Adenosine 5'-triphosphate (ATP) released by cells is sensed through activation of cell surface receptors cells to communicate (purinergic signalling) in both an autocrine and paracrine fashion.  Fast responses to ATP are mediated through activation of ligand-gated ion channels (P2X receptors) and slower effects through the activation of G protein coupled receptors (P2Y receptors).  In the cardiovascular system, specifically blood vessels, how purinergic signalling contributes to normal physiology and disease is poorly understood.

The project will address molecular mechanism by which purinergic signalling is used by blood cells and vascular endothelium in an autocrine fashion and how purinergic signalling influences interaction between blood cells and vascular endothelium.  The later is particularly important in cardiovascular disease such as atherosclerosis and stroke.

The student will receive training and supervision from Dr Samuel Fountain (School of Biological Sciences), Professor David Crossman and Dr Darren Sexton (Norwich Medical School) to address the following research questions:

1. What mechanisms underlie constitutive and agonist stimulated ATP release from human leukocytes and vascular endothelium, and what receptors are activated?

2. How does purinergic signalling influence the physical and communicative interaction between human leukocytes and vascular endothelium (stroke, atherosclerosis) or lung epithelium (asthma, COPD)?

The project will employ cell lines, peripheral blood taken from healthy patients, and leukocytes isolated by nasal lavage.  Training will include cell culture, cell sorted and analysis by FACS, patch-clamp electrophysiology, intracellular calcium measurements, molecular cloning, gene profiling and production of lentivirus for gene knockdown by small RNA interference. The student will be trained to isolate leukocytes subtypes using antibody labelling and cell sorting by FACS, how to make quantitative measurements of ion channel and GPCR activity by patch-clamp electrophysiology and by using fluorescent calcium reporter dyes, respectively, and how to knockdown gene expression using shRNA delivered by lentivirus.

The laboratory is extremely well funded by the BBSRC with many equipment and facilities in house including flow cytometry, electrophysiology, fluorescence readers and Class II biohazard facilities.  Funding is available for attendance at domestic and international scientific conferences.

Sivaramakrishnan V, Bidula S, Katikaneni D, Campwala H, Fountain SJ (2012). Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes. Journal of Cell Science (In Press).

Li J, Fountain SJ (2012).  Fluvastatin suppresses native and recombinant human P2X4 receptor activity.  Purinergic Signalling 8: 311-6.

Fountain SJ, North RA (2006).  A C-terminal lysine that controls human P2X4 receptor desensitization.  Journal of Biological Chemistry 22: 15044 – 15049.

Ward JR, West PW, Ariaans MP, Parker LC, Francis SE, Crossman DC, Sabroe I, Wilson HL (2010). Temporal IL-1beta secretion from primary human peripheral blood Monocytes by p2x7-independent and p2x7-dependent mechanisms. Journal of Biological Chemistry 285:23147-58, 2010.

Wilson HL, Varcoe RW, Stokes L, Holland KL, Francis SE, Dower SK, Surprenant A, Crossman DC (2007). P2X Receptor Characterisation and IL-1/IL-1Ra Release from Human Endothelial Cells. British Journal of Pharmacology 151:115-27,