A new side-effect of sufentanil: increased monocyte-endothelial adhesion

Background Opioids have been identified by the World Health Organization to be ‘indispensable for the relief of pain and suffering’. Side-effects, such as nausea, vomiting, postoperative delirium, and effects on breathing, of opioids have been well investigated; however, the influence of opioids on monocyte-endothelial adherence has never been reported. Therefore, we explored the effects of representative opioids, fentanyl, sufentanil, and remifentanil, on monocyte-endothelial adherence and the underlying mechanisms. Methods We built a cell adhesion model with U937 monocytes and human umbilical vein endothelial cells (HUVECs). Two kinds of connexin43 (Cx43) channel inhibitors, 18-α-GA and Gap 27, were used to alter Cx43 channel function in U937 monocytes and HUVECs, respectively, to determine the effects of Cx43 channels on U937-HUVEC adhesion. Subsequently, the effects of fentanyl, sufentanil and remifentanil on Cx43 channel function and U937-HUVEC adhesion were explored. Results When fentanyl, sufentanil and remifentanil acted on monocytes or endothelial cells, their effects on monocyte-endothelial adherence differed. When acting on U937 monocytes, sufentanil significantly increased U937-HUVEC adhesion which was associated with reduced release of ATP from Cx43 channels, while fentanyl and remifentanil did not have these influences. Although sufentanil could also inhibit Cx43 channel function in HUVECs, it had no effect on ATP release from HUVECs or U937-HUVECs adhesion. Conclusions We demonstrated that sufentanil application increases monocyte-endothelial adherence which was associated with reduced release of ATP from Cx43 channels in monocytes. This side-effect of sufentanil should be considered seriously by clinicians. Supplementary Information The online version contains supplementary material available at 10.1186/s12871-021-01487-3.


Cx43 over-expression
Cx43 was over-expressed in U937 monocytes with a pcDNA3.1-Cx43 vector (gift of Ryan Jensen and Peter M. Glazer) [1]. Transfection into U937 monocytes was carried out using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). After 72 h, western blotting was used to assess Cx43 expression.

Real-time polymerase chain reaction (RT-PCR) [2, 3]
Total RNA extracted from HUVECs and U937 monocytes using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). We used the NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific) to detect RNA quality and concentration. Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). Quantitative analysis was conducted with quantitative RT-PCR (qRT-PCR) using SYBR® Green Realtime PCR Master Mix According to the available reports, we noticed that the concentrations of these anaesthetics were different significantly in vitro study. For example, the concentration of fentanyl was 0.01 μM -150 μM (about 0.00336 μg/ml-50 μg/ml) [4][5][6]; the concentration of sufentanil was 0.5 ng/ml-50 ng/ml [7]; the concentration of remifentanil was 10 ng/ml-100 ng/ml [8,9]. Supplementary Figure 1 showed that fentanyl at the concentration of 50 μg/ml had cytotoxicity on HUVECs, so we chose

The original blots of western blotting
Supplementary Figure 3. The original blots of western blotting.

Effects of sufentanil on ATP and ADO release, and U937-HUVEC adhesion
when Cx43 was over-expressed on U937 monocytes.
Supplementary Figure 4a-c showed that with Cx43 over-expression on U937 monocytes, ATP and ADO release from U937 monocytes were increased significantly, but U937-HUVEC adhesion was increased (Supplementary Figure 4d). This phenomenon contradicted our results in Figure 1 that ATP and ADO could reduce U937-HUVEC adhesion. Our previous reports had demonstrated that Cx43 over-expression on U937 monocytes increased U937-HUVEC adhesion via modulating PKCα/NOX2/ROS signaling pathway [10]. Therefore, we speculated that the function of ATP reducing U937-HUVEC adhesion was reversed by Cx43 over-expression. Our results in this part also supported this speculation. With the application of sufentanil, ATP and ADO release from U937 monocytes were also depressed, and conversely, U937-HUVEC adhesion increased furtherly (Supplementary Figure 4).

Effects of sufentanil on U937-HUVEC adhesion when HUVECs were pretreated with or without TNF-α.
In Supplementary Figure 5, we studied the effect of sufentanil on U937-HUVEC adhesion under different physiological and therapeutic conditions. The results showed that at the condition of HUVECs untreated with TNF-α, U937-HUVEC adhesion was not changed, no matter U937 monocytes or HUVECs were pretreated with sufentanil ( Supplementary Figure 5a). In contrast, at the condition of HUVECs treated with TNF-α, sufentanil increased adhesion fraction when U937 monocytes were pretreated with sufentanil, but had no effects on adhesion fraction when HUVECs were pretreated with sufentanil ( Supplementary Figure 5b), which was coincident with our results in Figure 3. When U937 monocytes and HUVECs were both pretreated with sufentanil at the same time, adhesion fraction was also increased and the extent of increase was just the same as that U937 monocytes were pretreated with sufentanil

Cx37 and pannexin-1 are expressed on both U937 monocytes and HUVECs.
According to different reports, we notice that there is mainly pannexin-1 expressed in monocytes, which mediates ATP release [11,12]. Meanwhile, Cx37 channels are also considered to play an important role in regulating monocyte-endothelial adherence via regulating ATP release [13]. Therefore, we test pannexin-1 and Cx37 expression on U937 monocytes or HUVECs in this part. The results showed that Cx37 and pannexin-1 were expressed on both U937 monocytes and HUVECs.

Three main classes of opioid receptors, mu (MOR), kappa (KOR), and delta (DOR), were expressed on HUVECs and U937 monocytes and HUVECs.
We detected the 3 main classes of opioid receptors, mu (MOR), kappa (KOR), and

ATP release from HUVECs (the absolute ATP values).
We showed the absolute ATP values of HUVECs in Supplementary Figure 8

The selective antagonist of µ-opioid receptors, β-funaltrexamine, reversed effects of sufentanil on ATP release from U937 monocytes and U937-HUVECs adhesion.
In order to confirm the effects of µ-opioid receptors on U937-HUVECs adhesion, we used the selective antagonist of µ-opioid receptors, β-funaltrexamine, to pretreat U937 monocytes in Supplementary Figure 9. The results showed that βfunaltrexamine effectively reversed the influence of sufentanil on ATP release and U937-HUVECs adhesion, increasing ATP release from U937 monocytes (Supplementary Figure 9A)