) and family of organic anion transport (OAT) and OAT polypeptide (OATP)3,5.       1.2. Pathologies due to bile acid accumulation Liver disease Liver cholestasis is a condition of impaired bile flow resulting with the accumulation of BA in hepatocytes and blood serum6. This can lead to hepatocellular injury, fibrosis, cirrhosis and liver failure in chronic conditions7. Some of these accumulating BA are hydrophobic and toxic to the liver inducing inflammation and damaging bile ducts; increasing hepatocytes death6,8,9. The increased concentration of hydrophobic BA in the blood serum can induce oxidative damage in the kidneys, resulting in damaged endothelium and activating vasoactive mediators such as endothelin; which will reduce glomerular filtration rate and initiate renal failure10. However, BA conjugated with taurine or glycine are less hydrophobic and thus less toxic, although in high accumulating concentrations they can begin to induce cellular injury9.   Intrahepatic cholestasis of pregnancy ICP is a condition with increased BA in both maternal and fetal environment11-13. For the mother this can lead to pruritus and reduced quality of life11,14. However the fetus can be at increased risk of complications such as: fetal arrhythmia, preterm delivery and intrauterine fetal death compared to non-affected pregnancies11,14-17. Research shows a positive correlation between BA and an increased probability of complications15. This could be explained by data that demonstrated the cumulative increase in cholic and deoxycholic acid led to a concentration-dependent constriction of placental chorionic veins12,14. Vasoconstriction can impair the transport of nutrients to the fetus and result in difficulties. Research has also shown taurine conjugates for example taurocholate are predominately raised in ICP compared to non-ICP16,17.   Even with the data gathered, there is still no definite mechanism of the pathogenesis for these condition nor is it known how BA interact to induce the changes seen.   There has been numerous controversial data on whether BA affect smooth muscle (SM) cells in vascular tissue and whether an overall constriction or relaxation occurs. This project will discuss the mechanisms of SM contraction and relaxation, and explore how BA can influence vasomotor tone.   1.3. Mechanism of smooth muscle contraction Vascular contraction within SM cells can be initiated via different pathways, all eventually leading to the global increase of intracellular Ca2+ concentration (Ca2+i), which stimulates muscle contraction. Other independent pathways exist for example those dependent on membrane potential18, but these will not be the focus.   Increase in intracellular calcium The increase in Ca2+i can occur due to: release of calcium from intracellular stores including sarcoplasmic reticulum (SR) via inositol 1,4,5-trisphosphate (IP3) receptors and ryanodine receptors (RyR)19; and/or from extracellular calcium (Ca2+e) influx, which is mediated chiefly through L-type Ca2+ channels (LTCC)19-21.   IP3 receptor Agonists including endothelin-1 and phenylephrine (PE) interact with specific GPCR resulting in activation of phospholipase C (PLC)22. Activated PLC initiates the phosphatidylinositol pathway whereby the hydrolysis of phosphoinositide 4,5-bisphosphate (PIP2) results in the formation of IP3 and diacyclglycerol (DAG)21,23. IP3 substrates bind to IP3 receptors on SR enabling Ca2+ release into the cytosol21,24. DAG can also indirectly affect the release of Ca2+ via the activation of protein kinase-C (PKC) which has the ability to phosphorylate Ca2+ channels and  affect Ca2+ influx/efflux24. PKC can also indirectly stimulate SM contraction by phosphorylating myosin light chain kinase within the muscle25.   Ryanodine receptors Located on the SR and are modulated by various mediators including LTCC, Ca2+ and calmodulin26. The opening of LTCC enables Ca2+ to enter into the cytosol from extracellular space. RyR are able to detect the increase in Ca2+i and open its channel allowing further release of Ca2+ from the SR into the cytosol24,27. This shows the amplifying effect RyR can have on Ca2+i. However in high Ca2+i, the channel will close preventing further release of calcium from the SR26,27.     L-type Ca2+ channels LTCC opening allows a facilitated movement of extracellular calcium into the cell along Ca2+ concentration gradient24,25. An initial calcium influx is able to aid further release of calcium via RyR. LTCC opening can be triggered by various substrates e.g. PE and angiotensin-II. The contractile effect of PE in mice lacking the channel was significantly reduced compared to the force of contraction in mice with the channel present18. This indicates the importance of the Ca2+ and the channel for vasoconstriction.   Increased Ca2+i results in muscle contraction Calcium forms a complex with calmodulin. This complex activates myosin light chain kinase (MLCK), which phosphorylates myosin light chain (MLC) of myosin II23,28. Phosphorylated MLC allows the 2 contractile filaments in muscle; actin and myosin, to interact and induce cross-bridge cycle formation between them and therefore contraction of the muscle25,29. On the other hand, MLC phosphatase (MLCP) is involved in the removal of a phosphate group from MLC deeming it inactive. This promotes relaxation of the muscle; as no cross bridges occur between the contractile filaments25. The balance between MLCK and MLCP determines the level of contraction within the muscle.   Rho-kinase pathway can prevent MLCP from dephosphorylating MLC. Consequently a greater proportion of MLC remains phosphorylated and continues to maintain SM contraction22,25. Additionally, Rho-kinase also phosphorylates MLC23,30. This indicates the two processes Rho-kinase is able to stimulate SM contraction. Rho-kinase is activated by Rho, which can be stimulated by many agonist e.g. PE, endothelin-1 and thromboxane A222.   1.4 Channels, receptors and mediators involved in VSM and BA interaction ­L-type Ca2+ channels If BA are able to stimulate the opening of these channels whether it is direct or indirect, there will be in influx of Ca2+ which will result in contraction of the VSM. A study demonstrated BA are able to increase Ca2+i in a concentration-dependent biphasic method31. The initial increase is thought to be due to Ca2+ release from intracellular stores and the later persistent increase from the influx of extracellular in Ca2+ via LTCC. This increase in Ca2+ could elicit Ca2+ sparks which triggers various other pathways involved contraction of VSM. This data was true in bovine aorta and human umbilical vein; therefore suggesting a mechanism of vasoconstriction in ICP. Though this was found true for some BA and their conjugates; cholic acid however was unable to increase Ca2+i significantly31.       Endothelin Though there are many isoforms of endothelins, endothelin-1 (ET-1) is the most common and is secreted from the endothelium25. Whilst having an array of functions within different cell types, it is a potent vasoconstrictor of SM cells via interactions with ET-A receptor within VSM25,32-34. ET-A activation induces vasoconstriction by increasing Ca2+i via activation of: specific GPCR and consequently leading to an increase in PLC and therefore IP3 and DAG; stimulation of ryanodine receptors; activating Rho; and opening Ca2+ channels allowing extracellular influx24,25,32,34,35. Alternatively ET-1 can interact with ET-B receptor leading to vasodilation via ET-1 clearance32 and release of nitric oxides and prostacyclin25,35.   Nitric oxide (NO) NO plays an important role in vascular dilation and is important in maintaining basal tone of vessels. It is synthesized from enzymatic reaction of L-arginine conversion into NO using nitric oxide synthase (NOS). Endothelial NOS (eNOS) in its inactive form is bound to membrane protein caveolin and activated by caveolin detachment36. Activation of eNOS can occur from a number of mechanisms including the increase in Ca2+i, stimulating eNOS detachment from caveolin37,38; agonists stimulation e.g. acetylcholine (ACh) stimulates caveolin detachment38,39 as well as protein kinases phosphorylating eNOS37. NO binds to and activates enzyme guanylyl cyclase which increases the production of cyclic guanosine monophosphate (cGMP). cGMP is involved in vasodilation via reducing Ca2+i by limiting its release and increasing its uptake back into SR35. A study had found NO increases in the presence of BA within human umbilical endothelial cells compared to vessels with no BA addition31. Although, different BA affect NO differently, for example chenodeoxycholic acid and deoxycholic acid greatly enhanced NO; whereas cholic acid and TC did not significantly enhance NO31.   Acetylcholine (ACh) ACh is an agonist for muscarinic receptors (MR), of which there are 5 subtypes: M1-M540. The roles of each subtypes differ in different vascular beds. M2R and M4Rs are typically found respectively on the heart and brain, and are coupled with GPCR which stimulate inhibition of Ca2+ channels activation and therefore vascular relaxation41. Alternatively, M1R, M3R and M5R are linked to GPCR which are involved in increasing Ca2+i41. However M3R present on the endothelium induces vasodilation40 due to the activation of eNOS and NO production39,42. Some conjugated BA are known to interact with MR due to the similarities in structure to agonist ACh43,44. Whilst many have failed to focus on TC interaction with MR on the endothelium and VSM; other studies have shown TC ability to bind to M2R in neonatal rat cardiomyocytes45. Here TC reduces the rate of contraction and affects synchronicity, whilst acting as a partial agonist16,17,45. This can provide a possible explanation for the sudden intra-uterine fetal death and fetal dysrhythmias observed in ICP. For this reason, we will be looking at the effect of MR interaction with TC in regards to vascular contractility.   Thromboxane A2 Eicosanoids are derived from the catabolism of arachidonic acid (AA); a polyunsaturated fatty acid synthesised by membrane phospholipid breakdown by phospholipase A2. Free AA undergo various step-wise enzymatic reactions for the synthesis of different eicosanoids e.g. thromboxane A2 (TXA2) which can influence VSM tension. TXA2 is produced via catalytic action of cyclooxygenase (COX) enzymes35,46. COX is involved in the steps of AA breakdown into prostaglandin H2 (PGH2). PGH2 is then catalyzed into TXA2 by thromboxane synthase35,46,47. TXA2 acts on thromboxane-prostanoid (TP) receptor, a GPCR, to facilitate its actions. TP receptors are found on SM cells and the interaction with TXA2 results in the activation of Rho and PLC resulting in raised Ca2+i through the actions of IP3, DAG and PKC thus vasoconstriction28,35,46,48.   GR32191 is a potent thromboxane receptor antagonist. The blockade of the receptor means TXA2 is unable to stimulate constriction. Indomethacin is a known NSAID which inhibits COX enzymes therefore preventing the production of PGH2 and thus TXA2. Indomethacin within placental tissue has been shown to significantly reduce the production of TXA249. As well as COX inhibition, it can also provide inhibition of OAT. Studies have shown indomethacin to inhibit a range of OAT, although inhibition of OAT1 and OAT3 occurs with high affinity50-52.   Bile acid transport As mentioned previously, BA make use of transporters including the family of OAT and OATP. These transporters are predominantly found within the liver and kidney, although are present at other sites53; and eliminate organic anions including BA and drugs54. OAT3 has been identified as one of the many transporters involved in transportation of TC, ursodeoxycholic acid (UDCA), estrone-3-sulfate (E3S) and others55-57. Knockout of OAT3 had shown reduced or complete inhibition of these substrate uptake58. Some of the other transporters TC can also use include: OATP4, OATP1A1 and OATP1B, of which substrates such as UDCA, sulfobromophthalein sodium (BSP), and E3S use also59-62.   At present there is still controversial data regarding BA inducing constriction or dilation in vessel. Some studies show BA eliciting a concentration-dependent vasodilation in pre-constricted vessels2,63. Additionally, others found similar results with deoxycholic acid inducing relaxation after vessel had been incubated with noradrenaline or KCl64. However when this was repeated with cholic acid, TC and UDCA, there was no significant reduction found63. Many of the studies showing BA inducing relaxation had not used TC, the abundant BA in certain pathologies. Additionally studies had premeditated the use of constricted vessel, thereby meaning if BA were able to exhibit a small amount of constriction it would be masked by the vessel being pre-constricted.