MECHANISM OF PEPTIDE-INDUCED MAST-CELL DEGRANULATION - TRANSLOCATION AND PATCH-CLAMP STUDIES

Substance P and other polycationic peptides are thought to stimulate m ast cell degranulation via direct activation of G proteins. We investi gated the ability of extracellularly applied substance P to translocat e into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse tran scription-PCR whether substance P-specific receptors are present in th e mast cell membrane. To study translocation, a biologically active an d enzymatically stable fluorescent analogue of substance P was synthes ized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells wa s demonstrated using confocal laser scanning microscopy. The peptide w as shown to localize preferentially on or inside the mast ce:ll granul es using electron microscopic autoradiography with I-125-labeled all-D substance P and H-3-labeled substance P. Cell membrane capacitance me asurements using the patch-clamp technique demonstrated that intracell ularly applied substance P induced calcium transients and activated ma st cell exocytosis with a time delay that depended on peptide concentr ation (delay of 100-500 s at concentrations of substance P from 50 to 5 mu M) Degranulation in response to intracellularly applied substance P was inhibited by GDP beta S and pertussis toxin, suggesting that su bstance P acts via G protein activation. These results support the rec ently proposed model of a receptor-independent mechanism of peptide-in duced mast cell degranulation, which assumes a direct interaction of p eptides with G protein alpha subunits subsequent to their translocatio n across the plasma membrane.