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Extracellular nucleotides and nucleosides are important signalling molecules that exert a diverse range of physiological responses throughout the body. These chemical messengers often transduce their effects through interaction with specific cell surface receptors called purinoceptors. The P2Y purinoceptor family binds extracellular nucleotides, principally ATP, ADP, UTP and UDP. Conformational change of the P2Y purinoceptors upon ligand binding conveys a signal to intracellular heterotrimeric G proteins, which are activated, transducing the signal further downstream by acting at different effector enzymes to influence second messenger production. In this thesis I present the first pharmacological characterisation of a non-mammalian P2Y11 receptor orthologue, Xenopus laevis P2Y11 (XlP2Y11), and extend the pharmacological profile of the human P2Y11 receptor (hP2Y11). Second messenger assays were employed to record the intracellular cyclic AMP (cAMP) accumulation and Ca2+ mobilisation generated by both the human and Xenopus laevis P2Y receptors in response to various P2Y agonists and antagonists. In a manner similar to its human orthologue, I have shown that XlP2Y11 is activated by nucleotides and nucleotide analogues, mobilising calcium from intracellular stores, and increasing cAMP production through the activation of adenylyl cyclase. When compared to previously published data, XlP2Y11 exhibits a novel rank order of agonist and antagonist potency, revealing a receptor functionally similar but pharmacologically distinct from both the human and canine P2Y11 receptor orthologues (hP2Y11 and cP2Y11).

The Bcl-2 family of proteins regulates the mitochondrial apoptotic pathway and consists of both pro- and anti-apoptotic members. Bax-cc is present in the cytosol of healthy cells. Apoptotic stimuli induce the translocation of Bax-oc to the mitochondria leading to the permeabilisation of the outer mitochondrial membrane (OMM) and the release of downstream pro-apoptotic proteins from the intermembrane space (IMS). The mechanism by which Bax permeabilises the OMM remains unclear. Recent evidence suggests Bax alone might be sufficient to permeabilise the OMM. However, other models indicate the involvement of the mitochondrial permeability transition pore complex or the voltage-dependent anion channel (VDAC) present in the OMM. In this study Bax and C-terminally truncated Bax were expressed as GST-fusion proteins and were immobilised on agarose-GSH. The binding of mitochondrial membrane proteins that might be involved in the Bax mediated release of proteins from the IMS was investigated. The results showed that VDAC and the adenine nucleotide translocase (ANT) were retained by GST-Bax. Exogenous cyclophilin D (Cyp D) was added in the presence of VDAC and ANT and was also retained indicating that Bax interacts with the components of the permeability transition pore complex. The anti-apoptotic Bcl-2 protein Bcl-XL was expressed with a hexahistidine tag at its N-terminus. This was used to investigate its effects on Bax interaction with VDAC and ANT. The ANT ligands atractyloside and bongkrekic acid which promote and inhibit apoptosis respectively were shown to change the relative amounts of VDAC and ANT that bind GST-Bax. Apoptotic cell death has been identified in cardiomyocytes subjected to ischaemia. In this investigation cardiomyocytes transfected with GFP-Bax were treated with cyanide to simulate ischaemia and GFP-Bax translocation was observed using fluorescence microscopy. GFP-Bax co-immunoprecipitated with VDAC and ANT after translocation to mitochondria but not with Cyp D. The implications of these findings are discussed in this thesis.

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