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Chemerin Elicits Potent Constrictor Actions via Chemokine ‐ Like Receptor 1 ( CMKLR 1), not G ‐ Protein ‐ Coupled Receptor 1 ( GPR 1), in Human and Rat Vasculature

View Article: PubMed Central - PubMed

ABSTRACT

Background: Circulating levels of chemerin are significantly higher in hypertensive patients and positively correlate with blood pressure. Chemerin activates chemokine‐like receptor 1 (CMKLR1 or ChemR23) and is proposed to activate the “orphan” G‐protein‐coupled receptor 1 (GPR1), which has been linked with hypertension. Our aim was to localize chemerin, CMKLR1, and GPR1 in the human vasculature and determine whether 1 or both of these receptors mediate vasoconstriction.

Methods and results: Using immunohistochemistry and molecular biology in conduit arteries and veins and resistance vessels, we localized chemerin to endothelium, smooth muscle, and adventitia and found that CMKLR1 and GPR1 were widely expressed in smooth muscle. C9 (chemerin149–157) contracted human saphenous vein (pD2=7.30±0.31) and resistance arteries (pD2=7.05±0.54) and increased blood pressure in rats by 9.1±1.0 mm Hg at 200 nmol. Crucially, these in vitro and in vivo vascular actions were blocked by CCX832, which we confirmed to be highly selective for CMKLR1 over GPR1. C9 inhibited cAMP accumulation in human aortic smooth muscle cells and preconstricted rat aorta, consistent with the observed vasoconstrictor action. Downstream signaling was explored further and, compared to chemerin, C9 showed a bias factor=≈5000 for the Gi protein pathway, suggesting that CMKLR1 exhibits biased agonism.

Conclusions: Our data suggest that chemerin acts at CMKLR1, but not GPR1, to increase blood pressure. Chemerin has an established detrimental role in metabolic syndrome, and these direct vascular actions may contribute to hypertension, an additional risk factor for cardiovascular disease. This study provides proof of principle for the therapeutic potential of selective CMKLR1 antagonists.

No MeSH data available.


Characterization of small‐molecule CCX832 using cells expressing CMKLR1 or GPR1. Radiolabeled competition binding studies, with CCX832 (■) and unlabeled C9 (▼) as the competing ligands, revealed that CCX832 competed for all binding of radiolabeled C9 to CMKLR1 (A), but had no effect on binding to GPR1 (B). Data are expressed as a % of the total specific binding and show mean±SEM (n=3–6 independent replicates). pKi values are shown in Table 1. In β‐arrestin recruitment assays, increasing concentrations of CCX832 caused a rightward shift of C9 response at CMKLR1 (C) (pA2=8.32±0.04), but had no effect at GPR1 (D). Data are expressed as % of the maximum response to C13 in each assay and show mean±SEM (n=3 independent replicates). CMKLR1 indicates chemokine‐like receptor 1; GPR1, G‐protein‐coupled receptor 1.
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jah31763-fig-0008: Characterization of small‐molecule CCX832 using cells expressing CMKLR1 or GPR1. Radiolabeled competition binding studies, with CCX832 (■) and unlabeled C9 (▼) as the competing ligands, revealed that CCX832 competed for all binding of radiolabeled C9 to CMKLR1 (A), but had no effect on binding to GPR1 (B). Data are expressed as a % of the total specific binding and show mean±SEM (n=3–6 independent replicates). pKi values are shown in Table 1. In β‐arrestin recruitment assays, increasing concentrations of CCX832 caused a rightward shift of C9 response at CMKLR1 (C) (pA2=8.32±0.04), but had no effect at GPR1 (D). Data are expressed as % of the maximum response to C13 in each assay and show mean±SEM (n=3 independent replicates). CMKLR1 indicates chemokine‐like receptor 1; GPR1, G‐protein‐coupled receptor 1.

Mentions: To confirm the selectivity of reported CMKLR1 antagonist, binding and functional studies were carried out using the same cell lines. CCX832 competed for radiolabeled C9 binding to CMKLR1 (pKi=9.16±0.42; Figure 8A), but not GPR1 (Figure 8B). It blocked the C9‐mediated β‐arrestin recruitment at CMKLR1 (pA2=8.32±0.04, Figure 8C), but had no effect on the C9 response at GPR1 (Figure 8D).


Chemerin Elicits Potent Constrictor Actions via Chemokine ‐ Like Receptor 1 ( CMKLR 1), not G ‐ Protein ‐ Coupled Receptor 1 ( GPR 1), in Human and Rat Vasculature
Characterization of small‐molecule CCX832 using cells expressing CMKLR1 or GPR1. Radiolabeled competition binding studies, with CCX832 (■) and unlabeled C9 (▼) as the competing ligands, revealed that CCX832 competed for all binding of radiolabeled C9 to CMKLR1 (A), but had no effect on binding to GPR1 (B). Data are expressed as a % of the total specific binding and show mean±SEM (n=3–6 independent replicates). pKi values are shown in Table 1. In β‐arrestin recruitment assays, increasing concentrations of CCX832 caused a rightward shift of C9 response at CMKLR1 (C) (pA2=8.32±0.04), but had no effect at GPR1 (D). Data are expressed as % of the maximum response to C13 in each assay and show mean±SEM (n=3 independent replicates). CMKLR1 indicates chemokine‐like receptor 1; GPR1, G‐protein‐coupled receptor 1.
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jah31763-fig-0008: Characterization of small‐molecule CCX832 using cells expressing CMKLR1 or GPR1. Radiolabeled competition binding studies, with CCX832 (■) and unlabeled C9 (▼) as the competing ligands, revealed that CCX832 competed for all binding of radiolabeled C9 to CMKLR1 (A), but had no effect on binding to GPR1 (B). Data are expressed as a % of the total specific binding and show mean±SEM (n=3–6 independent replicates). pKi values are shown in Table 1. In β‐arrestin recruitment assays, increasing concentrations of CCX832 caused a rightward shift of C9 response at CMKLR1 (C) (pA2=8.32±0.04), but had no effect at GPR1 (D). Data are expressed as % of the maximum response to C13 in each assay and show mean±SEM (n=3 independent replicates). CMKLR1 indicates chemokine‐like receptor 1; GPR1, G‐protein‐coupled receptor 1.
Mentions: To confirm the selectivity of reported CMKLR1 antagonist, binding and functional studies were carried out using the same cell lines. CCX832 competed for radiolabeled C9 binding to CMKLR1 (pKi=9.16±0.42; Figure 8A), but not GPR1 (Figure 8B). It blocked the C9‐mediated β‐arrestin recruitment at CMKLR1 (pA2=8.32±0.04, Figure 8C), but had no effect on the C9 response at GPR1 (Figure 8D).

View Article: PubMed Central - PubMed

ABSTRACT

Background: Circulating levels of chemerin are significantly higher in hypertensive patients and positively correlate with blood pressure. Chemerin activates chemokine‐like receptor 1 (CMKLR1 or ChemR23) and is proposed to activate the “orphan” G‐protein‐coupled receptor 1 (GPR1), which has been linked with hypertension. Our aim was to localize chemerin, CMKLR1, and GPR1 in the human vasculature and determine whether 1 or both of these receptors mediate vasoconstriction.

Methods and results: Using immunohistochemistry and molecular biology in conduit arteries and veins and resistance vessels, we localized chemerin to endothelium, smooth muscle, and adventitia and found that CMKLR1 and GPR1 were widely expressed in smooth muscle. C9 (chemerin149–157) contracted human saphenous vein (pD2=7.30±0.31) and resistance arteries (pD2=7.05±0.54) and increased blood pressure in rats by 9.1±1.0 mm Hg at 200 nmol. Crucially, these in vitro and in vivo vascular actions were blocked by CCX832, which we confirmed to be highly selective for CMKLR1 over GPR1. C9 inhibited cAMP accumulation in human aortic smooth muscle cells and preconstricted rat aorta, consistent with the observed vasoconstrictor action. Downstream signaling was explored further and, compared to chemerin, C9 showed a bias factor=≈5000 for the Gi protein pathway, suggesting that CMKLR1 exhibits biased agonism.

Conclusions: Our data suggest that chemerin acts at CMKLR1, but not GPR1, to increase blood pressure. Chemerin has an established detrimental role in metabolic syndrome, and these direct vascular actions may contribute to hypertension, an additional risk factor for cardiovascular disease. This study provides proof of principle for the therapeutic potential of selective CMKLR1 antagonists.

No MeSH data available.