Abstract
Electronic cigarette refill liquids are commercially provided with a wide variety of flavoring agents. A recent study suggested that several common flavors may scavenge nitric oxide (NO) and cause endothelial dysfunction. It was the aim of the present study to investigate the effects of these flavors on NO/cyclic GMP-mediated signaling and vascular relaxation. We tested the flavoring agents for effects on Ca2+-induced cGMP accumulation and NO synthase activation in cultured endothelial cells. NO scavenging was studied with NO-activated soluble guanylate cyclase and as NO release from a NO donor, measured with a NO electrode. Blood vessel function was studied with precontracted rat aortic rings in the absence and presence of acetylcholine or a NO donor. Cinnamaldehyde inhibited Ca2+-stimulated endothelial cGMP accumulation and NO synthase activation at ≥0.3 mM. Cinnamaldehyde and diacetyl inhibited NO-activated soluble guanylate cyclase with IC50 values of 0.56 (0.54–0.58) and 0.29 (0.24–0.36) mM, respectively, and caused moderate NO scavenging at 1 mM that was not mediated by superoxide anions. The other compounds did not scavenge NO at 1 mM. None of the flavorings interfered with acetylcholine-induced vascular relaxation, but they caused relaxation of pre-contracted aortas. The most potent compounds were eugenol and cinnamaldehyde with EC50 values of ~0.5 mM. Since the flavors did not affect endothelium-dependent vascular relaxation, NO scavenging by cinnamaldehyde and diacetyl does not result in impaired blood vessel function. Although not studied in vivo, the low potency of the compounds renders it unlikely that the observed effects are relevant to humans inhaling flavored vapor from electronic cigarettes.
Highlights
Electronic cigarettes (e-cigs) vaporize liquids, typically containing propylene glycol, glycerol, nicotine, and flavoring compounds, at a temperature of 200–250 ̊C
The effect of A23187 was completely inhibited by the NO synthase (NOS) inhibitor NG-nitro-L-arginine (NNA), confirming activation of the endogenous nitric oxide (NO)/cyclic GMP (cGMP) pathway
Cinnamaldehyde reduced DEA/NO- and A23187-induced cGMP formation to about 35% of controls, while the other compounds had no significant effects. Based on these results we studied the concentration-dependent effects of cinnamaldehyde on A23187-stimulated formation of the endothelial NOS products NO and L-citrulline
Summary
L-[2,3,4,5-3H]arginine (57 Ci/mmol) and [α-32P]GTP (800 Ci/mmol) were from Perkin Elmer Life and Analytical Sciences (Vienna, Austria), 2,2-Diethyl-1-nitroso-oxyhydrazine (DEA/NO) was from Enzo Life Sciences (Lausen, Switzerland) obtained via Eubio (Vienna, Austria). All other chemicals, including the flavorings listed, were from Sigma-Aldrich (Vienna, Austria). Stock solutions of flavorings (1 M) were prepared in DMSO and diluted as required with deionized water. The following cinnamaldehyde containing liquids were obtained from happy liquid (Munich, Germany): Auszogne mit Zimt, Gluhwein, and Apfelstrudel. Porcine aortic endothelial cells were isolated as described [26] and cultured at 37 ̊C, 5% CO2 for up to 3 passages in Dulbeccos modified Eagles medium, containing 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 1.25 μg/ml amphotericin B. Cells were subcultured into 24-well plates (determination of cGMP formation) or 6-well plates (determination of L-citrulline formation)
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