1

Table of Contents List of Abbreviations and Acronyms..............................................................................................4 The low concentrations of oxygen in the muscles and joints primarily causes anaerobic respiration and lactic acid build up in the synoviocytes, cells lining the articular joints. . Acid sensing proteins respond to acidic conditions in extracellular spaces and may play a role in the development and pathology of rheumatoid arthritis involving diverse signaling and cellular functional responses. Determination of the exact signaling mechanism and expression of the acid sensing proteins may assist in developing management options for rheumatoid arthritis................................................................................................................................................6 Hypothesis.........................................................................................................................................6 Objective............................................................................................................................................6
Specific Objectives...................................................................................................6

Literature Review.............................................................................................................................7 Acid Sensing Ion Channels (ASICs)...............................................................................................9

ASIC3 co-expression with two nociceptive ion channels.........................................12 ASIC3 expression in small muscle afferents............................................................12 Metaboreception and the axon reflex.....................................................................12 Modulation by interacting proteins ........................................................................15 Modulation by RF-amide Neuropeptides.................................................................15

G- Protein coupled receptors (GPCRs)........................................................................................16 Transient receptor potential cation channel subfamily V member 1(TRPV1).......................17
The structure of TRPV1 consists of 6 trans-membrane domains. At the amino terminal there are sequences that mediate the CaM and PIP2 signalling pathways. ..............................................................................................................................18 Functionality .........................................................................................................19 Sensitization and desensitization...........................................................................19

Rheumatoid arthritis (RA)............................................................................................................22 Conclusion .....................................................................................................................................23 2

List of Abbreviations and Acronyms 7TM ASIC1 ASIC3 ASICs ASlC2 ASlC4 ATP Ca2+ CGRP CIPP DRG FLS GPCRs GPLR GRK MAGI-1b 7 transmembrane receptors Acid-Sensing Ion Channel 1 Acid sensing ion channel Acid sensing ion channels Acid sensing ion channel Acid sensing Ion channel Adenosine triphosphate calcium ion calcitonin gene-related peptide Channel-interacting PDZ protein Dorsal root ganglion Fibroblast - like synoviocytes G- protein coupled receptors G protein-linked receptors G protein coupled receptors kinase membrane-associated guanylate kinase with inverted orientation protein-1 MAPK mRNA Na+ NF200 Mitogen-activated protein kinase messenger ribonucleic acid sodium ion Neurofilament 200 4

NGF NHERF-1 NPAF NPFF P2X3 PGE2 PICK1 PSD-95 RA RANTES TRPV1

Nerve growth factor Na/H exchange regulatory factor-1 Neuropeptide FF/AF Receptors Neuropeptide FF-amide peptide precursor P2X purinoceptor 3 Prostaglandin E2 protein interacting with C-kinase 1 postsynaptic density protein 95 Rheumatoid arthritis Regulated on activation normal T cell expressed and secreted The transient receptor potential cation channel subfamily V member 1

The low concentrations of oxygen in the muscles and joints primarily causes anaerobic respiration and lactic acid build up in the synoviocytes, cells lining the articular joints. . Acid sensing proteins respond to acidic conditions in extracellular spaces and may play a role in the development and pathology of rheumatoid arthritis involving diverse signaling and cellular functional responses. Determination of the exact signaling mechanism and expression of the acid sensing proteins may assist in developing management options for rheumatoid arthritis.

Hypothesis Acid sensing proteins are expressed in human synoviocytes and contribute to the pathology of rheumatoid arthritis.

Objective To determine the pathological impact of acid sensing on proteins in rheumatoid arthritis

Specific Objectives
a.

Measure the expression of Acid Sensing Proteins in normal and RA human

synoviocytes. b. Measure specific acid induced cell signaling and functional response in

synoviocytes.
c.

Determine the effects of Acid Sensing Proteins inhibition on pathological responses

of synoviocytes.

Literature Review Introduction Rheumatoid arthritis (RA) is a symmetrically distributed disorder that is systemic autoimmune and chronic whose primary effect is on the joints of the feet and hands . Estimates on the pervasiveness of RA point to 0.5-1.5 percent of the entire global population according to Kobelt and Johnson (2008). Nonetheless, its common occurrence is in the ages starting fifty onwards although it can affect people at any ages especially those past the thirty year mark . Indeed, there are numerous theories whose credibility is indisputable, linking ageing and RAs onset. Nevertheless, the free radical theory of ageing is predominantly popular among all of them . The theory surmises that an accumulation of free radicals occurs throughout a lifespan while the immune system functions to inhibit the accumulation . On the other hand, further theoretical elucidations on RAs development in females with infertility complexities point to low progesterone and oestrogen levels among them due to their perceived frequent RA development in comparison to their counterparts whose progesterone and oestrogen levels are normal . Besides, Temprano et al (2011) contend that pregnancy lessens RAs activity and momentarily restores normal joint capsule function during gestation. The frequency of theories attempting to explain RAs development leads Clancy et al (2011) to term it a homeostatic imbalance that is normally termed an autoimmune disorder with unclear aetiology yet all in its progressiveness and severity. RAs characterization entails synovial cartilage and bone destruction and inflammation whose mediation is by prominent tumour-like synovial fibroblasts referred to as pannus expansion . Active synovium may need higher consumption of oxygen metabolically and joints that are chronically inflamed produce increased pressure following the huge joint effusion amount and the 7

consequent reduced supply of blood . Consequently, there are suggestions that the existence of rheumatoid synovial microenvironments is normally under hypoxic situations .Acttually studies in the past indicate that rheumatoid SFs are scidid, hypoxic and are chaaterised by increased lactae concentration anaerobic (Jurado, et al., 2008) his exhibits that an alteration to anaerobic metabolism happens in hypoxic synovium Several studies suggests that fibroblast-like synoviocytes (FLS) actively participate in RAs progression . Nonetheless, despite the production of MMPs and inflammatory cytokines by FLS are profuse in SF and the rheumatoid synovial microenvironments being hypoxic few studies investigate the role of acid sensing proteins in the development of RA (Jurado, et al., 2008). Cellular mechanisms for the testing of amino acids and proteins emerge to which suggests a role for each of the intracellular proteins. In additions, transporters of specific amino acids have a role in determination of the testing facilities. Determination of the exact signaling mechanism and expression of the acid sensing proteins may assist in developing management options for rheumatoid arthritis. The role played by cytokines in the process cannot be downplayed with TNF, IL-1 and IL-6 being the most notable cytokines. These cytokines also enhance the up regulation of adhesion molecules, induction of the inflammatory response and its mediators and activation of the osteoclasts apart from being chemotactic. In RA, CD4+ T cells, macrophages, and B cells intrude the synovium and at times their organisation into secretive lymphoid combines with germinal centres . The intimal linings hyperplasia is a result of marked rise in FLS and macrophage-like synoviocytes. In addition, degradative enzymes that are locally expressed including serine proteases, aggrecanases and metalloproteinases destruct the articular structures by digesting the extracellular matrix .

Figure 1. Comparing the normal and inflamed joints

Source: Rheumatory Arthritis (2012)

Acid Sensing Ion Channels (ASICs) Lamb, (2012) states that fluctions in nutrients differ from the normal portion to cancer cells and therefore may lead to future methods of cancer therapy. He suggests that use of nutitional sources can help cancer cells ready to tear up tomor to separate the tomor from the good cells. ASICs are cation channels that conduct and are permeable to Na+ at low rates and are activated by extracellular protons. However, ASC1a shows low calcium ion permeability . These channels are voltage insensitive/independent and occur in the neurons. There occur five major proteins of this family which are encoded by four genes recognised as ASIC1, ASIC2, ASIC3, and ASIC4. Out of the five proteins of ASICs genes, three of the transcribed proteins form ion channels, while the remaining two proteins are splice variants. ASCIs are expressed in the peripheral nervous system (PNS) while in the central nervous system the subunits ASlC1a, 2a and

4 are expressed. The ASICs mRNA is located outside the sensory neurons within the ganglia with the exception of ASIC3 which is found within the sensory neurons.

Figure 2: ASIC family of ion channels: ACCN1?? ACCN2?? ARE THEY CORRECT IN THIS
FIGURE? .

The activation of ASICs is mainly through the action of extracellular protons but ASIC1 and ASIC2 in particular, do not rely on the acidic pH formed by an increased H+ concentration for activation. Their activity and major characteristics are however achieved via association with other isoforms to form an active protein. Structurally similar to degenerins, ASICs were thought to be mechanosensitive ion channels degenerins , as they were expressed as mechanosensory specific skin endings in rodents .

10

Behavioural changes in touch sensitivity were noted in ASIC knockout mouse models as compared to others, . Trimeric in nature, ASICs are made up of a combination of different subunits. The subunit ASIC2b is non-functional on its own but is important in modulating channel activity when forming part of a heteromultimers. The ASIC4 subunit, on the other hand, has no known function. When the extracellular pH drops as a result of a high concentration of protons, ASICs are opened . The degenerin super family of ion channels, in which ASICs fall, are epithelial sodium channels , that are specific to sodium ions and are inhibited in function by the diuretic drugs such as amiloride. They are membrane spanning proteins that produce two regions with a large extracellular loop and an intracellular amino and terminal carboxyl groups. To form a functional ion channel, multiple ASICs subunits must assemble. ASIC1a is unique from the other forms in that it allows for the permeability of Ca2+in addition to Na+ thereby additionally allowing the entry of Ca2+ into the neurons. This however only occurs when the protein is in its homomeric form. Extracellular calcium ions can inhibit or stimulate the ion channels and thus also act in the regulation of their activity. The displacement of Ca2+ by H+ leads to the activation of ASIC3. The ions are displaced from a high affinity Ca2+ binding site located extracellular of the open pore . Variations of physiological pH thus play a role in the activation of ASICs. A transient inward triggered by the positively charged ions causes a rapid desensitisation and due to this property, ASICs are able to detect dynamic fluctuations in the pH of the system. As a functional role, ASIC3 during episodes of ischemic pain sense changes in the pH of the system that lead to lactic acidosis. This is due to the generation of large fluxes in current at the dorsal root ganglion of the sensory neurons that supply the nervous impulses to the heart , where 11

angina occurs. Also, small changes in the extracellular pH occurring in muscles due to metabolic stress, pH 7.4 to 7.0, are detected and responded to. The co-localisation of ASIC3 and ASIC2 occurs in the dorsal root ganglion sensory and these proteins are more commonly found in larger . However, when ASIC3 is co-expressed with calcitonin gene-related peptide (CGRP), it is found in large and small trigeminal ganglion neurons .

ASIC3 co-expression with two nociceptive ion channels Characterise nociceptive neurons carrying ASICs, P2X3 and ATP- gated ion channels were coexpressed . These were thought to be pain transducers and a capsaicin receptor of the transient receptor potential cation channel subfamily V member 1 (TRPV1) . An observed overlap between ASIC3 and P2X3 was noted, with co-expressed cells being large with P2X3 positive cells staining equally positive for ASIC3. This point towards there being a population of co-expressed sensory neurons with overlaps also noted between ASIC3 and TRPV1.

ASIC3 expression in small muscle afferents A necessitated low blood flow to the muscles may result in ischemic pain. However, as noted during thermoregulation and response to adrenaline boosts in the flight or fright response, reduced blood flow to the skin is without any pain.

Metaboreception and the axon reflex With a small change in pH, especially due to lactic acidosis build up during anaerobic metabolism in the muscles, causing the ASCIs to open up , , metaboreception is considered to be a 12

subset of nociception . Thus, compensatory changes in the peripheral tissues are triggered by low activity levels while high activity levels trigger the sensation of pain . To be considered as a sensor for ischemic pain, ASIC3 should ideally be located in the dorsal root ganglion afferents of the cardiac muscles which again should be enriched by sensory neurons that supply it as compared to the skin as ischemic pain is characteristic to muscles other than the skin. The overlap between ASIC3 and the vasodilator peptide CGRP is noted in a majority of AISC3 positive sensory neurons that innervate the muscles that also co-express CGRP. Lightly myelinated axons fibres co-express the three markers due to their expression of NF200, a marker of myelinated axons (Jurado, et al., 2008). The axon reflex, caused by the peripheral release of CGRP occurs due to vasodilation and extrvasations due to the activation of C fibres . The co-expression of all three proteins; ASIC3, TRPV1 and CGRP, on the afferent vessels of the arterial muscle might point to the fact that all three function as sensors and effectors in regulating localised flux of blood in response to elevated lactic acid levels and rising temperatures of the muscle. This co-expression thus assists the cells in detecting a broad range of acid sensitivities.

13

Figure 3: ASICs as displayed in the primary afferent nociceptors and in the spinal cord neurons (Molliver et al., 2005)

In the central and peripheral nervous systems, ASIC1 and ASIC3, respectively, play key roles together with extracellular modulators and interacting proteins. The activity of ASIC3 is upregulated by activators of the protein kinase C (PKC) pathway which are released during peripheral inflammation that leads to an elevation of mRNA transcript levels of ASIC3 in the dorsal root ganglion and the spinal cord. Protons released at either the pre or post synaptic membranes could activate .

14

Modulation by interacting proteins ASICs and their interacting protein have been associated with the function to control surface expression and the sub cellular distribution of ion channels. The PDZ binding motif of ASICs at their C termini interacts with several other PDZ motif containing proteins. The interaction between protein and C-kinase 1 (PICK1) that takes a part in the up-regulation of ASICs by protein kinase C , co-localizes and associates in the nervous system especially with ASIC1 and ASIC2 . ASIC3 association with proteins such as Channel-interacting PDZ protein (CIPP) increases or decreases the rate and level of surface expression of the ion channel without causing a significant change in its properties. This is due to heterologous expression systems . Integral membrane proteins such as stomatin expressed in sensory neurons associate with ASIC1a, ASIC2a, and ASIC3 and function to reduce the ion channel current and to increase the rate of desensitization of the channels .

Modulation by RF-amide Neuropeptides The potency of the H+ gated ion channels current by causing an increase in the peak amplitude or by slowing the inactivation of the ASICs is directly modulated by FMRF-amide at pH 7.4 and structurally related peptides on the channels . The neuropeptide FF/AF receptors (NPAF) and the neuropeptide FF-amide peptide precursor (NPFF), are expressed in the central nervous system and especially so in the spinal cord where they are in higher levels and their expression is enhanced during chronic inflammation. This modulation is in response to noxious acidosis that may occur when the channel is closed .

15

G- Protein coupled receptors (GPCRs) GPCRs referred to as seven-spanning transmembrane domain receptors or G proteinlinked receptors (GPLR) (Fig. 1). This protein family of transmembrane receptors recognize biomolecules in the extracellular space and initiate the activation of transduction signal pathways and appropriate cellular responses.
N4 H

P s am m ra e la m e b n

CO OH

Figure 4: Structure of GPCRs GPCRs consist of an intracellular C-terminal, 7 trans-membrane domains and an extracellular Nterminal. The G/ subunits attach near the carboxyl terminus

These receptors are activated when they bind their ligands which are normally peptides of varying sizes. GPCRs have two main principal transduction pathways, the cAMP and the phosphatidylinositol signal pathway . G protein coupled receptor has some units that are acid sensitive, namely, GPR4, GPR65, GPR68, and GPR132. These subunits, in the event of a fall in the extracellular pH to between 6.4-6.8, are activated as ASICs. These G protein receptors have been initially de-orpharnized as receptors of lipid messengers. Recently, GPR4, GPR68 (OGR1), GPR65 (TDAG8), and GPR132 (G2A) have been reported to be proton sensing receptors that activate either the phosphoinositol or cAMP pathways. It has been found out that LPC functions as an inverse agonist, and not as antagonist for GP132, because LPC inhibits the PH dependent activation of GPR132 in a dose dependent manner. 16

GPR4, GPR65 and GPR68 are proton sensing receptors. The cells that express GPR65 increase the level of cAMP (Lamb, 2012) in response to the neutral to the acidic extracellular pH. Due to this, cAMP accumulation is suppressed by pschosines inhibition of PH dependent effects in GPR68 and GPR4 expressing cell. This has also been observed to indicate that pschosine acts as an inverse agonist of GPR4, GPR65, and GPR68. Chemokines play a vital role in the pathology and treatment of arthritis by mediating chemotaxis and activation of the leukocytes in an immune response mechanism . Receptor antagonists to monocyte chemoattractant protein-1 (MCP-1) and RANTES affect the progression of the disease towards a positive outcome . These two chemokines and their receptors thus have an important role in arthritis by initiating and maintaining the local inflammatory process that enhances the recruitment of both monocytes and lymphocytes into the joints . Chemokine and 2 adrenergic receptors are examples of GPCRs which are turned off by kinases of the GRK . The GRK kinases switch off the receptors leading to a loss of the receptors responsiveness to its ligand, in a process referred to as homologous desensitization. The residues of serine/threonine on the carboxy tail of the intracellular loops of the receptor are phosphorylated by the kinases. The phosphorylated receptor residues act as docking sites for inhibitory proteins arrestins which potentially inhibit the coupling of the receptor and the G proteins . GRKs receptor substrates are embraced in a wide variety of functions including neurotransmission and immune responses, used to transduce signals through various intracellular second messengers . Transient receptor potential cation channel subfamily V member 1(TRPV1) The capsaicin and vanilloid receptor 1 protein is encoded by the TRPV1 gene in humans. This group of ion channels detects and regulates body temperature, and also provides sensations of high temperature (heat) and pain (nociception). Structurally, TRPV1 is 838 amino acids long with a 17

molecular weight of 95kDa. It is 6 transmembrane domains, a C-terminal cytosolic region that is 132 amino acids long, and an N-terminal region that is 432 amino acids long (Fig. 2).

P s amm r n la m e ba e

TP R bx o

N4 H CM a P 2 IP

CO OH

Figure 5: TRPV1 structure. The structure of TRPV1 consists of 6 trans-membrane domains. At the amino terminal there are sequences that mediate the CaM and PIP2 signalling pathways.

The TRPV1 is widely distributed across many tissues and is activated in a polymodal manner by both chemical and physical stimulators. Different TRPV1 activators are tabulated below.
Table 1: Physical and chemical activators of TRPV1

Activator Vanilloids Lipids Protons Cations Heat

Examples Capsaicin, olvanil, resiniferatoxin Anandamide, oleoyldopamine, endocannabinoid), N-arachidonoyl Dopamine, 18 20 carbon N-acylethanolamines, 12-hydroperoxyeicosatetraenoic acid H+ K+, Rb+, Cs+, Na+ and Li+ ions

18

Voltage Source:

Functionality The TRPV1 cation channel is non selective in nature and can be ideally stimulated by a wide range and different types of endogenous and exogenous stimuli , such as acidic conditions (increased H+ concentration), N-arachidonoyl dopamine, vanilloids, like capsaicin and heat , that leads to a painful and burning sensation . Located in the PNS nociceptive neurons, TRPV1 receptors transmit and modulate pain (nociception) as well as play a role in the integration of diverse painful stimuli .

Sensitization and desensitization During tissue damage, injury and in the inflammation process, prostaglandins and bradykinin, are released as inflammatory mediators into the system and they in turn lead to an increase in nociceptors sensitivity to noxious stimuli. This manifests as an increased sensitivity to the stimuli of pain or to the increased sensation of pain in response to non-pain stimuli. The phospholipase C (PLC) pathway is activated by these pro-inflammatory agents leading to the phosphorylation of TRPV1 by PKC leading to its sensitization. The cleavage of phosphatidylinositol 4, 5-bisphosphate (PIP2) by PLC results in then disinhibiton or TRPV1 and consequently contributes to the sensitivity of TRPV1 to noxious stimuli. Desensitization may however occur when a long exposure to the stimuli is affected due to decreased TRPV1 activity mediated by an increased intracellular Ca2+ due to its influx from the extracellular space. TRPV1 synthesis occurs in the dorsal root ganglia sensory neurons which are then transported along the central and peripheral axons to the spinal dorsal horn and to the skin and 19

viscera organs, respectively . TRPV1 is critical for a number of sensory issues in the skin, such as high temperature (heat), nociception or pain sensation, inflammatory hyperalgesia , allodynia, and neuropathic nociception (pain) . Capsaicin, used in the management of arthritic pain, works by temporarily desensitizing the receptor TRPV1 which, is also its only known receptor ,, and by reducing blood flow in capsaicin sensitive afferents , Despite its role in arthritis, the exact mechanisms by which TRPV1 is implicated are not fully understood. The sensation of pain leads to gene expression of the immediate genes in the sensory neurons. The extracellular-regulated kinase (ERK) pathway, part of the serine/threonine protein kinases of the MAPK family, links nocioception to altered gene expression by transducing the extracellular stimuli into through its signal pathway to achieve an intracellular transcriptional and post-translational response. After activation by mediators of cellular stress and inflammation, growth factors and mitogens, MAPK translocates into the nucleus where it binds to transcription factors and causes the regulation of relevant genes transcription to achieve many cellular activity, such as proliferation, and production of cytokines and other regulatory response factors during inflammation . The MAPK exist in numerous isoforms and are activated by a sequence of upstream phosphorylation and dephosphorylation reactions mediated by kinases, hence forming a complex biochemical cascade which is critical in the overall regulation of the inflammatory process . In respect to arthritis, ERK pathway inhibition results in an attenuated nociception and inflammation . The duration of arthritis is correlated with increased number ERK positive neurons in the spinal neurons and also this increase is noted in inflamed joint movement.

20

21

TABLE 2: summary of PH ranges, tissue distribution and inhibitors of acid sensing proteins.
ASP ASIC1a ASIC1b ASIC2a ASIC2b ASIC3 PH act 6.2-6.8 5.1-6.2 4.1-5.0 N/A 6.2-6.7 Expressed in PNS, brain, retina, taste cells, bone PNS, taste cells PNS, brain, retina, taste cells, bone PNS, brain, retina, taste cells PNS, brain, retina, taste cells, bone, inner ear, lung epithelial cells Plow in PNS, brain, spinal cord, retina, pituitary gland, inner ear CNS , non-neuronal tissues such as keratinocytes, mast cells, hair follicles, smooth muscle, bladder, liver, kidney, spleen and lungs To be completed Inhibitor Amiloride, NSAIDs Amiloride Amiloride N/A Amiloride, NSAIDs N/A

ASIC4 TRPV1

N/A

7.4-5.5

OGR1 (GPR68)

To be complet ed To be complet ed To be complet ed

To be completed

G2A

To be completed

To be completed

GPR65 (TDAG8)

To be completed

To be completed

Rheumatoid arthritis (RA) This is a long term illness causing inflammation of joints and any surrounding tissues. In the pathology of rheumatoid arthritis, pro-inflammatory cytokines have been noted due to their biological function as mediators of inflammation, cell activation and growth factors . The role of the nervous system in the pathogenesis of rheumatoid arthritis has been shown with the 2 adrenergic receptor antagonist delaying the onset and severity of joint pains and injury in arthritis .

22

Pro-inflammation of the nervous system in rheumatoid arthritis during clinical studies has shown that the distribution of the synovitis is symmetrical ,

Conclusion
The acid-sensing protein ASIC, G-protein-coupled receptors (GPCRs) and the TRPV1 receptor may play a critical role in the pathogenesis of RA through acid sensing, modulation of expression and release of hyaluronan, and induction of signalling pathways that aggravate inflammation. Understanding the molecular mechanisms that underline these processes is important as it can help to devise novel therapeutic agents for the treatment of RA.

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