Endothelin-1-induced contractile responses of human coronary arterioles via endothelin-A receptors and PKC-alpha signaling pathways

Abstract

Background: We investigated the contractile function in responses to endothelin-1 (ET-1) in the human coronary microvasculature as well as the roles of endothelin receptors and protein kinase C-alpha (PKC-alpha) in these responses.

Methods: Human atrial tissue was harvested from patients who underwent cardiac surgery pre- and post-cardioplegia (CP)/cardiopulmanory bypass (CPB). Microvascular constriction was assessed in pre- and post-CP/CPB samples in responses to ET-1, in the presence and absence of an endothelin-A (ET-A) receptor antagonist, an endothelin-B (ET-B) receptor antagonist, or a PKC-alpha inhibitor, respectively. The expression and localization of the ET-A and ET-B receptors were also examined using immunoblot and immunofluorescence photomicroscopy.

Results: The post-CP/CPB contractile response of coronary arterioles to ET-1 was significantly decreased compared with the pre-CP/CPB responses. The response to ET-1 was significantly inhibited in the presence of the ET-A antagonist BQ123 (10(-7)mol/L), but these values remained unchanged with the ET-B receptor antagonist BQ788 (10(-7)mol/L). Pretreatment with the PKC-alpha inhibitor safingol (2.5 x 10(-5) mol/L) reversed the ET-1 responses from contraction into relaxation. The total polypeptide levels of ET-A and ET-B receptors were not altered post-CP/CPB. Immunoblot and immunofluorescent staining displayed strong signals for ET-A receptors and relatively weak signals for ET-B receptors localized on coronary microvasculature.

Conclusion: CP/CPB decreases the contractile function of human coronary microvessels in responses to ET-1. ET-A receptors are predominantly localized in the human coronary microcirculation, whereas ET-B receptors seem to be less abundant. The contractile response to ET-1 is in part through the activation of ET-A receptors and PKC-alpha. These results suggest a role of ET-1-induced contraction in the vasomotor dysfunction after cardiac surgery.

Fig 1

Coronary microvascular vasoconstriction in response to endothelin-1 (ET-1) (A) pre- vs post-cardioplegic arrest /cardiopulmonary bypass (CP/CPB), (B) pre-CP/CPB vs pre-CP/CPB + BQ123 , (C) pre-CP/CPB vs pre-CP/CPB + BQ788, *P < 0.05.

Fig 2

Coronary microvascular vasoconstriction in response to endothelin-1 (ET-1) (A) post-cardioplegic arrest /cardiopulmonary bypass (post-CP/CPB) vs post-CP/CPB + BQ123, (B) post-CP/CPB vs post-CP/CPB + BQ788, *P < 0.05

Fig 3

Coronary microvascular vasoconstriction in response to Endothelin-1 (ET-1) (A) pre-cardioplegic arrest /cardiopulmonary bypass (pre-CP/CPB) vs pre-CP/CPB + safingol, (B) post-CP/CPB vs post-CP/CPB + safingol, *P < 0.05.

Fig 4

Representative immunoblot of human atria tissue. Lanes 1-2 loaded with 40 μg protein were developed for ET-A and ET-B receptor polypeptides, respectively, showing unaltered levels of ET-A and ET-B polypeptides after pre- vs post-cardioplegic arrest /cardiopulmonary bypass (CP/CPB).

Fig 5

Immunolocalization of ET-A and ET-B receptors (ET-AR and ET-BR) polypeptides in human coronary microvessels. Vessels were co-stained for smooth muscle α–actin and either (A) ET-AR, or (B) ET-BR antibody Matched negative controls for ET-AR or ET-BR are displayed below each row, indicating only α–actin signals in α–actin staining and merged images.

Fig 5

Immunolocalization of ET-A and ET-B receptors (ET-AR and ET-BR) polypeptides in human coronary microvessels. Vessels were co-stained for smooth muscle α–actin and either (A) ET-AR, or (B) ET-BR antibody Matched negative controls for ET-AR or ET-BR are displayed below each row, indicating only α–actin signals in α–actin staining and merged images.