Dexmedetonmidine Inhibits Vasoconstrction Of Human Pulmonary Arteries In Vitro

Dexmedetomidine,(DEX), a new agonist with high affinity to α2-adrenergic receptors.(α2/α1:1600/1), is used in clinical practice for its analgesic, sedative, sympatholytic and anxiolytic effects. The investigations about possible effects of DEX should not be limited only to sedation and anesthesia because (α2-adrenergic receptors are scattered throughtout the body.Some cardiovascular side effects of DEX also have been reported such as hypotension, hypertension and decreases in heart rate. DEX not only induces sympatholytic effects through central presynaptic α2-adrenoceptors, decreases arterial blood pressure and heart rate, but also has effects on peripheral blood vessels. Lots of study in vitro showed that the activation of postsynaptic α2-adrenoceptors causes both vasoconstriction (through the activation of α2-adrenoceptors on vascular smooth muscle cells) and vasodilatation (through the activation of α2-adrenoceptors on endothelial cells). In contrast to α1-adrenergic receptors,α2-adrenergic receptors were reported to be denser only in a number of tissues, such as human saphenous vein, femoral vein and gastroepiploic arteries tissue. It was not clear that how α2-adrenergic receptors denser in human pulmonary arteries or if DEX has effect on human pulmonary arteries. In this present study, we use isolated hunman pulmonary arteries rings to observe the effects of DEX on pulmonary vascular tone and deduce the possible mechanism for clinical drug-use guide. Materials and Methods:1Specimen SourceSpecimen were collected from patients undergoing pulmonary lobectomy for lung cancer in Guangdong General Hospital from Dec17,2012to Aug22,2013, ASA Ⅰ～Ⅱ. Normal lung tissue away no less than5cm from the cancer tissue edge was selected. The number of patients in the study was62(35males and27females, age range24-67years; including23.81%smokers). The patients did not have any clinical evidence of hypertension, pulmonary hypertension and diabetes, nor did they receive steroids and nonsteroidal anti-inflammatory drugs before surgery.Patients were informed the collection process and purpose of specimen. All subjects gave their written informed consent. This study was approved by the Medical Ethics Committee of Guangdong Province (No.2010079H).2Preparation of arterial ringsWithin15min of resection, the pulmonary arteries were isolated from the omentum in a dissecting chamber filled with cold Kreb’s solution. Fat and connective tissues were carefully removed under a binocular microscope, and a vascular ring (length,2mm,; diameter,1-1.5mm) was prepared from each artery for tension recording. Two stainless wires (each40μm in diameter) were introduced through the lumen of the vascular ring with each wire being fixed to the jaws of a myograph. The organ chamber was filled with5ml Kreb’s solution which was gassed with95%O2and5%CO2at37℃. Change in arterial tension were recorded isometrically by a Multi Myograph System (Danish Myo Technology, Aarhus, Denmark).Each ring was stretched gradually to a resting tension of1.8-2mN during a90min equilibration period to obtain optimal tension. Kreb’s solution was replaced in the chamber every20minutes and tension was readjusted to1.8-2mN. In some rings, endothelium was denuded by carefully inserting a small forceps into the lumen and gentle rolling the ring back and forth in the dissecting chamber.3Vascular reactivity testAfter the equilibration period,60mM of KCl was added to the baths and the contractile force was recorded. The vascular rings were then washed with Kreb’s solution at intervals of5min for4times to restore the basal tension. No more than3mN changes in vascular tone amplitude was considered poor contractibility and the arterial rings were discarded.4Endothelial integrity testEndothelial integrity in the pulmonary arterial rings was confirmed by at least80%relaxation in response to acetylcholine (ACh,1μm) at the plateau of5-HT (10μm). Sucessful endothelial removal was confirmed by a lack of vasorelaxant response to acetylcholine.To exclude any residual effect of Ach still present, the following experimental protocols were performed60minutes after the Ach trials. Only preparations that responded adequately to5-HT challenge (more than1mN) were used for further examinations. Every patient afforded1-4rings, and only1ring in each group came from the same patient. Each preparation was used in only1experimental protocol.5Effect of DEX on pulmonary arterial ringsDEX (0.1nm to1μm) was added to intact and endothelium-denuded rings cumulatively and a cumulative concentration-response curve (CRC) for DEX was obtained.6Effect of5-HT on pulmonary arterial rings5-HT (10nm to10μm) was added to intact and endothelium-denuded rings cumulatively, and the tension was expressed as a percentage of the initially contraction of5-HT (10μm). A cumulative concentration-response curve (CRC) for 5-HT was obtained. It was found that the first CRC to5-HT was not altered by the previous challenge with5-HT (10μm) but a consecutive CRC in the same preparation showed a depression compared to the initial curve. Therefore, a single CRC to an agonist was constructed in each preparation either in the absence or presence of a single concentration of an antagonist.7Effect of DEX on pulmonary vasoconstriction by5-HTRings were incubated for30min with DEX0.3nm, lnm, and3nm,and the changes in tension were measured. To determine the effect of DEX on contractility, a CRC to5-HT was obtained while maintaining DEX0.3nm, lnm, and3nm.8Effect of tools and endothelium on inhibition of DEX in pulmonary vasoconstrictionCumulative concerntrations of5-HT was added to intact rings incubated with DEX (3nm)in the presence or absence of L-NAME (100μm), yohimbine (50nm), and indomethacin (5μm) respectively. For endothelium-denuded rings, Cumulative concerntrations of5-HT was added without any tools after incubated with DEX(3nm).9Measurements of dataThe contraction was presented as a percentage of the initially contraction of5-HT (10μm). Emax was represented the maximal response percentage. EC50refereed to the concentrations of drugs that increased the maximal contraction by50%. pD2is the negative logarithm of the vasoconstrictor concemtration needed to produce half of the maximal contraction as determined by non-linear curve fitting (Graphpad Prism software,Version5.0).10Statistical analysisData are means±SEM of n experiments and analysed with Studen’s t-test or two-way ANOVA followed by Bonferroni pasthoc tests when more than two groups were compared. PD2and Emax is determined by non-linear regression curve fitting (Graphpad Prism software,Version5.0). P<0.05was considered statistically significant.Result1Effect of DEX on pulmonary arterial resting tensionDEX (0.1nm to1μm) had no direct effect on resting tension of intact pulmonary arteries. However, DEX (10nm to1μm) caused concentration-dependent contractions in endothelium-dunuded human pulmonary arteries.2Effect of5-HT on pulmonary arterial resting tension5-HT produced a concentration-dependent contraction either in endothelium-intact pulmonary arteries (pD2:6.11±0.05, Emax:102.10±1.96%) or endothelium-dunuded pulmonary arteries (pD2:6.10±0.07, Emax:107.40±3.20%).3Effect of DEX on pulmonary vasoconstriction by5-HTIn the rings with intact endothelium, incubation of dexmedetomidine (0.3,1,3nmol/L) significantly attenuated the Emax and pD2of5-HT induced vasoconstriction (pD2:5.94±0.03,Emax:79.96±1.31%). But There was no significant effect of dexmedetomidine on rings with denuded endothelium.4Effect of L-NAME on inhibition of DEX in pulmonary vasoconstrictionInhibitory effect of DEX (3nm) to5-HT induced contractions was abolished Incubation in the presence of L-NAME (100μm).5Effect of yohimbine on inhibition of DEX in pulmonary vasoconstrictionInhibitory effect of DEX (3nm) to5-HT induced contractions was abolished Incubation in the presence of yohimbine (50nm).6Effect of indomethacin on inhibition of DEX in pulmonary vasoconstrictionIndomethacin did not abolish the inhibitory effect of DEX (3nm) to5-HT induced contractions.Conclusion1.DEX affects pulmonary arteries in a biphasic manner. The vasoconstriction DEX induced through vascular smooth muscle was attenuated by vasodilation through endothelial.2.5-HT induced contractions in human pulmonary arteries effectively, which probably due to the localization of5-HT receptor subtype in pulmonary arteries.3.Dexmedetomidine could inhibited vasoconstriction effects of5-HT in human isolated pulmonary arteries, which was mediated probably through a2-adrenergic acceptor and NO released from endothelium.