Research interests include the pharmacology and biochemistry of G-protein-coupled receptors (particularly for adenosine, glutamate and cannabinoids) in isolated tissues and cultured cells; assay of cell signalling-associated enzymes (particularly relating to turnover of endocannabinoids, cyclic nucleotides, gasotransmitters such as NO and hydrogen sulphide, ATP); flavonoids as pharmacologically-active molecules.
Signalling convergence through Epac, exchange factors directly activated by cyclic AMP
Elevation of intracellular cyclic AMP levels leads to diverse cellular responses dependent on the cell type. An alternative route for cyclic AMP signalling has recently been identified; through Epac, exchange proteins directly activated by cyclic AMP. Epac1 and 2 are guanine nucleotide exchange factors for members of the Ras GTPase family, Rap1 and Rap2. These can activate an isoform of phospholipase C, PLC-ε, which generates inositol 1,4,5-trisphosphate and hence leads to elevation of intracellular calcium levels. Epac is also able to activate extracellular signal-regulated protein kinases (ERK, a mitogen-activated protein kinase) through B-Raf, leading to cellular proliferation.
We have identified two cell lines which exhibit a contrasting pattern of signalling evoked by adenosine receptors downstream from cyclic AMP elevation. It is our hypothesis that this divergence of signalling crosstalk is due to differential expression of members of the Epac pathway. We propose to expand further on these recent findings to identify the signalling pathways involved in Epac-evoked cellular responses in these cells. We intend to identify using qRT-PCR which messages for the relevant proteins are expressed in these cells. We will investigate real time calcium responses to receptor and Epac activation in these cell lines using a 96-well FlexStation. cAMP levels will be assessed in real time using novel fluorescent indicators. The signalling of Epac in these cells to ERK, as well as proliferative and differentiation influences will be investigated.
N-Arachidonoylglycine (NAGly) and GPR18 as regulators of pancreatic insulin secretion
The identification of a growing family of lipids often referred to as endocannabinoids/ endovanilloids and which are primarily N-acyl amides in structure has lead to important and novel insights into cell signalling, with the description of a number of such lipid molecules mediating a range of biological activity via G-protein coupled receptors (GPCRs). N-arachidonylglycine (NAGly) has recently been identified as an endogenous ligand for the orphan GPCR GPR18. In pancreatic beta-cells, NAGly has been shown to cause intracellular calcium mobilization and insulin release, which we hve confirmed locally. We hypothesise that NAGly and GPR18 may represent a novel endogenous signalling pathway to regulate pancreatic insulin secretion.
We will continue this investigation using molecular techniques to identify the involvement of GPR18 (qRT-PCR and siRNA), as well as making use of novel NAGly analogues in hormone release studies in pancreatic islets & beta-cell lines, confirming their activity at NAGly using rat and human recombinant receptors.