Hepatic and Pulmonary Clearance of Exogenous Vasoactive Intestinal Peptide in the Rat

Transcription

1 GASTROENTEROLOGY 77:55-60, 1979 Hepatic and Pulmnary Clearance f Exgenus Vasactive Intestinal Peptide in the Rat CHRISTOPHER S. HUMPHREY, PHILLIP MURRAY, AMIN M. EBEID, and JOSEF E. FISCHER The Department f Surgery, Massachusetts General Hspital, and Harvard Medical Schl, Bstn, Massachusetts J'25-labeled vasactive intestinal peptide (VIP) has been injected int the prtal and systemic circulatins f rats in an attempt t identify the distributin and fate f the circulating peptide. When VIP 1'25 was intrduced int the prtal circulatin radiactivity was cncentrated in the liver (415.5% ± 57.2 at 10 min--cunts per minute (cpm) per gram f tissue as percentage cpm per milliliter f plasma). Radiactivity in kidney and lung was 346.6% ± 37.4 and 136.4% ± 11.4, respectively. In cnti-ast, if the liver was bypassed by perfrming a prtacaval shunt r by injecting int the inferir vena cava, radiactivity was maximal in the lung (2,454.3% ± min after IVe injectin) with activity in the liver f nly 89.3% ± Analysis f the pattern f radiactivity in plasma and tissue extracts by gel filtratin chrmatgraphy shwed the presence f a number f fragments f smaller mlecular weight than VIP with a prgressive diminutin f the amunt f VIPlike radiactivity. Bth liver and lung have the capacity t cncentrate VIP frm the circulatin. Vasactive intestinal peptide released int the prtal circulatin is prbably taken up initially by the liver, and this may prevent subsequent uptake by pulmnary tissue. There is sme evidence t suggest that the liver and the lung may handle VIP in different ways. If this is s, the enhanced pulmnary extractin f VIP when the liver is bypassed may have sme significance fr the cardivascular cmplicatins f fulminant liver failure. Vasactive intestinal peptide (VIP) disappears rapidly frm the circulatin. Studies in human' and in Received August 8, Accepted February 6, Address reprint requests t: Jsef E. Fischer, M.D., Chairman, Department f Surgery, University f Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, Ohi by the American Gastrenterlgica! Assciatin /79/ $02.90 the dg 2 suggest that the half-life f VIP in the plasma may be as shrt as 1-3 min. Hwever, the site f clearance r inactivatin is uncertain. Results f investigatins that have used bilgic parameters t detect VIP have suggested that the liver may be a majr site fr inactivatin,3-5 yet a mre recent study that included direct measurement f VIP cncentratin by radiimmunassay2 is nt in agreement. Neither study cnsidered a pssible rle fr the lung. In the present study, we have determined thll fate f radiactively labeled VIP injected int bth systemic and prtal circulatins f the rat. Clumn chrmatgraphy has been used t identify breakdwn prducts f VIP in the plasma and in varius tissues. The results suggest that the lung may have a rle in the remval f VIP frm the circulatin. This may help t explain the discrepancies between the results f previus studies. Methds Adult male and female Sprague-Dawley rats weighing g were used in these experiments. The animals were allwed free access t fd and water. Vasactive intestinal peptide was btained as highly purified VIP frm Prfessr Viktr Mutt at the Karlinska Institute. Idinatin f the peptide with 1'25 (New England Nuclear, Bstn, Mass.) was carried ut by a chlramine-t methd similar t that described Hunter y and Greenwd 6 t a spec'ific activity f 350 p.ci/p.g. Separatin f VIP 1' 25 frm free 1' 25 was achieved by adding the reactin mixture t a cellulse clumn and washing the unreacted 1'25 thrugh with 0.3 M phsphate buffer. The VIP 1'25 was then eluted frm the clumn with a 12.5% slutin f bacitracin in buffer. Under certain circumstances, radilabeling may alter the metablic clearance f prteins and hrmnes. 7 8 Hwever, in the case f gastrin, Dckray et a1. 9 shwn that mnidinated gastrin has full bilgic activity, and it seems likely that altered metablism is the result f either

2 56 HUMPHREY ET AL. GASTROENTEROLOGY Vl. 77, N.1 substitutin f mre than ne idine per mlecule f peptide r xidatin damage. T guard against these pssibilities, the reactin between VIP and 1'25 was allwed t prceed fr nly 30 sec. This resulted in an incrpratin f idine int VIP f 35-40%. Any idinatins resulting in an incrpratin f >45% were discarded. Labeled peptide was used nly if the cntributin by free idine t the ttal activity was <5%. Each batch f labeled peptide was checked chrmatgraphically (see belw), and nly thse samples that eluted with a single peak f activity were used. This als allwed cnfirmatin that the activity f free idine was <5% f the ttal, because the idine eluted with the ttal vlume f the clumn. On a few ccasins the usual smth single peak f radiactivity assciated with VIP (see, fr example, Figure 3, upper graph) was bradened r cntained multiple peaks. This was t a t e n represent xidatin damage, and such idinatin batches were nt used. In a preliminary series f experiments designed t shw which tissues might be imprtant in clearing VIP frm the circulatin, 12 cnscius rats received apprximately 2 millin cunts f VIP 1'25 by tail vein injectin. This dse f VIP 1'25 cntained apprximately 4 pg f VIP, a small amunt cmpared with the nrmal circulating cncentratins f VIP f arund 30 pg/ml, and hence unlikely t cause saturatin f any physilgic clearance mechanisms. The rats were then killed by decapitatin, six at 10 min and six at 60 min after injectin. Prtins frm numerus rgans (see Results) were remved rapidly and frzen n dry ice, and samples f apprximately 0.5 g were weighed and transferred t glass tubes. The radiactivity in each sample, and in 1 ml f plasma, was determined by cunting fr 1 min in a scintillatin spectrmeter (Packard 5110 Packard Instrument C., Inc., Dwners Grve, 111.). An additinal six rats received injectins f 1'25 alne and were then treated in a similar manner. In a secnd series f studies, a cmparisn was made f the distributin f radiactivity in selected rgans-liver, lung, gastric fundus, mid ileum and kidney-after either systemic r prtal injectin f VIP 1'25. Rats were anesthetized with diethyl ether and submitted t midline lapartmy. Fr the systemic administratin, VIP 1'25 was injected directly int the inferir vena cava. Prtal administratin was by injectin int the prtal vein. The rats were exsanguinated by withdrawing bld frm the inferir vena cava (by a separate needle in the case f the systemically injected rats) at 2, 5, r 10 min. Prtins f the tissues were treated as befre. The numbers f rats in each grup are shwn in the results sectin. In sme f these latter experiments, additinal samples f tissue and plasma were prepared fr chrmatgraphic analysis. Peptide extractin frm the frzen tissues was carried ut by immersing the tissues in biling water t inactivate any peptidases, and then hmgenizing the biled tissue in 0.1 M frmic acid as described by Bryant et al.' The supernatant btained after separating the tissue pellet by centrifugatin was kept at -70 C until analyzed. Percentage recvery f radiactivity using this extractin prcedure was 81.9% and did nt vary frm tissue t tissue. Chrmatgraphy was perfrmed n 90-cm silicnized glass clumns (Kntes C., Vineland, N.J.) packed with Sephadex G 25 superfine gel (Pharmacia Inc., Piscataway, N.J.). One-half-milliliter aliquts f plasma r tissue extract were eluted with 0.3 M ptassium phsphate buffer (ph 7.4) at a flw rate f 15 ml/hr. Fractins f 1.5 ml were cllected and cunted fr radiactivity. The clumn was calibrated with Blue Dextran (Pharmacia) and 1'25 fr the vid and ttal vlumes. Each batch f labeled peptide used in the study was als run thrugh the clumn; the VIP 1'25 was added bth in phsphate buffer and in plasma. In the final experiment, three rats that had been submitted t end-t-side prtacaval shunt 2 wk previusly received VIP 1'25 int a distal radicle f the prtal vein. The animals were sacrificed at 5 min and the tissues examined as befre. Results The prprtin f the injected dse recvered in ttal rgans is related t the relative weights f these tissues. Examined in these crude terms, ten min after injectin, the percentage f radiactivity present in the liver was 7.7% f the injected dse, whereas the percentages in lung, kidney, and plasma, respectively, were 28.1%, 5.4%, and 14.3%. In rder t determine whether any tissues pssessed a particular capacity t cncentrate radiactivity, the activity f each sample was calculated as cunts per minute per gram f tissue and then expressed as a percentage f the radiactivity present in 1 ml f plasma taken frm the same animal at the same time. Mre than 97% f the activity present in the heparinized whle bld resided in the plasma. The pattern f distributin f radiactivity, expressed in this way, in varius rat rgans 10 and 60 min after tail vein injectin f VIP 1'25 is shwn in Figures 1 and 2. At 10 min after injectin, nly tw rgans shwed a greater cncentratin f radiactivity than OESOPHAGUS FUNDUS ANTRUM DUODENUM JEJUNUM ILEUM COLON LIVER PANCREAS BRAIN LUNG HEART KIDNEY BLADDER SPLEEN FEMUR FAT MUSCLE UTERUS 0.,1,. RADIOACTIVITY (cpm per q tissue as percentge activity In plasma) y// ' f--+-< / ) t : Figure 1. Distributin f radiactivity in rat rgans 10 min after tail-vein injectin f VIP 1'25. Radiactivity as cunts per minute per gram f tissue expressed as percentage radiactivity in 1 ml plasma. Mean values (n = 6) ± 1 S.E.M. Shaded area shws range f radiactivity fund 10 min after tail-vein injectin f 1'25 alne (n = 3). -" --.j CD <0

3 July 1979 HEPATIC AND PULMONARY CLEARANCE OF VIP IN THE RAT 57 OESOPHAGUS FUNDUS ANTRUM DUODENUM JEJUNUM ILEUM COLON LIVER PANCREAS BRAIN LUNG HEART KIDNEY BLADDER SPLEEN FEMUR FAT MUSCLE UTERUS RADIOACTIVITY (cpm per q. tissue as percentaqe activity in plasma)... l.. j j N :> t OJ.f (J1 I I (1) Figure 2. Distributin f radiactivity in rat rgans 60 min after tail-vein injectin f VIP 1'25. Details as in Figure 1. plasma: the lung and the kidney (mean values ± 1 S.E.M. f ± 165.6% and ± 44.6%, respectively). Ntable was the very lw activity in the brain (7.2 ± 0.65%) and the fairly unifrm distributin f radiactivity thrughut the gastrintestinal tract with a cncentratin f little mre than 50% that f the plasma. The pattern was quite unlike that fund in the rgans f the rats that were. killed 10 min after receiving 1'25 alne (Figure 1), which shwed the expected cncentratins f radiactivity assciated with free idine in the fundus and antrum f the stmach. In cntrast, at 60 min after injectin (Figure 2), the distributin f radiactivity in the VIP 1'25 injected rats was almst identical t that f the 1'25 injected rats. The nly difference between these tw 60-min grups was the greater cncentratin f activity in the lungs f the VIP 1'25 treated rats, althugh this was nt s great as that seen at 10 min. The amunts f tissue radiactivity present after systemic and prtal injectins f VIP 1'25 in the anesthetized rats are shwn in Table 1. The distributin f radiactivity in the five rgans examined after intracaval injectin was similar t that seen after tail vein injectin. The amunt f radiactivity cncentrated in the lung was in excess f % even at 2 min, and peak activity f almst 3,000% was apparent by 5 min. The amunt f radiactivity in the kidney increased prgressively ver the 10-min perid s that by 10 min, it was cncentrated t sme three times the plasma value. Althugh activity als increased in the liver and gastric fundus, it did nt exceed the plasma level in these rgans. A different pattern was bserved after intraprtal injectin, with peak activity f sme 400% the plasma level in the liver. The activity increased in the kidney ver the perid s that by 10 min it was cncentrated t abut 350%. Activity in the lung remained cnstant at abut 150%, whereas that in the fundus and ileum, althugh increasing, remained lwer than that in plasma. When VIP 1'25 was injected int the prtal system f rats wh had previusly undergne prtacaval shunt, the distributin f radiactivity was similar t that seen after systemic injectin. Thus, activity in the lung 5 min after injectin was 3,634.4% ± 1,073.1 that f plasma whilst in the liver the percentage f radiactivity was nly 118.8% ± Mean values fr percentage activity in the kidney, fundus, and ileum were 92.4 ± 13.2, 44.3 ± 6.2, and 47.6 ± 2.0, respectively. Analysis by gel filtratin chrmatgraphy f the plasma frm VIP 1'25 injected rats gave results as de- Table 1. Amunts f Radiactivity in Rat OrgansO at Varius Times After Systemic and Prtal Injectins f VIP 1'25 Time (min) n b Lung Liver Kidney Fundus Ileum Intracaval injectin 2 4 1, ±198.1 ±13.8 ±14.4 ±8.0 ± , , 37.9 ±86.2 ±7.5 ±14.4 ±3.1 ± , ±302.3 ±10.6 ±46.1 ±11.1 ±4.1 Intraprtal injectin ±16.2 ±130.2 ±64.9 ±4.7 ± ±26.6 ±74.2 ±64.1 ±3.6 ± ±11.4 ±57.2 ±37.4 ±10.8 ±8.8 a Radiactivity = cunts per minute per gram f rgan expressed as percentage f radiactivity in plasma. Mean values ± SEM. b n = number in grup. -

4 58 HUMPHREY ET AL. GASTROENTEROLOGY Vl. 77, N.1 picted in Figure q. When VIP 1'25 was incubated at 37 C with freshly drawn whle bld prir t running the separated plasma thrugh the clumn, the radiactivity eluted in tw peaks. The majr peak crrespnded t that btained when VIP 1'25 was run in buffer, and the minr peak eluted in the ttal vlume as indicated by calibratin with Thus, the labeled VIP was nt brken dwn in whle bld. In cntrast, evidence f breakdwn was seen in the plasma f rats injected with VIP 1'25. The appearance f a peak f activity in the vid vlume and an increase in the size f the V, peak were prbably caused by the liberatin f free idine (the V peak presumably representing carriage f 1'25 r a metablite by plasma prtein), as these peaks were als btained when plasma frm rats injected with 1'25 alne was run n the clumn (Figure 3, inset). Hwever, in additin t these features, the peak crrespnding t VIP 1'25 was cnsiderably reduced in 10,000 9,000 8,000 7, ,000. 5,000 E:::... <I.l 4,000 t:l c: h.. i:::: 0 CS,000 [ 5,000 4,000 I 3,000 IN VITRO IN VIVO FRACTION NUMBER Figure 3. Chrmatgraphic analysis f radiactivity in rat plasma after in vitr incubatin f VIP p25 with fresh whle bld (abve) and 10 min after injectin f VIP p25 (abve). Inset shws distributin f radiactivity in plasma 10 min after injectin f p25 alne. Fr details f chrmatgraphy, see text. V = vid vlume, Vi = ttal vlume.... <I.l...,1:: 3,000 2,500 1, <I.l t:l 2 MIN 0 t:: c:. 3,000 io MIN h.. 2,500 i::::... 1, O L - - L J- L FRACTION NUMBER Figure 4. Chrmatgraphic analysis f radiactivity in lung extracts 2 min (abve) and 10 min (belw) after systemic injectin f VIP p25 in the rat. magnitude, and a number f ther peaks were als apparent; usually at least tw mre, smetimes up t fur additinal discrete peaks f activity between V and V" There was n bvius difference between the plasma f systemically injected rats and that f thse receiving intraprtal injectins, The results f chrmatgraphy f the tissue extracts are shwn in Figures 4 and 5. Tw minutes after injectin (either. systemic r prtal), the majrity f the radiactivity in the lung extracts eluted in a single peak crrespnding t the elutin vlume f. VIP 1'25. By 10 min, this peak had bradened, which suggests that it cntained smaller peaks. A similar elutin pattern was bserved in liver extract after prtal injectin f labeled VIP. Hwever, the pattern f radiactivity in the liver when the VIP 1'25 had been given systemically mre clsely resembled the picture seen in plasma (Figure 5). Likewise, the radiactivity in kidney (Figure 5) and gastric fundus eluted in a way that was similar t that in plasma, irrespective f the rute f administratin. In an attempt t discver whether the smaller peaks f radiactivity present in plasma and tissues

5 July 1979 HEPATIC AND PULMONARY CLEARANCE OF VIP IN THE RAT 59 crrespnded t peptide fragments which still resembled VIP immunlgically, aliquts f plasma and tissue extract were incubated with VIP antiserum (raised in rabbits against highly purified prcine VIP cnjugated t bvine serum albumin") fr 5 days at 4 C. The radiactivity previusly eluting in peaks between V and V, then eluted entirely in the vid vlume, indicating that the fragments had been bund t the antibdy. The prprtin f radiactivity in the plasma that bund t antibdy varied frm 80% in plasma drawn 2 min after injectin t 51% at 5 min and 29% at 10 min. Using these figures t crrect the ttal cunts in the plasma, it was pssible t btain an estimate f the rate f clearance f VIP p25 frm the circulatin. The half-life f the labeled peptide was 2-5 min Cmparisn f this result with the half-life f unlabeled VIP reprted as 3.1 min in the dg 2 and min. in humans' suggests that labeling f VIP under the careful cnditins f these experiments is unlikely t have altered its metablic clearance significantly. Discussin The results f this study shw that when labeled VIP is injected int the circulatin f rats, the radiactivity is cncentrated in either lung r liver, depending upn the rute f administratin. Furthermre, the radiactivity has the elutin characteristics f VIP, at least fr up t 10 min after injectin, althugh evidence fr breakdwn int smaller fragments is apparent in the plasma and, t a lesser extent, in the liver and lung. Radiactivity is als cncentrated in the kidney, but chrmatgraphic analysis suggests that this is the result f uptake f breakdwn prducts in the plasma. The fact that the liver and lung bth have the ability t clear VIP frm the circulatin may explain sme previusly anmalus results. Thus, the discvery that cncentratins f VIP in prtal venus bld are higher than thse in peripheral systemic venus bld 12 r arterial bld13 is explicable n the basis f clearance by liver r lung r bth rgans. Similar reasning may explain the fact that cnstant infusins f VIP by the prtal and systemic rutes resulted in the same peak cncentratins. in: and subsequent rate f clearance frm, systemic venus bld. 2 A cmparisn f prtal with hepatic venus bld has nt been made. Granted that either rgan can cncentrate VIP, which is imprtant physilgically? VIP exists in bth endcrine cells '4 and nerve fibers '5 within the gastrintestinal tract. Release frm either f these sites will be int the prtal circulatin, and in these circumstances, the results f this study suggest that the liver may be f imprtance. T h is rsme e evi III 3,000 2,500 "::, i,500 c: '- III t::l " c: ::, C) '- "- ' J - KIDNEY 3,000 LIVER h:: '-.) 2,500 "'I: C) ,( W FRACTION NUMBER Figure 5, Chrmatgraphic analysis f radiactivity in extracts f kidney (abve) and liver (belw) after systemic injectin f VIP [125 in the rat. dence t suggest that the liver and the lung may deal with VIP in different ways. Thus, when the labeled peptide was injected int the prtal circulatin, the subsequent pulmnary uptake was much less than when the lungs were the first rgans t receive VIP. Furthermre, whereas the radiactivity recvered frm liver after prtal injectin had a large VIP cmpnent n clumn chrmatgraphy, the crrespnding picture in the liver after systemic injectin resembled the "breakdwn" pattern fund in plasma. Thus, it m be y that prir passage f VIP thrugh the liver alters the peptide in such a way as t minimize the subsequent uptake by the lungs. It is certainly pifficult t di.scunt sme srt f rle fr the liver in the metablism f VIP. In additin t the studies already mentined, any cncept f the fate f circulating VIP must recgnize the fact that patients and experimental animals with liver failure exhibit elevated cncentratins f VIP. '6,17 In this study, we have lked at VIP nly in terms f the distributin f radiactivity after injectin f labeled peptide. The analysis f plasma after incubatin with VIP antiserum indicates that at least sme f the breakdwn prducts are still antigenically similar t VIP, and the rate f clearance f VIP and these

6 60 HUMPHREY ET AL. GASTROENTEROLOGY Vl. 77, N.1 breakdwn prducts is f the same rder as the clearance f immunreactive VIP in dg and humans. Passage f VIP thrugh the liver r lungs might cnceivably result in altered bilgic activity f the peptide. We have nt investigated this aspect, but ther evidence suggests that the liver may affect sme prperties f VIP mre than thers. Thus, Kitamura et al.' reprted that the hyptensive and respiratry stimulating actins f VIP were diminished by prtal infusin, and Knturek et al. 5 fund that the actins f VIP as a pancreatic stimulant were similarly reduced. On the ther hand, Strunz et al. 2 were unable t shw any reductin in the inhibitry actins n gastric secretin when VIP was infused prtally. The idea that the hepatic and pulmnary metablism f VIP may be different has been suggested previusly by Kitamura et al.' These authrs reprted that the hyptensive prperties f VIP infused int the systemic arterial system were reduced when the peptide was delivered int the prtal system but enhanced if the VIP was delivered t the lungs via the right ventricle. They suggested that the ptentiatin f VIP effects by the lungs might be due t release f ther active agents r t further activatin, by metablism, f the VIP itself. Certainly the lung is knwn t metablize a number f ther vasactive cmpunds: the cnversin f angitensin I t II, the remval f 5-hydrxytryptamine frm the circulatin, and the inactivatin f nradrenaline and bradykinin. '8 The lungs d nt appear t catablize g a s trin, althugh the peptide may be sequestered in pulmnary edema fluid. '9 The demnstratin by this study f the uptake f VIP by pulmnary tissue cnfirlit's the bservatin f Krinek et al. 20 that labeled VIP binds t crude membrane preparatins f lungs. Althugh the presence f breakdwn prducts f VIP in the plasma and in tissues has been fund in assciatin with this uptake, direct evidence t implicate the lungs as the majr site f metablism is lacking. Hwever, if the lungs are respnsible fr a significant metablism f VIP, the bservatin f a cnsiderable uptake f prt ally injected VIP in previusly shunted rats assumes pssible clinical imprtance. Patients with liver disease are prne t the develpment f hyptensin and pulmnary edema The hyptensive prperty f VIp 3 and its pssible effect n pulmnary bld flw,23 particularly if these actins are augmented by virtue f increased pulmnary uptake, culd playa part in the genesis f these cmplicatins. References 1. Blm SR, Mitchell SJ, Dmschke S, et al: VIP infusin in man. Gut 18:A963, Strunz UT, Walsh JH, Blm SR, et al: Lack f hepatic inactivatin f canine vasactive intestinal peptide. Gastrenterlgy 73: , Said SI, Mutt V: Ptent peripheral and splanchnic vasdilatr peptide frm nrmal gut. Nature 225: , Kitamura S, Yshida T, Said SI: Vasactive intestinal plypeptide inactivatin in liver and ptentiatin in lung f anaesthetized dgs. Prc Sc Exp Bii Med 148:25-29, Knturek SJ, Dmschke S, Dmschke W, et al: Cmparisn f pancreatic respnses t prtal and systemic secretin and vasactive intestinal peptide. Am J PhysiI232:E , Hunter WM, Greenwd FC: Preparatin f l a b human grwth hrmne f high specific activity. Nature 194:495, l l e d Bersn SA, Yalw B, Schreiber SS, Pst J: Tracer experiments with 1'31 labelled human serum albumin: distributin and degradatin studies. J Clin Invest 32: , Izz JL, Bartlett JW, Rncne A, et al: Physilgical prcesses and dynamics in the dispsitin f small and large dses f bilgically active and inactive 131I-insulins in the rat. J Bii Chern 242: , Dckray GJ, Walsh JH, Grssman MI: Bilgical activity f idinated gastrins. Bichem Biphys Res Cmmun 69: , Bryant MG, Blm SR, Plak JM, et al: Pssible dual rle fr vasactive intestinal peptide as gastrintestinal hrmne and neurtransmitter substance. Lancet 1: , Ebeid AM, Murray P, Hirsch H, et al: Radiimmunassay f vasactive intestinal peptide. J Surg Res 20: , Ebeid AM, Murray P, Seters PB, et al: Release f VIP by calcium stimulatin. Am J Surg 133: , Schaffalitzky de Muckadell OB, Fahrenkrug J, Hlst n, et al: Release f vasactive intestinal plypeptide (VIP) by intradudenal stimuli. Scand J GastrenterI12: , Plak JM, Pearse AGE, Garaud J-C, et al: Cellular lcalisatin f a vasactive intestinal peptide in the mammalian and avian gastrintestinal tract. Gut 15: , Larssn L-I, Fahrenkrug J, Schaffalitzky de Muckadell 0, et al: Lcalisatin f vasactive intestinal plypeptide (VIP) t central and peripheral neurns. Prc Nat! Acad Sci USA 73: , Said SI, Falna GR, Den H, et al: Vasactive intestinal plypeptide: elevated levels in patients with hepatic cirrhsis. Clin Res 22:367, Ebeid AM, Escurru J, Seters PB, et al: Hepatic inactivatin f vasactive intestinal peptide in man and dg. J Surg Res 1978 (in press) 18. Bakhle YS, Vane JR: Pharmackinetic functin f the pulmnary circulatin. Pharmacl Rev 54: , Dent RI, Levine B, J<fmes JH, et al: Effects f islated perfused canine lung and kidney n gastrin heptadecapeptide. Am J PhysiI225: , Krinek JK, Tft DO, G VLW: Vasactive intestinal peptide (VIP) binding in different rgans in the rat. Gastrenterlgy 74:1051, Trewby PN, Williams R: Pathphysilgy f hyptensin in patients with fulminant hepatic failure. Gut 18: , Trewby PN, Warren R, Cntini S, et al: Incidence and pathphysilgy f pulmnary edema in fulminant hepatic failure. Gastrenterlgy 74: , Said SI, Kitamura S: Vasactive plypeptide dilates pulmnary vessels during avelar hypxia, histamine infusin r air breathing. Fed Prc 30:380, 1971