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Identification of a B2 bradykinin receptor expressed by PC12 pheochromocytoma cells.

ABSTRACT: We have used rat PC12 pheochromocytoma cells, a clonal cell line closely related to sympathetic neurons, to investigate reports that the bradykinin receptor expressed in the peripheral nervous system is distinct from the well-characterized B2 bradykinin receptor of smooth muscle. Although there have been reports that [Thi5,8,D-Phe7]bradykinin [where Thi is beta-(2-thienyl)alanine] is a full agonist at some sites in the peripheral nervous system, we find that in PC12 cells [Thi5,8,D-Phe7]bradykinin behaves as a competitive antagonist of bradykinin-stimulated phosphatidylinositol turnover. In particular, sufficient concentrations of [Thi5,8,D-Phe7]bradykinin completely block the increase in inositol bisphosphate and trisphosphate in response to 100 nM bradykinin; [Thi5,8,D-Phe7]bradykinin alone, at up to 10 microM, does not appreciably increase inositol bisphosphate and trisphosphate. In contrast to the absence of evidence for a distinctive neuronal receptor, we have found convincing evidence that the bradykinin receptor previously identified in smooth muscle is present in PC12 cells. Using the polymerase chain reaction, we have isolated a full-length cDNA encoding a bradykinin receptor that is expressed in PC12 cells and verified that its nucleotide sequence is identical except at a single position to that of the rat uterine B2 bradykinin receptor. When expressed in COS cells this uterine bradykinin receptor exhibits the same high affinity for [3H]bradykinin (Kd 4.4 nM), the same relative affinities for a series of kinin antagonists, and the same efficient coupling to phosphatidylinositol turnover (EC50 2.5 nM) as the receptor in PC12 cells. We interpret our data, and the findings of a number of pharmacological studies, as strengthening the view that the B2 receptor expressed in PC12 cells and in certain cells of the peripheral nervous system is identical to the receptor in rat uterine smooth muscle.

SUBMITTER: Nardone J

PROVIDER: S-EPMC43795 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Identification of a B2 bradykinin receptor expressed by PC12 pheochromocytoma cells.

Nardone J J Gerald C C Rimawi L L Song L L Hogan P G PG

Proceedings of the National Academy of Sciences of the United States of America 19940501 10

We have used rat PC12 pheochromocytoma cells, a clonal cell line closely related to sympathetic neurons, to investigate reports that the bradykinin receptor expressed in the peripheral nervous system is distinct from the well-characterized B2 bradykinin receptor of smooth muscle. Although there have been reports that [Thi5,8,D-Phe7]bradykinin [where Thi is beta-(2-thienyl)alanine] is a full agonist at some sites in the peripheral nervous system, we find that in PC12 cells [Thi5,8,D-Phe7]bradykin ...[more]

PMID: 8183922

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Project description:Bradykinin B2 receptor (B2R) and angiotensin-(1-7) Mas receptor (MasR)-mediated effects are physiologically interconnected. The molecular basis for such cross talk is unknown. It is hypothesized that the cross talk occurs at the receptor level. We investigated B2R-MasR heteromerization and the functional consequences of such interaction. B2R fused to the cyan fluorescent protein and MasR fused to the yellow fluorescent protein were transiently coexpressed in human embryonic kidney293T cells. Fluorescence resonance energy transfer analysis showed that B2R and MasR formed a constitutive heteromer, which was not modified by their agonists. B2R or MasR antagonists decreased fluorescence resonance energy transfer efficiency, suggesting that the antagonist promoted heteromer dissociation. B2R-MasR heteromerization induced an 8-fold increase in the MasR ligand-binding affinity. On agonist stimulation, the heteromer was internalized into early endosomes with a slower sequestration rate from the plasma membrane, compared with single receptors. B2R-MasR heteromerization induced a greater increase in arachidonic acid release and extracellular signal-regulated kinase phosphorylation after angiotensin-(1-7) stimulation, and this effect was blocked by the B2R antagonist. Concerning serine/threonine kinase Akt activity, a significant bradykinin-promoted activation was detected in B2R-MasR but not in B2R-expressing cells. Angiotensin-(1-7) and bradykinin elicited antiproliferative effects only in cells expressing B2R-MasR heteromers, but not in cells expressing each receptor alone. Proximity ligation assay confirmed B2R-MasR interaction in human glomerular endothelial cells supporting the interaction between both receptors in vivo. Our findings provide an explanation for the cross talk between bradykinin B2R and angiotensin-(1-7) MasR-mediated effects. B2R-MasR heteromerization induces functional changes in the receptor that may lead to long-lasting protective properties.

Project description:We here report the discovery and early characterization of Compound 3, a representative of a novel class of small molecule bradykinin (BK) B2 receptor antagonists, and its superior profile to the prior art B2 receptor antagonists Compound 1 and Compound 2. Compound 3, Compound 2, and Compound 1 are highly potent antagonists of the human recombinant B2 receptor (Kb values 0.24, 0.95, and 1.24 nM, respectively, calcium mobilization assay). Compound 3 is more potent than the prior art compounds and icatibant in this assay (Kb icatibant 2.81 nM). The compounds also potently inhibit BK-induced contraction of endogenous B2 receptors in a human isolated umbilical vein bioassay. The potencies of Compound 3, Compound 2, Compound 1, and icatibant are (pA2 values) 9.67, 9.02, 8.58, and 8.06 (i.e. 0.21, 0.95, 2.63, and 8.71 nM), respectively. Compound 3 and Compound 2 were further characterized. They inhibit BK-induced c-Fos signaling and internalization of recombinant human B2 receptors in HEK293 cells, and do not antagonize the venous effects mediated by other G protein-coupled receptors in the umbilical vein model, including the bradykinin B1 receptor. Antagonist potency of Compound 3 at cloned cynomolgus monkey, dog, rat, and mouse B2 receptors revealed species selectivity, with a high antagonist potency for human and monkey B2 receptors, but several hundred-fold lower potency for the other B2 receptors. The in vitro off-target profile of Compound 3 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor. Compound 3 showed a lower intrinsic clearance in the microsomal stability assay than the prior art compounds. With an efflux ratio of 1.0 in the Caco-2 permeability assay Compound 3 is predicted to be not a substrate of efflux pumps. In conclusion, we discovered a novel chemical class of highly selective and very potent B2 receptor antagonists, as exemplified by Compound 3. The compound showed excellent absorption in the Caco-2 assay, predictive of good oral bioavailability, and favourable metabolic stability in liver microsomes. Compound 3 has provided a significant stepping stone towards the discovery of the orally bioavailable B2 antagonist PHA-022121, currently in phase 1 clinical development.

Dataset Information

Identification of a B2 bradykinin receptor expressed by PC12 pheochromocytoma cells.

Publications

Identification of a B2 bradykinin receptor expressed by PC12 pheochromocytoma cells.

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