Endocrine Abstracts (2002) 3 P240

Expression and function of voltage-gated calcium channels in human pancreatic B-cells in health and disease

A Natarajan1, KE Cosgrove1, RM Shepherd1, RE O'Brien1, A-M Gonzalez1, KJ Lindley2, PE Clayton3, A Aynsley-Green2 & MJ Dunne1

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

Voltage-gated calcium channels (VGCC) play a fundamental role in the control of insulin secretion from pancreatic B-cells since they govern rises in cytosolic Ca2+ in response to depolarization-dependent agonists, such as glucose. Here, we have examined the function and expression of VGCC in human B-cells. We isolated tissue from patients with B-cell adenoma (AD) and Hyperinsulinism in Infancy (HI) following surgery and used transplantable human islets as controls. VGCC function was assessed by patch-clamp electrophysiology and cell imaging / microfluorimetry using fura-2. RNA was extracted using standard protocols and RT-PCR was performed to study the expression of the VGCC genes using specific oligonucleotide primers directed against Ca-v1.2, Ca-v1.3, Ca-v2.1, Ca-v2.2, Ca-v2.3, and the beta-2 and beta-3 subunits. Results: Whole-cell patch-clamp recordings and 40mM KCl-induced rises in the cytosolic Ca2+ concentration were used to reveal the presence of VGCC in control, AD and HI B-cells. By microfluorimetry techniques we estimated basal levels of cytosolic Ca2+ concentration in islets as: 80 +/- 9nM (n=677), 97 +/- 7nM (n=28), and 200 +/- 42nM (n=6), in control, HI and AD, respectively. Since, there were marked differences in the resting concentrations of Ca2+ in control and AD islets VGCC gene expression was examined by RT-PCR. In controls, we documented the presence of Ca-v1.2, Ca-v1.3, Ca-v2.1, and Ca-v2.2 but not Ca-v2.3, whereas both HI and AD expressed only Ca-v1.2 (strong) and Ca-v2.1 (weak). A differential pattern of gene expression was also found for the beta-subunits, since controls preferentially expressed the beta-2 regulatory subunit, where as AD B-cells expressed beta-3. In summary, these data document the functional expression of VGCCs in control and disease human islets and that there are differences in the patterns of VGCC subunit expression in B-cells from patients with hyperinsulinism with respect to controls.

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