Enhanced Ca 2+ -Sensing Receptor Function in Idiopathic Pulmonary Arterial Hypertension

Aya Yamamura,Amy Zeifman,Jason X.-J Yuan,Adriana M Zimnicka,Hisao Yamamura,Ruby A Fernandez,Ayako Makino,Qiang Guo,Nicole M Pohl,Kimberly A Smith,Hui Dong

doi:10.1161/circresaha.112.266361

Abstract

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca(2+)](cyt) and enhanced Ca(2+) influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). We examined whether the extracellular Ca(2+)-sensing receptor (CaSR) is involved in the enhanced Ca(2+) influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. In normal PASMC superfused with Ca(2+)-free solution, addition of 2.2 mmol/L Ca(2+) to the perfusate had little effect on [Ca(2+)](cyt). In IPAH-PASMC, however, restoration of extracellular Ca(2+) induced a significant increase in [Ca(2+)](cyt). Extracellular application of spermine also markedly raised [Ca(2+)](cyt) in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca(2+)-induced [Ca(2+)](cyt) rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca(2+)-mediated [Ca(2+)](cyt) increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. The extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca(2+) influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.

Highlights

Is an important stimulus for pulmonary vasoconstriction and vascular remodeling
Using animal models of pulmonary hypertension, our data showed that Ca2؉-sensing receptor (CaSR) expression and function were both enhanced in pulmonary arterial smooth muscle cells (PASMC), whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia
The extracellular Ca2؉-induced increase in [Ca2؉]cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca2؉ influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension. (Circ Res. 2012;111:469-481.)

Summary

Methods

Human PASMC were isolated from normal control subjects and patients with IPAH and chronic thromboembolic pulmonary hypertension (CTEPH).[8,20,21] Approval to use the human lung tissues and cells was granted by the UIC Institutional Review Board. Human PASMC were cultured in Medium 199 supplemented with 10% fetal bovine serum at 37°C. The cells at passages 5 to 8 were used for the experiments. We used freshly-dissociated PASMC from rats[22] and PASMC from mice.. [Ca2؉]cyt Measurement [Ca2ϩ]cyt was measured in PASMC using fura-2 and a Nikon digital fluorescent imaging system.[24] Cells were loaded with 4 ␮mol/L fura-2 acetoxymethyl ester (fura-2/AM) for 60 minutes at 25°C and [Ca2ϩ]cyt was measured using a ratiometric method at 32°C

Results

Discussion

Conclusion