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-

Abstract-Urotensin II (UII), which acts on the G protein-coupled urotensin (UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (Ϫlog EC50, mol/L:9.0Ϯ0.1). A second application of UII after 30 minutes elicited a reduced contraction (36Ϯ4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII ( 125 I-UII), Ϸ70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [t h ]: 5.6Ϯ0.2 minutes), but recycled quantitatively within 60 minutes (t h 31.9Ϯ2.6 minutes). UII-bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3-UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. Key Words: urotensin II Ⅲ vascular tone Ⅲ urotensin receptor Ⅲ recycling Ⅲ internalization U rotensin II (UII) is among the most potent mammalian vasoconstrictors identified so far. 1 UII was characterized as being 1 to 2 orders of magnitude more potent than endothelin-1 (ET-1) 2 and acts on the urotensin (UT) receptor, formerly known as GPR14. 3 Stimulation of the UT receptor results in a phospholipase C-mediated increase in cytosolic Ca 2ϩ and the activation of rho-kinase. 4,5 Both signaling pathways promote an increase in MLCK phosphorylation, resulting in a strong contraction of vascular smooth muscle cells. 6 In addition, stimulation of the UT receptor also results in an activation of tyrosine kinases, the mitogen-activated protein kinase (MAPK) p38, and extracellular signal regulated kinases (ERK)1/2. 6,7 UII-mediated vasoactive effects are variable and depend on the species and the disease state investigated. 8,9 In humans, UII peptide and human UT (hUT) receptor mRNAs are found in the heart (atrial and ventricular myocytes, fibroblasts) 10 and kidney (epithelia of tubules and ducts, renal capillary and glomerular endothelial cells). 11 Low levels of UII binding sites have been identified in the coronary arteries, kidney, left ventricle, and skeletal muscle. 12 A dose-dependent reduction of forearm blood flow in healthy subjects and vasoconstricto...

A 3.9-kb fragment of the genome of Pseudomonas putida H, containing the complete zwf-pgl-eda-operon, encoding glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase and 2-keto-3-deoxy-6-phosphogluconate-aldolase, respectively, and part of the divergently transcribed regulatory gene, hexR, was cloned and analyzed. The nucleotide sequences of these genes showed high similarities to the corresponding DNA sequences of P. putida KT2440 and also to sequences of Pseudomonas aeruginosa PAO1. Derivatives of strains H and KT2440, containing transcriptional lacZ fusions to P(zwf) were generated and used to study the expression of these operons. In both strains, this operon was induced by carbohydrates such as glucose, gluconate, fructose and glycerol. The transcription rate of the zwf-pgl-eda-operon was found to be about three times higher in the KT2440 background than in strain H. In both strains the induction of the zwf-pgl-eda-operon by carbohydrates during growth on carboxylic acids was not affected by carbon catabolite repression.