Upregulation of G protein‐couple receptor 68 (GPCR68) in pulmonary artery smooth muscle cells from animals and humans with pulmonary hypertension

Abstract

IntroductionPulmonary arterial hypertension (PAH) is a progressive and fatal disease with no cure. It is characterized by elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in part due to pulmonary vascular remodeling and sustained pulmonary vasoconstriction. The increased pulmonary arterial smooth muscle cell (PASMC) proliferation contributes to excessive pulmonary vascular remodeling while a rise in cytosolic free Ca2+concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation. G‐protein coupled receptors (GPCR), which are attractive pharmacological targets, are important regulators of pulmonary vascular tone and PASMC phenotype. Stimulation of GPCR leads to Ca2+ influx and increased [Ca2+]cytdue to store‐operated Ca2+ entry (SOCE) via inositol 1,4,5‐trisphosphate (IP3) production and receptor‐operated Ca2+entry (ROCE) via diacylglycerol (DAG) production. GPCR68 (or OGR1) is a proton‐sensing GPCR that responds to extracellular acidity and regulates a variety of cellular functions. As a Gq/11‐coupled receptor, GPR68 activation is known to lead to Ca2+ release from the intracellular stores in IP3‐dependent manner but the role of GPCR68 in the development of PAH is still unknown.MethodsLung, heart, brain, liver, and kidney tissues were isolated from control rats. Pulmonary hypertension model was induced by a single dose of monocrotaline (MCT) in rats. PASMC isolated from control or MCT rats and healthy subjects or patients with idiopathic pulmonary arterial hypertension (IPAH) were cultured in 5% fetal bovine serum (FBS) smooth muscle growth media and incubated in a humidified 5% CO2 atmosphere at 37°C. Total RNA was isolated from tissue or cells using TRIZOL method. mRNA expression of GPCR68 was quantified by RT‐PCR and qRT‐PCR. The data was analyzed using ImageJ and statistical analysis was performed using paired or unpaired Student’s t‐test.ResultsRT‐PCR analysis revealed that GPCR68 is expressed in brain, lung, heart, liver, and kidney in normal rat tissues (n=5). GPCR68 mRNA level was significantly higher in lung tissue and PASMC isolated from MCT‐treated rats compared with controls. Moreover, our data shows that GPCR68 is expressed in both human PASMC and pulmonary arterial endothelial cells. qRT‐PCR showed that GPCR68 was upregulated in PASMC isolated from IPAH patients compared with cells isolated from healthy subjects.ConclusionOur finding suggest that GPR68 is involved in the development and progression of PAH and may play an important role in Ca2+ signaling and enhanced [Ca2+]cyt in PASMC from animals and humans with PAH.Support or Funding InformationThis work was supported, in part, by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL125208, HL135807, and HL126609).