Changes of Kir4.1 and TASK-1 expression in rat Müller cell induced by SCH442416 at an elevated hydrostatic pressure in vitro

Objective

To evaluate the effect of adenosine receptor antagonist SCH442416 on the expression of Kir2.1, Kir4.1 and TASK-1 in rat Müller cell at an elevated hydrostatic pressure in vitro.

Methods

Thirty SPF Sprague Dawley rats were purchased from Shanghai Slack Laboratory Animals Ltd.Cultured Müller cells were divided into normal control group (n=6), 40 mmHg/24 hours (1 mmHg=0.133 kPa) group (n=6) and adenosine + SCH442416 intervention group(n=6). Müller cells were treated with 40 mmHg pressure for 24 hours in 40 mmHg/24 hours group, and Müller cells were treated with 40 mmHg pressure for 24 hours + 10 μmol/L adenosine + 100 nmol/L SCH442416 in adenosine + SCH442416 intervention group.The real-time PCR, Western blot, whole-cell patch-clamp recordings and immunohistochemistry were used to detect Kir2.1, Kir4.1 and TASK-1 expression and Müller cells Kir currents.The experimental procedures were in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory, and follow the 3R principle.

Results

Western blot assay showed that, following 40 mmHg pressure cultured for 24 hours, the expression of Kir4.1 and TASK-1 protein in Müller cell were significantly decreased by 38.6% and 52.6% compared with the normal control group, with significant differences between the two groups (both at P=0.000); Kir2.1 protein expression decreased by 14.7%, with insignificant difference between the two groups (P=0.082). Kir4.1 and TASK protein expression in adenosine + SCH442416 intervention group was increased by 60.7% and 61.4% compared with the 40 mmHg/24 hours group, with significant differences between the two groups (both at P=0.000); Kir2.1 protein expression in adenosine + SCH442416 intervention group was increased by 8.8% compared with the 40 mmHg/24 hours group, with insignificant difference between them (P=0.354). Real-time PCR assay showed that, following 40 mmHg pressure cultured for 24 hours, Kir2.1, Kir4.1 and TASK-1 mRNA expression in Müller cells were significantly decreased compared with the normal control group, with significant differences between the two groups (P=0.047, 0.001, 0.000); Kir4.1 and TASK-1 mRNA expression in Müller cells in the adenosine + SCH442416 intervention group was significantly increased compared with the 40 mmHg/24 hours group, with significant differences between the two groups (P=0.038, 0.030); however, there is no significant change in Kir2.1 mRNA expression (P=0.612).

Conclusions

SCH442416 upregulates the expression of Kir4.1 and TASK-1 mRNA and protein, but weakly affects the expression of Kir2.1.