Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 1387K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Lorenz, D. Articles by Bienert, M. Search for Related Content PubMed PubMed Citation Articles by Lorenz, D. Articles by Bienert, M. Social Bookmarking                            What's this?

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

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				R. Hayashi, S. Osada, M. Yoshiki, D. Sugiyama, I. Fujita, Y. Hamasaki, and H. Kodama Superoxide Production in Human Neutrophils Is Enhanced by Treatment with Transmembrane Peptides Derived from Human Formyl Peptide Receptor J. Biochem., June 1, 2006; 139(6): 981 - 988. [Abstract] [Full Text] [PDF]

				E. Zudaire, A. Martinez, M. Garayoa, R. Pio, G. Kaur, M. R. Woolhiser, D. D. Metcalfe, W. A. Hook, R. P. Siraganian, T. A. Guise, et al. Adrenomedullin Is a Cross-Talk Molecule that Regulates Tumor and Mast Cell Function during Human Carcinogenesis Am. J. Pathol., January 1, 2006; 168(1): 280 - 291. [Abstract] [Full Text] [PDF]

				Z. Marinova, V. Vukojevic, S. Surcheva, T. Yakovleva, G. Cebers, N. Pasikova, I. Usynin, L. Hugonin, W. Fang, M. Hallberg, et al. Translocation of Dynorphin Neuropeptides across the Plasma Membrane: A PUTATIVE MECHANISM OF SIGNAL TRANSMISSION J. Biol. Chem., July 15, 2005; 280(28): 26360 - 26370. [Abstract] [Full Text] [PDF]

				G. J. Graham, J. M. Stevens, N. M. Page, A. D. Grant, S. D. Brain, P. J. Lowry, and J. M. Gibbins Tachykinins regulate the function of platelets Blood, August 15, 2004; 104(4): 1058 - 1065. [Abstract] [Full Text] [PDF]

				R. Saban, N. P. Gerard, M. R. Saban, N.-B. Nguyen, D. J. DeBoer, and B. K. Wershil Mast cells mediate substance P-induced bladder inflammation through an NK1 receptor-independent mechanism Am J Physiol Renal Physiol, October 1, 2002; 283(4): F616 - F629. [Abstract] [Full Text] [PDF]

				X. Ferry, V. Eichwald, L. Daeffler, and Y. Landry Activation of {beta}{gamma} Subunits of Gi2 and Gi3 Proteins by Basic Secretagogues Induces Exocytosis Through Phospholipase C{beta} and Arachidonate Release Through Phospholipase C{gamma} in Mast Cells J. Immunol., November 1, 2001; 167(9): 4805 - 4813. [Abstract] [Full Text] [PDF]

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents