MOLECULAR MEDICINE REPORTS 8: 335-338, 2013 Concentration of tissue angiotensin II increases with severity of experimental pancreatitis HIROYUKI FURUKAWA1, ATSUSHI SHINMURA1, HIDEHIRO TAJIMA1, TOMOYA TSUKADA1, SHIN-ICHI NAKANUMA1, KOICHI OKAMOTO1, SEISHO SAKAI1, ISAMU MAKINO1, KEISHI NAKAMURA1, HIRONORI HAYASHI1, KATSUNOBU OYAMA1, MASAFUMI INOKUCHI1, HISATOSHI NAKAGAWARA1, TOMOHARU MIYASHITA1, HIDETO FUJITA1, HIROYUKI TAKAMURA1, ITASU NINOMIYA1; HIROHISA KITAGAWA1, SACHIO FUSHIDA1, TAKASHI FUJIMURA1, TETSUO OHTA1, TOMOHIKO WAKAYAMA2 and SHOICHI ISEKI2 1Department of Gastroenterological Surgery, Division of Cancer Medicine; 2Department of Histology and Embryology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8641, Japan Received January 23, 2013; Accepted May 30, 2013 DOI: 10.3892/mmr.2013.1509 Abstract. Necrotizing pancreatitis is a serious condition that Introduction is associated with high morbidity and mortality. Although vasospasm is reportedly involved in necrotizing pancreatitis, Acute pancreatitis, particularly the necrotizing type, is the underlying mechanism is not completely clear. In addition, associated with high morbidity and mortality. Necrotizing the local renin-angiotensin system has been hypothesized to pancreatitis is characterized by parenchymal non-enhance- be involved in the progression of pancreatitis and trypsin has ment on contrast-enhanced computed tomography images (1). been shown to generate angiotensin II under weakly acidic Although necrosis is irreversible, certain patients exhibiting conditions. However, to the best of our knowledge, no studies this pancreatic parenchymal non-enhancement recover have reported elevated angiotensin II levels in tissue with with normal pancreatic conditions (2). Vasospasm has been pancreatitis. In the present study, the concentration of pancre- implicated in the development of pancreatic ischemia and atic angiotensin II in rats with experimentally induced acute necrosis (3); however, the precise underlying mechanism is pancreatitis was measured. Acute pancreatitis was induced unclear. by retrograde injection of 6% sodium taurocholate into the The local pancreatic renin-angiotensin system (RAS) biliopancreatic duct. Control rats were sacrificed without may be a significant etiological factor of pancreatitis (4-9). injection into the biliopancreatic duct. The concentration of Angiotensin II receptors have been detected in the endothe- tissue angiotensin II was measured using the florisil method. lium of blood vessels and the epithelium of the pancreatic Angiotensin II concentration in tissues with acute pancreatitis ductal system (8). Previous studies have revealed that experi- measured at 3, 6, 12 and 24 h following taurocholate injection mental pancreatitis upregulates the RAS components in the were significantly higher than that of normal pancreatic tissue. pancreas (10,11). However, elevated local angiotensin II levels In addition, the concentration of angiotensin II increased in in pancreatitis tissues have not been reported. Trypsin may a time-dependent manner. The results demonstrated that the directly induce the generation of the pressor angiotensin II angiotensin II generating system is involved in the transition from angiotensinogen at a weakly acidic pH, independent of from edematous to necrotizing pancreatitis in experimental the action of angiotensin-converting enzyme (12,13). animals. We hypothesize that locally formed angiotensin II In the present study, pancreatic angiotensin II concentra- affects the microenvironment in pancreatitis. tion was measured in experimental animals with pancreatitis to evaluate the involvement of the local angiotensin II gener- ating system in acute pancreatitis. Materials and methods Correspondence to: Dr Hiroyuki Furukawa, Department Animal and experimental acute pancreatitis model. The of Gastroenterological Surgery, Division of Cancer Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 present study was conducted in compliance with the Division Takara-machi, Kanazawa, Ishikawa 920-8641, Japan for Animal Research Resources, Institute of Kanazawa E-mail: [email protected] University. The experiments and procedures were approved by the Animal Care and Use Committee of the Kanazawa Key words: pancreatitis, angiotensin II, trypsin, taurocholate University (Ishikawa, Japan). Male Wistar rats (weight, 250-350 g) were maintained on a 12-h light/dark cycle at an ambient temperature of 23˚C. 336 FURUKAWA et al: CONCENTRATION OF TISSUE ANGIOTENSIN II IN EXPERIMENTAL PANCREATITIS The rats were fasted with free access to water for 12 h prior to surgery. Rats were anesthetized with diethyl ether inhalation and laparotomy was performed using a midline abdominal incision. Acute pancreatitis was induced by retrograde injection of 6% sodium taurocholate (Wako Pure Chemical Industries, Osaka, Japan) into the biliopancreatic duct at a dose of 0.4 ml/kg body weight to induce acute pancreatitis. Time 0 indicates the time of injection of sodium taurocholate. The rats were divided into groups and sacrificed at 3, 6, 12, 24 or 36 h following the induction of acute pancreatitis. Control rats were sacrificed without injection into the biliopancreatic duct. Figure 1. Plasma amylase levels in rats with taurocholate-induced pancre- Sample collection and preparation. Blood samples were atitis were significantly higher than those of the control *( P<0.05). Data are collected by cardiac puncture for the measurement of plasma presented as the mean ± SD. amylase levels. Pancreatic tissue samples were fixed in 10% neutral‑buffered formalin and embedded in paraffin for histo- logical examination. Parallel samples of pancreatic tissue were Discussion prepared for the measurement of angiotensin II concentration by immediately freezing in liquid nitrogen, followed by storage Premature activation of trypsin within pancreatic acinar cells at -80˚C until the time of assay. by various proteases, which in turn are activated by ectopic activation of trypsinogen, is hypothesized to represent the Measurements of angiotensin II in tissues. Frozen tissue main etiological factor in pancreatitis. A number of studies in samples were homogenized at 4˚C in saline containing 0.1% acute pancreatitis have examined the mechanisms by which NHCl and 5% urinastatin. The homogenate was centrifuged at edematous injury progresses to necrosis. In experimental 10,000 x g for 30 min at 4˚C and the supernatant was collected models, pancreatic vascular perfusion failure has been impli- for radioimmunoassay of angiotensin II using the florisil cated in the development of pancreatitis. Spormann et al (15) method, as described previously (14). demonstrated that temporary ischemia significantly augmented the severity of pathology and Klar et al (16) revealed that Statistical analysis. Data were analyzed using the Welch's vasoconstrictor administration has adverse effects in experi- t-test. P<0.05 was considered to indicate a statistically signifi- mental acute pancreatitis. Intravascular thromboses have been cant difference. indicated to represent a significant factor (17). Although intra- venous thromboses were noted in the present study, arterial Results thromboses were not observed. Deterioration of the balance between nitric oxide and Plasma amylase levels. Plasma amylase levels were measured endothelins has also been hypothesized to account for the to confirm the successful induction of acute pancreatitis. development of necrotizing pancreatitis (18,19); however, the Following this induction, plasma amylase levels were association between this abnormal balance and trypsin, the elevated (Fig. 1). key molecule in the etiology of pancreatitis, is unclear. It has been hypothesized that an intrinsic tissue RAS may Histological examination. In the early stages of pancreatitis, be present in the pancreas (4). Chan et al (20) have demon- histological examination indicated subcapsular and interlob- strated activation of the pancreatic RAS in a rat model of ular edema, slight inflammatory neutrophilic infiltration and chronic hypoxia. Leung et al (10) revealed that experimental a small number of intralobular necrotic lesions. The extent of pancreatitis induced upregulation of several key RAS compo- inflammatory cell infiltration and areas of necrosis progressed nents in the pancreas. Several other studies have reported that with time. At 24 h following taurocholate injection, marked RAS blockers are effective in the treatment of experimental inflammatory and necrotic changes were noted (Fig. 2A‑E). In pancreatitis (21-23). addition, intravenous thromboses were observed, but there was Hypovascularity is a key characteristic of not only no evidence of arterial thrombosis (Fig. 2F). necrotizing pancreatitis but also pancreatic ductal cancer. The majority of pancreatic ductal cancers are hypovascular Angiotensin II concentration in experimental pancreatitis or avascular on angiography, but the angiography of surgi- tissue. Angiotensin II concentration was measured in pancre- cally resected specimens cannot always accurately confirm atic tissue samples obtained at 3, 6, 12, 24 and 36 h following poor circulation. Figarella et al (24) reported that cationic taurocholate injection. Up to 24 h, the angiotensin II concentra- trypsinogen is spontaneously converted to trypsin under tion was significantly higher in rats with taurocholate‑induced acidic conditions and Tajima et al (25) demonstrated that pancreatitis compared with controls