COMPARISON OF MODIFIED ENDOTHELIAL NITRIC OXIDE SYNTHASE FORMS IN HYPERTENSIVE AND NORMOTENSIVE RATS

Highlights

• Aortic endothelial cells and perivascular endothelial cells have distinct distribution of phosphorylated forms of endothelial nitric oxide synthase (eNOS)
• In hypertensive rats, aortic endothelial cells have increased levels of activated eNOS (Ser632 and Ser1177) whilst perivascular endothelial cells have elevated levels of inhibited eNOS (Ser117 and Thr495).
• Perivascular endothelial cells have increased levels of total and phosphorylated eNOS in comparison with aortic endothelial cells.

Abstract

Aim. To study the expression of endothelial nitric oxide synthase (eNOS) and its modified forms in the aortic endothelial cells and perivascular endothelial cells in hypertensive and normotensive rats.

Methods. The study included 12 male hypertensive ISIAH rats and 12 male normotensive Wistar rats at the 4 months of age. Following the cryosectioning, we performed an immunohistochemical staining of descending aorta using different antibodies to total eNOS and to phosphorylated eNOS (Ser117, Thr495, Ser632, Ser1177). Next, we scanned the whole slide images and conducted a semi-quantitative analysis of immunohistochemical signal in the aortic endothelial cells and perivascular endothelial cells using ImageJ software. Statistical analysis was carried out by Mann–Whitney U-test (when comparing eNOS expression in the endothelium of hypertensive and normotensive rats) and Wilcoxon matched-pairs signed-rank test (when comparing eNOS expression in the aortic endothelial cells and perivascular endothelial cells).

Results. Aortic endothelial cells and perivascular endothelial cells of hypertensive rats tended to the increase in total eNOS regardless of the antibody, although the staining pattern differed across the antibodies. Aortic endothelial cells of hypertensive rats had higher expression of eNOS phosphorylated at Ser632 and Ser1177 sites (activating phosphorylation) whereas perivascular endothelial cells displayed elevated levels of eNOS phosphorylated at Ser117 and Thr495 (inhibiting phosphorylation) and Ser632 (activating phosphorylation). Expression of total and phosphorylated eNOS in perivascular endothelial cells was significantly higher than in the aortic endothelial cells.

Conclusion. In comparison with normotensive Wistar rats, hypertensive ISIAH rats are characterised by an increased expression of activated eNOS in the aortic endothelial cells (Ser632 and Ser1177 phosphorylation) and by an elevated expression of inhibited eNOS (Ser117 and Thr395 phosphorylation) in the perivascular endothelial cells. Perivascular endothelial cells have a higher expression of total and phosphorylated eNOS than aortic endothelial cells.

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