Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function

Xiang-Qin Tian, Ke-Tao Ma, Xian-Wei Wang, Yang Wang, Zhi-Kun Guo, Jun-Qiang Si

Cell Physiol Biochem. 2018;49(2):706-716.

PMID: 30165368

Abstract:

Background/aims:




Calcium-activated chloride channels (CaCCs) regulate numerous physiological processes including cell proliferation, migration, and extracellular matrix secretion. T16Ainh-A01 and CaCCinh-A01 are selective inhibitors of CaCCs. But it is unknown whether these two compounds have functional effects on cardiac fibroblasts (CFs).













Methods:




Primary CFs were obtained by enzymatic dissociation of cardiomyocytes from neonatal rat hearts. Intracellular Ca2+ ([Ca2+]i) and Cl- ([Cl-]i) were measured using the fluorescent calcium indicators (Fluo-4 AM) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide respectively. The expression of anoctamin-1 (ANO1) and α-smooth muscle actin (α-SMA) was detected by quantitative RT-PCR, immunofluorescence, and western blotting. A hydroxyproline assay was used to examine collagen secretion. Cell proliferation, cell cycle distribution, and cell migration were assessed by Cell Counting Kit-8, flow cytometry, and Transwell assays, respectively.













Results:




ANO1 was preferentially expressed on the nuclear membrane and partially within intracellular compartments around the nucleus. T16Ainh-A01 and CaCCinh-A01 displayed different inhibitory effects on [Cl-]i in CFs. T16Ainh-A01 considerably decreased [Cl-]i in the nucleus, whereas CaCCinh-A01 reduced [Cl-]i in intracellular compartments around the nucleus, and both inhibitors exhibited a minimal effect on [Ca2+]i in CFs. ANO1 and α-SMA expression levels were significantly repressed by CaCCinh-A01. T16Ainh-A01 showed a marked inhibitory effect on the mRNA levels of ANO1 and α-SMA, but had a negligible effect on ANO1 at the protein level. T16Ainh-A01 and CaCCinh-A01 led to the significant repression of cell proliferation, cell migration, and collagen secretion in CFs.













Conclusion:




Our findings indicate that T16Ainh-A01 and CaCCinh-A01 have the potential to inhibit the proliferation and collagen secretion of CFs and may serve as novel anti-fibrotic therapeutic drugs in the future.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP162558523	1-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide		162558-52-3	Price
AP407587331	CaCCinh-A01		407587-33-1	Price
AP552309429	T16Ainh-A01		552309-42-9	Price