Myocardial ischemia-reperfusion induces upregulation of contractile endothelin ETB receptor in rat coronary arteries
Objective We have previously shown increased expression of endothelin ETB receptors in coronary arteries of ischemic heart disease patients. ETB receptors are normal physiological conditions primarily situated in the vascular endothelial cells mediating vasodilation, whereas only a limited part are situated in the vascular smooth muscle cells mediating vasoconstriction. This study aims to examine whether heart ischemia-reperfusion leads to upregulation of contractile ETB receptors in the smooth muscle layer of the coronary arteries and to investigate the signaling pathways involved in the putative ETB receptor upregulation.
Methods and Results Thirteen Sprague-Dawley male rats (body weight 260-410 g) were anaesthetized with Hypnorm-Midazolam and subjected to 15 min occlusion of left anterior descending coronary artery (LAD) followed by 22 h of reperfusion. The contractile response to the specific ETB receptor agonist Sarafotoxin 6c (S6c) (1 pM to 30 nM) was investigated after cumulative additions in a sensitive wire-myograph. LAD segments, situated in the post-ischemic area displayed significantly augmented ETB receptor mediated vasoconstriction (68±3% of maximal contraction, n = 7) compared to coronary arteries from the non-ischemic area (23±6 % of maximal contraction, n = 8) and sham operated rats (22±5% of maximal contraction, n = 5). Increased density of ETB receptors localized in the vascular smooth muscle layer was confirmed by immunohistochemistry in LAD segments from the post-ischemic area compared to the non-ischemic segments.
Interestingly, a rapid ETB receptor upregulation also appeared when coronary arteries from healthy non-operated rats were incubated for a short-term (7 h) in a physiological saline solution. We used this in-vitro model to study the signaling pathway involved in the ETB receptor upregulation. Incubation (7 h) of the coronary arteries in the presence of either the transcriptional inhibitor (Actinomycin D, 4µM) or the selective mitogen-activated protein kinase kinase (MEK1/2) inhibitor (U0126, 10 µM) significantly attenuated the S6c mediated response compared to vehicle. Immunohistochemical staining displayed enhanced phoshorylation of ERK1/2 after 4 h of incubation and demonstrated ETB receptors in the vascular smooth muscle cells after 7h of incubation.
Conclusion The results demonstrate that heart ischemia-reperfusion in rats induces an upregulation of contractile ETB receptors in the vascular smooth muscle cells in coronary arteries in the post-ischemic area. This study suggests that the upregulation of the ETB receptors depends on a transcriptional upregulation and involves the MEK/ERK type of MAPK.