الفهرس 
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Abstract
Summary : Data about the role of NO in intracellular Ca2+ level in pancreatic acinar cells are contradictory. The present study was therefore designed to investigate the possible interaction between intracellular Ca2+ and NO in rat pancreatic acinar cells, especially intracellular signaling events. I obtained following results, Nitric oxide donors SNP (0.1-100 µM) and NOR-3 (50-400 µM) induced Ca2+ oscillations in fluo-4-loaded acini, that appeared to be analogous to what we usually observe in acini stimulated with physiological secretagogues such as CCK-8. The NO donors-evoked Ca2+ oscillations were not abolished even in the absence of extracellular Ca2+ but totally disappeared when cells were pretreated with thapsigargin. Inhibition of guanylyl cyclase with 1 H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxaline-1-one (ODQ) attenuated Ca2+ oscillations evoked by SNP in the absence of extracellular Ca2+. Inhibitors of phospholipase C activity, U73122 and the IP3R blocker Xestospongin C, both abolished the SNP-induced Ca2+ response. Furthermore, I found that both CCK-8 and carbachol (CCh) induced NO production in DAF-2-loaded acinar cells and that an inhibitor of NO synthase, NG-monomethyl-L-arginine (L-NMMA), significantly reduced CCK-8-induced Ca2+ oscillation. These results indicate that NO mobilizes Ca2+ from internal stores through activation of guanylyl cyclase and resultant cGMP production. In addition, PLC activation of IP3 production is also suggested to be involved in Ca2+ mobilization via IP3 sensitive stores. However, whether or not IP3 is actually produced by NO has not yet been substantiated. The intracellular IP3 concentration ([IP3]i) was measured for better understanding underlying mechanisms with the help of pharmacological tools. The [IP3]i was measured using a Fluorescence Polarization technique (HitHunterTM). I found that varying concentrations of SNP elevated [IP3]i; this elevation was completely inhibited in the presence of sGC inhibitor, ODQ; varying concentrations of cGMP analogue, 8-Br-cGMP, also increased [IP3]i; the cGMP analogue-induced IP3 production was abolished by the pretreatment of either a PLC inhibitor, U73122, or a G-protein inhibitor, GP2A and KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase G (PKG), also abolished the IP3 production induced by 8-Br-cGMP. These results suggest that NO-induced [Ca2+]i increase is triggered by an increase in [IP3]i which is located at a downstream of intracellular cGMP elevation. In this intracellular pathway, each sGC, cGMP-dependent PKG, G-protein and PLC was suggested to be involved.
Furthermore, the effect of NO on basal amylase secretion was investigated and I found that NO has no effect on basal amylase secretion. Overall data imply a possible cross talk between intracellular Ca2+ and NO and would provide with a new insight into the intracellular signaling which is accelerated by NO. NO triggers [Ca2+]i increase via cGMP and IP3 in pancreatic acinar cells.