Impaired contractile response of human peripheral arterioles to thromboxane A-2 after cardiopulmonary bypass

Abstract

Background: We studied the contractile response of human peripheral microvasculature to thromboxane A-2 (TXA-2) before and after cardiopulmonary bypass (CPB), with and without the blockade of TXA-2 receptors, or the inhibition of phospholipase C (PLC), phospholipase A-2 (PLA-2) or protein kinase C (PKC)-α. We also examined the protein/gene expression and localization of TXA-2 receptors, TXA-2 synthase, PLC, and other TXA-2-related proteins.

Methods: Skeletal muscle arterioles (90-180 μm in diameter) were harvested pre- and post-CPB from patients (n = 28) undergoing cardiac surgery.

Results: The post-CPB contractile response of skeletal muscle arterioles to TXA-2 analog U-46619 was impaired compared with pre-CPB (P < .05). The presence of TXA-2 receptor antagonist SQ-29548 (10(-6)mol/L) prevented the contractile response to U-46619 (P < .05). Pretreatment with the PLC inhibitor U-73122 (10(-6)mol/L) significantly inhibited the U-46619-induced contractile response (P < .01). Administration of the PLA-2 inhibitor quinacrine (10(-6)mol/L) or PKC-α inhibitor safingol (2 × 10(-5)mol/L), however, failed to affect U-46619-induced contraction. Total protein levels and gene expression of TXA-2 receptors, and TXA-2 synthase of skeletal muscle, were not altered post-CPB. Confocal microscopy showed no differences in the expression of PLCβ-3 in the microcirculation. PLCβ-3 was localized to both smooth muscle and endothelium.

Conclusion: CPB decreases the contractile response of human peripheral arterioles to TXA-2 soon after cardiac surgery. This response may be in part responsible for the decrease in vascular tone, and accompanying hypotension sometimes observed after cardiac operations utilizing CPB.

Figure 1

(A) Peripheral arteriolar vasoconstriction in response to TXA-2 analog U-46619 before vs. after cardiopulmonary bypass (pre-CPB vs. post-CPB); *P < 0.05 vs. pre-CPB;; (B) Administration of U-46619 with or without TXA-2 antagonist SQ-29548 to microvessels of pre-CPB [U-46619 (pre-CPB) or SQ-29548 + U-46619 (pre-CPB)]; *P < 0.05, vs. U-46619 (pre-CPB); (C) Infusion of U-46619 with or without TXA-2 antagonist SQ-29548 to post-CPB vessels [U46619(post-CPB) or. SQ-29548 + U-46619 (post-CPB)]; P<0.05 vs. U-46619 (post-CPB); n = 8–11/group.

Figure 2

(A) Application of U-46619 to peripheral arterioles of pre-CPB with or without the presence of PLC inhibitor U-73122 [U-46619 (Pre-CPB) or U-73122 + U-46619 (pre-CPB), *P < 0.05 vs. U-46619 (pre-CPB); (B) Application of U-46619 to post-CPB peripheral arterioles with or without the presence of PLC inhibitor U-73122 [U-46619( post-CPB) or U-73122 + U-46619 (post-CPB)],*P <0.05 vs. U-46619 (post-CPB); n = 8–11/group.

Figure 3

(A) Exposure of U-46619 to peripheral arterioles of pre-CPB with or without the presence of PLA inhibitor, Quanacrine [U-46619 (Pre-CPB) or Quanacrine + U-46619 (pre-CPB)]; (B) Exposure of U-46619 to peripheral arterioles of post-CPB with or without the presence of Quanacrine [U-46619 (Post-CPB) or Quanacrine + U-46619 (post-CPB)] n = 7–11/group,

Figure 4

(A) Administration of U-46619 to the vessels from pre-cardioplegia followed by reperfusion (pre-CPB) samples in the absence or presence of PKC inhibitor safingol [U-46619 (pre-CPB) or Safingol + U-46619 (pre-CPB)], (B) Administration of U-46619 to the vessels from post-CPB samples in the absence or presence of PKC safingol [U-46619(post-CPB) or Safingol + U-46619 (post-CPB); n= 7–11/group.

Figure 5

Representative immunoblot of skeletal muscle tissue (top). Lanes 1-12 loaded with 40 μg protein were developed for TXA-2 receptor, and TXA-2 synthase. Immunoblot band intensity shows unaltered levels of TXA-2 receptor, and TXA-2 synthase after CPB (bottom); (n = 6/group).

Figure 6

Gene expression of TXA-2 receptor and TXA-2 synthase of the pre- and post-CPB skeletal-muscle samples (n = 5/group).

Figure 7

Immuno-localization of TXA-2 receptor, and PLCβ-3 polypeptides in human peripheral microvessels (n = 6). Vessels were co-stained for smooth muscle alpha-actin with PLCβ-3 antibody. Matched negative controls are displayed below each row of primary antibody.