Immunocytochemical Distribution of Trypsinogen and Pancreatic Secretory Trypsin Inhibitor in Normal and Neoplastic Tissues in Man

Immunoreactive trypsinogen and pancreatic secretory trypsin inhibitor (PSTI) were demonstrated in pancreas by means of an immunoperoxidase technique. They had the same distribution in acinar cells of ‘normal’ human exocrine pancreas tissues. Ductal adenocarcinoma tissue and pancreatic undifferentiated carcinoma contained neither antigen. Scattered ‘normal’-looking cells in the border area between normal and neoplastic tissue of both types of tumor stained positively for trypsinogen and for PSTI.     

Elimination of Pancreatic Secretory Trypsin Inhibitor from the Circulation

The elimination of human pancreatic secretory trypsin inhibitor (PSTI) from the circulation of man has been examined in two human volunteers. Serial samplings of blood and urine were made for 55 h, after a rapid intravenous infusion of ¹²⁵I-labeled human PSTI. The findings demonstrated a rapid initial elimination from the circulation. Within 30 min only 30% of the infused label remained (T½, 6 min). This was accompanied by the rapid appearance in the urine of radiolabel. Our results indicate that PSTI would prove a poor diagnostic marker for acute pancreatitis late in the course of the disease. This is opposed to the findings of Ogawa et al., who reported prolonged elevated circulating levels of PSTI weeks into the disease. However, they also noted rises of PSTI during acute pancreatitis in excess of 10 times the levels we have noted. Further exploration of population differences and the behavior of PSTI during acute pancreatitis are necessary to help resolve these findings.     

The role of trypsin, trypsin inhibitor, and trypsin receptor in the onset and aggravation of pancreatitis

Trypsin activity is properly suppressed in the pancreatic acinar cells under normal conditions. A small amount of trypsinogen is converted to active trypsin and inactivated by pancreatic secretory trypsin inhibitor (PSTI), thereby preventing damage to pancreatic acinar cells as a first line of defense. However, if trypsin activation (due to excessive stimulation of pancreatic acinar cells) exceeds the capacity of PSTI, a subsequent cascade of events leads to the activation of various proteases that damage cells. This can be interpreted as the main causative event of pancreatitis onset. Trypsin produced in and secreted from the pancreatic acinar cells activates protease activated receptor-2 (PAR-2), which is present at high densities on the luminal surfaces of pancreatic acinar cells and duct cells. Results of PAR-2 activation are the production of cytokines and the regulation of exocrine function via a negative feedback loop. Thus, the actions of trypsin, trypsin inhibitor (PSTI), and trypsin receptor (PAR-2) in the pancreas are strongly interconnected.     

Serum pancreatic secretory trypsin inhibitor in pancreatic diseases

To elucidate the diagnostic significance of serum pancreatic secretory trypsin inhibitor (PSTI) in pancreatic diseases, organ distribution of PSTI and abnormalities in serum PSTI were studied. The pancreas showed the highest content of PSTI, which was five times that of the stomach and almost 40 times that of the other organs. Serum PSTI and amylase were elevated in eight patients with acute pancreatitis, 27 and 11 patients of 47 with chronic pancreatitis, 31 and 13 of 36 with pancreatic cancer, and 67 and 62 of 109 with non-pancreatic disease, respectively. PSTI levels were more sensitive to the presence of pancreatic disease than were amylase levels. The specificities in serum of healthy controls and patients with non-pancreatic disease were similar for PSTI and amylase (69% vs 71%). In chronic pancreatitis and pancreatic cancer patients the efficiency of the PSTI assay was higher (P < 0.02) than the amylase assay (67% vs 63% for pancreatitis and 71% vs 66% for cancer). The sensitivity and efficiency of serum PSTI assay in chronic pancreatic diseases were superior to those of amylase.     

Elevated pancreatic secretory trypsin inhibitor levels during severe inflammatory disease, renal insufficiency, and after various surgical procedures

Elevated levels of immunoreactive pancreatic secretory trypsin inhibitor (PSTI) were found in serum from patients with perforated duodenal ulcer, bacterial peritonitis, urosepticemia, pneumonia, acute renal failure, and also after different surgical procedures. The extent of the trauma seemed to determine the maximal level of PSTI. The increase found paralleled the changes seen in the acute-phase protein antichymotrypsin. There was, however, almost no increase in trypsinogen, thought to be produced together with PSTI in the acinar cells of the pancreas. In conclusion, there is evidence that PSTI is probably also produced somewhere outside the pancreas, in agreement with recent immunohistochemical data. This production may be part of a general acute-phase reaction. Thus, PSTI may have a more general inhibitory function against trypsin-like protease release in tissue injury, instead of being a purely local trypsin inhibitor in the pancreatic gland.     

SPINK1基因突变与特发性慢性胰腺炎研究进展

丝氨酸蛋白酶抑制剂KazalⅠ型(SPINK1)基因突变的致病机制复杂,众多学者对其在特发性慢性胰腺炎(ICP)中的作用和临床特点进行了深入研究,中日韩在该方面的初步结果与欧美国家有所不同,仍需进一步探讨。     

A radioimmunoassay for rat pancreatic secretory trypsin inhibitor

A radioimmunoassay for the detection of pancreatic secretory trypsin inhibitor (PSTI) in the rat is described. The sensitivity of the assay enables the specific measurement of this inhibitor in the serum of normal rats. The average base-line value for multiple animal serum specimens taken from fasting female Wistar rats being fed conventional diets was 11.6 +/- 6.2 micrograms/l. The inhibitor existed in its free form in serum, and PSTI immunoreactivity increased significantly within 2 h of the induction of experimental pancreatitis. The present assay will facilitate the study of PSTI in experimental diseases of the pancreas.     

Peritoneal lavage with aprotinin in patients with severe acute pancreatitis

Summary Conclusion Although high-dose aprotinin given intraperitoneally to patients with severe acute pancreatitis seems to inhibit activated trypsin in the peritoneal cavity, the treatment has little effect on the balance between proteases and antiproteases. Plasma levels of leukocyte proteases were high in all the patients, indicating leukocyte activation to be an important feature of the pathophysiology of severe acute pancreatitis. A surprise finding was that the patients had higher peritoneal levels of pancreatic secretory trypsin inhibitor (PSTI) after the lavage procedure. Background Although most studies have shown protease inhibitor therapy to have little or no effect on acute pancreatitis, in an earlier study we found that very high doses of the protease inhibitor aprotinin given intraperitoneally to patients with severe acute pancreatitis seemed to reduce the need of surgical treatment for pancreatic necrosis. In the present study we have further analyzed plasma and peritoneal samples from the same patients to ascertain whether the aprotinin treatment affects the balance between proteases and endogenous antiproteases. Methods In a prospective double-blind randomized multicenter trial, 48 patients with severe acute pancreatitis were treated with intraperitoneal lavage. One group (aprotinin group,n=22) was also treated with high doses (20 million KIU given over 30 h) of aprotinin intraperitoneally. The remaining 26 patients made up the control group. The protease-antiprotease balance was studied by measuring immunoreactive anionic trypsin (irAT), cationic trypsin (irCT), complexes between cationic trypsin and alpha 1-protease inhibitor (irCT-α1PI), leukocyte elastase and neutrophil proteinase 4 (NP4), as well as the endogenous protease inhibitors, pancreatic secretory trypsin inhibitor (PSTI), alpha 2-macroglobulin (α 2M), alpha 1-protease inhibitor (α 1PI), antichymotrypsin (ACHY), and secretory leukocyte protease inhibitor (SLPI). Intraperitoneal levels were studied before and after the lavage procedure, and plasma levels were followed for 21 d. Results The control group had lower plasma levels of SLPI and analysis of peritoneal fluid showed the reduction of irCT-α 1PI to be more pronounced in the aprotinin group. None of the other variables measured differed significantly between the two groups. All patients had very high levels of leukocyte elastase and NP4 both in peritoneal exudate and in plasma. Peritoneal levels of PSTI were higher after the lavage procedure in contrast to the other measured variables that all showed lower peritoneal levels after the lavage.     

Transgenic expression of pancreatic secretory trypsin inhibitor-I ameliorates secretagogue-induced pancreatitis in mice

BACKGROUND & AIMS: Endogenous trypsin inhibitors are believed to inhibit protease activity if trypsin becomes inadvertently activated within the acinar cell. However, this action remains unproven, and the role of endogenous pancreatic trypsin inhibitors in acute pancreatitis is unknown. In this study, we tested whether increased levels of pancreatic secretory trypsin inhibitor-I (PSTI-I) in mice could prevent secretagogue-induced pancreatitis. METHODS: Rat PSTI-I expression was targeted to pancreatic acinar cells in transgenic mice by creating a minigene driven by the rat elastase I enhancer/promoter. Secretagogue-induced pancreatitis was achieved by 12 hourly intraperitoneal injections of caerulein. The severity of pancreatitis was assessed by measurements of serum amylase, histologic grading, and pancreas wet weight-to-body weight ratio. Trypsinogen activation and trypsin activity were measured in pancreatic extracts. RESULTS: Targeted expression of PSTI-I to the pancreas increased endogenous trypsin inhibitor capacity by 190% (P <.01) in transgenic vs. nontransgenic mice. Caerulein administration to nontransgenic mice produced histologic evidence of acute pancreatitis, and significantly elevated serum amylase and pancreas weight ratio. In caerulein-treated transgenic mice, the histologic severity of pancreatitis was significantly reduced. There was no difference in trypsinogen activation peptide levels between caerulein-treated transgenic and nontransgenic mice. However, trypsin activity was significantly lower in transgenic mice receiving caerulein compared with nontransgenic mice. CONCLUSIONS: These data demonstrate that the severity of secretagogue-induced pancreatitis is significantly ameliorated in mice with higher pancreatic levels of trypsin inhibitor. We propose that PSTI-I prevents pancreatitis by inhibiting the activity of trypsin, rather than by reducing trypsinogen activation.     

Appearance mechanism and molecular heterogeneity of serum pancreatic secretory trypsin inhibitor (PSTI)

Serum immunoreactive pancreatic secretory trypsin inhibitor (PSTI) was measured by RIA. Serum PSTI levels were elevated in case of acute pancreatitis (15 of 15 cases: 317.7 +/- 155.6 ng/ml: Mean +/- SE) or pancreatic carcinoma (16 of 25 cases; 71.8 +/- 17.1 ng/ml), and in those with chronic renal failure (6 of 6 cases: 412.8 +/- 98.2 ng/ml). The molecular heterogeneity of elevated serum PSTI n such diseases was studied using chromatofocusing column chromatography. The results showed that serum PSTI was free from trypsin(-ogen) and was composed of at least three molecular forms of different isoelectric points. Two major forms were eluted around pH 8.2 (peak I) and 7.5 (peak II), with one minor form around pH 6.9 (peak III) from the column. The relative ratio of three forms differed with the disease state. Peak I was high in patients with pancreatic carcinoma, and peak II was high in patients with acute pancreatitis.     

The measurement of serum immunoreactive pancreatic secretory trypsin inhibitor in gastrointestinal cancer and pancreatic disease

Summary The clinical usefulness of serum pancreatic secretory trypsin inhibitor (PSTI) in pancreatic disease and gastric and colorectal cancer has been examined. The results showed that serum PSTI in acute pancreatitis was significantly higher than in normal subjects and it was also raised in acute exacerbations of chronic pancreatitis. Although the sensitivities of serum PSTI, amylase and elastase I were similar, serum PSTI in necrotizing hemorrhagic pancreatitis was 2.7 times higher than in mild acute pancreatitis. Only a few patients with chronic pancreatitis showed increased concentrations and the mean value was near normal. The mean PSTI in patients with pancreatic and colorectal cancer was higher than normal, although that of gastric cancer was within normal limits. The sensitivity of serum PSTI measurements in patrents with these three malignant diseases was only about 30%. The results suggested that the measurement of serum PSTI could be useful in the diagnosis of acute pancreatitis, but of limited value in the diagnosis of other disease which we examined.     

The measurement of serum immunoreactive pancreatic secretory trypsin inhibitor in gastrointestinal cancer and pancreatic disease

The clinical usefulness of serum pancreatic secretory trypsin inhibitor (PSTI) in pancreatic disease and gastric and colorectal cancer has been examined. The results showed that serum PSTI in acute pancreatitis was significantly higher than in normal subjects and it was also raised in acute exacerbations of chronic pancreatitis. Although the sensitivities of serum PSTI, amylase and elastase I were similar, serum PSTI in necrotizing hemorrhagic pancreatitis was 2.7 times higher than in mild acute pancreatitis. Only a few patients with chronic pancreatitis showed increased concentrations and the mean value was near normal. The mean PSTI in patients with pancreatic and colorectal cancer was higher than normal, although that of gastric cancer was within normal limits. The sensitivity of serum PSTI measurements in patients with these three malignant diseases was only about 30%. The results suggested that the measurement of serum PSTI could be useful in the diagnosis of acute pancreatitis, but of limited value in the diagnosis of other disease which we examined.     

Trypsin inhibitor activities of fibroblasts increase with age of donor and are unaltered in familial Alzheimer''s disease

Increasing evidence suggests that proteases and their inhibitors play an important role in the etiology of ß-amyloidogenesis and Alzheimer's disease (AD). It is not clear, however, which proteases and protease inhibitors are responsible for the amyloidogenic proteolysis. Candidates include -1-antichymotrypsin, inter--trypsin inhibitor, and forms of ß-amyloid precursor protein (ßPP) bearing Kunitz protease inhibitor domains. As one approach to this question, we have determined the trypsin inhibitor activity of fibroblast-like cells from 10 familial AD subjects and 20 controls. The activity was quantitated by measuring remaining trypsin activity of reaction mixtures containing trypsin and cell lysates using a fluorogenic substrate and two physiologically distinct populations of fibroblasts: proliferating cells (grown in the presence of 16% serum) and quiescent cells (maintained in 0.1% serum). The remaining trypsin activities of crude protein extracts from proliferating and quiescent AD cultures were not significantly different from those of controls. Perhaps of more general interest to the biology of aging, however, was our finding that protease inhibitor activity increased with the age of the donor (p = 0.005).     

Pancreatic secretory trypsin inhibitor: More than a trypsin inhibitor

Kazal-type serine protease inhibitor is one of the most important and widely distributed protease inhibitor families. Pancreatic secretory trypsin inhibitor (PSTI), also known as serine protease inhibitor Kazal type I(SPINK1), binds rapidly to trypsin, inhibits its activity and is likely to protect the pancreas from prematurely activated trypsinogen. Therefore, it is an important factor in the onset of pancreatitis. Recent studies found that PSTI/SPINK1 is also involved in self-regulation of acinar cell phagocytosis, proliferation and growth of a variety of cell lines. In addition, it takes part in the response to inflammatory factor or injury and is highly related to adult type II citrullinemia.     

CCK-releasing activity of rat intestinal secretion: effect of atropine and comparison with monitor peptide

A bioassay for studying the cholecystokinin (CCK)-releasing activity of intraluminal protease-sensitive bioactive peptides was developed. In conscious rats, bile and pancreatic juice were chronically diverted from the proximal intestine to the ileum to cause chronic stimulation of CCK release and pancreatic protein secretion. CCK-releasing activity of test substances was assayed during transient inhibition of CCK release by intraduodenal sodium taurocholate (78 mumols/h). Intestinal secretion as a source of the putative trypsin-sensitive intestinal CCK-releasing peptide was obtained by rapid intestinal perfusion of isolated Thiry-Vella fistulae of jejunum in conscious rats, collected with or without atropine pretreatment. Partially purified rat pancreatic secretory trypsin inhibitor (PSTI, or "monitor peptide") was compared with ovomucoid trypsin inhibitor (OMTI) and with concentrated jejunal secretions for CCK-releasing activity and trypsin inhibitor activity. Concentrated, heat-treated jejunal secretions were the strongest stimulants of CCK release and pancreatic protein secretion in this model. OMTI had no CCK-releasing activity in this model, whereas a larger amount (approximately 5x, based on trypsin inhibitor activity) of PSTI weakly but significantly stimulated CCK release. CCK-releasing activity manifested by pancreatic protein secretion was equivalent in intestinal washes from atropine-treated and control Thiry-Vella fistula donor rats. Concentrated jejunal secretions had no trypsin inhibitory activity, indicating that the putative intestinal CCK-releasing peptide and "monitor peptide" are different substances.     

Elevated levels of serum pancreatic secretory trypsin inhibitor (PSTI) in patients with malabsorption syndrome

Serum pancreatic secretory trypsin inhibitor (PSTI) was measured by radioimmunoassay in 5 patients with malabsorption syndrome. The serum level of PSTI was elevated to 123.8 +/- 25.8 ng/ml (Mean +/- SE) in patients with malabsorption syndrome, which was significantly higher than the 16.6 +/- 0.7 ng/ml level seen in 116 healthy control subjects. Serum PSTI levels in 5 patients with malabsorption syndrome showed inverse correlations with serum levels of cholesterol, cholinesterase and amylase, and not with serum levels of vitamin E, carotene, apoprotein A-IV, albumin, nor with immunoreactive elastase 1, respectively. These results suggest that elevated levels of serum PSTI represent a state of malnutrition due to impaired intestinal absorption.     

Effects of pH on the formation and dissociation of the complex of trypsin and PSTI]

The effects of pH on the formation and dissociation of the complex of trypsin and PSTI was investigated using pancreatic juice of two patients. Pancreatic juice was eluted from a Sephadex G100 column with the buffer in different pH values, and the trypsin inhibitory activity in the eluate was measured. Under alkaline and neutral conditions, the complex of PSTI and trypsin was observed, while in acidic condition, dissociation of the complex was occurred. Molar ratio of PSTI and trypsin in the complex was found to be one mole/one mole. Human serum, alpha 1-antitrypsin, and alpha 2-macroglobulin dissociated the complex of PSTI and trypsin.     

Functional analysis of recombinant pancreatic secretory trypsin inhibitor protein with amino-acid substitution

Background: We hypothesized that mutation of the pancreatic secretory trypsin inhibitor (PSTI) gene may promote a predisposition to pancreatitis, possibly by reducing the inhibition of trypsin activity. Based on this hypothesis, we performed a biochemical analysis of recombinant PSTI protein. Methods: The trypsin inhibitory activity of the recombinant protein was analyzed. The activity of PSTI protein with a point mutation of the most common type: ³⁴Asn (AAT)-to-Ser (AGT)(101A>G N34S: N34S) in exon 3, was compared with that of the wild type. Results: The function of N34S PSTI remained unchanged under both the usual alkaline and acidic conditions compared with the wild-type PSTI. The calcium concentration did not affect the activity of recombinant PSTI. The trypsin susceptibility of the N34S protein was not increased either. Conclusions: Mechanisms other than the conformational change of PSTI associated with amino-acid substitution, such as abnormal splicing, may underlie the predisposition to pancretitis in patients with the N34S mutation. Received: January 10, 2002 / Accepted: May 17, 2002 Reprint requests to: M. Ogawa     

Adaptive response of the rat pancreas to dietary substrates: parallel regulation of trypsinogen and pancreatic secretory trypsin inhibitor

Chronic pancreatitis has been associated with malnutrition in alcoholic patients and malnourished juveniles. The composition of the diet, especially the protein content, regulates the synthesis of secretory proteins in the rat pancreas. Adaptive responses of the pancreas have shown that anionic proteases (e.g., trypsinogen) are upregulated during protein deprivation. We hypothesize that the (cationic) pancreatic secretory trypsin inhibitor (PSTI) is down-regulated after a protein-deficient diet. Low PSTI levels might cause a lack of protection from prematurely activated trypsin and therefore enhance the risk for pancreatic inflammation. Over a period of 1 month, rats were fed one of four isocaloric diets with a casein content varying from 0 to 82%. PSTI and trypsinogen mRNA remained fairly constant, irrespective of the diet composition. Trypsinogen and elastase secreted into pancreatic juice were upregulated after a protein-deficient diet relative to a control diet. Contrary to our hypothesis, PSTI was also upregulated. Parallel secretion of trypsinogen and PSTI appears to ensure protection against premature activation even under extreme dietary conditions.     

Gastric output of pancreatic secretory trypsin inhibitor is increased by misoprostol.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent protease inhibitor that also has growth promoting activity. It has recently been identified in the foveolar cells of the stomach, which secrete mucus. We examined the effects of the prostaglandin E1 analogue misoprostol on gastric PSTI output. Seven normal volunteers took part. An initial period of gastric aspiration was followed by four 40 minute periods of gastric perfusion at 5 ml/minute of: 0.14 mol/l saline, 0.17 mmol/l bicarbonate, bicarbonate with misoprostol 400 micrograms, and then bicarbonate again. All perfusates contained polyethylene glycol 4000 as a marker. Misoprostol increased median gastric secretion of PSTI from 11 to 33 micrograms/hour (p less than 0.05), producing concentrations in gastric juice six times higher than those found in jejunal juice and about 1/30 of the values seen in pancreatic juice. Median mucus secretion increased to a lesser extent from 29 to 38 mg/hour during misoprostol. There was no change in intragastric concentrations of protein or of epidermal growth factor during infusion of misoprostol. Infusion of pentagastrin (6 micrograms/kg/hour) had no effect on gastric secretion of mucus, PSTI, or protein. Human gastric mucus was degraded on incubation with trypsin in vitro and this was prevented by the addition of PSTI. These results suggest that gastric PSTI may protect the gastric mucus layer against refluxed pancreatic proteases. Increased output of PSTI during microprostol may contribute to the protective effect of this drug.