Thromboxane-induced contractile response of human coronary arterioles is diminished after cardioplegic arrest

Abstract

Background: We investigated the contractile response of human coronary microvasculature to thromboxane A-2 (TXA-2), with and without the blockade of TXA-2 receptors or the inhibition of phospholipase-C (PLC) or of protein kinase C-α (PKC-α) in the human coronary microvasculature before and after cardioplegia, followed by reperfusion (CP/Rep). Protein/gene expression and localization of TXA-2 receptors, TXA-2 synthase, PLC, and other TXA-2-related proteins was also examined.

Methods: Right atrial tissue was harvested before and after cold blood cardioplegia, followed by about 10 minutes of reperfusion, from 28 patients undergoing cardiac operations. Coronary arterioles (90 to 170 μm in diameter) were dissected from the harvested tissue.

Results: The post-CP/Rep contractile response of coronary arterioles to TXA-2 analog U-46619 was significantly impaired vs pre-CP/Rep (p<0.05). The TXA-2 receptor antagonist SQ-29548 (10(-6) M) prevented the contractile response to U-46619 (p<0.05). Pretreatment with the PLC inhibitor U73122 (10(-6) M) significantly inhibited the U-46619-induced contractile response (p<0.05). Administration of the PKC-α inhibitor safingol failed to affect U-46619-induced contraction. Total protein levels and gene expression of TXA-2 receptors, TXA-2 synthase, PLC-β3, phospho-PLC-β3, PLC-γ1, and phospho-PLC-γ1 were not altered after CP/Rep. Confocal microscopy showed no significant differences in the expression of TXA-2 receptors or PLC-β3 in the microcirculation. TXA-2 receptors and PLC-β3 were both present in smooth muscle and endothelium.

Conclusions: Cardioplegia/Rep decreases the contractile response of human coronary arterioles to TXA-2 soon after cardiac operations. The contractile response to the TXA-2 analog U-46619 is through activation of TXA-2 receptors and PLC.

Fig 1

(A) Coronary arteriolar vasoconstriction in response to the thromboxane A (TXA)-2 analog U-46619 before vs after cardioplegic ischemia, followed by reperfusion (pre-CP/Rep [white circles] vs post-CP/Rep [black circles]); *p < 0.05 vs pre-CP/Rep. (B) Administration of U-46619 with or without TXA-2 antagonist SQ-29548 to microvessels of pre-CP/Rep (U-46619 [pre-CP/Rep, white circles] or SQ-29548 + U-46619 [pre-CP/Rep, black squares]); *p < 0.05 vs U-46619 (pre-CP/Rep). (C) Infusion of U-46619, with or without the TXA-2 antagonist SQ-29548, to post-CP/Rep vessels (U-46619 [post-CP/Rep, black circles] or SQ-29548 + U-46619 [post-CP/Rep, black diamonds]); p < 0.05 vs U-46619 (post-CP/Rep); n = 6 to 8/group. The bars show the standard error of the mean.

Fig 2

(A) Administration of U-46619 to coronary arteriole before cardioplegic ischemia, followed by reperfusion (pre-CP/Rep), with or without the presence of the phospholipase-C (PLC) inhibitor U-73122 (U-46619 [Pre-CP/Rep, white circles] or U-73122 + U-46619 [pre-CP/Rep, white triangles]), *p < 0.05 vs U-46619 (pre-CP/Rep). (B) Perfusion of U-46619 to post-CP/Rep coronary arterioles, with or without the presence of the PLC inhibitor U-73122 (U-46619 [post-CP/Rep, black circles] or U-73122 + U-46619 [post-CP/Rep, black squares]),*p < 0.05 vs. U-46619 (post-CP/Rep); n = 6 to 8/group. The bars show the standard error of the mean.

Fig 3

(A) Addition of U-46619 to the vessels from samples before cardioplegic ischemia, followed by reperfusion (pre-CP/Rep), in the absence or presence of the protein kinase C (PKC) inhibitor safingol (U-46619 [pre-CP/Rep, white circles] or safingol + U-46619 [pre-CP/Rep, white diamonds]). (B) Addition of U-46619 to the vessels from post-CP/Rep samples in the absence or presence of PKC inhibitor safingol (U-46619 [post-CP/Rep, black circles] or safingol + U-46619 [post-CP/Rep, black diamonds]); n = 6 to 8/group. The bars show the standard error of the mean.

Fig 4

Representative immunoblot of human atria tissue. Lanes 1 to 12, loaded with 40 µg protein, were developed for (A, top) thromboxane A (TXA)-2 receptor and TXA-2 synthase (n = 6) and (B, top) phospholipase-C (PLC)-β3, phospho–PLC-β3, PLC-γ1, and phosphor–PLC-γ1 polypeptides (n = 6). Immunoblot band intensity shows unaltered levels of TXA-2 receptor, TXA-2 synthase, PLC-γ1, and PLC-β3 polypeptides after cardioplegic ischemia, followed by reperfusion (CP/Rep; bottom of Fig 4A and B), respectively. The bars show the standard error of the mean.

Fig 5

Immunolocalization of thromboxane A (TXA)-2 receptor, phospholipase-C (PLC)-γ1, and PLC-β3 polypeptides in human coronary microvessels (n = 6). Vessels were costained for smooth muscle α-actin with (A) TXA-2 receptor antibody or (B) PLC-β3. Matched negative controls are displayed below each row of primary antibody. (DAPI = 4’,6-diamidino-2-phenylindole.)