Academic literature on the topic 'Adenosine-signalling'

Adenosine 5′-triphosphate acts as an extracellular signalling molecule (purinergic signalling), as well as an intracellular energy source. Adenosine 5′-triphosphate receptors have been cloned and characterised. P1 receptors are selective for adenosine, a breakdown product of adenosine 5′-triphosphate after degradation by ectonucleotidases. Four subtypes are recognised, A1, A2A, A2B and A3 receptors. P2 receptors are activated by purine and by pyrimidine nucleotides. P2X receptors are ligand-gated ion channel receptors (seven subunits (P2X1-7)), which form trimers as both homomultimers and heteromultimers. P2Y receptors are G protein-coupled receptors (eight subtypes (P2Y1/2/4/6/11/12/13/14)). There is both purinergic short-term signalling and long-term (trophic) signalling. The cloning of P2X-like receptors in primitive invertebrates suggests that adenosine 5′-triphosphate is an early evolutionary extracellular signalling molecule. Selective purinoceptor agonists and antagonists with therapeutic potential have been developed for a wide range of diseases, including thrombosis and stroke, dry eye, atherosclerosis, kidney failure, osteoporosis, bladder incontinence, colitis, neurodegenerative diseases and cancer.

Adenosine is released in large amounts during myocardial ischemia and is capable of exerting potent cardioprotective effects in the heart. Although these observations on adenosine have been known for a long time, how adenosine acts to achieve its anti-ischemic effect remains incompletely understood. However, recent advances on the chemistry and pharmacology of adenosine receptor ligands have provided important and novel information on the function of adenosine receptor subtypes in the cardiovascular system. The development of model systems for the cardiac actions of adenosine has yielded important insights into its mechanism of action and have begun to elucidate the sequence of signalling events from receptor activation to the actual exertion of its cardioprotective effect. The present review will focus on the adenosine receptors that mediate the potent anti-ischemic effect of adenosine, new ligands at the receptors, potential molecular signalling mechanisms downstream of the receptor, mediators for cardioprotection, and possible clinical applications in cardiovascular disorders.

The mammalian cochlea is the sensory organ of hearing with a delicate, highly organised structure that supports unique operating mechanisms. ATP release from the secretory tissues of the cochlear lateral wall (stria vascularis) triggers numerous physiological responses by activating P2 receptors in sensory, supporting and neural tissues. Two families of P2 receptors, ATP-gated ion channels (P2X receptors) and G protein-coupled P2Y receptors, activate intracellular signalling pathways that regulate cochlear development, homeostasis, sensory transduction, auditory neurotransmission and response to stress. Of particular interest is a purinergic hearing adaptation, which reflects the critical role of the P2X2 receptor in adaptive cochlear response to elevated sound levels. Other P2 receptors are involved in the maturation of neural processes and frequency selectivity refinement in the developing cochlea. Extracellular ATP signalling is regulated by a family of surface-located enzymes collectively known as “ectonucleotidases” that hydrolyse ATP to adenosine. Adenosine is a constitutive cell metabolite with an established role in tissue protection and regeneration. The differential activation of A1 and A2A adenosine receptors defines the cochlear response to injury caused by oxidative stress, inflammation, and activation of apoptotic pathways. A1 receptor agonism, A2A receptor antagonism, and increasing adenosine levels in cochlear fluids all represent promising therapeutic tools for cochlear rescue from injury and prevention of hearing loss.

Background: There is clinical and experimental evidence for altered adenosine signalling in the fetoplacental circulation in pregnancies complicated by diabetes, leading to adenosine accumulation in the placenta. However, the consequence for fetoplacental vasocontractility is unclear. This study examined contractility to adenosine of chorionic vessels from type 1 diabetes mellitus, gestational diabetes mellitus and normal pregnancies. Methods: Chorionic arteries and veins were isolated from human placenta from normal, gestational diabetes mellitus and type 1 diabetes mellitus pregnancies. Isometric tension recording measured responses to adenosine and the thromboxane A2 analogue U46619 (thromboxane A2 mediates fetoplacental vasoconstriction to adenosine). Adenosine and thromboxane prostanoid receptor protein expression was determined by immunoblotting. Results: Adenosine elicited contractions in chorionic arteries and veins which were impaired in both gestational diabetes mellitus and type 1 diabetes mellitus. Contractions to potassium chloride were unchanged. Adenosine A2A and A2B receptor protein levels were not different in gestational diabetes mellitus and normal pregnancies. Contractions to U46619 were unaltered in gestational diabetes mellitus arteries and increased in type 1 diabetes mellitus arteries. Overnight storage of vessels restored contractility to adenosine in gestational diabetes mellitus arteries and normalized contraction to U46619 in type 1 diabetes mellitus arteries. Conclusion: These data are consistent with the concept of aberrant adenosine signalling in diabetes; they show for the first time that this involves impaired adenosine contractility of the fetoplacental vasculature.

Phototaxis and thermotaxis by Dictyostelium discoideum slugs on water agar were impaired by the presence in the agar of adenosine, which is a cyclic AMP receptor antagonist in aggregating amoebae. Caffeine, and presumably its analogue theophylline, inhibit cyclic AMP signalling in aggregating amoebae of D. discoideum. Both compounds perturbed slug behaviour in a similar manner to adenosine, as did both ammonium and sulphate ions. (NH4)3SO4 is known to perturb cyclic AMP binding to its receptor, and ammonia is an inhibitor of cyclic AMP signalling in aggregating amoebae. The receptor agonist, cyclic AMPS, disrupted slug organization and impaired phototaxis when present at concentrations high enough to saturate cyclic AMP receptors and compete effectively with endogenous cyclic AMP signals of similar magnitude to those observed during aggregation. Taken together with the considerable circumstantial evidence for cyclic AMP signalling in slugs, these results support a role for cyclic AMP signalling in slug behaviour.

Adenosine and adenosine triphosphate are involved in purinergic signalling which plays important role in control of immune system. Much data have been obtained regarding impact of purinergic signalling on dendritic cells, macrophages, monocytes and T lymphocytes, however less attention has been paid to purinergic regulation of B cells. This review summarizes present knowledge about ATP- and Ado-dependant signalling in B lymphocytes. Human B cells have been shown to express A2A­-R and A­3-R and each subtype of P2 receptors. Surface of B cells exhibits two antagonistic ectoenzymatic pathways, one relays on constitutive secretion and resynthesis of ATP while the second one depends on degradation of adenosine nucleotides to nucleosides and their subsequent degradation. Inactivated B cells remain under suppressive impact of autocrine and paracrine Ado however activated B lymphocytes increase ATP release and production. ATP protects B cells from suppressive impact of Ado and exerts pro-inflammatory impact on target tissues, it is also involved in IgM release. Ado synthesis however is related with optimal development, implantation and maintenance of plasmocyte population in bone marrow during primary immune response. Moreover Ado plays important role in immunoglobulin class switching which is a key mechanism of humoral immune response. Disruption of purinergic signalling is related with severe clinical implications. Impairment of Ado production in environment of B cells is one of the factors responsible for common variable immunodeficiency. List of evidence suggests also that dysfunction of immune system observed during diabetes may in part depend on disrupted ATP and Ado metabolism in B cells.

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

The release and clearance of adenosine are reasonably well-documented in the mature CNS but relatively little is known about how adenosine signalling changes during postnatal development. The activation of presynaptic A1 receptors (A1R) at cerebellar parallel fibre terminals is known to inhibit synaptic transmission and the expression of A1R has been observed in mature rat cerebellar slices. However its distribution during development or in relation to parallel fibre–Purkinje cell (PF-PC) synapses has not previously been described. In the mature cerebellum blockade of presynaptic A1R at PF-PC synapses enhances synaptic transmission suggesting an inhibitory adenosine tone and an extracellular purine tone is detectable with microelectrode biosensors under basal conditions. The active release of adenosine can be stimulated with trains of activity in the molecular layer of mature slices although this does not appear to be a source of the basal extracellular adenosine tone. This study used immunohistochemistry to determine the distribution of A1R at PFPC synapses in cerebellar slices at postnatal day 3 prior to PF-PC synapse formation, postnatal days 8-14 and postnatal days 21-28. This study also used cerebellar slices from rats at postnatal days 9-14 to investigate the pharmacological profile of the immature rat PF-PC synapse with electrophysiology and microelectrode biosensors. The immunohistochemistry suggests that A1R are widely distributed across Purkinje cell bodies and their dendrites and within the granule layer of the cerebellum and that its expression does not change during development. The same staining patterns were also observed prior to PF-PC synapse formation. Application of adenosine resulted in a variable A1R-mediated inhibition at immature PF-PC synapses. This did not appear to be gender-specific or correlated with age of rat and the synapses otherwise appeared identical in their properties. The comparison of log concentration-response curves generated for an A1R agonist suggested that some A1R may have a lower efficacy at this stage of development. Blockade of presynaptic A1R at immature PF-PC synapses suggested that an inhibitory adenosine tone is low or absent at this stage of development and is not the result of a low A1R expression or developmental differences in A1R efficacy. Inhibition of adenosine clearance via adenosine deaminase, adenosine kinase and equilibrative transporters had little effect on synaptic transmission suggesting that little adenosine is moving between the intracellular and extracellular spaces under basal conditions in immature slices. Active adenosine release measured by electrophysiology and microelectrode biosensors could be stimulated with hypoxia in immature slices but this was delayed and slower in comparison to the release observed in mature slices. Adenosine could not be actively released at immature PFPC synapses in response to electrical stimulation in the molecular layer.

Myocardial fibrosis contributes to the pathogenesis of diverse forms of cardiac hypertrophy. Recent evidence suggests a crucial role for purinergic signalling in the formation of tissue fibrosis. In particular, inhibition of adenosine A2A receptors (A2AR) in animal models for hepatic and skin fibrosis resulted in a reduction of fibrosis formation. The specific hypothesis of this investigation was that myocardial stress results in increased interstitial levels of adenosine, which activate cardiac fibroblasts via adenosine A2AR. Accordingly, the specific objective of this investigation was to delineate the role of adenosine A2AR signalling in the development of myocardial fibrosis. In vitro, isolated cardiac fibroblasts demonstrated a significant increase in collagen secretion into CCM upon specific A2AR stimulation. This stimulatory effect was inhibited by the addition of a specific A2AR inhibitor. In vivo, models for cardiac hypertrophy including the transgenic cardiac actin E99K hypertrophic cardiomyopathy model, and a transverse aortic constriction (TAC) model of pressure overload, were investigated as murine models for myocardial fibrosis (MF). In cardiac actin E99K hearts the occurrence of cardiac fibrosis was associated with ventricular dysfunction, as well as energetic perturbations. In addition, a direct linear correlation between myocardial collagen content and interstitial adenosine levels was found in actin E99K hearts upon microdialysis experiments. Crossbreeding of actin E99K and A2AR knock-out (KO) mice resulted in a significant reduction of myocardial collagen content and fibrosis in E99K heterozygous A2AR KO animals compared to E99K heterozygous A2AR wild-type (WT) animals. Further, adenosine A2AR KO mice undergoing transverse aortic constriction demonstrated significantly less fibrosis formation compared to constricted WT mice. This was associated with a significant rescue of cardiac function. In addition, pharmacologic adenosine A2AR inhibition using the antagonist ZM241385 demonstrated a partial rescue of myocardial fibrosis in both TAC and E99K animals. These data suggest that adenosine the A2AR plays a crucial role in the formation of myocardial fibrosis in a variety of cardiac pathologies and that this pathway is susceptible to pharmacologic modulation. A2AR manipulation may contribute to further understanding of pathophysiological pathways in the development and progression of cardiac disease and represents an excellent therapeutic target for clinically available A2AR antagonists.

African trypanosomiasis is caused by a unicellular eukaryote that parasitizes multicellular organisms and causes medically and economically important diseases in humans (Human African Trypanosomiasis) and their domestic animals (African Animal Trypanosomiasis). Incidence is currently declining due to the application of present chemotherapy, although the drugs are old, toxic, difficult to administer and in some cases expensive, and of diminishing efficacy due to resistance. However, this trend needs to be sustained with the discovery of new compounds active against the resistant strains. These new treatment options must meet the current pharmacological requirements, must be parasite specific and must be relatively cheap to produce. Pharmacological manipulation of phosphodiesterases (PDEs), which hydrolyse cyclic Adenosine Monophosphate (cAMP), have been extensively studied in humans and found to have great therapeutic effect. Kinetoplastid genomes code for the same set of cyclic nucleotide-specific class 1-type phosphodiesterases, with catalytic domains similar to those of human PDEs. The locus of Trypanosoma brucei PDEB1/2 was found to be essential, by either genetic manipulation or the use of the pharmacological inhibitor CpdA, but therapeutic exploitation of TbPDEB1 has so far been hampered by its catalytic domain similarity to human PDEs. However, investigating the unique downstream cAMP signalling cascade, which includes the recently identified cAMP Response Proteins (CARPs), could reveal potentially new trypanosome-specific therapeutic targets. In this study we show that single knockout (sKO) of the CARP genes causes a decreased susceptibility to CpdA, and that null mutants of CARP2-4 display significantly increased intracellular and extracellular cAMP levels. A double knockout (dKO) of CARP2 also shows a significant growth defect. Conversely, overexpression of CARP3 causes a growth delay in both WT s427 and CpdA resistant (R0.8) cells, when exposed to CpdA, and were more sensitive to CpdA compared to other CARP overexpressing cells as well as the WT s427 and R0.8 controls. These cells also have significantly higher intracellular and extracellular cAMP levels relative to the control lines and the other CARP overexpressors. In the cells overexpressing CARP3, whether WT s427 or R0.8, the cellular content of both CARP3 messenger Ribonucleic Acid (mRNA) and protein decreases extensively within 6 h of CpdA exposure. The CARP3 protein has domains that are indicative of a role in protein-protein interaction, signalling, regulation and degradation and probably undergo acylation. Some experimental confirmation of these traits was obtained, using Co-immunoprecipitation (Co-IP) and Mass Spectrometry (MS), with the identification of Adenylyl cyclase (AC) GRESAG4s (also found through RNAinterference Target Sequencing (RITseq) and confirmed by quantitative Reverse Transcription PCR), and proteasome regulatory proteins (PRNs) in addition to membrane and flagellar binding proteins, as potential interactors. Preliminary Immunofluorescence (IF) microscopy showed that CARP3 localizes to plasma membrane ad the flagellum, CARP2 to specific bodies/organelles in the cytosol and CARP1 in the cytosol. RNA sequencing of overexpressing CARP3 reveals differentially expressed proteins involved in cell cycle and cytokinesis as well as transport proteins with several transmembrane domains, consistent with the proposed acylated membrane localisation, and with interaction of CARP3 with membrane proteins and ACs (GRESAG4 isoforms).Thus CARP3 has the domains, interactions and localization consistent with a regulatory role in cAMP metabolism. Thus the CARPs and especially CARP3 are interesting biological molecules providing key new insights into signalling and the cell biology of the trypanosome.

Adenosine is precursor and a metabolic intermediate of adenosine triphosphate in energy transfer, and cyclic adenosine monophosphate in signal transduction. Recent studies have demonstrated that the role of adenosine in the body is much more than just structural as it can also behave as an important regulator of homeostatic functions. Adenosine signalling relies on the activation of the A₁, A₂A, A₂B and A₃ adenosine receptors. Through the development of pharmacological tools and genetic knockout mouse models of specific receptor subtypes, the involvement of these receptors in various physiological systems is quickly being established. This thesis investigates the function of adenosine in the digestive system and specifically how adenosine regulates the release of gastric and pancreatic peptides. With the use of a novel vascularly perfused isolated mouse stomach model and specific A₁ and A₂A receptor knockout animals, the role of adenosine on the release of somatostatin and ghrelin was determined. Lower concentrations of adenosine can inhibit the release of somatostatin and ghrelin via the activation of A1 receptors, while higher concentrations can stimulate their release via activation of A₂A receptors. Given the importance of somatostatin in regulating gastric acid secretion and motility, and ghrelin in regulating systemic energy balance, better understanding of how the release of these two peptides is regulated may reveal potential therapies for eating disorders, gastrointestinal dysfunctions and metabolic diseases. In the pancreas, adenosine was shown to regulate both insulin and glucagon secretion from the pancreatic islets. Studies presented in this thesis demonstrate that adenosine signalling interacts with the effects of the incretin hormone GLP-1 in the pancreas such that concomitant administration of adenosine and GLP-1 in the perfused pancreas induced greater insulin release than GLP-1 administration alone. Furthermore, A₁ receptor knockout mice exhibited more frequent pulses of insulin secretion, which may have contributed to their superior glucose tolerance compared to wild type control mice. These findings on the role of adenosine signalling in the pancreas may have implications in the etiology of diabetes mellitus. The involvement of adenosine signalling in the digestive tract further illustrates the importance of adenosine as a metabolic regulator in homeostasis.

Platelet activation is a critical physiological event, whose main role is to prevent excessive blood loss and repair vessel wall injuries. However, platelet activation must be controlled to prevent unwanted and exaggerated responses leading to the occlusion of the blood vessel. The endothelial-derived inhibitors prostacyclin (PGI2) and nitric oxide (NO) are known to play a critical role in the control of platelet activity, although the mechanism underlying their actions remains unclear beyond the triggering of cyclic nucleotides signaling pathways. The aim of this study was to improve our understanding of platelet regulation by cAMP signaling networks. We observed differences in cAMP signaling depending on the agonists used. Using phosphorylation of PKA substrates as a marker of PKA activity, it was observed that PKA substrates were phosphorylated and dephosphorylated at different time points in a unique temporal pattern. Consistent with this observation we found that individual PKA isoforms, PKA I and II, were localized in distinct subcellular compartments, with PKA I being identified as a lipid raft protein. Our experimental data suggest that the localization of PKA I to lipid rafts is mediated by interaction with A-kinase anchoring proteins (AKAPs). Additionally, PKA signaling events were reversed when potential PKA type I interactions with AKAPs were disrupted with competitive peptides. Using this approach we found that the redistribution of PKA I to lipid rafts facilitated the phosphorylation of GPIbβ and the inhibition of von-Willebrand factor-mediated aggregation. Our data also demonstrated for the first time that the chemical disruption of lipid rafts increased platelet sensitivity to PGI₂, through increased cAMP production and PKA activity. The mechanism by which this occurs may involve sequestering a population of adenylyl cyclase 5/6 to a location remote from Gαs. In conclusion, data presented in this thesis suggest differential roles of PKA subtypes in the regulation of platelet activity. This involves, at least in part, the localisation of PKA I into specific subcellular compartments through an interaction with AKAPs. The potential presence of PKAII-AKAP interactions and the identification of specific AKAPs will be the main aim of future work.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). One of the pathological hallmarks of MS is the T cells-mediated destruction of myelin sheath, which result in axonal damage and subsequent neurological dysfunction. Current MS therapies are focused on immunosuppression as they are aimed at limiting the entry of immune cells into CNS, thereby preventing neuroinflammation. Although these therapies have been shown to be potent disease-modifying agents they fail to prevent or reverse disease progression. Astrocytes, among CNS resident cells, has been recently suggested as alternative highly promising therapeutic targets because of the key role played by these cells in driving disease progression in MS. Indeed, thanks to a close contact between astrocyte end-feet and blood vessels, the crosstalk of astrocytes with encephalitogenic T cells is centrally implicated in MS pathogenesis (Ponath et al., 2018). In this view, we have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice accounts for a reduced demyelination and a milder experimental autoimmune encephalomyelitis (EAE), notwithstanding a higher frequency and pathogenicity of autoreactive T cells infiltrated within CNS highlighting the key role played by astrocytes in T cell modulation during EAE (Ulivieri et al., 2016). In the first part of this project we have investigated the molecular mechanism underlying the ability of ShcC/Rai-deficient astrocytes to generate an efficient T cell suppressive microenvironment in the pathological setting of EAE. At the beginning, we focused to study the ability of astrocytes to control the balance between extracellular ATP and adenosine in response to encephalitogenic T cells. We found that astrocytes respond to autoreactive T cells injury by enhancing the expression and activity of CD39 ectonucleotidase, responsible for the enzymatic hydrolysis of extracellular ATP into the immunosuppressive mediator adenosine, and that ShcC/Rai couples CD39 to its negative regulator RanBPM thereby limiting its activity. Accordingly, we measured high adenosine concentration in conditioned medium of Rai-/- astrocytes. As a result, T cells in the presence of microenvironment shaped by Rai knock-out astrocytes showed reduced proliferation and an up-regulation of inhibitory receptor CTLA-4, indicating that higher levels of adenosine are responsible to immunosuppression. We further characterized the impact of Rai on the protein composition of astrocytes-derived extracellular vesicles (EVs) in response to T cell-derived cytokines. Data obtained using a proteomic approach revealed that several proteins are differentially present in EVs released from Rai deficient or control astrocytes. Interestingly, enrichment analysis showed that these proteins participate in glutamate metabolism, in the control of protein folding and in the protection from oxidative stress suggesting that Rai controls pathways involved in brain homeostasis. Additionally, we examined functional polarization of astrocytes towards a neuroprotective (A2) or a neurotoxic (A1) phenotype. We show that Rai-/- astrocytes skew towards the A2 neuroprotective phenotype in response to encephalitogenic T cells both in vitro and in the EAE mouse model of MS by enhancing the activation of STAT3 transcription factor. In the second part of the project we have analyzed the role of ShcC/Rai in adenosine signaling in T cells. We identify a novel mechanism by which Rai in T cells dampens immunosuppressive effect of adenosine by inhibiting A2A receptor signalling interfering with the activation of transcription factor CREB. Characterization of molecular mechanism in a Jurkat T cell lines overexpressing Rai shows that Rai forms a complex with CREB upon A2AR triggering. In this respect, the phosphorylation/activation of CREB was significantly higher in Rai knock-out T cells compared with control following A2AR stimulation. Collectively, these data identify Rai/ShcC adaptor protein as critical regulator of astrocytes responses to T cells mediated neuroinflammation and highlight a new molecular mechanism to which Rai prevents establishment of an immunosuppressive program in T cells by limiting the transcriptional activity of nuclear factor CREB.

Protecting hearts from damage sustained during myocardial ischaemia and reperfusion remains an ongoing challenge. Despite successful findings with animal models, the task of trialling and effectively translating experimental findings from laboratory to patients has proven difficult, therefore treatments and clinical therapies are still urgently needed to protect the heart and improve cardiac functional outcome. Previous research has implicated adenosine and opioid receptor participation in the protective response preceding or following myocardial infarction, with evidence of potential cross-talk between receptors. This project aimed to investigate whether A1 adenosine (A1AR) and δ-opioid receptor (δ-OR) dependent cytoprotection and prosurvival kinase activation in murine hearts share common dependencies on „cross-talk‟ between both G-protein coupled receptor (GPCR) sub-types and whether these responses involve a common Matrix Metalloproteinase (MMP) and Epidermal Growth Factor Receptor (EGFR) dependent signalling pathway. This was achieved via four inter-related in vitro studies. Study 1: Healthy mouse hearts were cannulated in a Langendorff mode enabling the coronary circulation to be perfused. Hearts were subjected to 25 minutes of global (zero-flow) ischaemia followed by 45 minutes of aerobic reperfusion. The groups investigated included untreated control hearts, hearts receiving the selective A1AR agonist CCPA (± DPCPX, a selective A1AR antagonist, or BNTX, a selective δ-OR antagonist) and hearts receiving the selective δ-OR agonist BW373U86 (± DPCPX, a selective A1AR antagonist, or BNTX, a selective δ-OR antagonist). Agonists were applied 5 minutes pre-ischaemia and the antagonists were administered 10 minutes prior to agonist treatment. Cardiac functional outcomes were assessed via changes in coronary flow, left ventricular (LV) end diastolic pressure and pressure development, and ±dP/dt (± differentials of pressure change with time – indexing lusitropic and inotropic state). Cell death outcomes were also assessed via lactate dehydrogenase (LDH) release. Treatment with either the selective A1AR agonist CCPA, or the selective δ-OR agonist BW373U86, significantly reduced (p≤0.0001 vs. CTRL) LV end-diastolic pressure following ischaemia/reperfusion. Recovery of LV developed pressure (LVDP) was significantly increased following A1AR activation via CCPA (p≤0.0001 vs. CTRL) or δ-OR activation via BW373U86 (p≤0.001 vs. CTRL). Ventricular contractility (+dP/dt) and relaxation (-dP/dt) were also significantly improved with both CCPA (p≤0.0001 vs. CTRL, p≤0.01 vs. CTRL) and BW373U86 (p≤0.001 vs. CTRL, p≤0.001 vs. CTRL). Treatment with CCPA, but not BW373U86, significantly improved the recovery of coronary flow rate at the termination of reperfusion (p≤0.01 vs. CTRL). LDH release (corresponding to cell death) was significantly reduced by both CCPA and BW373U86 (p<0.05 vs. CTRL, p<0.05 vs. CTRL). A1AR or δ-OR inhibition, via the selective antagonists DPCPX and BNTX respectively (applied alone), did not significantly affect the recovery of functional outcomes or cell death relative to control. These results show that cardioprotection against ischaemic injury is induced with activation of A1ARs and δ-ORs, and that endogenous levels of receptor agonists may not be sufficient to induce this response. Protection with CCPA was abolished via cotreatment with either the selective A1AR antagonist DPCPX or the selective δ-OR antagonist BNTX. Conversely protection with BW373U86 administration was negated by co-treatment with either BNTX or DPCPX. This reveals that A1AR dependent cardioprotection is reliant on the activation of δ-ORs, and δ-OR mediated protection is dependent on A1AR activity, confirming essential cross-talk. Study 2: Perfused hearts from study 1 were snap-frozen in liquid nitrogen following the termination of reperfusion. Hearts were homogenised and fractioned to yield cytosolic proteins. Total and phosphorylated levels of Erk1/2 and Akt were subsequently assessed via western immunoblot. Both A1AR and δ-OR stimulation via CCPA and BW373U86 (respectively) did not significantly influence Erk1/2 phosphorylation. Akt phosphorylation, on the other hand, was increased by both CCPA and BW373U86; although only the latter effect achieved statistical significance (p<0.01 vs. CTRL). The A1AR antagonist DPCPX had minimal effect on Erk1/2 and Akt phosphorylation when applied alone. Alternatively inhibition of the δ-OR via BNTX, applied alone, was found to increase both Akt and Erk1/2 phosphorylation, a response in conflict with the existing literature. Co-treatment with the A1AR antagonist DPCPX or the δ-OR antagonist BNTX did not significantly influence Erk1/2 signalling compared to controls. Alternatively Akt phosphorylation was reduced by ~50% relative to control when hearts were co-treated with DPCPX or BNTX applied in conjunction with either the A1AR agonist CCPA or the δ-OR agonist BW373U86. These results imply that both A1ARs and δ-ORs together are necessary to induce protective Akt signalling during ischaemia/reperfusion with either receptor agonist. Study 3: To assess the roles of EGFRs and MMPs in A1AR and δ-OR responses, agonist studies with CCPA and BW373U86 were repeated with co-treatment with the EGFR antagonist AG1478 or the MMP inhibitor GM6001. Functional and cytoprotective outcomes were assessed in perfused hearts subjected to ischaemiareperfusion. The protective response observed with either A1AR and δ-OR stimulation was negated via co-treatment with either AG1478 or GM6001. A1AR and δ-OR dependent recovery of end diastolic pressure, LV developed pressure, +dP/dt and -dP/dt were all repressed via EGFR or MMP inhibition. Moreover, the cytoprotective response conferred by δ-OR activation was completely abolished via co-treatment with AG1478 or GM6001, providing evidence that adenosinergic and opioidergic protection within the myocardium involves an EGFR and MMP dependent pathway. Study 4: Western blot analysis was used to assess changes in Erk1/2 and Akt expression and phospho-regulation in hearts treated with CCPA or BW373U86 in the presence of AG1478 or GM6001. Due to time constraints, data collected previously in our lab was used in this research; therefore the effect of EGFR and MMP inhibition on A1AR dependent Erk1/2 and Akt signalling was assessed in whole heart rather than cytosolic fractions. In these hearts, administration of CCPA significantly elevated both Erk1/2 and Akt phosphorylation, a response negated via co-treatment with either AG1478 or GM6001 (p≤ 0.05 vs. CCPA). Infusion of the selective δ-OR agonist BW373U86 did not significantly alter Erk/1/2 expression or phosphorylation in cytosolic fractions. Despite this, co-treatment with AG1478 reduced Erk1/2 phosphorylation by ~50% compared to the agonist alone (p ≤ 0.05 vs. BW373U86), suggesting an EGFR dependent mechanism. Surprisingly co-treatment with GM6001 did not significantly influence δ-OR mediated Erk1/2 activity. Akt phosphorylation was increased by more than 60% with BW373U86 (p≤0.05 vs. CTRL) and this response was abolished via treatment with the selective EGFR antagonist AG1478 or the selective MMP inhibitor GM6001. This provides further evidence that adenosinergic and opioidergic protective signalling during ischaemia-reperfusion requires the activity of EGFRs and MMPs. Conclusions: As a whole, the present study confirms an essential interaction between ARs and ORs in the heart, with kinase signalling and tissue protection via either A1ARs or -ORs exhibiting common and essential dependencies on activity of both receptors. The basis of this intriguing response remains unclear, although we show that both receptors engage distal kinases (and cardioprotection) in an MMP/EGFR dependent manner, adding an additional level to this novel cross-talk. This research provides further insight into cardioprotective receptor interactions in the heart, potentially leading to the development of new pharmacotherapeutics and improved outcomes for cardiovascular disease patients. Further research is needed to clarify the mechanism behind adenosinergic and opioidergic cross-talk and cardioprotection, potentially exploring membrane signalling, temporal expression of kinases, existence of A1AR/δ-OR dimers/oligomers, and concepts such as dual agonism and potential signalling thresholds for protection.
Thesis (Masters)
Master of Medical Research (MMedRes)
School of Medical Science
Griffith Health
Full Text

This chapter on basic mechanisms of, and treatment targets for, bipolar disorder examines the cause and treatment of bipolar disorder from the perspective of intracellular signalling pathways implicated by the convergence of evidence from efficacious drugs, pathophysiology, and genetics and concludes that the unifying concept is calcium signalling. It discusses the pathways for cyclic adenosine monophosphate and inositol trisphosphate/calcium in the context of the action of drugs, with emphasis on lithium, the most effective true mood stabilizer. It proposes that the calcium signalling pathway and its components, such as channels, pumps, messengers, and enzymes, can explain both how dysfunction can affect neural activity and how this can be remedied by drugs. It argues for the central role of calcium, based on new evidence for the inositol depletion hypothesis and evidence of calcium dysregulation in peripheral and inducible pluripotent stem cells, as well as genome-wide association studies and drugs implicating a plasma membrane calcium channel.