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Evidence for autoregulation of cholecystokinin (CCK) secretion: Suppression of CCK-releasing peptide by somatostatin
A370
AGA ABSTRACTS
• INTRACELLULAR LOCALIZATION OF LYSOSOMAL CATHEPSIN B DURING EARLY ACUTE EXPERIMENTAL PANCREATITIS M.M. Lercb, H. Weidenbach, M.P. Lutz, E. Wolff, S. Turi, G. Adler. Department of Medicine I, Ulm University, Germany The redistribution of lysosomal hydrolases from their lysosomeenriched to a :zymogen granule-enriched subcellular fraction is a consistent observation in various animal models of acute pancreatitis. The underlying mechanism and the intxacellular target compartment of this redistribution, however, has remained a subject of controversy. Methods: We have investigated the initial phase (4h) of caerulein-induced pancreatitis (10~g/kg/h i.v.) in male Wistar rats (180230gm). After sacrifice under pentobarbital pancreatic tissue (3mm blocs) was fixed in 0.1% glutaraldehyde/4% formaldehyde. Ultrathin cryosections were double labelled for amylase and cathepsin B using immuno-gold techniques. Standardized EM-micrographs (x28.000) were evaluated morphometrically. Results: In acinar cells the absolute number of either zymogen granules (41.6+4 vs 41.4+3/100p 2) or cytoplasmic vacuoles (4.065:0.5 vs 3.08+0.5/100p 2) was not found to be different between pancreatitis and control animals. The relative density of cathepsin B-gold particles in pancreatitis was not found to be different in cytoplasmic vacuoles (3455 g/p2) or zymogen granules (8+1g/ia2) when compared to controls. The mean size of cytoplasmic vacuoles, on the other hand, increased from 0.2+0.02p 2 in controls to 1.9+0.22~2 in early pancreatitis. Zymogen granules size (0.86+0.04p 2) and amylase content remained unchanged. Conclusion: A small but distinct aniount of lysosomal cathepsin B is physiologically localized in zymogen granules. The zymogen granule content of cathepsin B does not increase during pancreatitis. The biochemical redistribution of lysosomal hydrolases to a subcellular fraction of greater density is morphologically paralleled by an increased size of cytoplasmic vacuoles. The probable mechanism for subcellular redistribution of lysosomai hydrolases is a disturbance of secretory vacuole condensation and maturation.
• FREQUENCY AND RISK FACTORS OF RECURRENT PAIN DURING REFEEDING IN PATIENTS WITH ACUTE PANCREATITIS.PRELIMINARY RESULTS OF A MULTIVARIATE MULTICENTER STUDY.~ , D Heresbach, A Pariente, A Boruchowicz, R Delcensede, B MiUat, J Moreau, L Le Bodic, A Sauvanet, P Bemades. H6pital Beaujon, 92118 Clichy Cedex, France, *APA. Refeeding a patient after an acute pancreatitis (Ap~)may cause recurrent pain whose frequency and risk factors have never been.studied. From 1/94 to 9/94, 91 consecutive patients (men : 65%).with an AP (biliary : 47 %, alcohol: 29 %, post-pancreatography: 3 %, miscellaneous : 2 1 % ) necessitating to stop feeding for more than 2 days were included in 10 centres. The diet during the refeeding period was the same in all centres but the decision to refeed the patient was left to the clinician. Recurrent pain occurred in 18 patients (20 %) including 3 AP, within 2 days after refeeding in 9. Using univariate analysis 'risk factors for recurrent pain were Balthazar's CT scan grade > D (72 %~vs 32 %; P < 0.007), postpancreatography AP (17 % vs 0 %; P < ~007), duration of painful period (12 days vs 6 ; P < 0.002). Factors not associated With an increased risk of recurrent pain were other causes of AP, sex, age, maximal serum amylase level (mean : 16 X normal range), Ranson's score'> 3 (34 %), maximal CRP level (116 rag/L), the presence of obvious chronic pancreatitis (12 %y, vomiting (62 %), nasogastric suction (53 %), artificial nutrition (53 %), pancreatic collection (25 %), Wirsung duct enlargement (5 %), time between the beginning of AP and refeeding (13 days) and between the end of pain and refeeding (6 days), serum amylase level just'before (1.8 X normal range) and the day after (1.4) refeeding. Using logistic regression, Balthazar's CT. scan grade (P < 0.02) and the duration of painful period (P < 0.002)Were the only factors indepenc]gntly associated with painffil relapse. Total hospital stay and hospital stay after refeeding were longer in patients with relapse (33 and 18 days, respectively) than in whose without (18 days and 7, P < 0~001 and 0.001, respectively). We eondude that recurrent pain occurs i n 2 0 % of patients with AP during refeeding. Recurrent pain is more frequent in patients with necrotic AP and doubles the duration of hospital stay.
* Supported by grants of LTM Latema Solvay.
GASTROENTEROLOGY, Vol. 108, No. 4
VALUE AND LIMITS OF EARLY ONSET BIOLOGICAL MARKERS IN THE PROGNOSIS OF ACUTE PANCREATIS (AP). J P Letourneurl, D Heresbachl, I Bahon3, R Fauchet 2, JF Bretagnel, Y Malledant3, M Gosselinl. Service de Gastroent6rologiel, d'H6matoImmunologic2 et de R6animation Chirurgicale3 CHRU Pontchaillou, 35033 Rennes, France. The prognosis of AP depends upon systemic complications and surinfection of pancreatic or extra-pancreatic tissue necrosis. Several biological markers have been evaluated in AP but most often independently or only from the onset of hosptitalization. The aim of this study was to determine the value of the C-reactive protein (CRP) and Interleukin-6 (IL6) and Interleukin-1 (ILl) as biological markers of complications or surlnfections of AP. Patients & methods : Thirty patients (23 M, 7 F, aged 59+3 years) hospitalised within the first 72 hours (h) (average 42_+3h) from onset of AP were included. Balthazar's score calculated on CT scan, was average 2.8_+0.4 (class A or B, n=ll; C, n=8; D or E, n=ll). Surinfection of abdominal collections (AP Inf+) occurred in 3 patients and a severe form (AP Sev+) defined by: renal or respiratory failure (n=3), death (n=2), hospital stay >21d (n=2) and sepsis (n=3), in 9 patients. Plasma CRP (nephelemetry), IL6 and ILl (Elisa) levels were measured every 12 h during 72 (h). Results : IL6 (N<10 ng/L) [ ILl (N<10 ng/L) Groups [ n peak 136-48h 160-72h [ 36-48h I 60-72h [ APSev- [ 21 108+-20° [ 81_+10° [ 76_+20° [ 19+6 [ 17_+3 ] APSev+ 9 644_+193° 387+74Q 340-2-1,12° 11+6 10+3 AVInf+13 400-2-_60*[422_+98* 1213_+1211 o* [ t-+1# I APInf- I 27 254+81" 1154_+39* 1152-+46 118_+4* I 16+_.2# [ non-parametric test °p<0.005: *p<0.05: #p<0.01. Plasma levels of CRP (data not shown) and IL6 peaked at 64_+4et 57-+4 h respectively (ns) after onset of symptoms.Among paUents with necrotic AP (n=19) only peak value of CRP (383-+59 vs 215_+28, p<0.05) and early ILl value (0i-_0vs 21+6 between 36-48h, p<0.05) significatly differed between patients with subsequent abdominal sepsis or sterile necrosis. Conclusions:Severe forms of AP are identified by an increase in CRP and 'IL6 levels within the first 72 h of onset. Amoflg patients withh necxotic AP, infection could be predicted by higher p e ~ CRP levels and also by lower ILl'levels.
• EVIDENCE FOR AUTOREGULATION OF CHOLECYSTOKININ (CCK) SECRETION: SUPPRESSION OF CCK-REI~ASING PEPTIDE BY SOMATOSTATIN. Y Li. YB Hao, C Owyang. Department of Internal Medicine, University of Michigan, Ann Arbor, MI. We bave shown that feedback regulation of CCK release by trypsin in rats is mediated by a CCK-releasing peptide (CCK-RP) secreted into the duodenum (AJP 256:G430, 1989). In anesthetized rats, diversion of bile pancreatic juice (BPJ) from the intestine results in marked increases m plasma CCK levels and pancreatic secretion which returns to basal after 5 hrsof diversion, suggesting that CCK may autoregulate its own secretion. We hypothesized that this phenomenon is mediated by increases in plasma somatostatin (SS) evoked by hypercholecystokinemia, and SS in turn inhibits CCK-RP secretion and lowers plasma CCK levels. To test this hypothesis, we showed that diversion of BPJ increases plasma CCK from a basal of 0.7_+0.12 pM to 9.2-+0.8 pM after 2 hrs, which was accompanied by increases in plasma SS levels from 150+12 fmol/ml to 262_+10 fmol/ml. The increase in SS was blocked by pretreatment with the CCK antagonist L364,718. At 5 hrs following diversion of BPJ, plasma CCK and SS decreased to basal levels which was accompanied by a lack of secretion of CCK-RP. To demonstrate that SS inhibits CCK release by inhibiting CCK-RP secretion, we employed a donor-recilSient rat model and showed that intraduodenal administration of intestinal perfusate containing the CCK-RP collected from a donor rat with BPJ diversion raised pancreatic pr0tein output by 2-fold and elevated plasma CCK levels to 7.5_+0.9 pM. The stimulatory effect of the intestinal perfusates was abolished when the "donor" rat was pretreated with SS (5 [.tg/kg/hr), which reproduced physiological plasma SS levels. This dose of SS completely inhibited any increase in plasma CCK levels evoked by BPJ diversion. To provide conclusive evidence that SS is the causative agent in lowering plasma CCK and pancreatic secretion, we showed that administration of a specific SS antagonist, cyclo [7-amino heptanoyl-Phe-D-Trp-Lys-Thr (Bzl)] (20 I.tg/kg/hr sc), prevented the drop in protein secretion and plasma CCK levels (7.8_+0.7 pM) following prolonged diversion of bile pancreatic juice. In contrast, the SS antagonist had no effect on the inhibitory action of bovine pancreatic polypeptide (100 pmol/kg/hr) on pancreatic secretion stimulated by 2,deoxy-D-glucose, indicating its specificity.on the action of SS. In conclusion, we have demonstrated that autoregulation of CCK sec~'etion occurs during diversion of BPJ and this is mediated by SS which inhibits the secretion of CCK-RP and lowers plasma CCK levels. This autoregulation mechanism may be important to preventprolonged hypercholecystokinemia which may cause pancreatitis.
AGA ABSTRACTS
• INTRACELLULAR LOCALIZATION OF LYSOSOMAL CATHEPSIN B DURING EARLY ACUTE EXPERIMENTAL PANCREATITIS M.M. Lercb, H. Weidenbach, M.P. Lutz, E. Wolff, S. Turi, G. Adler. Department of Medicine I, Ulm University, Germany The redistribution of lysosomal hydrolases from their lysosomeenriched to a :zymogen granule-enriched subcellular fraction is a consistent observation in various animal models of acute pancreatitis. The underlying mechanism and the intxacellular target compartment of this redistribution, however, has remained a subject of controversy. Methods: We have investigated the initial phase (4h) of caerulein-induced pancreatitis (10~g/kg/h i.v.) in male Wistar rats (180230gm). After sacrifice under pentobarbital pancreatic tissue (3mm blocs) was fixed in 0.1% glutaraldehyde/4% formaldehyde. Ultrathin cryosections were double labelled for amylase and cathepsin B using immuno-gold techniques. Standardized EM-micrographs (x28.000) were evaluated morphometrically. Results: In acinar cells the absolute number of either zymogen granules (41.6+4 vs 41.4+3/100p 2) or cytoplasmic vacuoles (4.065:0.5 vs 3.08+0.5/100p 2) was not found to be different between pancreatitis and control animals. The relative density of cathepsin B-gold particles in pancreatitis was not found to be different in cytoplasmic vacuoles (3455 g/p2) or zymogen granules (8+1g/ia2) when compared to controls. The mean size of cytoplasmic vacuoles, on the other hand, increased from 0.2+0.02p 2 in controls to 1.9+0.22~2 in early pancreatitis. Zymogen granules size (0.86+0.04p 2) and amylase content remained unchanged. Conclusion: A small but distinct aniount of lysosomal cathepsin B is physiologically localized in zymogen granules. The zymogen granule content of cathepsin B does not increase during pancreatitis. The biochemical redistribution of lysosomal hydrolases to a subcellular fraction of greater density is morphologically paralleled by an increased size of cytoplasmic vacuoles. The probable mechanism for subcellular redistribution of lysosomai hydrolases is a disturbance of secretory vacuole condensation and maturation.
• FREQUENCY AND RISK FACTORS OF RECURRENT PAIN DURING REFEEDING IN PATIENTS WITH ACUTE PANCREATITIS.PRELIMINARY RESULTS OF A MULTIVARIATE MULTICENTER STUDY.~ , D Heresbach, A Pariente, A Boruchowicz, R Delcensede, B MiUat, J Moreau, L Le Bodic, A Sauvanet, P Bemades. H6pital Beaujon, 92118 Clichy Cedex, France, *APA. Refeeding a patient after an acute pancreatitis (Ap~)may cause recurrent pain whose frequency and risk factors have never been.studied. From 1/94 to 9/94, 91 consecutive patients (men : 65%).with an AP (biliary : 47 %, alcohol: 29 %, post-pancreatography: 3 %, miscellaneous : 2 1 % ) necessitating to stop feeding for more than 2 days were included in 10 centres. The diet during the refeeding period was the same in all centres but the decision to refeed the patient was left to the clinician. Recurrent pain occurred in 18 patients (20 %) including 3 AP, within 2 days after refeeding in 9. Using univariate analysis 'risk factors for recurrent pain were Balthazar's CT scan grade > D (72 %~vs 32 %; P < 0.007), postpancreatography AP (17 % vs 0 %; P < ~007), duration of painful period (12 days vs 6 ; P < 0.002). Factors not associated With an increased risk of recurrent pain were other causes of AP, sex, age, maximal serum amylase level (mean : 16 X normal range), Ranson's score'> 3 (34 %), maximal CRP level (116 rag/L), the presence of obvious chronic pancreatitis (12 %y, vomiting (62 %), nasogastric suction (53 %), artificial nutrition (53 %), pancreatic collection (25 %), Wirsung duct enlargement (5 %), time between the beginning of AP and refeeding (13 days) and between the end of pain and refeeding (6 days), serum amylase level just'before (1.8 X normal range) and the day after (1.4) refeeding. Using logistic regression, Balthazar's CT. scan grade (P < 0.02) and the duration of painful period (P < 0.002)Were the only factors indepenc]gntly associated with painffil relapse. Total hospital stay and hospital stay after refeeding were longer in patients with relapse (33 and 18 days, respectively) than in whose without (18 days and 7, P < 0~001 and 0.001, respectively). We eondude that recurrent pain occurs i n 2 0 % of patients with AP during refeeding. Recurrent pain is more frequent in patients with necrotic AP and doubles the duration of hospital stay.
* Supported by grants of LTM Latema Solvay.
GASTROENTEROLOGY, Vol. 108, No. 4
VALUE AND LIMITS OF EARLY ONSET BIOLOGICAL MARKERS IN THE PROGNOSIS OF ACUTE PANCREATIS (AP). J P Letourneurl, D Heresbachl, I Bahon3, R Fauchet 2, JF Bretagnel, Y Malledant3, M Gosselinl. Service de Gastroent6rologiel, d'H6matoImmunologic2 et de R6animation Chirurgicale3 CHRU Pontchaillou, 35033 Rennes, France. The prognosis of AP depends upon systemic complications and surinfection of pancreatic or extra-pancreatic tissue necrosis. Several biological markers have been evaluated in AP but most often independently or only from the onset of hosptitalization. The aim of this study was to determine the value of the C-reactive protein (CRP) and Interleukin-6 (IL6) and Interleukin-1 (ILl) as biological markers of complications or surlnfections of AP. Patients & methods : Thirty patients (23 M, 7 F, aged 59+3 years) hospitalised within the first 72 hours (h) (average 42_+3h) from onset of AP were included. Balthazar's score calculated on CT scan, was average 2.8_+0.4 (class A or B, n=ll; C, n=8; D or E, n=ll). Surinfection of abdominal collections (AP Inf+) occurred in 3 patients and a severe form (AP Sev+) defined by: renal or respiratory failure (n=3), death (n=2), hospital stay >21d (n=2) and sepsis (n=3), in 9 patients. Plasma CRP (nephelemetry), IL6 and ILl (Elisa) levels were measured every 12 h during 72 (h). Results : IL6 (N<10 ng/L) [ ILl (N<10 ng/L) Groups [ n peak 136-48h 160-72h [ 36-48h I 60-72h [ APSev- [ 21 108+-20° [ 81_+10° [ 76_+20° [ 19+6 [ 17_+3 ] APSev+ 9 644_+193° 387+74Q 340-2-1,12° 11+6 10+3 AVInf+13 400-2-_60*[422_+98* 1213_+1211 o* [ t-+1# I APInf- I 27 254+81" 1154_+39* 1152-+46 118_+4* I 16+_.2# [ non-parametric test °p<0.005: *p<0.05: #p<0.01. Plasma levels of CRP (data not shown) and IL6 peaked at 64_+4et 57-+4 h respectively (ns) after onset of symptoms.Among paUents with necrotic AP (n=19) only peak value of CRP (383-+59 vs 215_+28, p<0.05) and early ILl value (0i-_0vs 21+6 between 36-48h, p<0.05) significatly differed between patients with subsequent abdominal sepsis or sterile necrosis. Conclusions:Severe forms of AP are identified by an increase in CRP and 'IL6 levels within the first 72 h of onset. Amoflg patients withh necxotic AP, infection could be predicted by higher p e ~ CRP levels and also by lower ILl'levels.
• EVIDENCE FOR AUTOREGULATION OF CHOLECYSTOKININ (CCK) SECRETION: SUPPRESSION OF CCK-REI~ASING PEPTIDE BY SOMATOSTATIN. Y Li. YB Hao, C Owyang. Department of Internal Medicine, University of Michigan, Ann Arbor, MI. We bave shown that feedback regulation of CCK release by trypsin in rats is mediated by a CCK-releasing peptide (CCK-RP) secreted into the duodenum (AJP 256:G430, 1989). In anesthetized rats, diversion of bile pancreatic juice (BPJ) from the intestine results in marked increases m plasma CCK levels and pancreatic secretion which returns to basal after 5 hrsof diversion, suggesting that CCK may autoregulate its own secretion. We hypothesized that this phenomenon is mediated by increases in plasma somatostatin (SS) evoked by hypercholecystokinemia, and SS in turn inhibits CCK-RP secretion and lowers plasma CCK levels. To test this hypothesis, we showed that diversion of BPJ increases plasma CCK from a basal of 0.7_+0.12 pM to 9.2-+0.8 pM after 2 hrs, which was accompanied by increases in plasma SS levels from 150+12 fmol/ml to 262_+10 fmol/ml. The increase in SS was blocked by pretreatment with the CCK antagonist L364,718. At 5 hrs following diversion of BPJ, plasma CCK and SS decreased to basal levels which was accompanied by a lack of secretion of CCK-RP. To demonstrate that SS inhibits CCK release by inhibiting CCK-RP secretion, we employed a donor-recilSient rat model and showed that intraduodenal administration of intestinal perfusate containing the CCK-RP collected from a donor rat with BPJ diversion raised pancreatic pr0tein output by 2-fold and elevated plasma CCK levels to 7.5_+0.9 pM. The stimulatory effect of the intestinal perfusates was abolished when the "donor" rat was pretreated with SS (5 [.tg/kg/hr), which reproduced physiological plasma SS levels. This dose of SS completely inhibited any increase in plasma CCK levels evoked by BPJ diversion. To provide conclusive evidence that SS is the causative agent in lowering plasma CCK and pancreatic secretion, we showed that administration of a specific SS antagonist, cyclo [7-amino heptanoyl-Phe-D-Trp-Lys-Thr (Bzl)] (20 I.tg/kg/hr sc), prevented the drop in protein secretion and plasma CCK levels (7.8_+0.7 pM) following prolonged diversion of bile pancreatic juice. In contrast, the SS antagonist had no effect on the inhibitory action of bovine pancreatic polypeptide (100 pmol/kg/hr) on pancreatic secretion stimulated by 2,deoxy-D-glucose, indicating its specificity.on the action of SS. In conclusion, we have demonstrated that autoregulation of CCK sec~'etion occurs during diversion of BPJ and this is mediated by SS which inhibits the secretion of CCK-RP and lowers plasma CCK levels. This autoregulation mechanism may be important to preventprolonged hypercholecystokinemia which may cause pancreatitis.