Cross-validated stable-isotope Dilution GC-MS and LC-MS/MS released from the endocannabinoid 2 arachidonoyl glycerol

2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC–MS and LC–MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8 -2AG; 10 µM) as the MAGL substrate and measure deuterium- labeled AA (d8 -AA; range 0–1 µM) as the MAGL product. Unlabelled AA (d0 -AA, 1 µM) serves as the internal standard. d8 -AA and d0 -AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC–MS/MS analysis or in anhydrous acetonitrile for GC–MS analysis. LC–MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M–H]− → [M–H – CO2 ]−, i.e., m/z 311 → m/z 267 for d8 -AA and m/z 303 → m/z 259 for d0 -AA. Prior to GC–MS analysis d8 -AA and d0 -AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC–MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M–PFB]−, i.e., m/z 311 for d8 -AA and m/z 303 for d0 -AA. The GC–MS and LC–MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0–1 µM) of d8 -AA measured by LC–MS/MS (y) and that by GC–MS (x) revealed a straight line (r2 = 0.9848) with the regression equation y = 0.003 + 0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8 -AA produced from d8 -2AG by hepatic MAGL activity. The formation of d8 -prostaglandin E2 by the consecutive catalytic action of recombinant MAGL on d8 -2AG and recombinant cyclooxygenase-2 (COX) on d8 -AA was demonstrated by GC–MS/MS

Pacemaker current inhibition in experimental human cardiac sympathetic activation: a double-blind, randomized, crossover study

Item does not contain fulltextHyperpolarization-activated, cyclic nucleotide-gated 4 (HCN4) channels comprise the final pathway for autonomic heart rate (HR) regulation. We hypothesized that HCN4 inhibition could reverse autonomic imbalance in a human model of cardiac sympathetic activation. Nineteen healthy men ingested oral metoprolol+reboxetine, ivabradine+reboxetine, or placebo+reboxetine in a double-blind, randomized, crossover fashion. We assessed HR, blood pressure (BP), stroke volume, and cardiac output during rest and profound orthostatic stress. HR variability, BP variability, and baroreflex sensitivity were analyzed. Metoprolol, but not ivabradine, decreased resting HR and BP. Ivabradine attenuated the HR increase to orthostatic stress, albeit to a lesser extent than metoprolol. Stroke volume and cardiac output at a given HR were significantly lower with metoprolol. Unlike metoprolol, ivabradine did not affect HR variability, BP variability, or baroreflex sensitivity. Ivabradine attenuates sympathetic influences on HR at the sinus node level, leaving myocardial sympathetic activation unopposed. Reversal of parasympathetic dysfunction by ivabradine appears limited

Allergen challenge increases anandamide in bronchoalveolar fluid of patients with allergic asthma

Preclinical studies suggest that the endocannabinoid anandamide restrains allergic airway obstruction and inflammation. Therefore, we applied saline or allergens via bronchoscopy into different lung segments of patients with allergic asthma. Twenty-four hours later, anandamide concentrations in bronchoalveolar lavage fluids had increased more than fourfold. Anandamide concentrations correlated with severity of airway inflammation. These observations suggest that allergen exposure specifically activates the pulmonary endocannabinoid system (ECS) in patients with allergic asthma