Altered coronary microvascular serotonin receptor expression after coronary artery bypass grafting with cardiopulmonary bypass

Abstract

Objective: We evaluated roles of serotonin 1B and 2A receptors, thromboxane synthase and receptor, and phospholipases A(2) and C in response to cardiopulmonary bypass.

Methods: Patients' atrial tissues were harvested before and after cardiopulmonary bypass with cardioplegia (n = 13). Coronary microvessels were assessed for vasoactive response to serotonin with and without inhibitors of serotonin 1B and 2A receptors and phospholipases A(2) and C. Expressions of serotonin receptor messenger RNA were determined with reverse transcriptase polymerase chain reaction. Expressions of serotonin receptors and thromboxane A(2) receptor and synthase proteins were determined with immunoblotting and immunohistochemistry.

Results: Microvessel exposure to serotonin elicited 7.3% +/- 2% relaxation before bypass, changing to contraction of -19.2% +/- 2% after bypass (P <.001). Additions of specific serotonin 1B receptor antagonist and inhibitor of phospholipase A(2) resulted in significantly decreased contraction, -8.6% +/- 1% (P < .001) and 2.8% +/- 3% (P = .001), respectively. Serotonin 1B receptor messenger RNA expression increased 1.82 +/- 0.34-fold after bypass (p = .044); serotonin 2A receptor messenger RNA expression did not change. Serotonin 1B but not 2A receptor protein expression increased after bypass by 1.35 +/- 0.7-fold (P = .0413). Thromboxane synthase and receptor expressions were unchanged after bypass. Serotonin 1B receptor increased mainly in arterial smooth muscle. There were no appreciable differences in arterial expressions of thromboxane synthase or receptor.

Conclusions: Serotonin-induced vascular dysfunction after cardiopulmonary bypass with cardioplegic arrest may be mediated by increased expression of serotonin 1B receptor and subsequent phospholipase A(2) activation in myocardial coronary smooth muscle.

Figure 1. Coronary microvascular response to serotonin in vitro

A. Diameter of microvessels before pre-contraction. There was no difference in baseline vessel diameter among the groups (p=0.4). B. Diameter of microvessels after pre-contraction. Vessels were pre-contracted with U46619, a thromboxane A2 analog. There was no difference among the groups prior to addition of serotonin (p=0.31). C. Serotonin response of vessels harvested before CP-CPB with and without selective 5-HT1B receptor blocker, SB224289. SB224289 administered in the presence of 5HT to the pre-CP-CPB microvessels resulted in a response similar to the 5HT alone (p= 0.44).

Figure 2. Serotonin-inducecd coronary microvascular response to 5-HT1B, PLC and PLA₂ inhibitors in vitro

A. Coronary microvascular response to serotonin demonstrates a relaxation response prior to cardiopulmonary bypass. After bypass the microvessels switched to a strong contraction response. After addition of a 5-HT1B receptor blocker (SB224289) the post bypass contraction response was significantly diminished. (* pre- to post-bypass p< 0.001) (+ post-bypass to post-bypass + antagonist p< 0.001). B. Microvascular response to serotonin in the presence of the PLC inhibitor, Quinacrine and PLA₂ inhibitor, U73122, prior to CP-CPB. (Quinacrine to 5HT alone p= 0.17 and U73122 to 5HT alone p= 0.38). Post-CP-CPB microvessels subjected to Quinacrine yielded a significant reduction in the contractile response as compared to the post-CP-CPB response with 5HT alone (* p< 0.001). The contractile response was not significantly changed when U73122 was applied to post-CP-CPB microvessels as compared to 5HT alone (p= 0.58).

Figure 3. 5-HT1B mRNA expression by RT-PCR

A. Expression of 5-HT1B mRNA was increased (* p= 0.044) fold after CP-CPB. B. Agarose- ethidium bromide gel (1%) showing the PCR amplification of 5HT-1B Post-CP-CPB as compared to pre-CP-CPB normalized to 1. GAPDH was used as a loading control. Data presented as fold change ± SEM.

Figure 4. 5-HT1B, TR and TS protein expression by western blotting

A. Expression of 5-HT1B receptor protein was found to increase after CP-CPB. The expression of TR and TS was not significantly changed the myocardial tissue. B. 5-HT1B receptor protein expression. Post-CP-CPB is compared to pre-CP-CPB normalized to 1 (* p= 0.041). C. Thromboxane receptor protein expression. (p= 0.62). D. Thromboxane synthase protein expression. (p= 0.18). Ponceau stain is used for loading/transfer monitoring. Data presented as fold change ± SEM.

Figure 5. Localization of 5-HT1B, 5-HT2A receptors and thromboxane receptor by Immunohistochemistry and immunofluorescence, respectively

A. Expression of 5-HT1B receptor is upregulated in the vascular smooth muscle cells (VSMC). B. Expression of 5-HT2A receptor is unchanged after CP-CPB. C. Expression of thromboxane receptor is unchanged between pre and post CP-CPB Magnification: immunohistochemoistry 400x, immunofluorescence 200x. (Large open arrow indicates VSMC, small black arrow indicates endothelium).

Figure 6. Summary of 5-HT receptor expression before and after cardiopulmonary bypass (CP-CPB)

The expression of 5-HT1B receptor is significantly increased after bypass and associated with vasoconstriction, while expression of 5-HT2A is unchanged after bypass. Serotonin (5-HT), along with TXA2, ADP and other mediators, are released from platelets. Platelets are activated after CP-CPB and degranulate releasing many substances. The endothelium also produces 5-HT. The 5-HT1B receptors are negatively coupled to adenylyl cyclase and the 5-HT2A receptors are positively coupled to phospholipase C. Serotonin activates PLA2 and may lead to further increases in TXA2. All of these signals influence the vascular smooth muscle cells (VSMC) to respond.