Does eNOS derived nitric oxide ameliorate the initiation of acute pancreatitis by increasing pancreatic blood flow?

The eftects of/eptin and Ob-Rb in pancreatitis were examined using genetically altered mice, which were either leptin dehclent (oh/oh) or leptin resistant (db/db), possessing a mutated lbrm of the leptm receptor. Pancreatitis was induced by hourly rejections of caerulein (50 ~g~4xg; ip) for 6 or 12 hours. The severity of pancreatitis was determined by quantitating changes in serum amylase, pancreatic edema, neutrophil sequestration in the pancreas and morphological evaluation of acinar cell necrosis. Expression ot Ob-Rb was evaluated by RTPCR Resuhs: Severity of acute pancreatitis was significantly increased m both ob/ob deficient attd db/db mice compared to the wild ~ype mice at both time points After 12 hours of caeruMn injections serum amylase increased from 1950 + 260 units (controls) to 18,650 + 2345 m wild type mice; 30250 +3560 (ob/ob) and 32750 +3590 in (db/db) mice Acinar cell necrosis increased from 18 + 3% in wild type to 26 + 2.5% in ob/ob deficiem mice and 245 + 4 0 % m db/db mice. Pancreatic edema was also higher in mice deficient m leptin compared to wild type mice after the induction of pancreatitis. Expression of leptin receptors in pancreas was significantly" higher afmr 6 and 12 hours of caerulein administration. Conclusion: The exacerbation oI the pancreatic injury in the absence of leptin or its receptors indicates that in pancreatitis, leptin plays an antidnflammatory sole. This is contrary to its pro-inflammatory effects in sewml other pathophysiological states, including colitis, and enterotoxin-mediated entr

Effect of anti-oxidant therapy on anti-oxidant levels and indices of lung injury.

Vadable(units) Amylase (lUlL) Selenium (microg#_)

Group t

(shin)

4954 505 (465564)

Group 2

(~)

Group 3

,(mS)

Group 4 (Eady Group 5 (Late

MAOX)

7461 (6537- 6673(3938- 7703(52469787) 7004) 8949) 460* (432-

469)

370" (335-

396)

MAOL

7798 (50988422)

468 (449-507) 472 (421-511)

7,0 (4.3. 6.7 (3,3.7,7) 12.3" (9.4- 10.5{8,4-14.0) 10.4) 13.71 2.31(2.13- 139"*(1.14- 1.86"(1.43- 1.72(1,1.2,49) MPO (units/g) 126 2,78) 1,59) 2.26) BAL protein 2189(1192- 240"(1141856(169(~lmL) 193 2809) 361) 189"* (24.427)_ 5076) O~a expressedas median(range),*P<0,05vs sham (group1); **P<0,05vs AP (group 2) (MannWhitney U-test), A~,m't~te (n~L)

6.5 (5.9-7,6)

Tl198 T1196

Serine Protease Inhibitor Prevents F-Actin Distribution in Rat Pancreatic Acinar Cells Induced by Cerulein Hyperstimulation Akira Matsukura, Taiichi Otarti, Yasuji Seyama, blasatoshi Makuuchi, Fred S. Gorelick

Does eNOS Derived Nitric Oxide Ameliorate the Initiation of Acute Pancreatitis by Increasing Pancreatic Blood Flow? Matthew Dimagno, Yfbai Hao, John Williams Chnng ()wyang

Previous studies have suggested that cemlein hyperstimulation has marked morphological effects on the pancreatic acinar cell cytoskeleton. These changes may modulate the secretory response and may induce acute pancreatitis (JCl 1990:86,1649). in Japan serine protease inhibitors; FUT-175 (nafamnstate mesilate) and FOY (gabexate mesilate), are administrated for treatment of acute pancreatitis in humans, and reported effective in reducing mortality. In addition, these serine pmtease inhibitors are prescribed to prevent endoscopic retrograde cholangiopancreaticography (ERCP)-induced acute pancreatitis (N Engl J Med 1996;335,919). Animal experiments using these serine protease inhibitors also show a beneficial effect (JCI 1988:81,229). We hypothesized that serine pmtease inhibitor prev'ents the apical cytuskeletal changes in acute pancreatitis. To examine this issue rats were subject to a continuous Iv" infusion of saline (control group), or cerulein hyperstimulation (5ug/ kg/h) for three hours. FUT-175 or FOY was administrated for 30 rain., then cerulein hyperstimulation was performed. The confocal microscopic study using phalloidin-TRITC showed the f-actin dismbution in the pancreatic acinar cells under the condition of cerulein hyperstimulation. However, f-actin was localized to the apical pole of the cell and very little appeaned to be associated with tbe basolateral cell surtace in the pretreated group with FUT175 or FOY as control group. These studies suggest that senne protease inbibitor may" prevent the apical cytoskeletal changes and may contribute to prevention oI acute pancreatins.

PURPOSE: Inadequate blood flow is ass~'iated with human arid experimental acute pancreatitis (AP) This association may be relined to the effects of NO on vascular endothelium, altfmngh this remains controversial, m part because three NOS isofornls exist: neuronal(nNOS), endothelial- (eNOS) and inducible- (iNOS) We have recemly shown that eNOSderived NO confers a protectiw' effect during the imtiation of cerulein AP We therefore iru,'estigated whether pancreatic blood flow (PBF) difters between eNOS KO mice and WT animals during the initiation of AP METHODS: In vivo AP was induced in mice by nne cerulein (50 ug&g IP) mjection~ Mice were sacrificed 30 minmes later. In vitro, isolated pancreatic acmi were hyperstmmlated 30 minutes w~th cerulein (100 riM) The NOS inhibitor I.-NNA was used m i,ivo (i0 u~"kg [P) and in vitio (100 uM). Mice with targeted deletion of the eNOS nNO8 or iNOS genes and respective controls (WT) were used in AP experiments. PBF measurements were obtained in anesthetized mice both pre- and 30 minutes postcerulein injection by intusing microspheres (BioPAl3 retrograde into the aorta by camrula, obtaining reference arterial blood samples and removing organs Microspheres carried different markers for each time point and were quantitated by neutron activation PBF was expressed as ml/mm/g ussue RESULTS: The initiation of AP in mice was established 30 minutes afier cerulein injection based on an early maximal increase in intraceflular pancreatic trypsin activw, redistribution of pancreatic acitmr actin cytoskeleton, and an increase in serum/ipase Pharmacologic NOS blocMde and eNOS deletion, but not nNOS or iNOS deletion, erfhanced the initiation in vivo ceruiein AP compared to W'T mice based on significantly greater increases in intracellular pancreatic trypsin activity and serum hpase. In contrast, NOS blockade and eNOS deletion had no effect on acmar trypsin activity in vitro. PBF was measured to investigate mechanisms for the effects of eNOS on AP. PBF in the WI" grunp increased 381 (+/-138) %, from 0.30 (+/-0.08) to 1.04 (+/-0.11) ml/min/ g tissue In contrast, the eNOS KO group showed an increase of only 57 (+t-11) %, from 0.15 (+/-004) to 0 2 2 (+/-0.06) nd/min/g tissue, CONCLUSION: Presence of eNOS is associated with greater PBF during AP Possibly, eNOS derived NO exerts a protective effect on the initiation of cerulein AP through interactions between acinar and non-adnar cell types, likely between acinar and endothelial cells to produce greater PBF.

Tl199 Maintenance of Intestinal Integrity"by Caspase Inhibitor Administration in Experimental Severe Acute Pancreatitis Takeo Yasuda, Yoshifumi Takeyama, Takashi Ueda, Makoto Shinzeki, Shinji Ki.stti, Naoki Matsumura, Yoshikazu Kuroda Although bacterial translocation including endotoxin translocation from gut is a dominant source for the infectious complications with severe acute pancreatitis, the molecular mechanism of bacterial translocation in acute pancreatitis has not been clarified. The breakdovm of the integrity of intestinal wall is believed to be implicated in bacterial translocation, and ",viii increase intestinal permeability. It has been recently reported that increase of intestinal permeability is correlated with the apoptosis ot the intestinal epifhefial cells The aim of this study was to investigate the changes of intestinal permeability and significance of apoptotic change of intestinal epithelial cells in bacterial tmnslocation associated with severe acute pancreatitis. Severe acute pancreatitis was induced by retrograde injection of 3% sodium deoxycholate into the biliopancreatic ducts ot male Wistar rats. A poly caspase inhibitor, Z-VAD-fmk, was injected in the peritoneal cavity (500 b~g eacD 2 rain prior to induction of severe acute pancreatitis. Six hours after induction, ileum was removed. Permeability of intestinal wall was evaluated ex vivo by measuring the leaked amount of FITCdextran (MW; 71,200), which was loaded into the lumens of the intestinal pouches. Permeability of ileum was significamly greater in the rats with pancreatitis than that in the rats with sham operation, and the caspase inhibitor significamly inhibited the increase of permeability. Moreover, the caspase inhibitor ameliorated morphological damage of intestinal mucosa due to pancreatitis, protected intestinal epithelial cells from apoptusis, and significantly lowered peripheral endotoxm concentration elevated in pancreatitis 18h after onset. Finally, the caspa~seinhibitor significantly- improved the mortality rate 24h after onset from 60.9 % to 28.6%. These results indicate that the caspase inhibitor reduces apoptusis of intestinal epithelial cells, and maintains the integrity of intestinal mucosa, and prevents endotoxin translocation in severe acute pancreatitis.

Tll97 lntravenons Anti-oxidant Modulation of End-organ Injury in Experimental Acute Panereatitis Jonathan ttardmau, Conor Shields |)avid ~hotield Fergus Rieley, Raymond McMahon, H Paul Redmond, Ajith Siriwardena Introduction: Oxidative stress mediates acinar injury m experimental acute pancreatitis (AP). Serum anti-omdants are depleted in human AP. TNs study tests the h'y~otbesis that antioxidant supplementation ameliorates experimental AP Methods: Male Spmgue-Dawley rats were used Studies conformed to the 1991 American Physiology Association guidelines for animal care Animals were' randomly allocated to one of 5 groups (n= 5/group), AP was induced by mtrapentoneal injection of l-arginine at 2 5mg/g. Group l(sham); Group 2 (AP) N-saline 5mL/kg alter induction of AP; Group 3 : 3 mVkg hypertonic (7.5~ saline (H~S)(treatment comparator: HT5 ameliorates end-organ injury in tbis model); Group 4 multiple anti-oxidant MAOX: 15 micro~kg seIenium, 30 micmg,qxg ascorbate and 300 mg/ kg n-acetytcysteine given at 6 and 30h; Group 5 IVlAOXcombination at 24 and 48h (delayed). Animals were sacrificed at 72h and blood analysed {or amylase, ascorbate and selenium levels, gronchoalveolar lavage (BAL) was analysed tbr protein content and lung tissue for myloperoxidase activity (MPO). Pancreatic and lmlg patvnchymal samples were examined qualitatively for histologic injury blind to group allocation. Results: L-arginine induced AP characterized by edema, nemrophd mfihration and acinar cell disruption and depletion m selettium levels. Early MAOX restored ascrobate, reduced pulmonary MPO and BAL protein (table) HTS also improved indices of lung i q u r y Early NIAOX reduced acinar calf swelling, &granulation and intihratiou ot parenchyma by" inflammatory cells Conclusion: In this model, early MAOX reduces indices of pulmonary injury arid downgrades histologic features of aeinar cell damage, These teatures were absent when intervention was delayed for 24 hours.

T1200 lntraperitoneal Administration of Oxygenated Perfluorochemical (PFC) Ameliorates Bacterial Translocation (BT) in Necrotizing Pancreatitis Makoto Shinzeki, Yoshifumi Takeyama, Takashi Ueda, Takeo Yasuda, Shinji Kishi, Naoki Matsumum, Yoshikazu Kuroda BT of intestinal flora is estimated as infectious soume in necrotizing pancreatitis. Recent reports suggest that microcirculatory disorder breaks intestinal integrity, and results in BT. PFC is an efficient oxygen carrier, and its intmperitoneal administration is reported to be beneficial on ischemia-reperfusion iNury of the intestine. Along these lines, we examined the effect of mtraperitoneal administration of oxygenated PFC in experimental acute pancreatitis. [Methods] Necrotizing pancreatitis was developed by retrograde injection of 3% deoxycholate into bifiopancreatic ducts of male Wistar rats. Immediately after induction of pancmatitis, lOml of oxygen-saturated PFC (perfiuorodecalin) was iNected into the peritoneal cavity

A-503

AGA Abstracts