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Article

Translocation and Patch-Clamp Studies

Dorothea Lorenz^*, Burkhard Wiesner^*, Josef Zipper^*, Anett Winkler^*, Eberhard Krause^*, Michael Beyermann^*, Manfred Lindau, and Michael Bienert^*

From the ^* Institute of Molecular Pharmacology, 10315 Berlin, Germany; and School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853-2501

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 µM). Degranulation in response to intracellularly applied substance P was inhibited by GDPβS 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 subunits subsequent to their translocation across the plasma membrane.

Key Words: substance P • exocytosis • confocal laser scanning microscopy • electron microscopy • capacitance

Abbreviations: ³H-SP, ³H-labeled SP; all-D-SP, D-amino acid analogue of SP; CLSM, confocal laser scanning microscopy; C_m, membrane capacitance; ES, external bath solution; FLUOS-SP, 5(6)-carboxyfluorescein-labeled Arg³,Orn⁷-SP; FLUOS-all-D-SP, 5(6)-carboxyfluorescein-labeled all-D-Arg³,Orn⁷-SP; GAPDH, glyceraldehyde-phosphate dehydrogenase; IS, intracellular or pipette solution; PLC, phospholipase C; PtX, pertussis toxin; ROI, region of interest; R_s, series resistance; RT-PCR, reverse transcription–PCR; SP, substance P

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