Study On Intercellular Calcium And Cell Secretion Affected By Native Polypeptides Somatostatin, Selysin And AIF-1

Native peptides play an important action in regulating a variety of cellular function. Neuropeptide and peptide hormones bind their special receptors and trigger a variety of cellular activities. Antibacterial polypeptides can form pore in cellular membrane and kill bacteria. Ca2+ binding proteins or polypeptides regulate intracellular Ca2+. In this paper,we study on intercellular calcium and cell secretory influenced by native polypeptides somatostatin, selysin and AIF-1.To investigate the role of extracellular somatostatin (SST) in insulin secretion from rat pancreaticβcells in the absence of extracellular Ca2+. Primary cultures of pancreaticβcells were prepared. The cells were loaded by incubation with fura2-AM in the bath solution. Intracellular Ca2+ ([Ca2+]i) was measured by dual-wavelength excitation (350/380 nm) microfluorometry using fura-2 as the Ca2+ indicator. The membrane capacitance (Cm) was examined by using the whole-cell patch clamp recording technique. Membrane capacitance measurements were performed using the'sine + DC'mode of the software lock-in extension in the Pulse software. The results showed that SST reduces Ca2+ current by depolarizing voltage ramp inβcell. In Ca2+-free medium, SST induced changes in [Ca2+]i in a sharp and transient increase. SST-induced Cm increment (ΔCm) indicate exocytosis from rat pancreaticβcells. SST stimulates insulin secretion via [Ca2+]i release from stores in rat pancreaticβcells.An 84-residue antibacterial polypeptide named selysin was purified from fly larvae of the Musca genus. Using Cm measurement, carbon fiber electrode and TIRFM, we found that selysin stimulates INS-1 cells secretion. Using microfluorometric measurement of calcium, we found that selysin can form pore in INS-1 cells and rat pancreaticβcells, and makes excellular Ca2+ influx. But selysin can not form pore in HEK293 cells and 5BI cells. The results imply that selysin can form pore via special receptors in cellular membrane. Studied on 6 components or mixed components, we found that all of them can form pore in INS-1 cells.Daintain/allograft inflammatory factor-1,originally identified from porcine intestine, is a EF-hand-containing Ca2+ binding protein with 146 amino acid residues. Using molecular biotechnological techniques, we constructed two recombinant eukaryotic expression vectors of daintain/AIF-1. In addition, the HeLa cells stably expressing daintain/AIF-1 were established, providing tools for further research on the functions of this protein. In HeLa cells,histamine can stimulates elevation of [Ca2+]i contributed by calcium release from calcium stores. Using microfluorometric measurement of calcium, we found that the elevation of [Ca2+]i stimulated by histamine was significantly reduced in daintain/AIF-1-expressing cells as compared with control cells. But the elevation of [Ca2+]i stimulated by Thapsigargin was significantly enhanced in daintain/AIF-1- expressing cells as compared with control cells, and the dynamics of the elevation of [Ca2+]i was changed.