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1 GASTROENTEROLOGY 1999;117: Nitric Oxide Increses Heptic Arteril Blood Flow in Rts With Crbon Tetrchloride Induced Acute Heptic Injury NORIKO TANAKA,* KATSUAKI TANAKA,* YOJI NAGASHIMA, MASAAKI KONDO,* nd HISAHIKO SEKIHARA* *Third Deprtment of Internl Medicine nd Deprtment of Pthology, Yokohm City University School of Medicine, Yokohm, Jpn Bckground & Aims: Little is known bout the chnges in heptic blood flow ssocited with cute heptic injury. The im of this study ws to investigte the effect of nitric oxide (NO) on heptic blood flow nd the severity of heptic injury in rts with crbon tetrchloride (CCl 4 )-induced cute heptic injury. Methods: Rts were pretreted with CCl 4 to induce cute heptic injury. Heptic blood flow ws mesured using rdioctive microsphere method. The role of NO in the regultion of heptic blood flow nd the severity of heptic injury ws investigted by dministering N G - nitro-l-rginine (L-NNA) nd minogunidine (AG). Plsm nitrite/nitrte levels, heptic NO synthse (NOS) ctivity, nd expression of heptic NOS messenger RNA (mrna) were mesured, nd histologicl exmintions were performed. Results: Heptic rteril nd portl venous blood flow ws incresed significntly by CCl 4, without ny chnge in men rteril pressure or crdic output. L-NNA nd AG dose-dependently decresed heptic rteril blood flow, with the highest dose resulting in complete blockde of heptic rteril diltion, but filed to chnge portl venous blood flow. Histologiclly, dministrtion of AG ggrvted the heptic injury in CCl 4 -treted rts. Plsm nitrite/ nitrte levels nd heptic NOS ctivity were incresed significntly by CCl 4 tretment. Inducible NOS mrna ws detected in CCl 4 -treted rts but not in the controls. Conclusions: The results of this study suggest tht the incresed heptic rteril blood flow in CCl 4 - induced cute heptic injury is medited by excessive NO production nd up-regulted by inducible NOS, which plys role in reducing heptic injury. Acute heptitis is histologiclly chrcterized by diffuse heptocyte degenertion nd necrosis without portl or periportl bnormlities. Previous studies hve shown tht totl heptic blood flow is firly norml 1,2 or incresed 3 in ptients with cute virl heptitis. We hve shown by color Doppler sonogrphy tht rteril vsodilttion occurs nd heptic rteril blood flow increses during the cute phse of heptitis in humns nd tht this is correlted with the speed of recovery from the disese. 4 However, the mechnism nd the role of this increse remin unknown. Among vrious vsodiltors, 5 nitric oxide (NO) 6 hs been suggested to be involved in the rteril vsodilttion in portl-hypertensive sttes 7 nd cirrhosis 8 in niml models. Production of NO by Kupffer cells nd heptocytes during endotoxemi nd inflmmtion hs lso been reported. 9,10 Recently, NO hs been proposed to regulte regionl blood flow by n oxygen grdient in tissues, 11 which implies tht occsionl hypoxi by liver injury my induce NO nd tht NO my regulte heptic circultion in cute heptitis. NO is produced from L-rginine either by constitutive or inducible NO synthses (NOS). 12 The constitutive isoforms of NOS re loclized predominntly in neuronl cells (nnos) nd endothelil cells (enos). Endotheliumderived NO produced by enos is n importnt modultor of vsculr tone. 13 Mcrophges nd vrious types of cells produce inducible NOS (inos) fter stimultion by lipopolyscchride nd cytokines, such s interleukin 1, tumor necrosis fctor, nd interferon gmm, 14 nd by sher stress. 15 It hs not yet been reported which isoforms of NOS re involved in the chnge in heptic rteril blood flow in cute heptic injury. We therefore ttempted to clrify the mechnism of regultion of heptic blood flow vi the NO-NOS system by using n NOS inhibitor, N G -nitro-l-rginine (L-NNA), 16 nd selective inos inhibitor, minogunidine (AG), 17 nd by mesuring heptic NO production nd three NOS isoforms in rts with crbon tetrchloride (CCl 4 )-induced cute heptic injury. The reltionship between NO production nd the severity of liver injury ws lso ssessed. Abbrevitions used in this pper: AG, minogunidine; CCl 4, crbon tetrchloride; CO, crdic output; enos, endothelil NOS; inos, inducible NOS; MAP, men rteril pressure; NADPH, reduced nicotinmide denine dinucleotide phosphte; L-NNA, N G -nitro-lrginine; nnos, neuronl NOS; NO, nitric oxide; NOS, nitric oxide synthse; PCR, polymerse chin rection by the Americn Gstroenterologicl Assocition /99/$10.00

2 174 TANAKA ET AL. GASTROENTEROLOGY Vol. 117, No. 1 Mterils nd Methods Animls Mle Sprgue Dwley rts weighing g were used. All nimls were housed individully in wire-bottomed stinless steel cges in temperture-controlled (23 2 C) environment with 12:12 hour light-drk cycles. Rts were llowed free ccess to lbortory chow nd wter until the night before the experiment, when lbortory chow ws withdrwn. All nimls received humne cre in complince with the institution s Guidelines for Animl Cre in Yokohm City University Animl Experimentl Center. Drugs 141 Ce microspheres were obtined from Du Pont Co. (Wilmington, DE). L-[ 14 C]rginine ws obtined from Amershm (Tokyo, Jpn). L-NNA nd AG were obtined from Sigm Chemicl Co. (St. Louis, MO). All the other regents were of nlytic grde. Hemodynmic Study Regionl tissue blood flow ws mesured by the reference smple microsphere technique 18 1 dy fter cnnultion to void ny effects of nesthesi 19 nd the stressor effect of restrinment. Cnnultion ws performed under pentobrbitl sodium nesthesi (30 mg/kg body wt). A ctheter filled with heprinized norml sline ws inserted into the right crotid rtery nd dvnced through the left ventricle under continuous pressure monitoring for injection of the rdiolbeled microspheres. Another ctheter ws inserted into the right femorl rtery for rteril pressure mesurements nd to obtin reference blood smples. Ech open end of the ctheters ws exposed in the posterior neck through the subcutneous tissue nd covered with plug. Ctheters were filled with heprin until the end of the study. The rts were then returned to their cges. One dy fter cnnultion, ll ctheters were connected to pressure trnsducer, nd the men rteril pressure (MAP) ws registered on multichnnel recorder under conscious nd unrestrined conditions. After obtining bseline mesurements of resting MAP, pproximtely 60, Ce-lbeled microspheres (15 3 µm dimeter) were injected into the left ventricle, nd reference smple ws withdrwn from the right femorl rtery over 60 seconds t rte of 1.2 ml/min. After injection of the microspheres, the rteril ctheter ws flushed with norml sline t rte equl to tht of withdrwl of the reference smple. The rts were then killed within 5 minutes with lethl dose of hexobrbitl. The following orgns were removed nd plced in individul tubes to determine the counts with gmm counter (Gmm 5500; Beckmn Jpn, Tokyo, Jpn): liver, spleen, stomch, pncres, mesentery, smll nd lrge intestine, nd both kidneys. The kidneys were used to monitor microsphere mixing; nimls in which greter thn 10% difference existed between estimted right nd left renl blood flow were excluded. Crdic output (CO) nd regionl blood flow were clculted from the 141 Ce counts by using the following formuls: CO(mL/min) nd Orgn Blood Flow (ml/min) Injected Rdioctivity Reference Blood Flow (counts/min) (ml/min) Reference Blood Rdioctivity (counts/min) Orgn Rdioctivity (counts/min) Reference Blood Flow (ml/min) Reference Blood Rdioctivity (counts/min) All vlues of flows were expressed s milliliters per minute per kilogrm. Heptic rteril blood flow ws clculted from the liver counts. Portl venous blood flow ws clculted indirectly s the sum of counts of the stomch, spleen, pncres, mesentery, nd smll nd lrge intestines. Experimentl Design of Hemodynmic Study Experiment 1: heptic hemodynmic study fter CCl 4 dministrtion. Two groups of 6 nimls ech were used to construct the time course (before nd 1, 3, nd 7 dys fter) of heptic rteril nd portl blood flow fter dministrtion of the drugs in group of CCl 4 -treted nd control rts. CCl 4 (500 µl/kg) ws dissolved in olive oil nd dministered intrperitonelly. Control nimls were injected with n equl volume of olive oil. In preliminry study, there were no differences in the results of the hemodynmic study between sline-dministered control rts nd olive oil dministered rts (dt not shown). Blood smples were obtined from rts when the hemodynmic studies were performed. Serum sprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) ctivities were mesured in n utonlyzer. Experiment 2: effects of L-NNA nd AG on heptic blood flow fter CCl 4 dministrtion. Four groups of 6 nimls ech were used to exmine the dose-response reltionships between L-NNA nd AG nd heptic rteril nd portl blood flow. Two groups of CCl 4 -treted (500 µl/kg) rts nd two groups of control rts were dministered different doses of L-NNA (0, 10, 100, nd 1000 µg/kg) nd AG (0, 10, 100, nd 1000 µg/kg). Hemodynmic studies were crried out 1 dy fter CCl 4 dministrtion. After 30-minute equilibrtion to obtin stble bseline redings, L-NNA or AG ws dministered intr-rterilly s slow bolus injection, nd the microspheres were injected 10 minutes lter.

3 July 1999 HEPATIC BLOOD FLOW IN ACUTE HEPATIC INJURY 175 Effects of AG Administrtion on Heptic Injury After CCl 4 Administrtion AG (0, 1000 µg/kg) ws dministered to two groups of CCl 4 -treted (500 µl/kg) rts (n 6) nd two groups of control rts (n 6). AG ws intrperitonelly dministered to the rts once fter CCl 4 tretment. One dy fter CCl 4 dministrtion, the livers were smpled nd fixed with 10% formlin for 24 hours. The tissue smples were embedded in prffin, cut into 4-µm-thick sections, nd exmined histologiclly. Blood smples were obtined from the rts when the livers were excised. Serum AST nd ALT ctivities were mesured in n utonlyzer. Mesurement of NO Formtion NO is highly unstble molecule with rpid turnover, mking direct mesurement difficult, nd its metbolic end products, nitrite/nitrte, were therefore quntittively determined. 20 Two groups of 6 nimls ech were used to construct the time course (before nd 1, 3, nd 7 dys) of plsm nitrite/ nitrte levels fter dministrtion of the drugs in group of CCl 4 -treted nd control rts. Blood smples were withdrwn from the ctheter before the liver ws removed. The plsm smples obtined were deproteinized by dding 0.2 ml of 35% sulfoslicyclic cid to 1-mL liquots before nlysis. The superntnt ws neutrlized with 5% NH 4 Cl/5% NOH fter centrifugtion (10,000g, 15 minutes t 4 C). Nitrite/nitrte levels were mesured with n utonlyzer (TCl-NOX1000; Tokyo Ksei Kogyo Co., Tokyo, Jpn). The nlysis ws bsed on the rection of nitrite with the Griess regent. Assy of NOS Activity NOS ctivity ws mesured by conversion of L-[ 14 C]rginine to [ 14 C]citrulline. 21 One dy fter CCl 4 dministrtion, the liver ws removed nd homogenized in buffer contining 50 mmol/l Tris-HCl (ph 7.4), 0.1 mmol/l EDTA, 2 µmol/l pepsttin A, 20 µmol/l phenylmethylsulfonyl fluoride, 1 mmol/l DL-dithiothreitol, nd 5 µmol/l leupeptin. After centrifugtion (15,000g, 4 C), n liquot of the superntnt ws dded to tube contining 200 µl of prewrmed (37 C) incubtion buffer contining 100 mmol/l HEPES, 10 mmol/l CCl 2, 100 µmol/l reduced nicotinmide denine dinucleotide phosphte (NADPH), 50 µmol/l H 4 B, 4 µmol/l flvin denine dinucleotide, 10 µg/ml clmodulin, 200 µmol/l L-rginine monohydrochloride, nd L-[ 14 C]rginine (0.5 µci/ ml). Rections were initited by the ddition of 100 µmol/l NADPH. After 10 minutes t 37 C, the rection ws terminted by the ddition of 50 µl of 0.6N trichlorocetic cid. Ech smple ws neutrlized with 200 µl of 2 mol/l HEPES (ph 7.5) nd pplied to columns contining 1 ml of Dowex AG 50W-X8, N form. Citrulline ws eluted with distilled wter. L-[ 14 C]citrulline ws quntified by liquid scintilltion counting. Protein concentrtions were mesured by the Lowry method. Reverse-Trnscription Polymerse Chin Rection Assys of NOS mrna Liver RNA ws extrcted by gunidium isothiocynte cid phenol extrction method s described by Chomczynski nd Scchi. 22 A smll liver smple ws homogenized with 300 µl solution D nd plced on ice to reduce degrdtion of RNA. Glycerol-3-phosphte dehydrogense messenger RNA (mrna) ws used to confirm successful RNA extrction from liver specimens nd s positive control for the polymerse chin rection (PCR) ssy. Complementry DNA (cdna) ws synthesized in 10 µl of solution contining 1 µl RNA smple, 0.5 µmol/l of ech deoxynucleotide triphosphte, 50 mmol/l Tris-HCl (ph 8.3), 75 mmol/l KCl, 3 mmol/l MgCl 2, 1 µmol/l of rndom hexmer, nd 200 U of Moloney murine leukemi virus reverse trnscriptse (GIBCO BRL Life Technologies Inc., Githesburg, MD) t 37 C for 1 hour. The cdna thus obtined ws heted t 98 C for 10 minutes to inctivte the reverse trnscriptse nd ws then rpidly cooled. Synthesized cdna ws used for the PCR mplifiction. In brief, synthesized cdna ws dded to n mplifiction mixture contining 3 U Tq DNA polymerse (Perkin Elmer Cetus, Norwlk, CT), 1 µmol/l of ech primer, 300 µmol/l of ech deoxynucleotide triphosphte, nd PCR buffer (70 mmol/l Tris-HCl, 10 mmol/l [NH 4 ] 2 SO 4, 2 mmol/l MgCl 2, 1 mmol/l dithiothreitol, 100 µg/ml bovine serum lbumin, nd 0.1% Triton X-100, ph 8.8). The mplifiction mixture ws mplified by 35 cycles of denturtion for 1 minute t 95 C, nneling for 30 seconds t 62 C, nd extension for 1 minute 30 seconds t 72 C. The primers for nnos were: sense 5 -ACGTCTGACAAGCTGGTGACCAA-3 nd ntisense 5 -CGTATGGTATCTGTGTCCTTCAG-3. The primers for inos were: sense 5 -ATGGCTTGCCCCTGGAAGTTTCTC-3 nd ntisense 5 -GCAGATATGCTGGAACATTTCTGA-3. The primers for enos were: sense 5 -TACGGAGCAGCA- AATCCAC-3 nd ntisense 5 -CAGGCTGCAGTCCTTT- GATC The glycerol-3-phosphte dehydrogense sense nd ntisense primer were 5 -TCCCATCACCATCTTCCA- GG-3 nd 5 -CATGAGTCCTTCCACGATAC-3, respectively. Six-microliter liquots of the finl PCR product were subjected to electrophoresis in 2% grose gel, nd the bnds were visulized by ethidium bromide stining. Sttistics All dt re expressed s the men vlue SD. Dt for the time course studies were first ssessed by two-wy nlysis of vrince; sttisticl evlution between groups ws then performed by Fisher s lest significnt difference method for multiple comprisons. The Student t test ws used to compre the mens of the rest of the dt when pproprite. A P level of 0.05 ws ccepted s sttisticlly significnt.

4 176 TANAKA ET AL. GASTROENTEROLOGY Vol. 117, No. 1 Tble 1. Bseline Vlues of Systemic Hemodynmic Vribles nd Heptic Blood Flow in CCl 4 -Treted Rts Groups Before 1 dy 3 dys 7 dys MAP (mm Hg ) Controls CCl 4 -treted rts CO (ml/min) Controls CCl 4 -treted rts Heptic rteril blood flow (ml/min) Controls CCl 4 -treted rts Portl venous blood flow (ml/min) Controls CCl 4 -treted rts AST (U/L) Controls CCl 4 -treted rts ALT (U/L) Controls CCl 4 -treted rts NOTE. Dose of CCl 4, 500 µl/kg. Vlues re mens SD; n 6. P 0.01 compred with control rts. Results Heptic Hemodynmic Study After CCl 4 Administrtion Tble 1 shows the time courses of the chnges in MAP, CO, heptic rteril, portl venous blood flow, nd AST nd ALT levels in CCl 4 -treted nd control rts. There were no significnt differences in bseline vlues of MAP nd CO between the CCl 4 -treted nd control rts fter CCl 4 dministrtion. Heptic rteril blood flow in the CCl 4 -treted rts ws significntly higher thn in the controls. It incresed more thn 5-fold nd peked 1 dy fter CCl 4 dministrtion. It hd decresed by 3 nd 7 dys fter injury, but still remined significntly higher thn in the controls. Portl venous blood flow ws lso significntly higher in the CCl 4 -treted rts thn controls nd, 1 nd 3 dys fter CCl 4 dministrtion, ws more thn twice the bseline level in the CCl 4 -treted rts. However, it declined to level tht ws not significntly different from the controls by 7 dys fter injury. Neither heptic rteril blood flow or portl venous flow chnged in control rts. AST nd ALT levels were significntly higher in CCl 4 -treted rts thn in controls. AST nd ALT levels peked 1 dy fter CCl 4 dministrtion, then declined to control levels by 7 dys fter injury. Effects of L-NNA nd AG on Heptic Blood Flow After CCl 4 Administrtion Tbles 2 nd 3 show the chnges in MAP, CO, nd heptic rteril nd portl venous blood flow in CCl 4 - treted nd control rts fter vrious doses of L-NNA nd AG. MAP nd CO did not show ny significnt chnges fter the vrious doses of L-NNA nd AG in either the CCl 4 -treted or control rts, nd there were no significnt differences between the two groups. Heptic rteril nd portl venous blood flow in the control rts showed no significnt chnges fter the vrious doses of L-NNA or AG; however, in the CCl 4 -treted rts, the incresed heptic rteril blood flow ws reduced by both L-NNA nd AG in dose-dependent mnner. At the highest dose of L-NNA (1000 µg/kg) nd AG (1000 µg/kg), heptic rteril blood flow decresed to the level of control rts, showing complete inhibition of the increse. The incresed portl venous blood flow fter CCl 4 dministrtion ws unchnged by the vrious doses of L-NNA nd Tble 2. Bseline Vlues of Systemic Hemodynmic Vribles nd Heptic Blood Flow in CCl 4 -Treted Rts Treted With L-NNA 1 Dy After CCl 4 Administrtion Dose of L-NNA ( g/kg) Groups MAP (mm Hg ) Controls CCl 4 -treted rts CO (ml/min) Controls CCl 4 -treted rts Heptic rteril blood flow (ml/min) Controls CCl 4 -treted rts Portl venous blood flow (ml/min) Controls CCl 4 -treted rts NOTE. Dose of CCl 4, 500 µl/kg. Vlues re mens SD; n 6. P 0.01 compred with control rts.

5 July 1999 HEPATIC BLOOD FLOW IN ACUTE HEPATIC INJURY 177 Tble 3. Bseline Vlues of Systemic Hemodynmic Vribles nd Heptic Blood Flow in CCl 4 -Treted Rts Treted With AG 1 Dy After CCl 4 Administrtion Dose of AG ( g/kg) Groups MAP (mm Hg ) Controls CCl 4 -treted rts CO (ml/min) Controls CCl 4 -treted rts Heptic rteril blood flow (ml/min) Controls CCl 4 -treted rts Portl venous blood flow (ml/min) Controls CCl 4 -treted rts NOTE. Dose of CCl 4, 500 µl/kg. Vlues re mens SD; n 6. P 0.01 compred with control rts. AG nd remined significntly higher thn the level in the controls. Effects of AG Administrtion on Heptic Injury After CCl 4 Administrtion Figure 1 shows effects of AG on CCl 4 -induced cute heptic injury. CCl 4 dministrtion cused centrilobulr heptocyte degenertion (Figure 1C), s evidenced by swelling of the cytoplsm nd pyknosis of the nuclei, findings never observed in the control livers (Figure 1A). AG lone never cused this type of tissue injury in the control rts (Figure 1B). AG tretment fter CCl 4 cused more severe heptocyte dmge (Figure 1D). In ddition to more extensively degenerted heptocytes, Figure 1. Photomicrogrphs of the liver 24 hours fter AG injection in CCl 4 -treted nd control rts. (A) Norml control rts nd (B) AG-treted control rts hd no significnt chnges in the liver. (C) Centrilobulr heptocytes were dmged in CCl 4 -treted rts, nd (D) AG tretment fter CCl 4 dministrtion showed more severe heptocyte degenertion with inflmmtory infiltrtes (H&E; originl mgnifiction 179 ).

6 178 TANAKA ET AL. GASTROENTEROLOGY Vol. 117, No. 1 Tble 4. Effects of AG on Serum AST nd ALT Activities in CCl 4 -Treted Rts Groups Tretment AST (U/L) ALT (U/L) Controls None AG CCl 4 -treted rts None (500 µl/kg) AG NOTE. Vlues re mens SD; n 6. P 0.01 compred with CCl 4 -treted control rts. it incresed monocyte inflmmtory infiltrtion. Tble 4 shows the effects of AG on serum AST nd ALT ctivities of CCl 4 -treted nd control rts. The levels were significntly incresed by AG dministrtion in CCl 4 -treted rts (P 0.05). AG tretment did not chnge these indices in control rts. Chnges in Plsm Nitrite/Nitrte Genertion After CCl 4 Administrtion Tble 5 shows the time courses of chnges in plsm nitrite/nitrte concentrtion fter CCl 4 dministrtion. The plsm nitrite/nitrte concentrtion ws significntly higher in the CCl 4 -treted rts thn in controls t both 1 nd 3 dys fter CCl 4 dministrtion. It ws 2-fold higher in CCl 4 -treted rts thn in controls nd peked t 1 dy fter CCl 4 dministrtion. It then declined to the control level by 7 dys fter injury. Chnges in the NOS Activity of Heptic Tissue After CCl 4 Administrtion The NOS ctivity of heptic tissue in CCl 4 -treted rts ws significntly higher thn in the controls ( vs nmol min 1 mg of protein 1 ; P 0.01) 1 dy fter CCl 4 dministrtion. NOS mrna Expression Figure 2 shows typicl ethidium bromide stined grose gel of the reverse-trnscription PCR mplifiction products for nnos, inos, nd enos mrna in liver tissues from control nd CCl 4 -treted rts 1 dy fter CCl 4 dministrtion. nnos mrna ws detected in both groups; however, semiquntittive PCR nlysis, in which RNA levels were checked every fifth cycle of PCR, showed no differences between the two groups (dt not shown). inos mrna ws undetectble in control rts but it ws detected in the CCl 4 -treted rts. enos mrna ws undetectble in both groups. Discussion We show in this study tht CCl 4 tretment incresed heptic rteril blood flow pproximtely 6 8- fold nd portl venous blood flow pproximtely 3-fold, without ny chnges in MAP or CO. It is known tht regionl rteril blood flow increses t sites of inflmmtion due to n incresed demnd for oxygen relted to tissue recovery. Most of the oxygen uptke is used for DNA nd protein synthesis in heptocytes during liver regenertive processes. 24 Therefore, the greter oxygen supply provided by incresed heptic rteril blood flow my promote liver regenertion fter cute heptic injury. We lso showed tht L-NNA nd AG decresed heptic rteril blood flow in dose-dependent mnner in CCl 4 -induced cute heptic injury, without ny chnge in MAP or CO levels, nd blocked heptic rteril diltion completely t the highest dose. In ddition, heptic NO production ws found to be incresed by CCl 4 tretment, which mens tht NO regultes heptic rteril blood flow nd plys n importnt role in the severity of liver tissue under these conditions. Endothelil cells express both the presumbly constitutive enos nd inos under certin inflmmtory conditions. 25 Therefore, induction of NO production by both inos nd enos my cuse vsodilttion in CCl 4 - induced cute heptic injury. We performed n experiment using AG to scertin which NOS isoform is involved in the increse of heptic rteril blood flow. inos mrna, mong the three mjor NOS isoforms, ws most strongly expressed in CCl 4 -induced cute heptic injury, compred with the norml conditions in our study. The decresed heptic rteril blood flow cused by AG nd the fct tht inos mrna ws most strongly expressed suggested tht NO production induced by inos cused the increse in heptic rteril blood flow in CCl 4 -induced cute heptic injury. The significnce of NO in inflmmtion remins uncler nd is still controversil. Induction of inos by Tble 5. Seril Determintion of Plsm Nitrite/Nitrte Concentrtions in Control nd CCl 4 -Treted Rts After CCl 4 Administrtion Before 1 dy 3 dys 7 dys Controls ( mol/l) CCl 4 -treted rts ( mol/l) NOTE. Dose of CCl 4, 500 µl/kg. Vlues re mens SD; n 6. P 0.01 compred with control rts.

7 July 1999 HEPATIC BLOOD FLOW IN ACUTE HEPATIC INJURY 179 Figure 2. Reverse-trnscription PCR nlysis of nnos, inos, nd enos in liver tissue from control nd CCl 4 -treted rts 1 dy fter CCl 4 dministrtion. Ethidium bromide stined grose gel of the mplifiction products showed uniform levels of glycerol-3-phosphte dehydrogense expression, used s control (dt not shown). Bnd sizes (rrows) were s follows: nnos, 353 bse pirs; inos, 675 bse pirs; enos, 806 bse pirs. lipopolyscchride contributes to the pthogenesis of septic shock, leding to orgn destruction, including in the liver. 26 Therefore, excessive production of NO induced by inos in inflmmtion my be involved in tissue injury nd cuse profound vsodilttion, thereby reducing regionl blood flow. On the other hnd, Chmulitrt et l. 27 implied tht NO produced in experimentl CCl 4 plus lipopolyscchride-treted rts plys protective role in the metbolism nd removl of CCl 4.In nother study using perfused rt livers, NO improved microcircultion nd led to decresed heptic dmge in ethnol-induced heptic injury. 28 These studies hve suggested tht NO plys protective role in heptic injury. We found no chnges in CO or MAP, which suggests tht NO production under our experimentl conditions ws insufficient to reduce regionl blood flow. The selective NOS inhibitor AG incresed the severity of CCl 4 -induced heptic injury, which suggests tht NO plys protective role t the doses we used. In this study, neither heptic rteril blood flow, portl venous blood flow, nor MAP were influenced by tretment with L-NNA or AG under norml conditions. Gelderen et l. 29 showed tht MAP ws incresed by N G -monomethyl-l-rginine (1 mg/kg) dministrtion in rts; however, Wng et l. 30 showed tht MAP ws not incresed by L-NNA (1 mg/kg) nd tht the vsoconstriction in response to L-NNA (16 mg/kg) hd the lest influence on the liver, with no significnt chnge in heptic blood flow. At the lower dose of drug ( 1 mg/kg) in our study, L-NNA would not be expected to ffect the systemic hemodynmic vribles or heptic blood flow of control nimls. We hve ssumed tht NO does not contribute to the bsl tone of the heptic rtery under norml conditions. In recent study, however, NO ws relesed becuse of sher stress to modulte vsoconstriction, nd NO ws found to ply regultory role in heptic blood flow under norml conditions. 31 AG hs been reported not to chnge MAP 32 within the rnge of doses we used. Portl venous blood flow is simply the sum of the outflow of the extrheptic splnchnic orgns, 33 which mens tht it represents splnchnic rteril blood flow. In our study, intestinl nd mesenteric blood flow ws incresed in CCl 4 -induced cute heptic injury, but ws unffected by L-NNA nd AG. This mens tht NO does not contribute to the increse in portl venous blood flow in CCl 4 -induced cute heptic injury rts. It lso suggests tht inos produced by injured heptic tissue contributes to increse in heptic rteril blood flow, not portl venous blood flow, becuse NO induced by enos should increse both heptic rteril nd portl venous blood flow. Mny substnces re known to influence the mechnism tht regultes splnchnic blood flow, 5 but further studies re required to clrify this process. In conclusion, this study hs provided evidence tht heptic rteril blood flow nd portl venous blood flow re gretly incresed in rts with CCl 4 -induced heptic injury nd tht the mechnism of this increse in heptic rteril blood flow my involve the NO-iNOS system. We re convinced of the importnce of the heptic rtery nd the role of NO in cute heptic injury; however, further studies re needed to clrify the role of heptic rteril blood flow nd NO in cute heptic injury. References 1. Preisig R, Rnjin JG, Sweeting J, Brdley SE. Heptic hemodynmics during virl heptitis in mn. Circultion 1966;34: Burkle JS, Gliedmn ML. Externl recording method for estimting heptic blood flow with the use of rdiogold. Gstroenterology 1959;36: Lundbergh P, Strndell T. Chnges in heptic circultion t rest, during nd fter exercise in young mles with infectious heptitis compred with controls. Act Med Scnd 1974;196: Tnk K, Mitsui K, Morimoto M, Numt K, Inoue S, Tkmur Y, Msumur M. Incresed heptic rteril blood flow in cute virl heptitis: ssessment by color Doppler sonogrphy. Heptology 1993;18:21 27.

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