|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1994 : ¥1,400,000 (Direct Cost : ¥1,400,000)
The mechanisms of exocytosis from rat peritoneal mast cells induced by mastoparan (MP) and substance P (SP), an amphiphilic peptide isolated from wasp venom, and a neuropeptide isolated from mammals, respectively, were investigated to elucidate whether MP and SP cause exocytosis to directly activate GTP-binding regulatory proteins (G proteins) in these cells. MP and SP induced non-lytic beta-hexosaminidase release from mast cells at concentrations of 1-30 muM,and these effects were prevented by pertussis toxin, indicating the involvement of pertussis toxin-sensitive G proteins in the secretion. The presence of extracellular Ca^<2+> was not essential for peptide-induced secretion, but it increased the kinetics and maximal response of the secretion, and the potency was decreased in its presence. Replacing the hydrophobic amino acid residues of MP with Ala significantly decreased the stimulation of beta-hexosaminidase release and the activation of G_i. Lys residue (s) was required for MP to stimulate beta-hexosaminidase release and activate G_i. These results demonstrated the importance of hydrophobic and positively charged side chains for MP to stimulate target molecules in mast cells and G_i in vitro. Both hydrophobic and positively charged amino acid residues were also required the activation of mast cells and G_i by SP.[Lys^<10>, Leu^<13>] MP was the most potent analog that stimulated beta-hexosaminidase release without causing cell lysis, indicating that this peptide is the most useful chemical stimulator of the mast cells. However, this peptide only slightly activated G_i. The possibility of direct activation of a G_i like protein by MP and SP in peritoneal mast cells in discussed based upon the structure-activity correlation between exocytosis from these cells and G_i-protein activation in vitro by MP,SP and their analogs.