HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995 AASLD A B S T R A C T S 249A

5 6 9 ROLE OF ENDOTHELIN-1 IN HEPATIC MICROCIRCULATION AND CHOLESTATIC LIVER INJURY INDUCED BY ENDOTOXIN T Huan¢'. T Nishida. A Seivama*. H Kosaka*. W Kamiike. Y Morffnoto, H Kazuo, E Hamada, and H Matsuda. First Dept. of Surgery and First Dept. of Physiology*, Osaka University Medical School, Osaka, Japan.

Cholestatic liver injury is observed in septic patients and is induced by endotoxin. Endothelin-1 (ET-I) is also induced by endotoxin. The purpose of this study is to investigate the role of ET-I in hepatic microcirculation and liver injury induced by endotoxin. [MATERIALS & METHODS] Male Sprague-Dawley rats, that were anesthetized with pentobarbital, were mechanically ventilated and monitored arterial blood pressure (BP), and were continuously infused with lipopolysaccharide (LPS) from E. coli (0.8 mg/kgha) for 7 h. BQ485, an endothelin A receptor antagonist (3 mg/kg/h), was infused intravenously. Controls were infused with saline (2.5 ml/h) or BQ485. BP, arterial blood gas, bile flow rate, plasma levels of AST and total bilirubin (TB) were monitored. For evaluation of hepatic microcirculation, reflectance spectrophotomeWy and dual-spot in vivo microspectroscopy were used. [RESULTS] Both controls showed no significant change in BP, hepatic ATP level, plasma AST and TB levels. Administration of LPS, which slightly increased plasma levels of AST and TB without affecting BP, decreased bile flow rate and hepatic ATP level compared with controls. Addition of LPS+BQ485 greatly increased plasma levels of AST and TB, and markedly decreased bile flow rate (20%) and ATP levels (60%). After 5 h infusion, BP gradually decreased and it became to 70 mmHg after 7 h infusion. Reflectance spectrophotometry and dual-spot in vivo microspectroscopy after 4 h infusion revealed no changes in hepatic microcirculation of controls; mild decrease in sinusoidal blood velocity and oxygenation of hemoglobin was observed in LPS infused-rats, marked decrease in LPS+BQ485 infused-rats. [CONCLUSION] These results suggested that blockade of endothelin A receptor deteriorated hepatic microcirculation and injury induced by endotoxin.

570 ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR IN REGULATION OF HEPATIC VASCULAR TONE: IN RELATION TO NITRIC OXIDE M Oshita, N Hayashi, T I-Iiiioka, Y Sasaki, K Kasahara, H Fusamoto and T Kamada. First Dept. of Medicine, Osaka University School of Medicine, Osaka 565, Japan.

Enodothelium-derived hyperpolarizing factor (EDHF), distinct from nitric oxide (NO), is one of the factors to be responsible for endothelium-derived relaxation in some vessels. EDHF hyperpolarized vascular smooth muscle by activating membrane K + channels, which lead to vasodilatation. In this study, to clarify the role of EDHF in hepatic circulation, we examined its effect on phenylephrine (PHE)- induced vasoconstriction in perfnsed rat liver. METHODS: Rat livers were perfused with Krebs-I-lenseleit buffer in a non-recirculating system at a constant flow rate. PHE was injected into the influent and portal pressure (PP) was monitored continuously as an indicator of vasoconstriction. The degree of vasoconstriction was indexed by the area under the curve of PHE-induced increase in PP (AUC) and change in PP 10 rain after PHE injection (PPI0). RESULTS: (1) When PHE (10-10-10 -5 moles) was injected into the influent, PP was increased dose-dependently, reaching maximal levels at 1-3 rain and thereafter, decreased gradually and returned to the baseline (escape phenomena). (2) Infusion of either an nonselective K + channel blocker, tetraethyl- ammonium (TEA) (lmM) or an NO inhibitor, Nt°-nitro-L-arginine (LNA) (100p.M) did not increase AUC and PPI0. (3) But, simultaneous infusion of TEA and LNA suppressed escape phenomena and enhanced AUC and PP10 significantly (see Table). CONCLUSIONS: Inhibition of actions of both EDHF and NO enhanced hepatic vasoconstriction. These results suggest that EDHF and NO have an important role in the control of hepatic vascular tone and may equally contribute to escape phenomena in PHE-induced vasoconstriction.

<Effect of TEA and LNA on AUC and PP10 in PHE (10-Smoles) injection> PIlE only PIIE+TEA PHE+LNA PHE+TEA+LNA

AUC (¢mmo x rain) 97+9 a 108+5 a 1184"11 146+9 PP10 (==mo) 2.2-'~.3 a 2.9+=0.2 a 2.5:]:0.@ 5.32:0.6

(ap<0.01, bp<0.02, compared to value of PHE+TEM+LNA group)

571 REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE DURING LIVER INJURY. DC Rockcv and JJ Chunk. Liver Center Laboratory and the Dept. of Medicine, Univ? of California, San Francisco.

In liver, nitric oxide (NO) has been proposed to have a variety of physiologic functions, including anti-mi'crobial, ischemia-reperfusion and sinusoida-I blooo now regulatory ettects. NO induces relaxation of ljpocytes (stellate, Ito, or fat-storing cells), perisinusoidal cells with teatures simila(, to vasoregulatory p ericytes, tiecause ot this property, NO has been impficateu in smusoidal bloou now regulation in chronic liver disease associated with portal hypertension. The aim of this study was to determine how the inducible isof-orm of NO synthase (iNOS) is regulatea in specific hepatic cells during different forms of experimental liver injury. Methods: Elver injury was induced in inflammatory (bile duct ligation) and toxic (carbon tetrachloride, ,CCl4),inj, ury models in male Sprague- t)awley rats. Lipocytes, enoomedm ana r~upffer cells were isolated by gradient qentofdgatmn and centrifugal elutriation, iNOS mRNA was quantitated wire a specific cDNA by RNase protection assay and protein was detected by immunoblot, iNOS was also localized by immunocytochemistry of fresh frozen tissue. Nitrite, a stable end product of NO, was m~sure~l 24_hours after culture of freshly cells isolated from iniured livers. Kesults: In the bile auct ligation model of injury, iNOS mRNA increased over 20-fold at 24 hours in Kupffer cells 5ut declinect thereafter and was not detectable after 8 days. Similar induction was detected in sinusoidal endothelial cells after 24 hours, and iNOS mRNA remained elevated (up to 25% of peak) 8 days after bile duct ligation. Lipecyte iNOS mRNA followed a pattern similar to, but of lesser magnitude than, that for KuEffer cells. In CC14 induced liver inju~, iNOS mR2qA was detected within 24 hours of exposure in Sinusoidal en;5othelial cells, but was essentially undetectable in other cell types. In chronic CCIa iniury (with portal hypertension), this iNOS mRNA was undetectable in all ceql types, lmmunocytochemistry revealed expression of iNOS-when present-primarily in perisinusoidal locations or in rare cells within expandedportal triads (both models), iNOS protein (by immunoblot) and nitrite proouction paralleled iNOS mRNA expression in both experimental models. Conclusion: Differential regumtion of iNOS rhRNA in intlammatory and toxic models of experimental liver injury., as well as in different cells types is conspicuous. For example, in the bile duct ligation model of injury, iNOS was prominent in slnusoidal endothelial cells, whereas in chronic liver injury induced by CC14, iNOS induction was absent, These data bring into question me importance of NO as a regulator of blood flow in some forms of liver disease, yet emphasize the potential role of NO in other forms of chronic liver disease. The physiologic relevance of differential iNOS mRNA and protein expression m different cell types requires further investigation.

572 PROLONGED INCREASE OF PORTAL PRESSURE BY KUPFFER CELL (KC)- DERIVED REACTIVE OXYGEN SPECIES (ROS). M Bilzer, G Paum,qartner, AL Gerbes. Department of Medicine II, Klinikum Grol~hadem, Ludwig-Maximilians- University of Munich, Munich, Germany

Activated KC secrete O2 into the sinusoidal space which yields H202 and other toxic products, such as hydroxyl radicals. These ROS can cause vasoconstriction in various organs. Their effect on the hepatic circulation, however, remains to be determined. Therefore, we investi- gated the effect of KC-derived and KC independent ROS on the he- patic circulation. Methods: Livers of male Sprague-Dawley rats (250 g) were perfused with Krabs-Henseleit buffer, gassed with 95% 02 / 5% CO2 (n=5). KC were activated by infusion of zymosan (ZY)(150 pg/ml) from 40-46 min without (n=6) or with administration of katalase (15(~ U/ml) or superoxi- de dismutase (SOD)(50 U/ml) plus katalase (150 U/ml)(n=4 each). In order to generate 0 2 in the vascular space without concomitant KC activation, Xanthin (XA, 0.5 mM) / Xanthin-Oxidase (XO, 60 mU/ml) were infused for 15 min. In further experiments XA/XO were infused together with katalase (150 U/ml). H202, the product of O2 - dismutation, was infused for 15 min (50-1000 pM, n=3-4 each). Results: (x+SD). During KC activation by ZY portal pressure (cm H20) increased transiently from 4.0i-0.2 to 18.3+~.8 and remained elevated until 60 rain after termination of ZY infusion (6.6:t:0.7)(p<0.05). SOD/katalase or katalase alone did not affect maximal increase of portal pressure, but during further perfusion portal pressure returned to baseline (4.1+0.9 vs. 4.2+0.5). Infusion of XA/XO resulted in a re- versible increase of portal pressure to 12.5+9.7, which was markedly blunted by katalase (7.3:1:1.4), Administration of H202 increased portal pressure in a dose-dependent fashion above 50 pM, reaching half- maximal effects at 200 pM and maximal effects (9.8+9.6) at 500 pM. Conclusions: Activation of KC results in a prolonged increase of por- tal pressure which may be related to the release of O2 with subse- quent dismutation to H202.