Studies on the intracellular regulatory proteins in pancreatic exocrine secretion

의학과/박사[한글] 본 실험은 Camostat 처치후 비대된 이자의 조직에서 채취한 분산 선포세포를 이용하여 분비자극 물질에 대한 효소 분비반응을 관찰하고, Camostat 또는 분비 자극 물질 처치에 의하여 초래되는 단백 및 인단백 변화를 관찰함으로써 이자 외분비 기능에 영향을 미치는 세포내 신호변환체계와 세포내 조절 단백의 변화를 규명하고자 하였다. Camostat(200mg/kg)은 4일간 1일 2회 위내 투여하였고, 마지막 투여 12시간뒤 이자를 적출, 검색하여 다음과 같은 결과를 얻었다. 1. 체중 100g당 이자 무게는 대조군에 비해 camostat 처치군에서 126%로 증가하였으나, 단위 단백당 amylase 활성은 현저히 감소하여 camostat 처치군에서는 대조군의 41%에 불과하였다. 2. 분리된 선포세포의 amylase 기초유리는 대조군과 camsotat 처치군간에 차이가 없었으나, CCK-8 또는 carbachol 자극시 최대 amylase 유리율은 대조군에 비하여 camostat 처치군에서 각각 65%, 46%로 감소하였다. Secretin 자극시 amylase 유리율은 camostat 처치 의 영향을 받지 않았다. 3. Camostat 처치군에서 24-28kD, pl 4.5-8.5 단백의 양이 대조군에 비해 증가되었으며, 25kD, pl 5.0 단백의 인산화 중가가 관찰되었다. 4. CCK-8 또는 secretin 자극시 34kD, pl 4.7 단백의 인산화가 증가되었고, 이는 대조군 및 camostat 처치군 모두에서 관찰되었다. 5. 선포세포내 tyrosine 인산화 단백은 camostat 처치군에서 전반적으로 증가되었다. CCK-810-10 M 투여시 대조군에서는 tyrosine 인산화가 중가되었으나, camostat 처치군에서는 오히려 감소되었다. Secretin 자극은 tyrosine 인산화에 뚜렷한 영향을 미치지 않았다. 이상의 결과로 보아 CCK-8 또는 secretin은 이자 선포세포내 34kD, pl 4.7 단백의 인산화를 특이하게 증가시키므로 이 단백의 인산화 과정에는 phospholipase C 및 adenylate cyclase가 모두 관여할 것으로 생각된다. 또한 camostat를 장기간 투여하였을 때, tyrosine 인산화가 증가하는데, 이는 CCK 수용체를 매개하는 자극 분비과정에 serine/throsine protein kinase 뿐만 아니라 protein tyrosine kinase/phosphatase의 기능적 연관도 시사한다. [영문] Secretagogue-induced changes of intracellular proteins were evaluated in dispersed pancreatic acini. The rats were treated with camostat(FOY-305, 200 mg/kg body wt., P.0.) for 4 days and dispersed pancreatic acini were prepared at 12 hours after the last treatment. To observe the profile of intracellular phosphoproteins, acini were incubated in a buffer containing 32P, and the 32P-labeled Phosphoprotein profiles were analyzed by two-dimensional gel electrophoresis. The effects of secretagogues on the exocrine function of dispersed acini were estimated by measuring amylase relase. Following results were obtained. 1. After camostat treatment, pancreatic weight per 100g body weight increased to 126% relative to the control group, while amylase activity per milligram protein decreased to 41% of control group. The amylase release from dispersed pancreatic acini was increased by secretagogues dose-dependently. 2. The maximum release of amylase from camostat-treated acini stimulated by CCK-8 or carbachol decreased markedly(65% and 46%, of control, respectively), while the amylase release by secretin was similar to the control. 3. The group of intracellular proteins(24kD, pI 4.5∼ 8.5) was increased in quantity by camostat. 4. CCK-8 or secretin increased phosphorylation of a Protein(34kD, pI 4.7) in both camostat-treatedand control groups. 5. CCK-8 increased tyrosine phosphorylation in the acini of control rats. However, in camostat-treated rats, the basal level of tyrosine phosphorylation was increased which was rather decrersed by CCK-8. Secretin did not change the level of tyrosine phosphorylation in acini. These results indicate that both phospholipase C and adenylate cyclase induce phosphorylation of an intracellular acinar protein (34kD, pI 4.7) and camostat treatment increases the basal level of tyrosine phosphorylation in acinar cells, and this suggests that not only serine/threonine protein kinase but also protein tyrosine kinase/phosphatase are involved in the process of stimulation-secretion coupling via CCK receptors.restrictio

(The) influence of dietary cholesterol and ursodeoxycholic acid treatment on serum and bile cholesterol levels

의학과/석사[한글] 담석의 형성기전 및 원인에 대해서는 아직도 많은 논란이 있으며, 그중 특히 유전적, 식이적 요인에 대한 생체 또는 동물실험 결과가 보고되고 있다. 콜레스테롤 담석이 많은 구미에서는 담석형성에 미치는 고콜레스테롤식이의 영향에 대한 연구가 많이 실시되었으며 고콜레스테롤식이가 담즙내 콜레스테롤 분비의 증가를 유발하는 것이 이상 담즙 형성의 한 원인이 된다고 하였다. Chenodeoxycholic acid 및 ursodeoxycholic acid는 담즙내 콜레스테롤 생성을 감소시켜 담석 형성을 억제한다고 하였고 이를 이용한 임상연구에서도 담석의 용해에 효과가 있음이 보고 되었다. 따라서 이번 연구에서는 담석증 환자에 있어서 고콜레스테롤식이 및 ursodeoxycholic acid가 담즙내 롤레스테롤 분비에 미치는 영향을 측정하고자 하였다. 담석증 수술을 받은 21명의 환자를 대상으로 저콜레스테롤식이(400mg/일), 고콜레스테롤식이(1000mg/일), 고콜레스테롤식이 및 ursodeoxycholic acid투여의 3군으로 분류하였으며, 각 환자에서 각각 혈청 및 담즙 콜레스테롤 농도를 측정하여 다음과 같은 결론을 얻었다. 1. 담석증 환자의 수술전 혈청 콜레스테롤치를 간 담도계 질환이 없는 대조군과 비교할 때 의의있는 차이가 없었다. 2. 수술 전후에 따르는 혈청 콜레스테롤 농도의 변동은 볼 수 없었다. 3. 저콜레스테롤식이는 혈청 콜레스테롤 농도를 감소시키고 고콜레스테롤식이는 혈청 콜레스테롤 농도를 증가시키는 경향을 보였으나 통계학적 의의는 없었다. 4. 고콜스테롤식이를 주면서 ursodeoxycholic acid를 투여한 환자의 혈청 콜레스테롤 농도는 고콜레스테롤 단독 투여군의 농도보다 낮은 양상을 나타내었으나 통계적 의의는 없었다. 5. 고콜레스테롤식이는 담즙 콜레스테롤을 의의있게 증가시켰으나 고콜레스테롤식이를 주면서 ursodeoxycholic acid를 투여한 환자에서는 담즙 콜레스테롤 분비 증가가 미약하였다. 이상의 성적으로 보아 혈청 및 담즙 콜레스테롤 농도는 음식내 콜레스테롤 함량에 따라 변동될 수 있으며 ursodeoxycholic acid는 고콜레스테롤식이로 증가된 담즙내 콜레스테롤 농도를 낮출 수 있다고 생각된다. [영문] There is some debate on the pathogenesis of gall stone formation and numerous factors, especially genetic and dietary factors, were reported as a potential for the development of gall stone. In order to define the possibility of abnormal lithogenic bile formation by high cholesterol diet in patients with gall stone and the effect of ursodeoxycholic acid on the biliary cholesterol secretion, the serum and bile cholesterol levles were measured in 21 patients with gall stone under the challenge of high cholesterol diet and ursodeoxycholic acid. The results obtained are summarized as follows. 1. There was no significant difference of serum cholesterol concentration between gall stone patients and control. 2. The serum cholesterol concentration was little changed after operation. 3. The serum cholesterol concentration were tended to be increased by high cholesterol diet and to be decreased by low cholesterol diets but there was no statistical significance between them. 4. The ursodeoxycholic acid (UDCA) treatment tended to decrease the serum cholesterol concentration in high cholesterol diet group, but there was no statistical significance. 5. The cholesterol concentration in bile was significantly elevated after high cholesterol diet, and this elevation was not significant by concomitant treatment of ursodeoxycholic acid (UDCA). By these findings it can be suggested that the cholesterol concentration in serum and bile are influenced by dietary cholesterol, and ursodeoxycholic acid could well affect the high cholesterol diet induced elevation of biliary cholesterol.prohibitio

Studies on intracellular regulatory proteins of pancreatic exocrine secretion

CCK and cholinergic agonist stimulate enzyme release from the pancreatic acini via G-protein-mediated activation of phospholipase C. In contrast secretin and related peptides increase the level of cAMP and activate cAMP-dependent protein kinase. Camostat, a synthetic protease inhibitor, causes pancreatic hypertrophy and hyperplasia by increasing the CCK release. In this study, the secretagogue-induced changes of intracellular proteins were examined in the dispersed pancreatic acini of rats with or without camostat treatment. Camostat (FOY-305, 200 mg/kg, p.o.) was given for 4 days twice daily and the dispersed acini were prepared at 12 hours after last treatment. The profiles of intracellular phosphoproteins were analyzed by two-dimensional gel electrophoresis after incubating the acini with 32P. The amylase release from the dispersed acini was measured. The pancreatic weight was increased to 126% of control, while amylase activity per mg acinar protein decreased to 41% of control. The maximum response of amylase release from dispersed acini to CCK-8 or carbachol was markedly decreased (65% or 46% of control, respectively). The group of intracellular proteins (24 kD, pI 4.5 ~ 8.5) was increased in quantity by camostat. CCK-8 or secretin increased phosphorylation of a protein (34 kD, pI 4.7) in camostat-treated as well as control rats. CCK-8 increased tyrosine phosphorylation in the acini of control rats. However, in camostat-treated rats, the basal level of tyrosine phosphorylation was increased and it was rather decreased by CCK-8. Secretin had no effect on the level of tyrosine phosphorylation in acini. These results indicate that both phospholipase C and adenylate cyclase induce phosphorylation of an intracellular acinar protein (34 kD, pI 4.7) and camostat treatment increases the basal level of tyrosine phosphorylation in acinar cells. And these results suggest that not only serine/threonine protein kinase but also protein tyrosine kinase/phosphatase are involved in the process of CCK receptor mediated stimulation-secretion coupling

Two-day fasting prior to intestinal ischemia-reperfusion injury on bacterial translocation in rats

Purpose: The aim of this study is to verify the effect of two-day fasting prior to intestinal ischemia-reperfusion (I/R) injury on bacterial translocation (BT). Materials and Methods: Mail SpragueDawley rats were divided into four groups: group 1, control rats that underwent sham operation only; group 2, rats fasted for two days prior to sham operation; group 3, rats that underwent occlusion of mesenteric vessels for 30 min followed by reperfusion for 4 hr; and group 4, rats fasted for two days prior to the same intestinal I/R injury as in group 3. In all groups, E. coli labeled with 99mTc were inoculated into the terminal ileum. Two hr after inoculation of E. coli, the rats were killed. A segment of ileum was obtained for histological examination and samples of mesenteric lymph nodes (MLNs), liver, lung, blood, and spleen were obtained for radioactivity determination. Results: There were no significant differences in the intestinal mucosa and radioactivity of all samples between groups 1 and 2. Group 3 showed significantly shorter mucosa and villi, and higher radioactivity of samples, except for MLNs, compared to group 1. Group 4 showed similar mucosa and villi, but significantly higher radioactivity of samples, except for MLNs, compared to group 3. Conclusion: Two-day fasting without I/R injury does not cause mucosal change and BT, but in cases following intestinal I/R injury, two-day fasting increases the susceptibility of BT to systemic organs in rats. © 2011 Informa Healthcare USA, Inc

Expression of E-cadherin in pig kidney

E-cadherin; Kidney; Pi

Nitric oxide synthesis in the adult and developing kidney

Nitric oxide (NO) is synthesized within the adult and developing kidney and plays a critical role in the regulation of renal hemodynamics and tubule function. In the adult kidney, the regulation of NO synthesis is very cell type specific and subject to distinct control mechanisms of NO synthase (NOS) isoforms. Endothelial NOS (eNOS) is expressed in the endothelial cells of glomeruli, peritubular capillaries, and vascular bundles. Neuronal NOS (nNOS) is expressed in the tubular epithelial cells of the macula densa and inner medullary collecting duct. Furthermore, in the immature kidney, the expression of eNOS and nNOS shows unique patterns distinct from that is observed in the adult. This review will summarize the localization and presumable function of NOS isoforms in the adult and developing kidney

Mucosal protective effect of PGI2 on canine small bowel auto-transplantation

Purpose: We designed this experimental study to assess the mucosal protective effects of continuous prostaglandin I2 (PGI2) infusion after canine small bowel autotransplantation. Materials and Methods: Six Mongrel dogs were randomly divided two groups: PGI2 (n = 3) and control (n = 3). The small bowel from jejunum to ileum was obtained, including the mesenteric vascular pedicle. After cold flushing ex vivo, the harvested segment was preserved in an icebox for 3 hours. Thereafter we reimplanted the harvested intestinal segment. While completing the anastomosis, PGI2 (50 μg) was slowly infused through the mesenteric artery in the PGI2 group versus the same volume of saline in the control group. At 1, 3 and 6 days after autotransplantation, we obtained blood samples, and at 6 days, small bowel segments. Endotoxin and interleukin 6 (IL-6) levels were measured and all histologic specimens stained with hematoxylin-eosin H-E were reviewed by a pathologist to grade mucosal damage as: mild (1 point), moderate (2 points), or severe (3 points) change. Results: Mean basal serum endotoxin levels were similar in both groups the PGI2 groups versus control group were 0.216 ± 0.018 versus 0.223 ± 0.040 EU/mL, respectively. However, on day 3 after the operation, the PGI2 group showed much decreased levels of serum endotoxin compared to control levels: 0.349 ± 0.196 versus 0.842 ± 0.446 EU/mL. The mean concentration of serum IL-6 on day 1 after operation among the PGI2 versus control group were 32.13 ± 7.13 pg/mL versus 36.96 ± 3.65 pg/mL. The histologic scores at 6 days after the operation were PGI2 group versus control group: 1.33 versus 1.66 (P = NS). Conclusion: Continuous infusions of PGI2 through the mesenteric artery after the canine small bowel autotransplantation may protect the small bowel mucosal barrier. © 2012 Elsevier Inc

Prevention of primary nonfunction after canine liver allotransplantation: The effect of gadolinium chloride

Effective suppression of Kupffer cell function is believed to contribute to the prevention of preservation/reperfusion injury. In this study, effect of gadolinium, a synthetic Kupffer cell suppressant, on the reperfusion injury was examined using a canine partial liver transplantation model. About a 70% partial liver segment was harvested and reimplanted in a mongrel recipient dog weighing 20 to 25 kg. Gadolinium chloride (10 mg/kg) was infused via the cephalic vein 24 hours before harvest of the partial liver (gadolinium group, n = 5). Serum aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and morphologic grading of graft were compared with those of a control group (n = 5). Statistical analysis was done with an independent t-test. Average total ischemic time was 4 hours and 27 minutes. At 1 hour after reperfusion, there were no significant differences in AST, ALP, or LDH levels, or pathologic scores. At 48 hours after reperfusion, AST (P =. 03) and LDH (P =. 05) levels were significantly lower in the gadolinium group. Kupffer cell blockade using gadolinium chloride may be effective to reduce ischemia reperfusion injury, but the effect is not evident at an early stage of reperfusion