Fig 1.
Chemical structure of CaSR antagonist (calcilytic) and agonist (calcimimetic).
(A) The chemical structures of calcilytics, or negative allosteric modulators of CaSR, NPS2143 and Calhex 231. (B) The chemical structure of a calcimimetic, or a positive allosteric modulator of CaSR, R568.
Fig 2.
Excessive cell proliferation in PASMCs from IPAH patients.
The proliferation rates of PASMCs from normal subjects, IPAH patients, and CTEPH patients were analyzed using quantitative colorimetric assay based on MTT test for cellular viability. When PASMCs were subcultured in 96-well plates, the cell number was made up to approximately 1×104 cells per well. Summarized data show the proliferation rates of PASMCs from normal subjects, IPAH patients, and CTEPH patients. Data were obtained from 8 experiments. **p<0.01 vs. normal PASMCs.
Fig 3.
Inhibitory effect of NPS2143 on enhanced proliferation of PASMCs from IPAH patients.
The effects of NPS2143, which is widely used as a calcilytic or negative allosteric regulator of CaSR, on cell proliferation were examined in normal, IPAH, and CTEPH PASMCs. Summarized data (a) show the effect of NPS2143 on cell viability at the concentration range between 100 nM and 10 μM in normal (A), IPAH (B), and CTEPH (C) PASMCs. Dose-response curve (b) of cell proliferation at 72 h for NPS2143 in normal (A), IPAH (B), and CTEPH (C) PASMCs. The absorbance at several concentrations of NPS2143 was normalized by the value in the absence of NPS2143 (as 100%). The IC50 value of NPS2143 for the proliferation of IPAH-PASMCs was 2.64 μM and the Hill coefficient was 1.00. Data were obtained from 5~13 experiments. *p<0.05 or **p<0.01 vs. control (100%).
Fig 4.
Attenuation of cell proliferation by Calhex 231 in PASMCs from IPAH patients.
The effects of Calhex 231, a calcilytic structurally-unrelated to NPS2143, on cell proliferation were examined in normal, IPAH, and CTEPH PASMCs. Summarized data (a) show the effect of Calhex 231 on the cell viability at the concentration range between 100 nM and 10 μM in normal (A), IPAH (B), and CTEPH (C) PASMCs. Dose-response curve (b) of cell proliferation at 72 h for Calhex 231 in normal (A), IPAH (B), and CTEPH (C) PASMCs. The absorbance at several concentrations of Calhex 231 was normalized by the value in the absence of Calhex 231 (as 100%). The IC50 value of Calhex 231 for the proliferation of IPAH-PASMCs was 1.89 μM and the Hill coefficient was 1.39. Data were obtained from 5~8 experiments. **p<0.01 vs. control (100%).
Fig 5.
Enhancement of cell proliferation by a calcimimetic in PASMCs from IPAH patients.
The effects of R568, a calcimimetic or positive allosteric regulator of CaSR, on cell proliferation were examined in normal, IPAH, and CTEPH PASMCs. Summarized data (a) show the effect of R568 on the cell viability in the concentration range between 100 nM and 10 μM in normal (A), IPAH (B), and CTEPH (C) PASMCs. Dose-response curve (b) of cell proliferation at 48 h for R568 in normal (A), IPAH (B), and CTEPH (C) PASMCs. The absorbance at several concentrations of R568 was normalized by the value in the absence of R568 (as 100%). The EC50 value of R568 for the proliferation of IPAH-PASMCs was 0.33 μM and the Hill coefficient was 2.10. Data were obtained from 5~11 experiments. *p<0.05 or **p<0.01 vs. control (100%).
Fig 6.
Effect of CaSR modulators on cell proliferation using BrdU incorporation assay.
The proliferation rates of PASMCs from normal subjects, IPAH patients, and CTEPH patients were analyzed using quantitative colorimetric assay based on the BrdU incorporation method for cell proliferation. The effects of NPS2143 (A), Calhex 231 (B), and R568 (C) on cell proliferation were examined in normal, IPAH, and CTEPH PASMCs. Summarized data (a) show the effect of CaSR modulators at a concentration of 3 μM on cell proliferation at 48 (for R568) or 72 (for NPS2143 and Calhex 231) h in normal, IPAH, and CTEPH PASMCs. Dose-response curve (b) of cell proliferation for NPS2143, Calhex 231, and R568 in IPAH-PASMCs. The absorbance was normalized by the value in the absence of drug (as 100%). The IC50 values of NPS2143 and Calhex 231 for the proliferation of IPAH-PASMCs were 1.48 and 0.62 μM, respectively. The EC50 value of R568 was 0.34 μM. Data were obtained from 7~14 experiments. *p<0.05 or **p<0.01 vs. control (100%).