Endocrine Abstracts (2002) 3 P99

Expression of purinergic receptors in human insulin-secreting cells in health and disease

A-M Gonzalez1, RM Shepherd1, A Lee1, K Hussain2, PE Clayton3, A Aynsley-Green2, KJ Lindley2 & MJ Dunne1

1Department pf Biomedical Science, Sheffield University, Sheffield, UK; 2Institute of Child Health, University College London, London, UK; 3Manchester Children's Hospital, Manchester University, Manchester, UK.

ATP-evoked signalling events are known to promote release of insulin from pancreatic beta-cells in a Ca2+-dependent manner. In rodent beta-cells and insulin-secreting cell lines, this is mediated by purinergic receptors and there is evidence for the involvement of both P2X and P2Y subtypes. Here,we have used human insulin-secreting cells to examine the expression of purinoceptors in control and disease tissue. Intact islets and isolated beta-cells were obtained from control human donors (n=3) and from patients with either Hyperinsulinism in Infancy (n=3, HI) or adenoma (n=3, AD) following surgery (with permission). Experiments were also undertaken with the human insulin-secreting cell line, NES2Y. We used Fura-2 loaded cells to examine ATP-evoked cytosolic Ca2+ signals ([Ca2+]i) by microfluorimetry and cell imaging, and studied the expression of P2Y and P2X mRNA subtypes by RT-PCR and immunofluorescence. Results: ATP (0.1mM) consistently elevated [Ca2+]iin control, HI, AD and NES2Y beta-cells, whereas RT-PCR experiments showed that the expression of purinoceptor subtypes was variable across the different tissues. Thus, control islets expressed P2X4 and P2Y1 subtypes but not P2Y2 or P2Y4; HI islets and AD beta-cells expressed both P2X4 and P2Y1, yet only the adenoma tissue also expressed P2Y2 mRNA. NES2Y beta-cells, a proliferating cell line derived from a patient with HI, expressed P2Y2 and P2Y4, but not P2Y1 mRNA subtypes. NES2Y beta-cells were also found to express P2X4 mRNA, yet these cells failed to respond to the P2X4 receptor agonist ADP-beta-S (0.1mM, n=21) and no protein was found to be localized on the cell surface (by immunofluorescence using a specific P2X4 antibody). Summary: These studies reveal that whilst control and human islets of Langerhans obtained from patients with hyperinsulinism respond to ATP through changes in [Ca2+]i signals, purinergic receptor expression is altered.

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