Abstract
Norepinephrine (NE) is the naturally occurring adrenergic agonist that is released in response to hypotension, and it is routinely administered in clinical settings to treat moderate to severe hypotension that may occur during general anesthesia and shock states. Although NE has incontestable beneficial effects on blood pressure maintenance during hypotensive conditions, deleterious effects of NE on endothelial cell function may occur. In particular, the role of reactive oxygen species (ROS) and NADPH oxidase (Nox) on the deleterious effects of NE on endothelial cell function have not been fully elucidated. Therefore, we investigated the effects of NE on ROS production in rat lung microvascular endothelial cells (RLMEC) and its contribution to cell death. RLMEC were treated with NE (5 ng/mL) for 24 hours and ROS production was assessed by CellROX and DCFDA fluorescence. Nox activity was assessed by NADPH-stimulated ROS production in isolated membranes and phosphorylation of p47phox; cell death was assessed by flow cytometry and DNA fragmentation. Caspase activation was assessed by fluorescent microscopy. Nox1, Nox2, and Nox4 mRNA expression was assessed by real-time PCR. NE increased ROS production, Nox activity, p47phox phosphorylation, Nox2 and Nox4 mRNA content, caspase-3 activation, and RLMEC death. Phentolamine, an α1-adrenoreceptor antagonist, inhibited NE-induced ROS production and Nox activity and partly inhibited cell death while β-blockade had no effect. Apocynin and PEGSOD inhibited NE-induced caspase-3 activation and cell death while direct inhibition of caspase-3 abrogated NE-induced cell death. PEG-CAT inhibited NE-induced cell death but not caspase-3 activation. Collectively, these results indicate that NE induces RLMEC death via activation of Nox by α-adrenergic signaling and caspase-3-dependent pathways. NE has deleterious effects on RLMECs that may be important to its long-term therapeutic use.
Highlights
Norepinephrine (NE) is the endogenous catecholamine released by sympathetic nerve fibers in the vascular wall in response to acute reductions in blood pressure
reactive oxygen species (ROS) production was measured in a realtime manner using CellROX® probe (Thermo Fisher Scientific) in rat lung microvascular endothelial cells (RLMEC) stimulated or not with NE
NE increased ROS production (NE = 86350 ± 9380 RFU vs. vehicle = 24677 ± 4712 RFU) in RLMEC, an effect inhibited by both PEGSOD and PEG-CAT (19138 ± 2531 and 25710 ± 9278, respectively) (Figures 1(d) and 1(e))
Summary
Norepinephrine (NE) is the endogenous catecholamine released by sympathetic nerve fibers in the vascular wall in response to acute reductions in blood pressure. Studies in rats indicate that typical pressor doses of NE or epinephrine can alter pulmonary capillary integrity leading to accumulation of lung water and pulmonary edema [1, 2]. These reports reveal an important and yet unexplored area: the potential long-term side effects of the use of catecholamines in clinical settings. The lung endothelium uptakes approximately 30% of the circulating NE, making this organ an important target for NE
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