EVIDENCE FOR THE INVOLVEMENT OF CGMP AND PROTEIN-KINASE-G IN NITRIC OXIDE-INDUCED APOPTOSIS IN THE PANCREATIC B-CELL LINE, HIT-T15

Intracellular production of nitric oxide (NO) is thought to mediate th e pancreatic B-cell-directed cytotoxicity of cytokines in insulin-depe ndent diabetes mellitus, and recent evidence has indicated that this m ay involve induction of apoptosis. A primary effect of NO is to activa te soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intrac ellular function has been defined for islet cGMP. Here me demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-cell li ne HIT-T15 in a dose- and time-dependent manner. This response was sig nificantly attenuated by micromolar concentrations of a specific inhib itor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 mu M ) and dibutyryl cGMP (300 mu M) were able to fully relieve this inhibi tion. In addition, incubation of HIT-T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to eit her GSNO or cGMP analogues. This effect was dose-dependent over the co ncentration range of 10-250 nM. Overall, these data provide evidence t hat the activation of apoptosis in HIT-T15 cells by NO donors is secon dary to a rise in cGMP and suggest that the pathway controlling cell d eath involves activation of PKG.