Regulation of eNOS expression in HCAEC cell line treated with opioids and proinflammatory cytokines.

Background: Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular function and homeostasis. eNOS activity maintains normal vascular tone, regulates leukocyte-endothelial cell interactions, inhibits platelet aggregation, limits smooth muscle cell proliferation and influences cardiac myocyte contractility. The loss of endothelium-derived NO has been shown to result in serious cardiovascular abnormalities, which may indicate eNOS involvement in the origin/development of cardiovascular disorders.

Aim: Evaluation of eNOS mRNA level in the endothelium of human coronary arteries upon opioids treatment (mediators of ischaemic preconditioning) and after incubation with proinflammatory cytokines (stress stimuli).

Methods: Different concentrations of beta-endorphin, endomorphin-1 and endomorphin-2 (alone or in combination with the opioid receptor blocker naloxone) as well as different concentrations of cytokines alone (IL-1beta, TNF-alpha) or in combination were applied to in vitro cultured human coronary artery endothelial cells (HCAEC). After 24 hrs incubation, the cells were harvested, mRNA extracted and relative quantification of eNOS mRNA was conducted using real-time PCR.

Results: Opioid treatment altered eNOS expression; however, none of the changes reached a statistically significant level. As for proinflammatory cytokines, both TNF-alpha and IL-1beta substantially down-regulated the eNOS mRNA level (p <0.05). When applied in combination, these cytokines lowered eNOS mRNA even further (p <0.05). The effect was independent of the cytokine combination used.

Conclusions: It was demonstrated that proinflammatory cytokines exert a substantial and statistically significant negative effect on eNOS mRNA level in human coronary artery endothelial cells in in vitro culture. Unfortunately, we were unable to demonstrate significant changes within the eNOS mRNA pool upon opioid treatment. These results indicate that opioids (basal release) do not affect eNOS expression in normal in vitro culture conditions but might do so upon stress stimuli.