Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETEndothelins (ET-1, ET-2, and ET-3) 1 are important regulators in the vascular system. They act via two receptors: the endothelin A (ET A ) and endothelin B (ET B ) receptors (1, 2). Although human ET A and ET B receptors share 59% amino acid sequence identity (exceeding 75% at the cytoplasmic face), both receptor subtypes couple to different G proteins and differ in their ligand-induced internalization and intracellular trafficking. Whereas the ET A receptor stimulates G proteins of the G q/11 and G 12/13 families, the ET B receptor activates mainly G proteins of the G i and G q/11 families (3, 4). Whether ET B receptors also stimulate proteins of the G 12/13 family is still controversial and may depend on expression levels or cell types investigated (5, 6). Upon ligand binding, both receptor subtypes are rapidly desensitized by phosphorylation through the G protein-coupled receptor kinase type 2 (7). Following internalization via caveolae and/or clathrin-coated pits, the ET A receptor is recycled back to the cell surface (8, 9). In contrast, the ET B receptor is exclusively internalized via a clathrin-dependent pathway and transported to late endosomes and lysosomes (9, 10).The ET A receptor is mainly expressed in vascular smooth muscle cells. Its activation elicits a long-lasting contraction via an increase in cytosolic Ca 2ϩ concentrations and activation of Rho proteins (11,12). The ET B receptor is predominantly expressed in endothelial cells and stimulates the release of NO and prostacyclin, thereby causing relaxation of vascular smooth muscle cells (13). In addition, ET A and ET B receptors are co-expressed in numerous cells, e.g. astrocytes, cardiomyocytes, epithelial cells of the choroid plexus and the anterior pituitary, and certain vascular smooth muscle cells (14 -16). In disease states, such as atherosclerosis and hypercholesterolemia, vascular smooth muscle cells co-express ET A and ET B receptors (17). Because atypical ligand binding was observed for cells co-expressing ET A and ET B receptors, e.g. astrocytes, epithelial cells of the anterior pituitary, or vascular smooth muscle cells, it was suggested that the two receptor subtypes form heterodimers (15,16,18). For example, in epithelial cells of the anterior pituitary, ET B receptor-selective ligands such as sarafotoxin 6c, ET-3, and IRL1620 were competitors of 125 I-ET-1 binding only in the presence of the ET A receptor-selective antagonist BQ123 (16). In astrocytes, ET A and ET B receptors cooperatively control ET-1 clearance, because only the combi-

The influence of the peptide-to-cell ratio and energy depletion on uptake and degradation of the cell-penetrating peptides (CPPs) MAP (model amphipathic peptide) was investigated. The intracellular concentration of the CPPs, MAP and penetratin was monitored while varying the number of cells at fixed peptide concentration and incubation volume, or changing the concentration and incubation volume at fixed cell number. The uptake of CPPs was shown to be dependent on the peptide/cell ratio. At given peptide concentration and incubation volume, the intracellular concentration of peptide increased with lower cell number. At given cell number, doubling of the incubation volume increased intracellular peptide concentration to a similar extent as the doubling in incubation concentration. From a practical view, this means that the peptide/cell ratio has at least the same importance for the uptake of CPPs as the used peptide concentration. No influence of the peptide/cell ratio was found for the cellular uptake of peptide nucleic acid (PNA), or a non-amphipathic MAP analogue, investigated in parallel for comparison purposes. Energy depletion resulted in significantly reduced quantities of intracellular fluorescence label. Moreover, we show that this difference is mainly due to a membrane-impermeable fluorescent-labelled degradation product, which is lacking in energy-depleted cells. The mechanism of its generation is not likely to be endosomal degradation of endocytosed material, as it is not chloroquine- or brefeldin-sensitive.

Endothelins, which exist in three isoforms, exert their action via two endothelin receptor subtypes: (i) the endothelin A (ET A ) 1 and (ii) the endothelin B (ET B ) receptor (1, 2). Both receptors belong to the large family of GPCRs. The ET A receptor is predominantely expressed in smooth muscle cells, and its stimulation causes a long-lasting vasoconstriction (3). The ET B receptor is mainly expressed in endothelial cells, and its activation results in a transient vasodilatation (4).The ET B receptor displays several unique properties, which are not shared by the ET A receptor or other members of the GPCR family. For example, the ET B receptor binds the ligand ET1 almost irreversibly (5). The receptor-ligand complex is resistant to acid washes and 2% SDS and even survives SDSpolyacrylamide gel electrophoresis at low temperature (6). In living cells, internalized ET1-ET B receptor complexes remain stable for more than 2 h. This is remarkable, as the ligandreceptor complex is transported into late endosomal/lysosomal compartments within 30 min (7,8). The tight binding of ET1 to the ET B receptor is evolutionary conserved, since it was also observed for the ET B receptor of calf, dog, mouse, and guinea pig (9). Mutagenesis studies revealed that aspartate 75 and proline 93 within the extracellular N terminus of the human ET B receptor are important determinants for the formation of a stable ligand/receptor complex (9). The physiological significance of this tight receptor/ligand association is still not understood. Recently it was shown that the ET B receptor is crucially involved in the regulation of circulating ET1 plasma levels: blockade of the ET B receptor causes a significant increase in ET1 plasma levels (10, 11). Thus, the tight binding of ET1 to the ET B receptor and the transport of the receptor/ligand complex into lysosomal compartments could provide a molecular basis for the efficient removal of ET1 from the circulation. The ET B receptor possesses a cleavable signal peptide (comprising 26 amino acids), which is removed in the ER lumen during receptor biosynthesis (6,12,13). In addition to the removal of the signal peptide, the N terminus of the mature protein undergoes proteolytical cleavage. Protein analysis of purified ET B receptors from human placenta revealed two different isoforms: (i) a full-length receptor comprising 416 amino acids (after removal of the signal peptide) and starting with glutamate 27, and (ii) an N-terminal-truncated ET B receptor, comprising 378 amino acids and starting with serine 65 (6, 14). The existence of the two ET B receptor isoforms has been demonstrated for the dog, pig, and calf receptor, indicating that the proteolytical processing of the mature ET B receptor is evolutionary conserved (12,(15)(16)(17). The proteolytically released Nterminal peptide fragment harbors the only N-linked glycosylation. Thus, the N-terminal proteolysis yields unglycosylated ET B receptors.So far, the mechanism and physiological significance of the N-terminal proteolysis are unknown. In o...

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