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itric Oxide-Mediated Effects on Liver Blood Flow

.I. Gustafsson, M. Wallin, D.S. Delbro, and S. Friman

ABSTRACT

We investigated whether blockade of nitric oxide synthase by the arginine analog L- NAMEcould affect peripheral liver blood flow (PLBF) or hepatocyte integrity (serum ALT) ineither a control series or in a series subjected to mild reduction of liver blood flow bytemporary clamping of the hepatic artery (HA). Anesthetized rats were arranged for meanarterial pressure (MAP) recordings via a carotid artery, drug injections, and bloodsampling via a jugular vein, and monitoring of PLBF using a laser Doppler flowmeter. Inseries 1, the rats received either L-NAME (30 mg/kg i.v.) or NaCl. L-NAME caused asignificant decrease in PLBF and an increase in MAP compared to NaCl; ALT did notdiffer. In series 2, L-NAME (30 mg/kg i.v.) or NaCl was administered at the beginning ofthe experiment. After 60 minutes of equilibration, the HA was clamped for 60 minutesthen unclamped for another 60 minutes. As in series 1, the L-NAME group hadsignificantly lower PLBF and higher MAP than the NaCl group. Occlusion of the HAresulted in significantly greater reduction in PLBF in the NaCl versus the L-NAME group.Upon unclamping, there was no difference in ALT levels, PLBF, or MAP. To conclude,NO displayed a positive tonic effect on liver blood flow, reduction of which with L-NAME

did not aggravate mild ischemia/reperfusion injury in this model.

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HE VASCULAR SUPPLY to the liver ensures aconstant blood flow with uniform perfusion, mainly

or metabolic reasons, and to maintain cardiovascular ho-eostasis.1,2 Liver circulation is regulated by controlling (1)

ortal vein flow; (2) hepatic arterial inflow, by the rate ofdenosine wash-out (ie, hepatic arterial buffer response);nd (3) direct regulation of hepatic microcirculation at theinusoidal level.1,3 The last site of regulation has recentlyeen recognized to be important under physiologic andathophysiologic conditions. Circulatory disturbances ofhe liver occur in a number of hepatic disorders, includingschemia/reperfusion injury, possibly leading to remotergan failure.3–5 The effector cells responsible for the localegulation of sinusoidal perfusion are the hepatic stellateells, which possess contractile properties.3 Nitric oxideNO) is an important mediator for the regulation of liverlood flow both in vivo and in vitro.6–9 In the current studyonducted with anesthetized rats, we investigated NO-ediated tonic regulation of peripheral liver blood flow

PLBF) and hepatocyte integrity.

ETHODS

he study design was approved by our animal ethics committee.xperiments were performed with female Wistar rats weighing 200

o 250 g. The rats were anesthetized with pentobarbitone (60 mg/kg g

041-1345/05/$–see front matteroi:10.1016/j.transproceed.2005.09.135

338

.p., supplemented when necessary). The right carotid artery andugular vein were cannulated for mean arterial blood pressureMAP) recording and drug administration, respectively. The liveras exposed by a midline incision. The hepatic artery (HA) wasrranged for subsequent temporary clamping. A laser Doppleriniprobe with an adhesive miniholder was placed on the surface

f the liver for monitoring of PLBF. The incision was closed withlips. Body temperature was thermostatically maintained at 38°C byheating pad. Upon completion of surgery, the rats were assigned

o one of the following four study groups (n � 6 to 9 in each). Inroups 1 and 2, 1 hour upon the completion of surgery, either NaClgroup 1) or the nitric oxide synthase (NOS) antagonist, L-NAME30 mg/kg) was injected i.v. and the animals were monitored fornother hour. In groups 3 and 4, immediately upon the completionf surgery, either NaCl (group 3) or L-NAME (group 4) wasdministered as above. After 1 hour of drug equilibration, the HAas clamped for 1 hour, and thereupon unclamped, and thenimals were monitored for another hour. MAP and PLBF werestimated for the last 30 minutes of each 60-minute period, after

From the Transplantation and Liver Surgery Unit, Dept ofurgery, Sahlgrenska University Hospital, Göteborg, Sweden.Address reprint requests to Dr B.I. Gustafsson, Transplantation

nd Liver Surgery Unit, Department of Surgery, Sahlgrenska Uni-ersity Hospital, S-413 45 Göteborg, Sweden. E-mail: bengt.

ustafsson@surgery.gu.se

© 2005 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 37, 3338–3339 (2005)

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hich serum ALT was determined. After the experiment, thenimals were humanely killed by an overdose of pentobarbitone.tatistical analyses were performed by the Mann-Whitney U testor unpaired data presented as mean values � S.E. A P value �.05as considered statistically significant.

ESULTS

n the first series (groups 1 and 2), the effects of L-NAMEnd NaCl on MAP, PLBF, and ALT were compared.itro-L-arginine methyl ester (L-NAME) caused a signifi-ant increase of MAP (�20 � 4.5%, P � .005) and aecrease in PLBF �24 � 6.9%, P � .002), whereas ALT levelsid not differ significantly (Table 1). In the second seriesgroups 3 and 4), clamping of the HA resulted in aignificantly greater reduction in PLBF after NaCl (�31 �.1% versus �21 � 2.7%, P � .03; Table 2). However, uponnclamping, there were no significant differences in MAP,LBF, or ALT values.

ISCUSSION

he current findings clearly demonstrate that pharmaco-ogic blockade of NO generation results in a reduction inLBF. However, it was unclear whether this effect was due

o systemic vasoconstriction or locally altered hepaticirculation. However, to judge from previous studies, it isighly likely that there is a direct NO-mediated control ofLBF. Interestingly, L-NAME did not add to the mildepatic injury as caused by HA clamping/unclamping,uggesting that portal vein flow is unimpaired by NOSntagonism and may compensate for the reduction iniver oxygenation as caused by the clamping.

Table 1. Effect of L-NAME, No Clamping of the HA

Groups 1 and 2 PLBF MAP ALT

-NAME vs NaCl �24 � 6.9% �20 � 4.5% NS

at

P � .002 P � .005

In conclusion, we demonstrate a tonic NO-dependentontrol of hepatic microcirculation. Blockade of NO pro-uction did not result in any exaggeration of the mildepatic injury in this in vivo model of ischemia/reperfusion.

EFERENCES

1. Lautt WW, Greenway CV: Conceptual review of the hepaticascular bed. Hepatology 7:952, 1987

2. Lautt WW, Schafer J, Legare DJ: Hepatic blood flow distri-ution: consideration of gravity, liver surface, and norepinephrinen regional heterogeneity. Can J Physiol Pharmacol 71:128, 19933. Clemens MG, Zhang JX: Regulation of sinusoidal perfusion:

n vivo methodology and control by endothelins. Semin Liver Dis9:383, 19994. Chun K, Zhang J, Biewer J, et al: Microcirculatory failure

etermines lethal hepatocyte injury in ischemic/reperfused rativers. Shock 1:3, 1994

5. Lentsch AB, Kato A, Yoshidome H, et al: Inflammatoryechanisms and therapeutic strategies for warm hepatic ischemia/

eperfusion injury. Hepatology 32:169, 20006. Mittal MK, Gupta TK, Lee FY, et al: Nitric oxide modulates

epatic vascular tone in normal rat liver. Am J Physiol 267:G416,9947. Ayuse T, Brienza N, Revelly JP, et al: Role of nitric oxide in

orcine liver circulation under normal and endotoxemic conditions.Appl Physiol 78:1319, 19958. Zhang B, Borderie D, Sogni P, et al: NO-mediated vasodila-

ion in the rat liver. J Hepatol 26:1348, 19979. Yang W, Benjamin IS, Alexander B: Nitric oxide modulates

Table 2. Effect of L-NAME, Clamping/Unclamping of the HA

Groups 3 and 4Clamping Unclamping

PLBF PLBF, MAP, ALT

aCl �31 � 3.1%P � .03 NS

-NAME �21 � 2.7%

cetylcholine-induced vasodilatation in the hepatic arterial vascula-

ure of the dual-perfused rat liver. Acta Physiol Scand 171:413, 2001