Effects of inhibiting nitric oxide biosynthesis on the systemic and splanchnic circulation of rats with portal hypertension

Transcription

1 Br. J. Phrmcol. (1992), 15, lv,'-. Mcmilln Press Ltd, 1992 ffects of inhibiting nitric oxide biosynthesis on the systemic nd splnchnic circultion of rts with portl hypertension M.P. Pizcuet, J.M. Pique, J. Bosch, l*b.j.r. Whittle & *S. Moncd Heptic Hemodynmics Lbortory, Liver Unit, Hospitl Clinic i Provincil, University of Brcelon, Spin, nd *Wellcome Reserch Lbortories, Beckenhm, Kent 1 The effects of inhibiting endogenous nitric oxide (NO) synthesis with N-monomethyl-L-rginine (L- NMMA) on the systemic nd splnchnic circultion hve been investigted in rts with experimentl chronic portl hypertension, nesthetized with ketmine. 2 Portl hypertension ws induced by prtil portl vein ligtion, 2 weeks prior to study. This procedure induced reduction in systemic rteril blood pressure (MAP), n increse in crdic output s mesured by rdiolbelled microspheres, reduction in peripherl nd splnchnic vsculr nd n incresed portl venous inflow (PVI) nd portl pressure, s compred to control non-ligted rts. 3 L-NMAA (6.25 nd 5mgkg-', i.v.) dose-dependently incresed MAP, reduced crdic output nd PVI, nd incresed peripherl nd splnchnic vsculr. With (SOmgkg- 1), PVI nd the vsculr s returned to vlues comprble to those determined in control non-ligted nesthetized rts under resting conditions. 4 Porto-collterl ws lso incresed by these doses of, wheres portl pressure ws unchnged. The increse in renl blood flow nd decrese in renl vsculr lso seen in portl-hypertensive rts ws reversed by (5 mg kg- 1). 5 These effects of (5mgkg- 1) were inhibited by prior dministrtion of L-rginine (3 mg kg- t, i.v.). 6 These findings indicte tht the chronic hyperdynmic circultory chrcteristics following portl vein stenosis cn be ttenuted by. Thus, the excessive formtion of endogenous NO my be implicted in the pthogenesis of the hemodynmic disturbnces nd splnchnic vsodilttion ssocited with chronic portl hypertension. Keywords: Portl hypertension; splnchnic circultion; nitric oxide; N-monomethyl-L-rginine () Introduction Chronic portl hypertension is ssocited with hyperdynmic circultion, chrcterized by incresed blood flow nd reduced vsculr in the splnchnic nd systemic circultion (Vorobioff et l., 1983; Krvetz et l., 1986). Such hyperdynmic circultion observed in ptients (Lebrec et l., 1983; Bosch et l., 1988) is reproduced in experimentl models of portl hypertension such s tht induced by chronic prtil portl vein stenosis in the rt (Vorobioff et l., 1983; Blnchet & Lebrec, 1982; Benoit et l., 1984; Krvetz et l., 1986). The mechnisms underlying the splnchnic vsodilttion under such conditions my reflect the reduced sensitivity of the vsculr tissue to vsoconstrictor meditors (Kiel et l., 1985; Pizcuet et l., 199) or the relese of vsodiltor fctors, such s glucgon (Benoit et l., 1984; 1986; Krvetz et l., 1988). The biologicl ctivity of endothelium-derived relxing fctor, (DRF, Furchgott & Zwdzki, 198; Furchgott, 1984) cn be ttributed to endogenous nitric oxide (NO) formed by vsculr endothelil cells (Plmer et l., 1987; Ignrro et l., 1987; Khn & Furchgott, 1987; Kelm et l., 1988). The biosynthesis of NO from its substrte mino cid, L-rginine (Plmer et l., 1988) is inhibited by N- monomethyl-l-rginine () in endothelil cells nd vsculr tissue in vitro (Plmer et l., 1988b; Rees et l., 1989). Furthermore, studies with in vivo hve implicted NO in the physiologicl regultion of systemic rteril blood pressure in the rbbit, rt nd guine-pig (Rees et l., 1989b; Compton et l., 1989; Whittle et l., 1989; Aisk et l., 1989), of peripherl vsculr tone in mn (Vllnce et l., 1989,b) nd of blood flow in the rt gstric, mesenteric nd renl vsculr beds (Pique et l., 1989; Grdiner et l., 199; Wlder et l., 1991). Author for correspondence. While reduction in endogenous NO biosynthesis my be involved in the pthogenesis of some forms of hypertension (Moncd et l., 1991), excessive formtion of NO my contribute to crdiovsculr disese conditions involving reduced vsculr. In the present study therefore, the possible contribution of NO to the ltered systemic nd splnchnic circultion of the chronic portl hypertensive rt induced by prtil portl vein ligtion hs been investigted by use of. Methods Animl preprtion The study ws performed in mle Sprgue-Dwley rts, in which portl hypertension ws induced by the ligtion of the portl vein, s previously described in detil (Chojkier & Groszmnn, 1981; Krvetz et l., 1988). In brief, rts were nesthetized with ketmine (loomgkg-1, i.m.), the portl vein isolted nd stenosis creted by single ligture of 3- silk plced round both the portl vein nd 2 guge blunt tipped needle. The needle ws then removed from the ligture, thus leving clibrted constriction of the portl vein. The rts were llowed to recover nd hd free ccess to wter nd food until the dy of the study, 2 weeks lter, when the portl hypertension syndrome hd fully developed. The portl hypertensive nimls were divided in 4 groups: vehicle control group tht received n intrvenous bolus injection of sline (1 ml; n = 9; body weight g); second group tht received t dose of 6.25mg kg-1, i.v. (n = 9; weight g); third group tht received t dose of 5mg kg-1, i.v. (n = 8, weight g) nd fourth group in which L-rginine (3mgkg-1, i.v.; Sigm Chemicl Compny, Poole, Dorset, prepred s the cette) dissolved in isotonic sline ws

2 NO AND RAT PORTAL HYPRTNSION 185 dministered 5 min before dministrtion of (5mgkg -1, n = 7, weight g). These doses were chosen from preliminry dose-response studies on the effects of (1.25-1mgkg-') on systemic rteril blood pressure (MAP) in portl hypertensive rts nesthetized with ketmine, s those tht gve submximl nd ner mximl responses. (6.25mg kg-1) ws the lowest dose tht returned MAP to vlues comprble to those in control nonligted nimls. In n dditionl study using control non-operted rts without portl ligtion, one group received the vehicle, sline (n = 8, weight g) while in further group, (5mgkg-1, i.v.; n = 8, weight g) ws dministered. s the cette, prepred in the Dept. Medicinl Chemistry, Wellcome Reserch Lbs, ws dissolved freshly in isotonic sline before use. Hemodynmic studies The techniques used for the hemodynmic mesurements hve been described previously in detil (Groszmnn et l., 1982; Krvetz et l., 1986; Pizcuet et l., 1989). The rts were nesthetized with ketmine HCI (1mgkg-1, i.m.). The left ventricle ws ctheterized vi the right crotid rtery with P-5 tubing. Another P-5 ctheter ws plced in the left femorl rtery for rteril pressure mesurements nd for blood withdrwl. Through 2-cm midline incision, the portl vein ws ctheterized vi the ileocolic vein with tip chilled tubing. After verifiction tht free bckflow of blood ws obtined, the ctheter ws fixed with cynocrylte glue nd the bdomen wll ws closed with silk sutures. This ctheter ws subsequently used for portl pressure mesurements. Another P-5 ctheter ws plced in the right trium vi the left externl jugulr vein nd used for tril pressure meurement nd drug infusion. All ctheters were connected to pressure trnsducers, clibrted before ech experiment, nd blood pressures were registered on multichnnel recorder (Lectromed MT6-PX). The zero reference point ws estblished 1 cm bove the operting tble. Rectl temperture ws mintined t C throughout the study. After obtining bseline mesurements of resting MAP, right tril pressure nd portl pressure, sline or (1 ml) ws dministered s slow bolus injection through the ctheter in the internl jugulr vein. Ten minutes lter, these pressures were mesured gin nd crdic output nd regionl blood flows were mesured by rdioctive microsphere technique, s previously described (Krvetz et l., 1986; Pizcuet et l., 1989). A reference blood smple ws obtined from the femorl rtery ctheter over 75 s period t rte of 1 ml min- using continuous withdrwl pump. Approximtely 5 x 14 microspheres lbelled with 141Ce ( pm dimeter; specific ctivity: 1 mci g'; New nglnd Nucler, Boston, MA, U.S.A.) were injected into the left ventricle, 15s fter beginning the blood withdrwl. At the end of the experiments, the nimls were injected intrvenously with sturted KCl. The bdominl orgns were dissected, blotted, weighed, cut into smll pieces, nd plced in counting tubes. The rdioctivity (c.p.m.) of ech orgn ws determined in gmm-scintilltion counter (Pckrd, 8c). The interference of Cr rdioctivity (energy window: 24 to 4 kev) ws corrected using Cr nd Ce stndrds. Crdic output (ml min-1) ws clculted s follows: Injected rdioctivity (c.p.m.) Reference blood flow (ml min ') x Reference blood rdioctivity (c.p.m.) Regionl blood flows were clculted s: Orgn blood flow (ml min 1) = Orgn rdioctivity (c.p.m.) Reference blood flow (ml min 1) x Reference blood rdioctivity (c.p.m.) Portl venous inflow (PVI), which represents the totl blood flow entering the portl venous system, ws clculted s the sum of blood flow to stomch, spleen, smll nd lrge intestines, pncres nd mesentery. Resistnce in ech vsculr bed ws clculted from the rtio between perfusion pressure (P) nd blood flow (Q) of ech vsculr territory. For the clcultion of totl peripherl vsculr, P ws the vlue for men rteril pressure minus the right tril pressure nd Q ws the crdic output, in the clcultion of splnchnic vsculr, P ws the vlue for men rteril pressure minus the right tril pressure nd Q ws the crdic output; in the clcultion of splnchnic vsculr, P ws men rteril pressure minus portl pressure nd Q ws the PVI. For the clcultion of portocollterl, P ws portl pressure minus right tril pressure nd Q ws the PVI. Dt nlysis All results re expressed s men + stndrd error of the men. Student's t test for pired nd non-pired dt nd the nlysis of vrince with contrsts were used in the sttisticl nlysis of the results. Significnce ws tken t P <.5. Results Two weeks fter ligtion of the portl vein, the chronic portl hypertensive nimls under resting conditions exhibited chrcteristic hemodynmic disturbnces. Thus, there ws significntly (P <.5) decresed men rteril pressure (MAP), incresed crdic output, nd reduced totl peripherl vsculr with splnchnic vsodilttion, s shown by lowered splnchnic vsculr s compred with nesthetized control non-operted rts (Tbles 1 nd 2). Renl blood flow ws lso significntly incresed with Tble 1 Men rteril pressure (MAP) nd portl pressure (PP) under resting conditions nd fter the intrvenous dministrtion of N-monomethyl-L-rginine (, 5mgkg- ) or sline in control nd chronic portl hypertensive rts; the effect of pretretment with L-rginine (L-Arg; 3 mg kg- 1, i.v.) on the ctions of in the portl ligted rts is lso shown MAP (mmhg) PP Control rts Resting Sline Resting ± * ± Portl hypertensive rts Resting Sline Resting + L-Arg 1 5t 13 ± 5t 99 ± 6t 146 ± 7* ** t t 11 ±.3t t t Results, shown s MAP or PP (mmhg) under resting conditions or following sline or drug dministrtion in control non-operted rts or following chronic portl vein ligtion, re men + s.e.men of 8-9 rts, where significnt difference from corresponding resting vlues is given s * P <.1 nd from the resting vlues in control rts s t P <.5. Inhibition of the effects of by L-rginine is shown s ** P <.1.

3 186 M.P. PIZCUTA et l. Tble 2 ffects of sline or NG monomethyl-l-rginine (, 5mgkg-') on systemic nd splnchnic hemodynmics in control nd portl hypertensive rts: the effect of pretretment with L-rginine (L-Arg; 3mgkg-1, i.v.) on the responses to in the portl ligted rts is lso shown Crdic output (mlmin-1 1g-') Peripherl vsculr (mmhg ml -P min -l 1 g -1) Splnchnic vsculr (mmhgml-'min-' 1g-1) Control Vehicle (n = 8) (n = 8) Portl hypertensive Vehicle + L-Arg (n = 9) (n = 8) (n = 7) * t * t * t ** * ** Results, shown s the vlue of ech prmeter per 1 g body weight following intrvenous dministrtion of vehicle (sline) or or L-rginine nd in control non-ligted or chronic portl hypertensive rts, re expressed s the men + s.e.men of (n) studies. Significnt difference from the corresponding control non-ligted group is shown s * P <.5 nd from the vehicle (sline) group in the portl hypertensive rts s t P <.5. Significnt inhibition by L-rginine of the ctions of is shown s ** P <.5. decrese in renl vsculr (Tble 3). Portl venous inflow ws likewise significntly elevted, s ws portl pressure, while porto-collterl ws unchnged (Tbles 1 nd 3). ffects of NG-monomethyl-L-rginine in portl hypertensive rts In portl hypertensive rts, intrvenous dministrtion of (6.25 nd 5mgkg-') induced dose-dependent significnt increse in MAP with n increse of mmhg (n = 8) t the highest dose (Figure 1). The chnge in MAP ws observed within 1 min following dministrtion of, reched its mximl vlues fter 5-1min, nd ws mintined for the durtion of the study. The increse in MAP ws ccompnied by decline in crdic output, which ws significnt t the higher dose of (Figure 1). (6.25 nd 5mg kg- 1) dose-dependently nd significntly incresed totl peripherl vsculr nd splnchnic vsculr (Figure 2). At the higher dose, cused significnt (P <.5) reduction in portl venous flow nd increse in porto-collterl, but there ws no chnge in portl pressure induced by either dose of (Figure 3). In the renl circultion, (SOmg kg- ') induced significnt decrese in renl blood flow nd n increse in renl vsculr (Figure 4). Comprison of the crdiovsculr prmeters following dministrtion of the low dose of (6.25mgkg-1), indicted tht MAP in these portl hypertensive rts ws comprble to those in control nimls under resting conditions ( nd mmhg, respectively P <.5). Furthermore, following this dose of, crdic output nd totl peripherl vsculr were likewise returned to vlues not significntly different from those in control nimls under resting conditions. Following dministrtion of (SOmgkg-1), the vlues for ll the crdiovsculr prmeters mesured in the portl hypertensive rts were not significntly different from those determined in control nimls under resting conditions (Tbles 2 nd 3), except for MAP, porto-collterl nd portl pressure which were significntly higher (Tbles 1 nd 3). ffects of L-rginine Pretretment of the portl hypertensive rts with L-rginine (3mgkg 1, i.v.) 5min before (Smgkg- '), bolished (P <.1) its ctions on MAP nd ll of the crdiovsculr prmeters mesured (Tbles 1, 2 nd 3). L-Arginine Tble 3 ffects of sline or NG monomethyl-l-rginine (, 5mg kg -1) on portl nd renl hemodynmics in control nd portl hypertensive rts: the effect of pretretment with L-rginine (L-Arg; 3mgkg- ', i.v.) on the responses to in the portl ligted rts is lso shown Portl venous inflow (mlmin-1 1g-) Porto-collterl (mmhgml-'min-' 1g-') Renl blood flow (mlmin-1 1g-1) Renl vsculr (mmhg ml-'min-1 1g-) Control Vehicle (n = 8) (n= 8) Portl hypertensive Vehicle + L-Arg (n = 9) (n = 9) (n = 7) * t t t * 3.4 ±.3t ** t * t ** ** ** Results, shown s the vlue of ech prmeter per 1 g body weight following intrvenous dministrtion of vehicle (sline) or or L-rginine nd in control non-ligted or chronic portl hypertensive rts, re expressed s the men + s.e.men of (n) studies. Significnt difference from the corresponding control non-ligted group is shown s * P <.5 nd from the vehicle (sline) group in the portl hypertensive rts s t P <.5. Significnt inhibition by L-rginine of the ctions of is shown s **P <.5.

4 NO AND RAT PORTAL HYPRTNSION CD I, b 16 Con l b 4- Con CD I ) I i.v Co 2 Con mg kg-' i.v. Figure 1 ffects of NG-monomethyl-L-rginine (, 6.25 nd 5mgkg-1, i.v.) on () systemic rteril blood pressure (mmhg) nd (b) crdic output (ml min-' 1g-l body weight) in the portl hypertensive rt, 2 weeks fter portl ligtion. Results re shown s the men of 8-9 experiments in ech group, where verticl lines show s.e.men. Significnt difference from the resting vlues is shown s *P <.5; **P <.1. I 1* - Con mg kg-' i.v. Figure 3 ffects of NG-monomethyl-L-rginine (, 6.25 nd 5mgkg-', i.v.) on () portl venous inflow (mlmin' 1g` body weight) (b) portl pressure (mmhg) nd (c) porto-collterl (mmhg ml-'min-' 1g` body weight) in the portl hypertensive rt, 2 weeks fter portl ligtion. Results re shown s the men of 8-9 experiments in ech group, where verticl lines show s.e.men. Significnt difference from the resting vlues is shown s * P < C) '.. I : C) 45 -_ b- bii Con 6. O Con b ) CD 4.5 I 3. I 1.5 Con mg kg-' i.v. Figure 2 ffects of NG-monomethyl-L-rginine (, 6.25 nd 5mgkg-1, i.v.) on () totl peripherl vsculr nd (b) splnchnic vsculr (mmhg ml - 'min 1 g` body ' weight) in the portl hypertensive rt, 2 weeks fter portl ligtion. Results re shown s the men of 8-9 experiments in ech group, where verticl lines show s.e.men. Significnt differences from the resting vlues is shown s * P <.5; ** P <.1. Con mg kg Figure 4 ffects of N-monomethyl-L-rginine ( 6.25 nd 5mgkg-', i.v.) on () renl blood flow (mlmin' 1g` body weight) nd (b) renl vsculr (mmhg ml- 'min - g l) in the portl hypertensive rt, 2 weeks fter portl ligtion. Results re shown s the men of 8-9 experiments in ech group, where verticl lines show s.e.men. Significnt differences from the resting vlues is shown s * P <.5. i.v.

5 188 M.P. PIZCUTA et l. (3mg kg-, i.v.) lone cused n initil inconsistent nd trnsient fll in MAP but there ws no significnt effect on MAP when mesured 5min fter dministrtion, t the time of dministrtion (P >.5, n = 5). ffects of NG-monomethyl-L-rginine in control rts From preliminry dose-response studies with (6.25-1mg kg- 1) in ketmine-nesthetized control non-ligted rts, dose of (5mgkg-', i.v.) ws selected for comprison of its ctions on the hemodynmic prmeters with those in the portl hypertensive rt. (5mgkg-1) significntly incresed resting MAP by mmhg (n = 8, P <.5) in these control rts, lthough this ws significntly (P <.5) less thn tht observed in the portl hypertensive rts with this dose. As with the portl hypertensive rts, did not ffect portl pressure in control rts (Tble 1). However, unlike the findings in the portl hypertensive rts, the ctions of this dose of on crdic output, portl venous inflow or porto-collterl did not rech sttisticl significnce (Tbles 2 nd 3). Likewise, the overll chnges in totl peripherl nd splnchnic vsculr did not rech significnce lthough did significntly reduce renl blood flow nd renl vsculr (Tbles 2 nd 3). Discussion Prtil ligtion of the portl vein in the rt induces n initil elevtion of portl pressure nd nd decresed venous inflow (Sikuler et l., 1985). However, 8 to 14 dys lter, extensive collterls re formed, leding to the portosystemic shunting of over 9% of the blood entering the portl venous system (Chojkier & Groszmnn, 1981). This brings bout decrese of portl to level comprble to its resting vlue in non-ligted rts, while substntil increse in venous inflow contributes to the sustined increse in portl pressure (Vorobioff et l., 1983; Benoit et l., 1984; Sikuler et l., 1985; Krvetz et l., 1986; 1988). The elevted inflow into the portl system is promoted by the ccompnying splnchnic nd renl vsodilttion, which is reflected by fll in totl peripherl, splnchnic nd renl vsculr, with reduction in systemic rteril blood pressure (BP) nd incresed crdic output (Groszmnn et l., 1982; Sikuler et l., 1985, Benoit et l., 1986; Krvetz et l., 1988). All of these crdiovsculr chnges were confirmed in the present study in the rt following 2-week prtil portl ligtion. The processes underlying the splnchnic vsodilttion in chronic portl hypertension my involve reduced vsculr sensitivity to endogenous vsoconstrictors (Richrdson & Withrington, 1976; Kiel et l., 1985; Pizcuet et l., 199) or n increse in circulting levels of vsodiltor fctors, possibly due to their decresed heptic metbolism s result of portosystemic shunting (Benoit et l., 1984; 1986; Krvetz et l., 1988). Although glucgon hs been implicted (Benoit et l., 1986; Krvetz et l., 1988), studies with infusions of this peptide suggest tht it could only ccount for 3-4% of the observed splnchnic vsodilttion observed in portl hypertensive rts (Benoit et l., 1984; 1986). The present findings using suggest NO is likely cndidte for the meditor of vsculr events following chronic portl ligtion. Intrvenous dministrtion of dose-dependently elevted BP in the portl hypertensive rt, s observed previously in the conscious or nesthetized normotensive rt (Compton et l., 1989; Whittle et l., 1989; Grdiner et l., 199). This ws ccompnied by decrese in the crdic output nd n increse in totl peripherl, splnchnic nd renl vsculr to vlues not significntly different from those observed under resting conditions in control nonligted rts. In the control non-ligted rts, lthough BP ws incresed by, s ws renl vsculr, the overll chnges in totl peripherl vsculr or totl splnchnic did not rech sttisticl significnce. However, recent preliminry studies using similr rdiolbelled microsphere techniques in non-operted rts hve demonstrted differentil chnges in the vrious splnchnic vsculr res following this dose of (Pizcuet et l., 1991). Thus, incresed vsculr in the stomch, pncres, spleen nd mesenteric bed, but not in the smll intestine nd colon, indicting different sensitivities to the ctions of this NO-synthse inhibitor in these splnchnic vsculr beds (Pizcuet et l., 1991). In contrst, in portl hypertensive rts, significnt increses in vsculr were observed with in ll of these vsculr beds (Pizcuet et l., 1991), s supported by the current findings of incresed totl splnchnic. Previous studies with in normotensive chroniclly instrumented conscious rts hve lso demonstrted reduction in regionl blood flow, including vsoconstrictor ctions on the mesenteric nd renl vsculr beds (Grdiner et l., 199), while reductions in blood flow to the gstric mucos (Pique et l., 1989) nd renl cortex (Wlder et l., 1991) hve been observed in the nesthetized rt. In previous studies in this model, reduction in portl inflow by /J-drenoceptor blockde, or the dministrtion of somtosttin ws ccompnied by smll but significnt fll in the elevted portl pressure (Krvetz et l., 1988; Pizcuet et l., 1989; Pique et l., 199). In the current study, the splnchnic vsoconstriction induced by ws ssocited with reduction in portl venous inflow in the ligted rts, but portl pressure ws not significntly modified. This my be the consequence of the concurrent increse in portocollterl following dministrtion of, which is likely to reflect venoconstriction in the portocollterl vsculture. The crdiovsculr ctions of in the portl hypertensive rts were bolished by pretretment with L- rginine, s previously demonstrted in normotensive rts (Compton et l., 1989; Whittle et l., 1989; Grdiner et l., 199). The dose of L-rginine used hd itself no significnt effect on resting BP, mesured 5 min fter dministrtion in these portl hypertensive rts. Thus, the mechnism by which could normlize BP, s well s vsculr nd portl venous inflow, presumbly reflects ttenution of the locl synthesis of NO from L-rginine, suggesting n exggerted locl production of NO in these vsculr beds under such conditions. Furthermore, by comprison with control non-ligted rts, portl hypertensive rts ppered to be more sensitive to the crdiovsculr effects of. A detiled comprtive evlution of the sensitivity of these vsculr beds to, nd other NO synthesis inhibitors under such conditions is hence wrrnted in future studies. The present findings with lso suggest tht the portosystemic collterl vsculture, which is developed from preexisting venous chnnels (Vorobioff et l., 1983), is cpble of ctively synthesizing NO. This contrsts with the findings of reltively low production of NO by venous tissue under bsl conditions (Vllnce et l., 1989b). The fctors tht regulte substrte vilbility nd enzyme ctivity involved in endogenous NO synthesis in these vsculr beds re not yet fully known. Furthermore, lthough the clcium-dependent synthse responsible for NO production by endothelil cells is constitutive enzyme, n inducible enzyme tht forms NO from L-rginine nd which is lso inhibited by, hs lso been described in these cells (Rdomski et l., 199). An inducible NO synthse, identified originlly in the mcrophge (Hibbs et l., 1988; Mrlett et l., 1988; Stuehr et l., 1989) hs lso been detected in vsculr smooth muscle, lung nd liver tissue (Busse & Mulsch, 199; Knowles et l., 199; Rees et l., 199). Furthermore, it hs been suggested recently tht the vsculr formtion of NO by this inducible enzyme my contribute to the hyperdynmic

6 NO AND RAT PORTAL HYPRTNSION 189 stte seen in cirrhosis (Vllnce & Moncd, 1991). It will therefore be importnt to investigte the enzymic chrcteristics of NO formtion in endothelil cells nd smooth muscle of the splnchnic vsculr beds s well s in the newly developed porto-collterl venous system under the conditions seen in portl hypertension. Should n inducible NO synthse be involved in this systemic nd splnchnic vsodilttion, then its selective ttenution would offer novel nd specific pproch to the correction of vsculr tone nd blood flow under such pthologicl conditions with miniml effects on the physiologicl ctions of NO formed by the constitutive enzyme. The present findings tht the hyperdynmic circultory chrcteristics following portl vein stenosis cn be ttenuted by, thus provide initil evidence tht n excessive formtion of NO is implicted in the pthogenesis of the hemodynmic disturbnces ssocited with chronic portl hypertension. This work ws supported in prt by grnt from F.I.S.S. (Spin; 91/374). References AISAKA, K., GROSS, S.S., GRIFFITH, O.W. & LVI, R. (1989). N - methyl-l-rginine, n inhibitor of endothelium-derived nitric oxide synthesis, is potent pressure gent in the guine pig: does nitric oxide regulte blood pressure in vivo? Biochem. Biophys. Res. Commun., 16, BNOIT, J.N., BARROWMAN, J.A., HARPR, S.L., KVITYS, P.R. & GRANGR, D.N. (1984). Role of humorl fctors in the intestinl hyperemi ssocited with chronic portl hypertension. Am. J. Physiol., 247, G486-G493. BNOIT, J.N., ZIMMRMAN, B., PRMN, A.J., GO, V.L.W. & GRANGR, D.N. (1986). Role of glucgon in splnchnic hyperemi of chronic portl hypertension. Am. J. Physiol., 251, G674-G677. BLANCHT, L. & LBRC, D. (1982). Chnges in splnchnic blood flow in portl hypertensive rts. ur. J. Clin. Invest., 12, BOSCH, J., GINS, ARROYO, V., NAVASA, M. & RODS, J. (1988). Heptic nd systemic hemodynmics nd the neurohumorl systems in cirrhosis. In: The Kidney in Liver Disese. ed. pstein, M. pp Bltimore: Willims & Wilkins. BUSS, R. & MULSCH, A. (199). Induction of nitric oxide synthse by cytokines on vsculr smooth muscle cells. FBS Lett., 275, CHOJKIR, M. & GROSZMANN, R.J. (1981). Mesurement of portl systemic shunting in the rt by using gmm-lbelled microspheres. Am. J. Physiol., 24, G371-G375. COMPTON, A.M., GARDINR, S.M., BNNTT, T., MONCADA, S. & PALMR, R.M.J. (1989). Hemodynmic effects of N - monomethyl-l-rginine () in conscious Long vns rts. Br. J. Phrmcol., 98, 523P. FURCHGOTT, R.F. (1984). The role of endothelium in the response of vsculr smooth muscle to drugs. Annu. Rev. Phrmcol. Toxicol., 24, FURCHGOTT, R.F. & ZAWADZKI, J.V. (198). The obligtory role of endothelil cells in the relxtion of rteril smooth muscle by cetylcholine. Nture, 288, GARDINR, S.M., COMPTON, A.M., BNNTT, T., PALMR, R.M.J. & MONCADA, S. (199). Control of regionl blood flow by endothelium-derived nitric oxide. Hypertension, 15, GROSZMANN, R.J., VOROBIOFF, J. & RILY,. (1982). Mesurement of splnchnic hemodynmics in portl-hypertensive rts: ppliction of y-lbelled microspheres. Am. J. Physiol., 242, G156-G16. HIBBS, J.R. Jr., TAINTOR, R.R., VAVRIN, Z. & RACHLIN,.M. (1988). Nitric oxide: cytotoxic ctivted mcrophge effector molecule. Biochem. Biophys. Res. Commun., 157, IGNARRO, L.J., BUGA, G.M., WOOD, K.S., BYRNS, R.. & CHAUDHURI, G. (1987). ndothelium-derived relxing fctor produced nd relesed from rtery nd vein is nitric oxide. Proc. Nti. Acd. Sci. U.S.A., 84, KLM, M., FLISCH, M., SPAHR, R., PIPR, H-M., NOACK,. & SCHRADR, J. (1988). Quntittive nd kinetic chrcteriztion of nitric oxide nd DRF relesed from cultured endothelil cells. Biochem. Biophys. Res. Commun., 154, KHAN, M.T. & FURCHGOTT, R.F. (1987). Additionl evidence tht endothelium-derived relxing fctor is nitric oxide. In Phrmcology. ed. Rnd, M.J. & Rper, C. pp New York: lsevier. KIL, K.W., PITTS, V., BNOIT, J.N., GRANGR, D.N. & SHPHRD, A.P. (1985). Reduced vsculr sensitivity to norepinephrine in portl hypertensive rts. Am. J. Physiol., 248, G192-G195. KNOWLS, R.G., MRRTT, M., SALTR, N. & MONCADA, S. (199). Differentil induction of brin, lung nd liver nitric oxide synthse by endotoxin in the rt. Biochem. J., 27, KRAVTZ, D., SILUKR,. & GROSZMANN, R.J. (1986). Splnchnic nd systemic hemodynmics in portl hypertensive rts during hemorrhge nd blood volume restitution. Gstroenterology, 9, KRAVTZ, D., BOSCH, J., ARDRIU, M.T., PIZCUTA, M.P., CASMIT- JANA, R., RIVRA, F. & RODS, J. (1988). ffects of somtosttin on splnchnic hemodynmics nd plsm glucgon in portl hypertensive rts. Am. J. Physiol., 254, G322-G328. LBRC, D., BATAILL, C., BRCOFF,. & VALLA, D. (1983). Hemodynmic chnges in ptients with portl venous obstruction. Heptology, 3, MARLTTA, M.A., YOON, P.S., IYNGAR, R., LAF, C.D. & WISHNOK, J.S. (1988). Mcrophge oxidtion of L-rginine to nitrite nd nitrte: Nitric oxide is n intermedite. Biochemistry, 27, MONCADA, S., PALMR, R.M.J. & HIGGS,.A. (1991). Nitric Oxide: Physiology, pthophysiology nd phrmcology. Phrmcol. Rev., 43, PALMR, R.M.J., FRRIG, A.G. & MONCADA, S. (1987). Nitric oxide relese ccounts for the biologicl ctivity of endothelium-derived relxing fctor. Nture, 327, PALMR, R.M.J., ASHTON, D.S. & MONCADA, S. (1988). Vsculr endothelil cells synthesize nitric oxide from L-rginine. Nture, 333, PALMR, R.M.J., RS, D.D., ASHTON, D.S. & MONCADA, S. (1988b). L-rginine is the physiologicl precursor for the formtion of nitric oxide in endothelium-dependent relxtion. Biochem. Biophys. Res. Commun., 153, PIQU, J.M., WHITTL, B.J.R. & SPLUGUS, J.v. (1989). The vsodiltor role of endogenous nitric oxide in the rt gstric microcircultion. ur. J. Phrmcol., 174, PIQUt, J.M., PIZCUTA, M.P., PRZ AYUSO, R.M. & BOSCH, J. (199). ffects of proprnolol on gstric microcircultion nd cid secretion in portl hypertensive rts. Heptology, 12, PIZCUTA, M.P., D LACY, A.M., KRAVTZ, D., BOSCH, J. & RODS, J. (1989). Proprnolol decreses portl pressure without chnging portl-collterl in cirrhotic rts. Heptology, 1, PIZCUTA, M.P., CASAMITJANA, R., BOSCH, J. & RODS, J. (199). Decresed systemic vsculr sensitivity to norepinephrine in portl hypertensive rts. Role of hyperglucgonism. Am. J. Physiol., 258, G191-G195. PIZCUTA, M.P., PIQU, J.M., BOSCH, J., FRNANDZ, M., WHITTL, B.J.R. & MONCADA, S. (1991). Involvement of endogenous nitric oxide in the regultion of the rt splnchnic circultion. Gstroenterology, 1, A24. RADOMSKI, M.W., PALMR, R.M.J. & MONCADA, S. (199). Glucocorticoids inhibit the expression of n inducible, but not the constitutive, nitric oxide synthse in vsculr endothelil cells. Proc. Ntl. Acd. Sci. U.S.A., 87, RS, D.D., PALMR, R.M.J. & MONCADA, S. (1989). Role of endothelium-derived nitric oxide in the regultion of blood pressure. Proc. Ntl. Acd. Sci. U.S.A., 86, RS, D.D., PALMR, R.M.J., HODSON, H.F. & MONCADA, S. (1989b). A specific inhibitor of nitric oxide formtion from L-rginine ttenutes endothelium-dependent relxtion. Br. J. Phrmcol., 96, RS, D.D., CLLK, S., PALMR, R.M.J. & MONCADA, S. (199). Dexmethsone prevents the induction by endotoxin of nitric oxide synthse nd the ssocited effects on vsculr tone: n insight into endotoxin shock. Biochem. Biophys. Res. Commun., 173, RICHARDSON, P.D.I. & WITHRINGTON, P.G. (1976). The inhibition by glucgon of the vsoconstrictor ctions of nordrenline, ngiotensin, nd vsopressin on the heptic rteril vsculr bed of the dog. Br. J. Phrmcol., 57, SIKULR,., KRAVTZ, D. & GROSZMANN, R.J. (1985). volution of portl hypertension nd mechnisms involved in its mintennce in rt model. Am. J. Physiol., 248, G618-G625. STUHR, D.J. & NATHAN, C.F. (1989). Nitric oxide. A mcrophge

7 19 M.P. PIZCUTA et l. product responsible for cytostsis nd respirtory inhibition in tumour trget cells. J. xp. Med., 169, VALLANC, P. & MONCADA, S. (1991). Hypothesis: Induction of nitric oxide synthse in the vsculture underlies the hyperdynmic circultion of cirrhosis. Lncet, 337, VALLANC, P., COLLIR, J. & MONCADA, S. (1989). ffects of endothelium-derived nitric oxide on peripherl rteriolr tone in mn. Lncet, ii, VALLANC, P., COLLIR, J. & MONCADA, S. (1989b). Nitric oxide synthesised from L-rginine medites endothelium dependent diltion in humn veins in vivo. Crdiovsc. Res., 23, VOROBIOFF, J., BRDFLDT, J. & GROSSZMANN, R.J. (1983). Hyperdynmic circultion in portl hypertensive rt model: primry fctor for mintennce of chronic portl hypertension. Am. J. Physiol., 244, G52-G57. WALDR,., THIMRMANN, C. & VAN, J.R. (1991). The involvement of endothelium-derived relxing fctor in the regultion of renl corticl blood flow in the rt. Br. J. Phrmcol., 12, WHISTL, B.J.R., LOPZ-BLMONT, J. & RS, D.D. (1989). Modultion of the vsodepressor ctions of cetylcholine, brdykinin, substnce P nd endothelin in the rt by specific inhibitor of nitric oxide formtion. Br. J. Phrmcol., 98, (Received July 26, 1991 Accepted September 17, 1991)