The Study Of Mechanism Of Relaxation To Propofol In Isolated Rat Intrapulmonary Arteries

Intravenous anesthetic propofol has been widely used in clinic anesthsia and in intensive care unit. Propofol is anesthesiologists’favorite anesthetic agent for its quick onset, short-acting, rapid emergence from general anesthesia with minimal side effects. Besides its multiple anesthetic advantages, propofol exerts numbers of non-anesthetic effects, like antioxidant properties, neuro-protection, enhancing immunomodulatory activity, modulating platelet aggregation et al. Circulatory suppression occurs after administration of propofol, which may involve in decreasing myocardial contractility and peripheral vascular resistance. Tons of studies have shown that propofol has direct effects on vessels, but the precise mechanism is not fully understood. Vasodilation effects of propofol have been demonstrated in several in vitro studies on blood vessels, including porcine coronary artery, rat aorta, pulmonary, coronary and renal artery, and human omental artery and fetal placental vessels. On the contrary, studies have also demonstrated that propofol increased rat pulmonary vascular resistance, and attenuated acetylcholine-induced pulmonary vasodilation. Based on these studies, it is suggested the effect and mechanism of propofol may vary with species and location of different vascular beds. Literature above deduced the possible mechanisms of propofol effecting vascular smooth muscle from different angles, including endothelium-dependent or endothelium-independent, involvement of potassium and calcium channels, increasing or decreasing myofilament Ca2+sensitivity and effecting vascular tone after agonist activating (eg. α-adrenoreceptor activation). But most of the researchers agree that the involvement of calcium plays a crucial role in vasoconstriction or vasodilation caused by propofol. Propofol has no effects on normal vascular baseline, but it influences vascular resistance on pathological vessels or vessels treated with vasoactive agents. In clinic anesthetic practice, besides the slight unhealthy patients, anesthesiologists also face patients with hemodynamic instability, impaired vital organ function and controlling vascular reactivity to sustain tissue blood flow with drugs. Therefore,-understanding the mechanisms of popofol involved in regulation of vascular tone would be helpful for anesthesiologists. In this present study, we use isolated rat secondary pulmonary artery rings to observe effects of propofol on pulmonary vascular tone and deduce the possible mechanism, and provide laboratory data to guide clinical drug-use.Materials and Methods:1. Preparation of artery ringsSPF, healthy adult male SD rats (provided by the animal laboratory of Sun Yatsen University) ranging from200-300gram were anesthetized by intraperitoneal injection of pentobarbital sodium (150mg/Kg). The cardio-pulmonary tissue was placed into a container fulfilled with ice-cold Kreb’s solution. Second order of intrapulmonary small arteries were removed and cut into several rings about1-2mm in length. Each ring was mounted in the chamber of a Multi Myograph System with two wires passing through the lumen. Each chamber contained5ml Kreb’s solution that bubbled with95%02plus5%CO2constantly. The room temperature maintained at37℃throughout the duration of the experiment. After an equilibration period of60min, each ring was stretched to an optimal tension of2mN, each ring was contracted by60mmol/L K+at30min intervals until the two consecutive contraction were performed. Contractile ability of each ring was confirmed by good contracting exposed to60mmol/L K+solution. The Kreb’s solution in chambers was changed every15min during the equilibration. In some rings, the endothelial layer was mechanically disrupted by gently rubbing the luminal surface with a tiny wire back and forth several times. Its functional removal was verified by lack of a relaxant response to1μmol/L acetylcholine. In order to make sure that the highest concentration of DMSO at1:500did not affect the U46619-or high K+-induced vessel tone, several rings were contracted by U46619or high K+, then DMSO at1:500concentration was added into the chamber.2. Effects of propofol on vessels contracted by different vasoconstrictorsRings were contracted by60mmol/L high K+solution(n=6),100nmol U46619(n=6),3μmol/L5-hydroxytryptamine(n=4) or1μmol Phenylephrine(n=4), the contractile responses were recorded, if the response was more than3mN,the cumulative doses of propofol(1to300μmol/L) were added to the chambers, if not, the vasoconstrictors were cumulatively added into the chambers to make sure the does is enough to cause vasoconstriction.3. The role of endothelium on vsondilation effect of propofolEndothelium-intact ring(n=5) was contracted with100nmol/L U46619, then propofol(1-300μmol/L)was cumulatively added in the absence and presence of1nmol/L NG-nitro-L-arginine methyl ester(L-NAME). Endothelium-denuded rings (n=5) were preconstricted with100nmol/L U46619. Propofol was added as described above.4. The role of Ca2+on vasodilation effects of propofolSome rings (for separated experiment, n=5)were rinsed three times in a Ca2+-free solution containing500μmol/L EGTA, then incubated in a Ca2+-free,60mmol/L K+-containing solution(without or with propofol,20min pre-incubation).The ability of propofol to modulate Ca2+influx via the L-type Ca2+channels was evaluated by examining concentration-dependent responses to CaCl2(0.01to3mmol/L) in the absence or presence of propofol(10to300μmol/L). Other rings were preconstricted with60mmol/L K+solution to open the voltage-gated Ca+channels, followed by an addition of1μmol/L nifedipine to block L-type voltage-gated Ca2+channels. After the tone returned to basal line, which is an indication that most, if not all, of the L-type voltage-gated channels were blocked, the rings were recontracted with100nmol/L U46619. Cumulative dose (1to300μmol/L) of propofol was added to the chamber.5. Measurements of dataThe contraction was presented as percentage of the contractions to60mmoI/L K+or100nmol/L U46619. Emax was represented the maximal response percentage. EC50refereed to the concentrations of drugs that reduced (or increased) the maximal contraction by50%. The negative logarithm of the dilator (or contractor) concentration that resulted in half of the maximal relaxation or contraction (pD2) was calculated. Curves were analyzed by non-linear curve fitting using Graphpad software (Version3.0).6. Data analysisSPSS13.0was used as analyzing software. Results are mean±S.E.M of n arterial rings. Paired student’s t-test was used to assess the effects of propofol on preconstricted rings in the absence or presence of L-NAME. Independent student t-test was used to analyze the effects of propofol on preconstricted rings with or without endothelium. One-way ANOVA followed by LSD test was used when more than two groups were compared. P<0.05was considered significant.Results:1. Effects of different vasoconstrictors on isolated rat intrapulmonary arteries: 60mmol/L high K+or100nmol/L U46619caused strong contraction on isolated second order of rat intrapulmonary arteries, but the effects of5-HT or Phe were very weak. Rings which had great contractile response to high K+solution, even exposed to very high concentrations of5-HT(10μmol/L) or Phe(30μmol/L), had little contractile response.2. The-effects of propofol on non-receptor-dependent and receptor-dependent vasoconstrictors:Propofol relaxed both high K+(non-receptor-dependent vasoconstrictor) and U46619(receptor-dependent vasoconstrictor) preconstricted rings in a concentration dependent manner. The maximal relaxant effects of propofol on high K+-preconstricted rings was (97.57±2.05)%, pD2was4.38±0.08,in U46619-preconstricted rings, Emax was (88.18±10.33)%, pD2was4.15±0.27.3. The role of endothelium on propofol-induced relaxation:Propofol induced relaxation on U46619-mediated contraction in both endothelium-intact and endothelium-denuded rings in a concentration-dependent manner.1μmol/L L-NAME incubation did not affect propofol-induced maximal relaxtion in endothelium-intact rings, but affect the value of pD2.(relaxation:82.60±22.15%in control,77.62±26.58%in L_NAME, n=5,P=).213; pD2:4.21±0.26in control,4.01±0.28in L-NAME,n=5,P=0.012).Propofol induced similar degree of inhibition of U46619-preconstricted rings both endothelium-intact and-denuded.(relaxation:82.60±22.15%with endothelium and86.27±18.37%without endothelium, P=0.783;pD2:4.21±0.26with endothelium and4.41±0.36without endothelium, P=0.343).4. Effect of propofol on Ca2+channels:Different concentrations of propofol(10to300μmol/L) were tested on contractions in membrance-depolarized rings. Cumulative addition of CaCl2induced contractions, in Ca2+-free,60mmol/L K+-containing solution in the absence (n=5) and presence of propofol (10to300μmol/L, n=5). Propofol inhibited CaCl2-induced contraction with progressive reduction of maximal contraction with increasing concentrations (p=0.000), but the pD2value between groups had no significant difference. Propofol at100and300μmol/L almost inhibited CaCl2-cantraction.Rings preconstricted with60mmol/L K+could be fully reverse by1μmol/L nifedipine, which indicated the L-type Ca2+channel was fully inhibited. High concentration of K+in extracellular bath causes membrane depolarization, which open voltage-gated L-type Ca2+channels, resulting in vascular contraction. After the inhibition of L-type Ca2+channels with nifedipine, subsequent addition of U46619could still induce contraction. Cumulative addition of propofol(1to300μmol/L) caused a concentration-dependent inhibition of U46619-induced contraction(Emax=88.97±5.60%).Conclusion:(1) Isolated rat intrapulmonary arteries have great contractile response when they are exposed to high K+solution and U46619.(2) Propofol induced both non-receptor dependent and-receptor dependent contraction.(3) Propofol induced U46619preconstricted pulmonary rings in an endothelium-independent manner.(4) The possible mechanism may involve in inhibition of influx of extracellular Ca2+via VOCC and ROCC.