Mechanism of Peptide-induced Mast Cell Degranulation Translocation and Patch-Clamp Studies

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription–-PCR whether substance Pspecific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptorand energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125 Ilabeled all-D substance P and 3 H-labeled substance P . Cell membrane capacitance measurements using the patchclamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100–500 s at concentrations of substance P from 50 to 5 m M). Degranulation in response to intracellularly applied substance P was inhibited by GDP b S and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein a subunits subsequent to their translocation across the plasma membrane. key words: substance P • exocytosis • confocal laser scanning microscopy • electron microscopy • capacitance i n t r o d u c t i o n Mast cells stimulated with immunoglobulin E/antigen release a variety of preformed mediators (e.g., histamine) by exocytosis. They can, however, also be triggered to undergo exocytosis by nonantigenic stimuli. Polycationic substances, such as the neuropeptides substance P (SP) 1 (Repke and Bienert, 1987) or neuropeptide Y (Gundemar and Håkanson, 1991), or the venom peptides mastoparan and mast cell degranulating peptide (Mousli et al., 1989; Landry et al., 1992), and naturally occurring or synthetic polyamines such as spermine (Purcell et al., 1996) and compound 48/80 (Paton, 1951) stimulate the release of vasoactive and inflammatory compounds from mast cells. The peptidergic stimulation of mast cells has raised the question of whether other peptide-secreting cells in the vicinity of mast cells are capable of stimulating mast cells. In this context, it is interesting to note that mast cells have been found in close proximity to peptidergic neurones, suggesting a probable functional relationship between the immune and nervous systems (McKay and Bienenstock, 1994; Marshall and Waserman, 1995; Purcell and Atterwill, 1995) mediated by neuropeptides. The mechanism by which cationic peptides and polyamines stimulate mast cell exocytosis remains unclear. It is generally accepted that heterotrimeric GTPbinding proteins (G proteins) are involved in the stimulation of mast cell degranulation by basic secretagogues. Thus, application of substance P and other cationic secretagogues transiently increases intracellular Ca 2 1 concentrations through G protein-mediated activation of phospholipase C (PLC), and pretreatment of mast cells with pertussis toxin abolishes both inositol-3-phosphate production and peptide-induced mast cell deAnett Winkler’s present address is Addiction Research Group, MaxPlanck-Institute for Psychiatry, Kraepelin-Strasse 2, D-80804 München,