Arrestin-independent internalization and recycling of the urotensin receptor contribute to long-lasting urotensin II-mediated vasoconstriction

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 (-log EC50, mol/L:9.0+/-0.1). A second application of UII after 30 minutes elicited a reduced contraction (36+/-4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII ((125)I-UII), approximately 70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [t(h)]: 5.6+/-0.2 minutes), but recycled quantitatively within 60 minutes (t(h) 31.9+/-2.6 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. The continuous externalization of rUT receptors provides the basis for repetitive and lasting UII-mediated vasoconstriction

Detection of angiotensin II in supernatants of stimulated mononuclear leukocytes by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass analysis

Angiotensin II (Ang II) is the major vasoactive component of the renin-angiotensin system. Several components of the renin-angiotensin system have been demonstrated in different tissues. Whereas the roles of tissue and renal renin-angiotensin system have been studied in detail, much less is known on whether the corpuscular elements of circulating blood contribute to Ang II production. Here we examined whether, in addition to vasculature, blood cells also contribute to the circulating Ang II levels. Mononuclear leukocytes were obtained from healthy subjects and were incubated. The resulting supernatant was chromatographed using different chromatographic methods. The vasoconstrictive effects of aliquots of the resulting fractions were tested. Each fraction with a vasoconstrictive effect was analyzed by mass spectrometry. In one fraction with a strong vasoconstrictive effect, Ang II was identified. Mononuclear lymphocytes produced Ang II in amounts sufficient to stimulate Ang II type 1 receptors. Moreover, in mononuclear leukocytes, renin as well as angiotensin-converting enzyme mRNA expression was detectable by RT-PCR. These findings demonstrate that mononuclear leukocytes are a source of Ang II. Ang II secretion by these cells may play a significant role in humoral vascular regulation. In conclusion, the isolation of Ang II in supernatants of mononuclear leukocytes adds a further physiological source of Ang II to the current view of angiotensin metabolism. The quantitative role of lymphocyte-derived Ang II secretion compared with the other sources of Ang II should be defined further, but the release found under the present conditions is at least sufficient to elicit vasoconstrictive effects

THE COMPLETE SEQUENCE OF A 40-KDA ACTIN-MODULATING PROTEIN FROM THE EARTHWORM LUMBRICUS-TERRESTRIS

GIEBING T, Hinssen H, DHAESE J. THE COMPLETE SEQUENCE OF A 40-KDA ACTIN-MODULATING PROTEIN FROM THE EARTHWORM LUMBRICUS-TERRESTRIS. EUROPEAN JOURNAL OF BIOCHEMISTRY. 1994;225(3):773-779.The complete primary structure of a 40-kDa actin-modulating protein from the earthworm Lumbricus terrestris is presented. A muscle-specific cDNA library of the earthworm was constructed and screened with a specific DNA probe obtained by polymerase chain reaction considering information from peptide sequencing. A full-length clone with a coding region of 1098 bp was isolated. The deduced polypeptide sequence of 366 amino acids (41457 Da) reveals the segmental structure typical of both the 40-kDa and 80-kDa actin-modulating proteins. Prominent similarities to the 80-kDa protein gelsolin especially exist with respect to the first segment and to the C-terminal segment. The comparatively high nucleation efficiency of the earthworm actin modulator is probably determined by its third segment which seems to enable the earthworm actin modulator to bind a second G-actin molecule more tightly than other previously described 40-kDa modulators

The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems

1. Several peptidic urotensin-II (UT) receptor antagonists exert ‘paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells. 2. BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4–11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)≫GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human≫rat UT receptor). 3. The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. 4. In contrast to the ‘low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4–5-fold less than seen with urantide). Since GSK248451 (1 mg kg(−1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases