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 P-specific 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 receptor- and 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 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp 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 microM). Degranulation in response to intracellularly applied substance P was inhibited by GDPbetaS 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 alpha subunits subsequent to their translocation across the plasma membrane.
Lorenz
D
D
Institute of Molecular Pharmacology, 10315 Berlin, Germany. lorenz@fmp-berlin.de
Wiesner
B
B
Zipper
J
J
Winkler
A
A
Krause
E
E
Beyermann
M
M
Lindau
M
M
Bienert
M
M
D000818:Animals; D001345:Autoradiography; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects* / Q000502:physiology; D003594:Cytoplasmic Granules / Q000187:drug effects* / Q000378:metabolism; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D006636:Histamine Release / Q000187:drug effects / Q000502:physiology; D008297:Male; D008407:Mast Cells / Q000502:physiology* / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013373:Substance P / Q000235:genetics / Q000494:pharmacology*
D012333:RNA, Messenger; D013373:Substance P; D019204:GTP-Binding Proteins; D002118:Calcium