Kinuko Kuwata

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.

Further Evidence for Endothelin as an Important Mediator of Pancreatic and Intestinal Ischemia in Severe Acute Pancreatitis

Introduction Severe acute pancreatitis is occasionally associated with pancreatic and intestinal necrosis. Mesenteric vasoconstriction is one of the most probable types of pathogenesis of these complications. Aim To investigate the involvement of endothelin-1 (ET-1), a potent vasoconstrictor. Methodology and Results Plasma ET-1 concentrations were extremely high in patients with pancreatic and/or diffuse intestinal necrosis. ET-1 mRNA was demonstrated in the rat pancreas, and the production of ET-1 protein by human umbilical vein endothelial cells was enhanced by tumor necrosis factor-&agr;, thrombin, and protease-activated receptor-2–activating peptide. Administration of ET-1 in vivo induced mesenteric arterial spasm and decreased pancreatic and intestinal blood flow. Conclusion These results suggest the following: ET-1 is produced in and around the pancreas, mainly by endothelial cells, in severe acute pancreatitis; in the inflammatory setting, cytokines, activated thrombin and trypsin, may stimulate ET-1 production in a paracrine fashion; produced ET-1 may exaggerate the splanchnic microcirculation; and progressive ischemia may lead to necrosis of the pancreas and intestine.

Genetic mutations in exons 3 and 4 of the pancreatic secretory trypsin inhibitor in patients with pancreatitis.

Purpose. We hypothesized that mutations in the pancreatic secretory trypsin inhibitor (PSTI) gene could promote autodigestion, leading to acute or chronic pancreatitis. Our investigation involved mutation analysis of the PSTI gene in patients with acute or chronic pancreatitis. Methods. Mutation analysis for the PSTI gene was performed in patients with acute or chronic pancreatitis. Unrelated healthy volunteers and family members of a chronic pancreatitis patient with point mutations in the PSTI gene were also analyzed. Results. Two types of single-point mutation in the PSTI gene were observed in one patient with chronic pancreatitis: 34Asn (AAT)-to-Ser (AGT) (101 A > G N34S: N34S) in exon 3, and 67Arg (CGC)-to-Cys (TGC) (199 C > T R67C: R67C) in exon 4. No mutations with amino-acid substitution were found in other patients or in the volunteer group. In the patient with the PSTI gene mutations, no additional mutations were observed in the cationic trypsinogen gene. The family study revealed that the mother and a maternal uncle were homozygotes for the N34S mutation, while the father and brother were compound heterozygotes for the N34S and R67C mutations. The uncle (N34S/N34S) showed clinical manifestations of pancreatitis, but the other family members did not. Conclusions. The N34S mutation may cause a predisposition to pancreatitis, with incomplete penetrance. However, with the limited information available, it is not known whether the R67C mutation promotes pancreatitis.

Mutational analysis of the pancreatic secretory trypsin inhibitor gene in familial and juvenile pancreatitis in Japan

Background: Mutations in the pancreatic secretory trypsin inhibitor (PSTI) gene have been reported in patients suffering from chronic pancreatitis. The aim of the present study was to investigate whether similar gene mutations are present in familial and juvenile pancreatitis patients in Japan. Methods: All four exons of the PSTI gene and their flanking intronic sequences were amplified by polymerase chain reaction and sequenced for 37 familial pancreatitis patients (24 families) and 15 juvenile pancreatitis patients, distributed throughout Japan. Results: Three types of exonic mutation in the PSTI gene were observed. The N34S mutation was found in six familial pancreatitis patients (three families) and in one juvenile pancreatitis patient, and the R67C mutation was found in one familial pancreatitis patient and one juvenile pancreatitis patient. We also found a 272C>T mutation in the 3′ untranslated region of exon 4 in one familial pancreatitis patient and four juvenile pancreatitis patients. It should be noted that the N34S mutation was cosegregated with two intronic mutations, specifically, IVS1-37T>C and IVS3-69insTTTT. Conclusions: The same set of N34S mutations (N34S + IVS1-37T>C + IVS3-69insTTTT) that exists in other countries was found in the PSTI gene in Japanese familial and juvenile pancreatitis patients. Another unique mutation (R67C) was also observed in two patients; 272C>T was suggested to be a normal polymorphism.

Dynamic Aspects of Granulocyte Activation in Rat Severe Acute Pancreatitis

We demonstrated dynamic aspects of granulocyte activation in rat severe acute pancreatitis, which was induced by cerulein and aggravated following lipopolysaccharide (LPS) injection. Pancreatitis induced by cerulein increased intracellular elastase activity of granulocytes in the blood. However, significant systemic cytokinemia was not provoked under such conditions. After induction of severe pancreatitis by LPS, intracellular elastase activity of circulating granulocytes decreased markedly and immediately. This decrease occurred simultaneous to induction of systemic hypercytokinemia and granulocyte migration into the lung. Overall results imply that: (1) circulating granulocytes are activated by induction of mild pancreatitis; (2) activation of granulocytes is mediated by factors other than systemic cytokinemia, such as locally produced cytokines; (3) those priming granulocytes immediately and significantly migrate from the circulation into the extravascular space by induction of endotoxemia; and (4) migration of granulocytes, in turn, may be mediated by systemic cytokinemia.

Significance of trypsin inhibitor gene mutation in the predisposition to pancreatitis

Abstract Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed a hypothesis: if the function of PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis, and performed mutational analysis of PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were suggested to be associated with the predisposition to pancreatitis. N34S mutation was co-segregated with two intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the function of recombinant N34S protein, we could not demonstrate the loss of function of this protein. Intronic mutations rather than N34S itself (IVS1-37T>C+N34S+IVS3-69insTTTT complex) may be associated with the decreased function of PSTI. Alternatively, increased digestion of N34S in vivo may be applicable. As for R67C, the conformational alteration of the protein by forming intramolecular or intermolecular disulfide bonds with 67 Cys was strongly suggested. These results, along with the brand new findings in PSTI knockout mice, suggest that genetic mutation of PSTI is one of the important mechanisms for predisposition to pancreatitis by lowering the trypsin inhibitory function.

The effect of TNF-α converting enzyme inhibitors on cytokine response in acute pancreatitis

Abstract Tumor necrosis factor-α (TNF-α) is known to play a central part in the pathogenesis of acute pancreatitis. TNF-α and its two receptors (p55 and p75 TNF receptors) have shown to be expressed at various sites in the body. TNF-α is initially expressed as a 26-kDa membrane-associated perform, which is proteolytically processed to a 17-kDa secreted mature form. Recently, TNF-α converting enzyme (TACE) has been identified as a membrane-bound TNF-α activating enzyme. Hence, it is expected that the newly devised TACE inhibitor, Y39083, will be a useful candidate for the treatment of inflammatory disorders provoked by TNF-α, including acute pancreatitis. Y39083 suppressed TNF-α production in vitro and in vivo significantly. However, the production of other cytokines, such as IL-1β, IFNγ and IL-6, in vivo were not. The blockade of secreted TNF-α production by Y39083 could not improve the organ injury. These results suggest that the blockade of activities of secreted TNF-α production is not sufficient to suppress systemic cytokine reaction and distant organ injury.

Dynamic aspects of granulocyte activation in acute pancreatitis

Abstract We demonstrated the dynamic aspects of granulocyte activation in rat severe acute pancreatitis which was induced by cerulein and aggravated following lipopolysaccharide (LPS) injection. Pancreatitis induced by cerulein increased intracellular elastase activity of granulocytes in the blood. However, significant systemic cytokinemia was not provoked under such conditions. After induction of severe pancreatitis by LPS, intracellular elastase activity of circulating granulocytes decreased markedly and immediately. The timing of this decrease was concomitant with induction of systemic hypercytokinemia and granulocytes migration into the lung. Overall results provide the following implications: (1) circulating granulocytes are activated by the induction of mild pancreatitis; (2) the activation of granulocytes is mediated by other factors than systemic cytokinemia, such as locally produced cytokine; (3) those priming granulocytes immediately and significantly migrate from the circulation into extravascular space by the induction of endotoxemia; (4) the migration of granulocytes, in turn, may be mediated by systemic cytokinemia.

Endothelin is involved in pancreatic and intestinal ischemia during severe acute pancreatitis

Abstract Introduction: Severe acute pancreatitis is occasionally associated with pancreatic and intestinal necrosis. Vasoconstriction of the abdominal artery is one of the most probable pathogeneses of these complications. Hence, we investigated the involvement of endothelin-1 (ET-1), a potent vasoconstrictor, in the pathogenesis of acute pancreatitis. Materials and methods: (1) Plasma samples from patients with acute pancreatitis were analyzed. (2) Production of ET-1 mRNA in rat pancreas and ET-1 protein by human umbilical vein endothelial cells (HUVECs) in vitro was analyzed. (3) After the administration of ET-1, pancreatic and intestinal blood flow was analyzed in rat. Results: (1) Plasma ET-1 concentrations were extremely high in patients with pancreatic and diffuse intestinal necrosis. (2) ET-1 mRNA was demonstrated in rat pancreas after the induction of pancreatitis, and the production of ET-1 protein by HUVECs was enhanced by TNF-α, thrombin, and trypsin receptor-activating peptide. (3) Administration of ET-1 induced mesenteric arterial spasm and resultant decreased splanchnic blood flow. Conclusions: These results suggest that (1) ET-1 is produced in and around the pancreas, mainly by endothelial cells, in severe acute pancreatitis; (2) in the inflammatory setting, cytokines, thrombin, and trypsin may stimulate ET-1 production through paracrine fashion; (3) produced ET-1 may exaggerate the splanchnic microcirculation; and (4) progressive ischemia may lead to necrosis of the pancreas and intestine.

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

Abstract Introduction: The pancreatic secretory trypsin inhibitor (PSTI) is thought to be a specific inactivation factor of intrapancreatic trypsin activity. N34S mutation of PSTI was reported in chronic pancreatitis patients from all over the world including us, and is thought to be one of the predisposing factors for pancreatitis. The N34S mutation located close to the reactive lysine–isoleucine site (K41∼I42) might lead to a decreased inhibitory capacity. N34S is also in complete linkage with other intronic sequence variants such as IVS1-37T→C and IVS3-65insTTTT. Materials/Methods: Based on the hypothesis above, we performed biochemical experiments. (I) Activity of recombinant PSTI protein with N34S was compared to that of the wild type. (II) Molecular weight of PSTI from a patient with N34S was analysed by Western blotting to check the possibility of abnormal splicing (e.g. splice-out of exon 4). Results: (I) The function of N34S PSTI remained unchanged compared to wild-type PSTI. (II) The molecular weight of PSTI protein from a patient with N34S was not altered. Conclusion: We could not detect any functional or configurational abnormalities of N34S PSTI protein by the two experiments mentioned above. Other factors, such as translational abnormality by intronic mutation, may be associated with the predisposition to pancreatitis.