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Hepatocyte growth factor (HGF) stimulates fetal gastric epithelial cell growth in vitro
A982
AGA ABSTRACTS
• HEPATOCYTE GROWTH FACTOR (HGF) STIMULATES FETAL GASTRIC EPITHELIAL CELL GROWTH 1N VITRO. Wuyi Kong, Laurence F. Yee, and Sean J. Mulvihill. Gastrointestinal Research Laboratory, Department of Surgery, University of California San Francisco. HGF, the most potent hepatocyte mitogen known, has recently been found to stimulate growth of other epithelial cells• Its effect on fetal gastric epithelial ceils is unknown. In this study, we compared the mitogenic effects of graded doses of HGF (gift of Dr. R. Schwall, Genentech) to that of other known growth factors (EGF, TGFo% and IGF-1) On fetal rabbit gastric epithelial cells in short and long term culture• Methods: 56 fetal rabbits were sacrificed between gestational days 22-26 (term is 31). Fetal rabbit gastric epithelial cells were purified by mechanical dissociation and • selected culture• For short term cultures, cell growth was measured by 3Hthymidine DNA incorporation after 24 hours of treatment. For long term culture, cell growth was measured by cell counting after 10 days of treatment• Results: In short term culture, HGF stimulated 3H-thymidine incorporation with a threshold dose of I0 pM and maximal dose of 1O0 pM. Threshold effects ofEGF, TGFot and IGFT1 were observed at 0.1 pM, 0.1 pM, and 10 nM, respectively. Maximal effects were observed at 100 pM for EGF and TGFo~. Maximal 3H-thymidine incorporation was 3•6_+0.7 times basal for HGF, 4.3_+0.4 for EGF, and 3.6-+0.4 for TGF~x, respectively• Maximal effect oflGF-1 was not attained, even at a concentration of 100 nM. In long term culture, the cell purification method Yielded more than 90% epithelial cells after 10 days. Significant increases in cell number were observed at 1 nM concentrations ofHGF, EGF, and TGFct, and 20% rabbit amniotic fluid. None of the individual factors, however, increased cell growth as high as that of 10% fetal bovine serum. Conclusions: 1) HGF stimulates 3H-thymidine uptake and cell proliferation in fetal rabbit gastric epithelial cells in vitro. 2) This effect is comparable to that observed in response to EGF and TGFa, and superior to the effect of IGF-1. We conclude that HGF may regulate fetal gastric epithelial cell growth.
• ROLE OF EPIDERMAL GROWTH FACTOR (EGFI AND TRANSFORMING GROWTH FACTOR ALFA (TGFa) IN GASTRIC ADAPTATION T O ASPIRIN (ASA}. P.Ch. Konturek, H. Ernst, T. Brzozowski, E.G. Hahn, d. Stachura, S.J. Kont urek. Med. Dept I, Univ. Erlangen-NuPnberg, Erlar~en, Germany & Inst. Physiol. & Pathomorphol. Univ. Sch. Med., Kl"akow, Poland. Gastric mucosa exhibits the ability t O adapt to ulcerogenic ~ction of ASA but the mechB-~ism of this effect is unknown. Excessive mucosal cell proliferation was described in the ASA-adapted stomach but the mediators of these changes have not been defined. This report was designed to determine the expression of EGF and TGFcc in mucosal sections stained tmmunohistochemically for EGF and TGF~z (all antibodies from Oncogenel and cell proliferation by bromodeoxyuridine lBrdU) uptake in intact and ASA-adapted mucosa. The intensity of cytoplasmic staining for EGF or TGF~ was graded from 0 (minimal} to 3.0 (maximal staining) and assessed accordin~ to different regions of the oxyntic glands (top, neck and base). ASA (i00 mg/kg) in 0.2 M HCl or vehicle was administered intragastrically (ig} either once OF every day during four consecutive days. After single exposure to acidified ASA the area of gastric lesions averaged about S7 mm 2, the expressiol/ of TGF~ and EGF was negligible and the BrdU staining was absent. After four repeated ASA insults, the area of gastric erosions "was reduced by about 87%. EGF expression, which in the intact mucosa was negligible (0.18+0.2), increased by about 4 folds in the ASA-adapted mucosa (0.8-+0.21. The highest EGF expression was found at the top and the neck of gastric glands (1.0+0.3 and smaller at the gland base (O.S+O.I). The EGF expression was not affected b y four daily i.g. application of vehicle. The expression of TGF~ in the intact non-adapted mucosa vehicle-treated mucosa was small [1.0+0.1) and similar to that in the ASA-adapted mucosa (i. 3-+0.2). The Br-dU uptake after four ASA insults 'was remarkable increased from about 0.2-+02 at day 0 to about I0.2-+i. 8 of labelled nuclei per microscopic field~ We conclude that the In~jor mee h ~ ~d~d~: ~ ~ ~1~,111 ~a~m~zcel adaptation to ASA is an excessive ~ e ~ j s i o n ~)f F-~F &Sut" not TGFc~) and the stimulation of mucosa~Zeem~wg°ro'~m~'~ balance the cell loss due to irrit~%%,Lon,l:tv,ASA.
GASTROENTEROLOGY, VOI. 108, NO. 4
• EXPRESSION OF TGFo~ AND EGF IN GASTRIC M U C O S A AFTER EXPOSURE TO STRESS IN RATS. P. Konturek*, H. Ernst*, T. Brzoz0wskL, E.G. Hahn* and SJ Konturek. *Dept. Med. I, Univ. Erlangen-Nuremberg, FRG and Inst. Physiol., Univ. Sch. Med., Krakow, Poland. The mechanism of stress-induced gastric ulcerations and mucosal heali,g after stress remains controversial. Transforming growth factor alpha (TGF(xI and epidermal growth factor (EGF) stimulate mucosal growth and cell proliferation and protect the gastric mucosa against injury via action on trleir common receptor (EGFR). The aim of this stu~ was to assess the expression of TGF~. EGF and EGFR in the gastric mucosaafter single exposureto stress. Material and Methods:25 rats were exposedto water restraintstress for 3,5h and then sacrific~ at 0, 2, 4, 6 and 12h. Each grouo included at least 5 rats. The number of stress lesions were counted and sections obtainedfrom the stomachwere stained immunohistochemically for proliferating ceil nuclear antigen (PCNA), TGF(~, EGF and EGFR (all antibodiesfrom Oncogene).At least 300 consecutiveceils were counted in each section to determine the rate of proliferation by PCNA index. The intensity of cytoplasmic staining for TGF(~and EGF were graded (0,1,2,3) by examinationof 300 consecutive ceils per section. In addition, we evaluatedthe numberof EGFR positive calls. All semiquantitative data were assessed according tO different regions of the gastric gland (top, neck and base). DNA synthesis was measured in the mucosat scraping by in vitro incorporationof 3H-thymidine into DNA. Results:The numberof ulcerationsat Ohwas about 15+3 and then graduallydeclined to about 4 ± 2 at 12h after stress: Labelling index for PCNA showed significant increase at 2h afterr stress and reached its highest level at 6h after exposure to stress. There was almost no staining for EGF immediately (0 h) after stress compared to the control (staining index, x=0.26). Staining intensity for EGF then increased reaching its peak at 6h after stress(x=0,93) and then declined at 12 h after stress. EGFRexpression increasedfrom 0 h, reaching peak at 4 h and then gradually declining from 6 h to t2 h. In contrast, immunostainingfor TGF(~increasedgradually from 0 to 12 h after stress with predominant staining of supedicial epithelial cells. At 0 h DNA synthesis fell by about 350 compared to intact gastric mucosa and then started to rise after 12 h after stress. Conclusions:This study providesan evidencethat there is a time-dependentincrease in TGF(~, EGF and EGFR expression in the gastric mucosa after single exposureto stress and this is accompanied by increased mucosal cell proliferation ano DNA synthesis. These findings suggest an important role of growth factors in the mucosel repair by cell proliferation and DNA synthesis after exposureto stress.
AMILORIDE SENSITIVE Na + CHANNEL EXPRESSION IN THE ILEUM AFTER TOTAL COLECTOMY IN RATS. K. Koyama, I. Sasaki, Y. Funayama, H. Naito, T. Tsuchiya, M. Unno, M.Takahashi, K. Fukushima, C. Shibata, S. Matsuno, Y. Suzuki*. 1st Dep. of Surgery, Tohoku University, School of Medicine, Sendal; * Dep. of Physiology, Shizuoka University, Shizuoka, Japan." Background and Aim: Clinical study have shown that after total colectomy, profuse and dehydration occur initially, but gradually subside over a period of several months. The mechanism for this gradual recovery is not clear, but may involve the ability of the gut to absorb water and sodium. It was recently reported that in normal rats, mRNA for the amiloride:Sensiti';,e sodium channel (rENaC) is detectable in distal colon but not in the small intestinel). Since rENaC is thought to play a key role in the control of intestinal Na+ absorption, we tested the hypothesis that after c01ectomy, rENaC expression in the remaining intestine would be up-regulated. We examined the effect of total colectomy with ileoanal anastomosis on amiloride-sensitive Na+ channel mRNA levels in rats. Methods: Fifty,five male Sprague-Dawley rats (250-300 g) were used. Total colectomy with ileoanal anastomosis (IAA) or sham surgery was performed under peutobarbital anesthesia. Urinary and fecal Na+ excretion; and plasma aldosterone levels were monitored for 14 and 28 days after IAA. At the end of the study ~periods, animal were anesthetized, intestinal mucosa was collected, and total mucosal RNA was isolated. Expression of rENaC was examined by Northern blot analysis. Results: Daily urinary output of Na+ was significantly decreased and fecal output of Na+ was significantly increased both 14 and 28 days after IAA. These changes appear to recover back towards normal by the end of the 28 days, although there was no significant difference in values between i4 days ahd 28 days after IAA. Plasma aldosterone levels were significantly elevated for 28 days after surgery. Expression of rENaC mRNA was not detected in before surgery and sham operative group but it was obviously detected bt~th 14 days and 28 days post-surgery. Conclusion: Total colectomy produced induction of rENaC expression; this may play a role in ihcreases in intestinal Na + absorption after colectomy. Reference:l) Cecilia M. Canessa, et al.: Amiloride-sensitive epitherial Na+ channel is made of three homologous subunits. Nature, 367;463-467,1994.
AGA ABSTRACTS
• HEPATOCYTE GROWTH FACTOR (HGF) STIMULATES FETAL GASTRIC EPITHELIAL CELL GROWTH 1N VITRO. Wuyi Kong, Laurence F. Yee, and Sean J. Mulvihill. Gastrointestinal Research Laboratory, Department of Surgery, University of California San Francisco. HGF, the most potent hepatocyte mitogen known, has recently been found to stimulate growth of other epithelial cells• Its effect on fetal gastric epithelial ceils is unknown. In this study, we compared the mitogenic effects of graded doses of HGF (gift of Dr. R. Schwall, Genentech) to that of other known growth factors (EGF, TGFo% and IGF-1) On fetal rabbit gastric epithelial cells in short and long term culture• Methods: 56 fetal rabbits were sacrificed between gestational days 22-26 (term is 31). Fetal rabbit gastric epithelial cells were purified by mechanical dissociation and • selected culture• For short term cultures, cell growth was measured by 3Hthymidine DNA incorporation after 24 hours of treatment. For long term culture, cell growth was measured by cell counting after 10 days of treatment• Results: In short term culture, HGF stimulated 3H-thymidine incorporation with a threshold dose of I0 pM and maximal dose of 1O0 pM. Threshold effects ofEGF, TGFot and IGFT1 were observed at 0.1 pM, 0.1 pM, and 10 nM, respectively. Maximal effects were observed at 100 pM for EGF and TGFo~. Maximal 3H-thymidine incorporation was 3•6_+0.7 times basal for HGF, 4.3_+0.4 for EGF, and 3.6-+0.4 for TGF~x, respectively• Maximal effect oflGF-1 was not attained, even at a concentration of 100 nM. In long term culture, the cell purification method Yielded more than 90% epithelial cells after 10 days. Significant increases in cell number were observed at 1 nM concentrations ofHGF, EGF, and TGFct, and 20% rabbit amniotic fluid. None of the individual factors, however, increased cell growth as high as that of 10% fetal bovine serum. Conclusions: 1) HGF stimulates 3H-thymidine uptake and cell proliferation in fetal rabbit gastric epithelial cells in vitro. 2) This effect is comparable to that observed in response to EGF and TGFa, and superior to the effect of IGF-1. We conclude that HGF may regulate fetal gastric epithelial cell growth.
• ROLE OF EPIDERMAL GROWTH FACTOR (EGFI AND TRANSFORMING GROWTH FACTOR ALFA (TGFa) IN GASTRIC ADAPTATION T O ASPIRIN (ASA}. P.Ch. Konturek, H. Ernst, T. Brzozowski, E.G. Hahn, d. Stachura, S.J. Kont urek. Med. Dept I, Univ. Erlangen-NuPnberg, Erlar~en, Germany & Inst. Physiol. & Pathomorphol. Univ. Sch. Med., Kl"akow, Poland. Gastric mucosa exhibits the ability t O adapt to ulcerogenic ~ction of ASA but the mechB-~ism of this effect is unknown. Excessive mucosal cell proliferation was described in the ASA-adapted stomach but the mediators of these changes have not been defined. This report was designed to determine the expression of EGF and TGFcc in mucosal sections stained tmmunohistochemically for EGF and TGF~z (all antibodies from Oncogenel and cell proliferation by bromodeoxyuridine lBrdU) uptake in intact and ASA-adapted mucosa. The intensity of cytoplasmic staining for EGF or TGF~ was graded from 0 (minimal} to 3.0 (maximal staining) and assessed accordin~ to different regions of the oxyntic glands (top, neck and base). ASA (i00 mg/kg) in 0.2 M HCl or vehicle was administered intragastrically (ig} either once OF every day during four consecutive days. After single exposure to acidified ASA the area of gastric lesions averaged about S7 mm 2, the expressiol/ of TGF~ and EGF was negligible and the BrdU staining was absent. After four repeated ASA insults, the area of gastric erosions "was reduced by about 87%. EGF expression, which in the intact mucosa was negligible (0.18+0.2), increased by about 4 folds in the ASA-adapted mucosa (0.8-+0.21. The highest EGF expression was found at the top and the neck of gastric glands (1.0+0.3 and smaller at the gland base (O.S+O.I). The EGF expression was not affected b y four daily i.g. application of vehicle. The expression of TGF~ in the intact non-adapted mucosa vehicle-treated mucosa was small [1.0+0.1) and similar to that in the ASA-adapted mucosa (i. 3-+0.2). The Br-dU uptake after four ASA insults 'was remarkable increased from about 0.2-+02 at day 0 to about I0.2-+i. 8 of labelled nuclei per microscopic field~ We conclude that the In~jor mee h ~ ~d~d~: ~ ~ ~1~,111 ~a~m~zcel adaptation to ASA is an excessive ~ e ~ j s i o n ~)f F-~F &Sut" not TGFc~) and the stimulation of mucosa~Zeem~wg°ro'~m~'~ balance the cell loss due to irrit~%%,Lon,l:tv,ASA.
GASTROENTEROLOGY, VOI. 108, NO. 4
• EXPRESSION OF TGFo~ AND EGF IN GASTRIC M U C O S A AFTER EXPOSURE TO STRESS IN RATS. P. Konturek*, H. Ernst*, T. Brzoz0wskL, E.G. Hahn* and SJ Konturek. *Dept. Med. I, Univ. Erlangen-Nuremberg, FRG and Inst. Physiol., Univ. Sch. Med., Krakow, Poland. The mechanism of stress-induced gastric ulcerations and mucosal heali,g after stress remains controversial. Transforming growth factor alpha (TGF(xI and epidermal growth factor (EGF) stimulate mucosal growth and cell proliferation and protect the gastric mucosa against injury via action on trleir common receptor (EGFR). The aim of this stu~ was to assess the expression of TGF~. EGF and EGFR in the gastric mucosaafter single exposureto stress. Material and Methods:25 rats were exposedto water restraintstress for 3,5h and then sacrific~ at 0, 2, 4, 6 and 12h. Each grouo included at least 5 rats. The number of stress lesions were counted and sections obtainedfrom the stomachwere stained immunohistochemically for proliferating ceil nuclear antigen (PCNA), TGF(~, EGF and EGFR (all antibodiesfrom Oncogene).At least 300 consecutiveceils were counted in each section to determine the rate of proliferation by PCNA index. The intensity of cytoplasmic staining for TGF(~and EGF were graded (0,1,2,3) by examinationof 300 consecutive ceils per section. In addition, we evaluatedthe numberof EGFR positive calls. All semiquantitative data were assessed according tO different regions of the gastric gland (top, neck and base). DNA synthesis was measured in the mucosat scraping by in vitro incorporationof 3H-thymidine into DNA. Results:The numberof ulcerationsat Ohwas about 15+3 and then graduallydeclined to about 4 ± 2 at 12h after stress: Labelling index for PCNA showed significant increase at 2h afterr stress and reached its highest level at 6h after exposure to stress. There was almost no staining for EGF immediately (0 h) after stress compared to the control (staining index, x=0.26). Staining intensity for EGF then increased reaching its peak at 6h after stress(x=0,93) and then declined at 12 h after stress. EGFRexpression increasedfrom 0 h, reaching peak at 4 h and then gradually declining from 6 h to t2 h. In contrast, immunostainingfor TGF(~increasedgradually from 0 to 12 h after stress with predominant staining of supedicial epithelial cells. At 0 h DNA synthesis fell by about 350 compared to intact gastric mucosa and then started to rise after 12 h after stress. Conclusions:This study providesan evidencethat there is a time-dependentincrease in TGF(~, EGF and EGFR expression in the gastric mucosa after single exposureto stress and this is accompanied by increased mucosal cell proliferation ano DNA synthesis. These findings suggest an important role of growth factors in the mucosel repair by cell proliferation and DNA synthesis after exposureto stress.
AMILORIDE SENSITIVE Na + CHANNEL EXPRESSION IN THE ILEUM AFTER TOTAL COLECTOMY IN RATS. K. Koyama, I. Sasaki, Y. Funayama, H. Naito, T. Tsuchiya, M. Unno, M.Takahashi, K. Fukushima, C. Shibata, S. Matsuno, Y. Suzuki*. 1st Dep. of Surgery, Tohoku University, School of Medicine, Sendal; * Dep. of Physiology, Shizuoka University, Shizuoka, Japan." Background and Aim: Clinical study have shown that after total colectomy, profuse and dehydration occur initially, but gradually subside over a period of several months. The mechanism for this gradual recovery is not clear, but may involve the ability of the gut to absorb water and sodium. It was recently reported that in normal rats, mRNA for the amiloride:Sensiti';,e sodium channel (rENaC) is detectable in distal colon but not in the small intestinel). Since rENaC is thought to play a key role in the control of intestinal Na+ absorption, we tested the hypothesis that after c01ectomy, rENaC expression in the remaining intestine would be up-regulated. We examined the effect of total colectomy with ileoanal anastomosis on amiloride-sensitive Na+ channel mRNA levels in rats. Methods: Fifty,five male Sprague-Dawley rats (250-300 g) were used. Total colectomy with ileoanal anastomosis (IAA) or sham surgery was performed under peutobarbital anesthesia. Urinary and fecal Na+ excretion; and plasma aldosterone levels were monitored for 14 and 28 days after IAA. At the end of the study ~periods, animal were anesthetized, intestinal mucosa was collected, and total mucosal RNA was isolated. Expression of rENaC was examined by Northern blot analysis. Results: Daily urinary output of Na+ was significantly decreased and fecal output of Na+ was significantly increased both 14 and 28 days after IAA. These changes appear to recover back towards normal by the end of the 28 days, although there was no significant difference in values between i4 days ahd 28 days after IAA. Plasma aldosterone levels were significantly elevated for 28 days after surgery. Expression of rENaC mRNA was not detected in before surgery and sham operative group but it was obviously detected bt~th 14 days and 28 days post-surgery. Conclusion: Total colectomy produced induction of rENaC expression; this may play a role in ihcreases in intestinal Na + absorption after colectomy. Reference:l) Cecilia M. Canessa, et al.: Amiloride-sensitive epitherial Na+ channel is made of three homologous subunits. Nature, 367;463-467,1994.