Experimental medicine and laboratory investigations M. Treggiari-Venzi a, E. R. Schiffera, J.-A. Romand b, M. Lickera, D. R. Morel a a
Division of Anaesthesiological Investigations; b Division of Surgical Intensive Care; Department of Anaesthesia, Pharmacology and Surgical Intensive Care, University Hospital of Geneva
Schweiz Med Wochenschr 2000;130:608–16 Peer reviewed article
Hepatic haemodynamic changes following inhibition of endothelium-derived relaxing and hyperpolarising factors in anaesthetised miniature pigs1
Summary The influence of endothelium-dependent vasodilatation in regulating the hepatic circulation has been investigated by intraportal infusion of inhibitors of either endothelium-derived relaxing factor (NG-nitro-L-arginine-methyl-ester [L-NAME]) or of endothelium-derived hyperpolarising factor (ATP-dependent K+-channel inhibitor, glybenclamide) in barbiturate anaesthetised miniature pigs. Intraportal infusion of acetylcholine (5.5 µg kg–1 min–1 over 2 min) produced a selective 3-fold increase in hepatic artery and coeliac trunk blood flow, while mesenteric, portal, systemic, and pulmonary vascular beds remained unchanged. Intraportal L-NAME or glybenclamide did not reduce the hepatic artery and coeliac trunk flows but increased systemic and mesenteric vascular resistances. The acetylcholine-induced hepatic artery vasodilatation was partially blocked by 59%, 76% and 66% by L-NAME, at 30, 100, and 300 mg/kg respectively. Glybenclamide
pretreatment up to 3 mg/kg did not modify acetylcholine-induced vasodilatation of the hepatic artery and coeliac trunk. Furthermore, prior cyclooxygenase inhibition did not alter the hepatic vascular response to acetylcholine. These results suggest that, in contrast to what is observed in large vessels, the hepatic vascular tree may not be entirely regulated by nitric oxide under basal conditions, but nitric oxide is released readily upon stimulation with acetylcholine, a response that is largely but incompletely blocked by L-NAME pretreatment. Neither basal vascular tone nor acetylcholineinduced vasorelaxation are mediated by the opening of glybenclamide-sensitive K+ channels in the hepatic circulation in pigs. Keywords: EDRF (endothelium-dependent relaxing factor); EDHF (endothelium-dependent hyperpolarising factor); acetylcholine; nitric oxide; ATP-sensitive K+ channels; hepatic artery blood flow; splanchnic haemodynamics
1 Supported by a grant from the Carlos and Elsie De Reuter Foundation no 192.
list of abbreviations
EDRF = endothelium-derived relaxing factor ERHF = endothelium-derived hyperpolarising factor flows: CTBF = coeliac trunk blood flow HABF = hepatic artery blood flow MABF = mesenteric artery blood flow PVBF = portal vein blood flow L-NAME = NG-nitro-L-arginine-methyl-ester MAP = mean arterial pressure MPAP = pulmonary artery pressure
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Correspondence: Miriam Treggiari-Venzi Division of Pulmonary and Critical Care Medicine Box 359762 Harborview Medical Center University of Washington School of Medicine 325 Ninth Avenue Seattle, WA 98104, USA e-mail:
[email protected] [email protected]
Schweiz Med Wochenschr 2000;130: Nr 17
Experimental medicine and laboratory investigations
Résumé L’influence de l’endothélium dans la régulation de la circulation hépatique a été étudiée après l’infusion intraportale de l’inhibiteur de l’«endothelium-derived relaxing factor» (NGnitro-L-arginine-methyl-ester [L-NAME]) ou de l’«endothelium-derived hyperpolarising factor» (l’inhibiteur de canaux potassiques ATPdependant, glybenclamide) dans un model porcin anesthésié par des barbituriques. L’infusion intraportale d’acétylcholine (5,5 µg kg–1 min–1 en 2 minutes) triple le débit de l’artère hépatique et du tronc céliaque, tandis que les débits mésentériques, portes, systémiques et pulmonaires n’étaient pas changés. L’injection intraportale de L-NAME ou glybenclamide n’a pas modifié les débits de l’artère hépatique et du tronc céliaque, mais a augmenté les résistances vasculaires systémiques et mésentériques. La vasodilatation de l’artère hépatique induite par l’acétylcholine a été partiellement bloqué de 59%, 76% et 66% en présence de L-NAME, aux doses de 30, 100, et 300 mg/kg, respectivement.
Le pré-traitement par du glybenclamide jusqu’à 3 mg/kg n’a pas modifié la vasodilatation induite par l’acétylcholine au niveau de l’artère hépatique et du tronc céliaque. De même, l’inhibition préalable de la cyclooxygenase n’a pas modifié la réponse vasculaire hépatique à l’acétylcholine. Ces résultats suggèrent que la régulation du tonus vasculaire basal de l’artère hépatique ne dépend pas uniquement de la synthèse du NO, contrairement à ce qui est observé dans d’autres vaisseaux artériels. Cependant, ce réseau vasculaire peut libérer ce médiateur rapidement après stimulation par l’acétylcholine. Enfin, ni le tonus vasculaire de base ni la vasodilatation induite par l’acétylcholine dérivent de l’ouverture des canaux potassiques sensibles au glybenclamide. Keywords: EDRF (endothelium-dependent relaxing factor); EDHF (endothelium-dependent hyperpolarising factor); acétylcholine; NO; canaux potassiques sensibles à l’ATP; débit de l’artère hépatique; hémodynamique splanchnique
Nitric oxide, a lipophilic short-life molecule produced in basal conditions by endothelial cells, is an endothelium-dependent relaxing factor (EDRF) possessing special properties which make it different from a classical mediator [1, 2]. Nitric oxide is able to activate the cytosolic guanylate cyclase enzyme to produce cyclic guanosine monophosphate, the second intracellular messenger inducing vascular smooth muscle relaxation. It has been shown that in large vessels [3], as well as in coronary [4] and pulmonary circulation [5], nitric oxide may account for the local regulation of basal vascular tone, being continuously released by constitutive nitric oxide synthase [6]. However, some smaller calibre vessels, i.e. femoral, mesenteric and renal arteries, have been shown to be less dependent on nitric oxide production
for the regulation of their basal vascular tone [7]. It has been postulated that in these vessels an endothelium-derived hyperpolarising factor (EDHF) is involved for control of basal tone [8–10]. ATP-sensitive K+ channels represent one of these possible systems and their hyperpolarisation may play a role in basal tone regulation [11]. The hepatic artery is a small calibre vessel that possesses a special regulation, able to adapt its blood flow according to portal vein supply and to the requirements of liver clearance [12]. The present study was designed to assess the respective importance of the nitric oxide pathway and ATP-sensitive K+ channels in mediating the hepatic artery response when these systems are stimulated by the endotheliumdependent vasodilator acetylcholine.
Introduction
Methods Animal preparation The experimental protocol conformed to the Guiding Principles in the Care and Use of Animals and was approved by the Ethical Committee for Animal Research of our institution. Adult miniature pigs of either sex weighing 25 to 28 kg were anaesthetised with halothane, intubated with a cuffed endotracheal tube, mechanically ventilated using a Siemens 900 respirator, and anaesthesia was maintained by continuous i.v. barbiturate (sodium thiopental, 10 µgkg–1 h–1) and opiate infusion (fentanyl 10 µg kg–1 h–1) and supplemented
with inhaled nitrous oxide (70% in oxygen). Muscle relaxation was provided with i.v. pancuronium bromide. Tidal volume and respiratory rate were adjusted to maintain the animals in normocarbic conditions. Body temperature was maintained constant with the use of a heating fan. Acid-base balance and arterial oxygenation was checked at regular intervals. Halothane inhalation was stopped at the end of the surgical preparation. Intravascular catheters were placed into the thoracic aorta and right atrium for mean arterial pressure (MAP) and central venous pressure measurement. A Swan-Ganz flow-directed thermodilution
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Experimental medicine and laboratory investigations catheter was inserted into the pulmonary artery for measurement of cardiac output, mean pulmonary artery pressure (MPAP) and capillary wedge pressure. Through a median laparotomy the animals were surgically instrumented with transit time ultrasonic blood flow probes (Transonic System, Inc., Ithaca, NY), placed as follows: a 6R-Sil probe around the portal vein and a 2R-Sil probe around the hepatic artery (PVBF and HABF respectively); two additional transit-time flow probes (2R-Sil or 3R-Sil, in accordance with the vascular diameter) were used for mesenteric artery (MABF) and coeliac trunk blood flow (CTBF) monitoring, placed at 2 cm from their origin. After splenectomy a catheter was inserted in the splenic vein and advanced into the portal vein for measurement of portal pressure and for administration of study drugs. Finally, a second 7F Swan-Ganz flow-directed thermodilution catheter was inserted through an external jugular vein and pulled into one of the retro-hepatic veins for measurement of hepatic sinusoidal wedged pressures. Correct position of the cannula tip in the main stem of one of the hepatic veins was verified during surgery. A 2-hour period was allowed for haemodynamic stabilisation before starting the protocol.
Haemodynamic measurements Vascular pressures were continuously measured using calibrated pressure transducers (Honeywell, Zurich, Switzerland). Cardiac output was recorded as the mean of triplicate determinations by thermodilution injecting 5 ml of 4 °C saline (Edwards Laboratories Model 9520A). Mean portal, hepatic, coeliac and mesenteric blood flows were continuously measured with transit-time flowmeters (T101CDS, Transonic system, Inc., Ithaca, NY, USA). Vascular pressures and blood flows were recorded on an 8-channel recorder (Kontron W+W, model 408, Basel, Switzerland), as well as connected via an analogue-digital computer (SICMU, Geneva, Switzerland) to an ALR AT-386 microcomputer (CPI, Geneva, Switzerland) for online data acquisition. Systemic (SVR) and pulmonary (PVR) vascular resistance were calculated by standard formula, hepatic arterial vascular resistance (HAVR) by dividing the difference between MAP and hepatic venous pressure by HABF, coeliac (CTVR) and mesenteric (MAVR) vascular resistance by dividing the difference between MAP and mean portal venous pressure by CTBF or MABF respectively.
Schweiz Med Wochenschr 2000;130: Nr 17
Protocol The study protocol consisted of measuring the regional haemodynamic changes induced by sequential intraportal administration of acetylcholine (5.5 µg kg–1 min–1) over 2 minutes through a ratecontrolled infusion pump (Harvard apparatus, model 2604, Millis, MA, USA), at baseline (control) and after subsequent injections of either intraportal L-NAME or glybenclamide. After the control acetylcholine infusion, 6 miniature pigs were treated with intraportal short course infusions, over 2 minutes, of increasing doses of LNAME (30, 100 and 300 mg/kg) at 30-minute intervals. Five other animals were treated with intraportal infusion of increasing doses of glybenclamide (0.1, 0.3, 1.0 and 3 mg/kg). Three additional animals were pretreated with an intraportal infusion of the cyclooxygenase inhibitor indomethacin (1.5 mg/kg, over 2 min) before intraportal infusion of 100 mg/kg L-NAME. After the administration of every single drug dose, the haemodynamic response to acetylcholine (5.5 µg kg–1 min–1) was reexamined. Data were acquired during each sequential injection until stabilisation as follows: during acetylcholine at control, during L-NAME 30 mg/kg, during acetylcholine infusion after L-NAME 30 mg/kg, and the same sequence was repeated for the other L-NAME doses. A similar procedure was conducted for the glybenclamide treated animals.
Drugs The following drugs were used: acetylcholine (Fluka Ltd, Buchs, Switzerland), L-NAME, glybenclamide (Sigma Chemical Company Ltd, Buchs, Switzerland) and indomethacin (Merck Sharp & Dohme Pharmaceuticals, Glattbrugg/Zurich, Switzerland).
Data analysis Means ± SE values of each 0.5-second time point of every treatment group were calculated and plotted for the 300-second online recording period. For statistical comparisons baseline values were obtained from averaging the first 30 seconds of recording and compared with the maximum and minimum value recorded using one-way ANOVA for repeated measures, followed by Duncan’s post hoc test if p
