RT Journal Article
SR Electronic
T1 Fluorescent Ligands of the Bradykinin B<sub>1</sub> Receptors: Pharmacologic Characterization and Application to the Study of Agonist-Induced Receptor Translocation and Cell Surface Receptor Expression
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 159
OP 168
DO 10.1124/jpet.108.149724
VO 329
IS 1
A1 Bawolak, Marie-Thérèse
A1 Gera, Lajos
A1 Morissette, Guillaume
A1 Bouthillier, Johanne
A1 Stewart, John M.
A1 Gobeil, Lise-Andrée
A1 Lodge, Robert
A1 Adam, Albert
A1 Marceau, François
YR 2009
UL http://jpet.aspetjournals.org/content/329/1/159.abstract
AB Unlike the widely distributed and preformed B2 receptors, the bradykinin B1 receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B1 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-ϵ-aminocaproyl-Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-bradykinin] and the agonist B-10378 [5(6)CF-ϵ-aminocaproyl-Lys-des-Arg9-bradykinin]. B-10376 has a Ki of 10 to 20 nM to displace [3H]Lys-des-Arg9-bradykinin from rabbit or human recombinant B1 receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA2, 7.49). B-10378 was a full agonist at the naturally expressed B1 receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition Ki of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B1 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 B1 receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B1 receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B1 receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry). The American Society for Pharmacology and Experimental Therapeutics