Attenuated Vasodilator Effectiveness of Protease-Activated Receptor 2 Agonist in Heterozygous par2 Knockout Mice

Abstract

Studies of homozygous PAR2 gene knockout mice have described a mix of phenotypic effects in vitro and in vivo. However, there have been few studies of PAR2 heterozygous (wild-type/knockout; PAR2-HET) mice. The phenotypes of many hemi and heterozygous transgenic mice have been described as intermediates between those of wild-type and knockout animals. In our study we aimed to determine the effects of intermediary par2 gene zygosity on vascular tissue responses to PAR2 activation. Specifically, we compared the vasodilator effectiveness of the PAR2 activating peptide 2-furoyl-LIGRLO-amide in aortas of wild-type PAR2 homozygous (PAR2-WT) and PAR2-HET mice. In myographs under isometric tension conditions, isolated aortic rings were contracted by alpha 1-adrenoeceptor agonist (phenylephrine), and thromboxane receptor agonist (U46619) and then relaxation responses by the additions of 2-furoyl-LIGRLO-amide, acetylcholine, and nitroprusside were recorded. A Schild regression analysis of the inhibition by a PAR2 antagonist (GB-83) of PAR2 agonist-induced aortic ring relaxations was used to compare receptor expression in PAR2-WT to PAR2-HET. PAR2 mRNA in aortas was measured by quantitative real-time PCR. In aortas contracted by either phenylephrine or U46619, the maximum relaxations induced by 2-furoyl-LIGRLO-amide were less in PAR2-HET than in the gender-matched PAR2-WT. GB-83 was 3- to 4-fold more potent for inhibition of 2fly in PAR2-HET than in PAR2-WT. PAR2 mRNA content of aortas from PAR2-HET was not significantly different than in PAR2-WT. Acetylcholine- and nitroprusside-induced relaxations of aortas from PAR2-HET were not significantly different than in PAR2-WT and PAR2 knockout. An interesting secondary finding was that relaxations induced by agonists of PAR2 and muscarinic receptors were larger in females than in males. We conclude that the lower PAR2-mediated responses in PAR2-HET aortas are consistent with evidence of a lower quantity of functional receptor expression, despite the apparently normal PAR2 mRNA content in PAR2-HET aortas.

Aspergillus fumigatus induction of IL‐33 expression in chronic rhinosinusitis is PAR2‐dependent

“…Because PAR2 was specifically upregulated in CRSwNP inflamed mucosa, we examined the role of PAR2 in the A. fumigatus ‐mediated induction of IL‐33 in CRSwNP SNECs. GB83 is a reversible PAR2‐selective antagonist that previously has been found to inhibit peptide agonist‐ and protease‐induced activation of PAR2 . GB83 was preincubated with confluent CRSwNP SNECs at increasing concentrations for a minimum of 2 hours at 37°C prior to the addition of A. fumigatus extract, and cells were collected 24 hours later for RNA analysis.…”

Sexual dimorphism in PAR2-dependent regulation of primitive colonic cells

“…All primers used have PCR efficiency > 90%. Hprt and Gapdh were used as reference genes since these genes have already been used in experiments where PAR 2 or GSK3 expression/activity varied [15, 20–22]. The delta Ct was calculated (Microsoft Excel software) from the means of reference gene and target gene duplicates.…”

“…Indeed, the PAR 2 -GSK3β pathway controlling ISC survival may pave the way to inflammation and cancer where GSK3β is overactivated [13]. Further, PAR 2 activity is known to control the expression of cell cycle genes [14] and has been shown to display sexual dimorphism in vasodilatation and pruritus [15, 16]. The primary organoid model was chosen to investigate a potential sexual dimorphism in PAR 2 -dependent regulation of ISC.…”

Heat shock protein‐27 and sex‐selective regulation of muscarinic and proteinase‐activated receptor 2‐mediated vasodilatation: differential sensitivity to endothelial NOS inhibition

“…The mechanisms for vasodilatation of the female‐derived tissues that persists in the presence of either L‐NAME or ODQ acting alone, but is blocked in the combined presence of L‐NAME and ODQ, remain to be determined. The resistance to L‐NAME for PAR2‐induced relaxation in the female tissues may relate to differences between the PAR2 responsiveness of male and female‐derived murine vascular tissue reported previously (Hennessey and McGuire, ). It is also possible that the effects may relate in some way to a constitutive activation of protein kinase G triggered either by residual NO or, as suggested by Greenstein, Nelson and colleagues (Khavandi et al ., ), by oxidative stress.…”

“…Top is equal to the maximum contraction (E max ), and log EC 50 is the agonist concentration that produces 50% of the maximum response. Bottom is equal to the resting tone in the absence of any contractile agonist (Hennessey and McGuire, ). Data points in the figures represent the mean ± SEM (bars in figures) of five or more independent experiments done on tissue preparations from eight or more different animals.…”