Ketamine reduces nitric oxide biosynthesis in human umbilical vein endothelial cells by down-regulating endothelial nitric oxide synthase expression and intracellular calcium levels* Ruei-Ming Chen, PhD; Ta-Liang Chen, MD, PhD; Yi-Ling Lin, MS; Tyng-Guey Chen, MD; Yu-Ting Tai, MD K etamine, a widely used intra- venous anesthetic agent, is clinically applied as an in- ducer of anesthesia in criti- cally ill patients because it has more sta- ble hemodynamics than barbiturates or inhaled anesthetic agents (1). Ketamine has relatively high clearance, which ac- counts for the short elimination half-life of 3 hrs in adults (2). Induction of an- esthesia with ketamine is usually associ- ated with increases in cardiac output, ar- terial blood pressure, and heart rate (3). Traditionally, the primary mechanism for explaining ketamine-induced cardiovas- cular stimulation is its modulatory effects on central nervous system and sympa- thetic nervous system outputs (4, 5). In addition, several lines of evidence have shown that ketamine can directly sup- press myocardial and vascular smooth muscle (6, 7). Recently, an endothelium- dependent mechanism was reported to be involved in ketamine-induced vasoregu- lation (8, 9). Endothelial cells can synthesize and secrete a variety of vasoregulatory factors that participate in the modulation of vas- cular tone (10). Nitric oxide (NO), syn- thesized from L-arginine by a calcium- dependent endothelial NO synthase (eNOS) in endothelial cells, is one of the critical relaxing factors (11). Endogenous formation of NO in the vascular system is critical for regulating multiple physio- logic functions (12). An imbalance in NO production will result in progression of disease. In endothelial cells, there are two major mechanisms involved in the regu- lation of NO production (11, 13). One is via the modulation of eNOS gene expres- sion. Changes in eNOS expression can directly affect endothelial NO production (11). The other mechanism is through modulation of intracellular calcium con- centrations. The activity of eNOS is cal- cium dependent (11). A decrease in intra- cellular calcium levels has been shown to reduce the association of the Ca 2+ / calmodulin complex with eNOS and, se- quentially, to inhibit NO synthesis (13). Recently, the roles of NO in ketamine- induced vasoregulation were evaluated. In the canine pulmonary artery, ket- amine and etomidate were shown to in- hibit endothelium-dependent relaxation via inhibition of endothelial intracellular calcium concentrations responsible for receptor activation rather than by direct suppression of NO and endothelium- derived hyperpolarizing factor activities (8). Meanwhile, Nagase et al. (14) re- ported that the ketamine-induced atten- uation of cerebral vasodilation in hyper- capnia is reversed by an L-arginine or *See also p. 1162. From the Graduate Institute of Medical Sciences, Department of Anesthesiology, Wan-Fang Hospital, College of Medicine, Taipei Medical University, Taipei, Taiwan (RMC, YLL, TGC, YTT); and Taipei City United Hospital, Ren-Ai Campus and Taipei Medical Univer- sity, Taipei, Taiwan (TLC). Supported, in part, by the National Science Council (NSC93-2314-B-038-045 and NSC93-2745-B-038- 002-URD), Taipei, Taiwan. Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/01.CCM.0000163246.33366.51 Objective: Ketamine, an intravenous anesthetic agent, can modulate vascular tone. Nitric oxide (NO), constitutively produced in endothelial cells, contributes to vasoregulation. In this study, we attempted to evaluate the effects of ketamine on NO biosyn- thesis in human umbilical vein endothelial cells and its possible mechanism. Design: Controlled laboratory study Settings: Research laboratory in a universal hospital. Subjects: Human umbilical vein endothelial cells prepared from human umbilical cord veins were exposed to 1, 10, 100, and 1000 M ketamine for 1, 6, and 24 hrs. Measurements and Main Results: Exposure to 1, 10, and 100 M ketamine for 1, 6, and 24 hrs was not cytotoxic to human umbilical vein endothelial cells. However, ketamine at 1000 M significantly caused cell apoptosis. A therapeutic concentration of ketamine (100 M) time-dependently reduced the levels of nitrite in human umbilical vein endothelial cells. Immunoblot analysis revealed that ketamine time-dependently decreased endothelial NO synthase protein production in human umbilical vein endothe- lial cells. Results of an assay by reverse-transcription polymerase chain reaction showed that ketamine significantly inhibited levels of endothelial NO synthase messenger RNA. Ketamine time-de- pendently reduced bradykinin-enhanced intracellular calcium concentrations. Analysis by confocal microscopy further demon- strated the suppressive effects of ketamine on bradykinin-in- duced calcium mobilization. Conclusions: A clinically relevant concentration of ketamine can reduce NO biosynthesis. The suppressive mechanisms occur not only by pretranslational inhibition of eNOS expression but also by a posttranslational decrease in endothelial NO synthase activ- ity due to a reduction in intracellular calcium levels. (Crit Care Med 2005; 33:1044 –1049) KEY WORDS: ketamine; human umbilical vein endothelial cells; nitric oxide; endothelial nitric oxide synthase expression; intra- cellular calcium 1044 Crit Care Med 2005 Vol. 33, No. 5