Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides

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Abstract
In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide‐induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA‐derived model peptides. Studies on structure–activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence‐labeled Arg3,Orn7‐SP and its D‐amino acid analog, all‐D‐Arg3,Orn7‐SP, as well as by electron microscopic autoradiography using 3H‐labelled SP and 125I‐labelled all‐D‐SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin‐treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl‐KLALKLALKALKAALKLA‐amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA‐based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy‐dependent and ‐independent contributions, is not well understood, KLA‐derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP‐ and KLA‐derived peptides are discussed in the context of the current literature. Copyright © 2004 John Wiley & Sons, Ltd.