Fluorescent ligands of the bradykinin B1 receptors: pharmacologic characterization and application to the study of agonist-induced receptor translocation and cell surface receptor expression.
Unlike the widely distributed and preformed B(2) receptors, the bradykinin B(1) receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B(1) receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-epsilon-aminocaproyl-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-bradykinin] and the agonist B-10378 [5(6)CF-epsilon-aminocaproyl-Lys-des-Arg(9)-bradykinin]. B-10376 has a K(i) of 10 to 20 nM to displace [(3)H]Lys-des-Arg(9)-bradykinin from rabbit or human recombinant B(1) receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA(2), 7.49). B-10378 was a full agonist at the naturally expressed B(1) receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition K(i) of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B(1) receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B(1) receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B(1) receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B(1) receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).
Bawolak
Marie-Thérèse
MT
Centre de Recherche en Rhumatologie et Immunologie, Room T1-49, Centre Hospitalier Universitaire de Québec, Quebec, QC, Canada.
Gera
Lajos
L
Morissette
Guillaume
G
Bouthillier
Johanne
J
Stewart
John M
JM
Gobeil
Lise-Andrée
LA
Lodge
Robert
R
Adam
Albert
A
Marceau
François
F
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D001920:Bradykinin / Q000031:analogs & derivatives / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D008024:Ligands; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D011817:Rabbits; D043783:Receptor, Bradykinin B1 / Q000819:agonists* / Q000096:biosynthesis / Q000187:drug effects*; D011956:Receptors, Cell Surface / Q000819:agonists / Q000096:biosynthesis / Q000187:drug effects
C510436:B-9958; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008024:Ligands; D043783:Receptor, Bradykinin B1; D011956:Receptors, Cell Surface; D001920:Bradykinin