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P-239 Interaction of the microtubule cytoskeleton with endocytic vesicles and cytoplasmic dynein in cultured rat hepatocytes
$96
Posters / International Hepatology Communications 3 Suppl. (1995) $37-S169
P-237 NEUTROPHIL-MED1ATED SINUSOIDAL CELL INJURY AFTER EXTENDED HEPATECTOMY IN CHOLESTATIC RATS FOLLOWED BY EXTERNAL DRAINAGE M.Ohtsuka, M.Miyazaki, H.Itoh, T.Kaiho,
S.Ambiru,
A.Togawa,
K.Sasada, M.Shiobara, Y.Shimizu, S.Yoshioka, H.Yoshidome, H.Omoto, A.Katoh, S.Nakamura, A.Okuno, Y.Nukui, S.Nozawa, H.Yoshitomi, N.Nakajima, Y.Kondo* 1st Dept. of Surgery and 2nd Dept. of Pathology*, Chiba Univ. School of Medicine, Chiba, Japan Liver failure after extended hepatectomy occurs more frequently in the patients with obstructive jaundice. We hypothesized that such a pathophysiological condition might be mediated by polymorphonuclear neutrophils (PMNs). Serum concentration of cytokine-induced neutrophil chemoattractant (CINC), which is one of the rat interleukin-8 family and a major mediator of PMN infiltration and activation in rats, and PMN accumulation in the hepatic sinusoids increased in 2-week-cholestatic rats. These changes were improved, but still persisted, when cholestasis was relieved by external drainage for 5 days, At this phase, serum alanine aminotransferase
(ALT) and purine nucleoside phosphorylase (PNP)
activities, however, remained almost unchanged. Following about 80% hepatectomy, CINC concentration surged much higher at 12 hours after hepatectomy, along with PMN accumulation. In addition, PNP activities tended to be raised and PNP/ALT ratio was significantly greater in cholestatic rats at 48 hours after hepatectomy. These results suggest that activated PMNs might contribute to the occurrence of sinusoidal cell injury after extended hepatectomy in cholestatic rats followed by external drainage.
P-239 XNTE~CTION OF THE MICROTUBULE CYTOSKELETON WITH ENDOCYTIC VESICLES AND CYTOPLASMIC DYNEIN IN CULTURED RAT HEPATOCYTES H. Oda*,+, R. J. Stockert+, P. M. Novikoff+, P. Satir+, S. Kuroki*, K. Chijiiwa*, M. Tanaka*, and A. W. Wolkoff+ *Dept. of Surgery I, Kyushu Univ., Fukuoka, Japan; +Liver Research Center, Albert Einstein Coll. of Med., Bronx, U.S.A. A requirement of mierotubules (MTs) in receptor-medaated endocytosis (RME) has been well known, but the molecular basis for this requirement is unclear. To elucidate this question, we quantified endocytic vesicles and the MT motors, cytoplasmic dynein and kinesin, which bind to MTs during RME of ASOR by overnight cultured rat hepatocytes. Following a 60 rain single wave of RME of ~=~I-ASOR,hepatocytes were homogenized at 4°C and centrifuged at 40,000 x g. The supematant was incubated at 37°C for 30 min with 20 HM taxol to polymerize endogenous tubulin followed by an additional 30 min incubation at 37°C with or without ATP or other nudeotides. The mixture was centrifuged at 16,000 x g to sedimentMTs and associated endocytic vesicles andproteius. Ligand (L) or receptor (R) -contaimng endocyfic vesicles, dynein, and kinesin were quantified in the MT-pellet by automdiogram or Western blot. In the presence of 5 mM ATP, binding of L-containing vesicles to MTs was reduced to 50.4 % _+ 1.4 % of control (p < 0.001) while that of Rcontaining vesicles was unchanged (97.5 % _+ 1.9 %). For various nudcotides, dynein binding to MTs corrdated significantly with binding of L-containing vesicles (r = 0.77), but not with binding of R-contaiuing vesicles (r = 0.06). Kinesin binding to MTs did not correlate with binding of endocytic vesicles containing either L or R. Analysis of endocytic vesicles that had been released from MTs by ATP revealed that Lcontaining vesicles were immunoprecipitated by monoclonal antibody to dyneia, but not by antibody to kinesin. Confocal microscopy showed that L-contaiuing endosomes colocalized on MTs with dyneia. These results support the view that in RME, L-contaiuing vesicles become bound to MTs by dyaein. While R-containing endosomes remain attached to MTs in an ATP-independeut manner, L-containing domains might be moved away towards pericentrosomal lysosomes by this motor molecules.
P-238
~ ' r r ' r c - r OF BILE ACIDS ON PHAGOCYTIC ACTIVITY ON K U P F F R CELl, M. Ftmaoka, M. Komatsu, T. Kuramitsu, d. Kato, T. ()no, T. Hoshino. T. lshii. I. To2,oshima, O. M a s a m t m e 1st Dept. of Medicine, A k i t a Univ. of Medicine 'Akita, J a p a n We a t t e m p t e d to examine how bile acids effect on phago~,tic activity and movement of Kupffer cell. Cells were isolated from m a l e Spraque-Dawley s t r a i n r a t s by pertizsion of the liver with collagenase a n d pronase-E a n d purified by percoll density gradient centrifugation, a n d seeded on a 24 m m cover slip at a concentration of I x 105 After 48 h culture x~ilh 3 ml GIT ( W A K e ) without sertnn, cells were incubated with taurocholic acid (TCP0, taurodeox),cholic acid (TDCA). taurochenodeoxycholic acid (TCDCA), gtycholitbocholic acid (GLC), or tauroursedeoxTcholic acid (TUDCA) at desired censentration for 3 h. and then 1.'3 × 11lTlatex particles (3pro d i a m e | e r . Polyscience) were added to each medium..M'ter 90 mira the n u m b e r of latex particles incorporated in a Kupffer cell were d e t e r m i n e d by counting 200 cells per cover slip. Phagocytic activity was significantly inhibited at the concentrations over 500 ~tM TDU.A a n d TCDCA ur over 100 IIM GLC. contraly enhanced at the concentralions up to 1.5 mM TCA a n d TUDCA. Ruffling m o v e m e n t s a n d dynamic scene of phagrocytusis of latex parlicles were observed by video- e n h a n c e d differential interterencecontrast (DIC) m i c r o s c o p y . TDCA, TCDCA, and GLC inhibited ruffling m o v e m e n t s a n d i n t r a c e l l u l a r organelle m o v e m e n t s di Kupffer ceils, they were thickened a n d formed filopodia. Contraly TUDCA e n h a n c e d m o v e m e n t s of ruffles a n d intracellular organelle, such as m i t o c h o n t h i a or cytoplasmic vesicles, in conclusion, a d m i n i s t r a t i o n of UDCA m a y play a p a r t in prevention of bacterial inl~ction in cholestasis.
P-240 QUANTITATIVE EVALUATION OF THE ARTERIAL HEMODYNAMICS OF WELL-DIFFERENTIATED HEPATOCELLULAR CARCINOMA BY ENHANCED ULTRASONO-GRAPHY Y.Mlyajimal)2), S.Sakaguchil), K.Tohara, J.Ohara, M.Tanaka, N.Hatono, T.Ueki, S.Nakabayashi, T.Yao and M.Okumura2) Dept. of Gastroenterology, Chikushi Hasp., Fukuoka Univ., Chikushino, Japan 1) and 1st Dept. of Int. Med., Fukuoka univ. , Fukuoka, Japan2) The enhanced ultrasonography(EU) injecting CO2 microbubbles into a hepatic artery showed excellent sensitivity in the detection of the arterial flow in a small hepatocellular carcinoma(HCC). However, well-differentiated HCC(wHCC) with fatty change and/or clear cells were demonstrated hyperechoic on a plain ultrasonogram. In these wHCC, it is difficult to judge whether they were enhanced by CO2 or not. We attempted to make quantitative evaluation of the intensity of the hepatic mass-echoes and their surrounded parenchyma in course of time after injection of CO2 using the Freeland system. Histologically diagnosed 77 hepatic masses less than 2cm in diameter were examined by EU and analyzed the intensity of the echoes of them using Freeland system. We defined Enhancement Index(El) as the slope ratio of the tumor to parenchyma calculated from their time intensity curves by this system. In 36 wHCC, E1(1.3+0.6) was significantly higher (p<0.01) than that(0.5_+0.6) of 6 borderline lesions and that(O.5_+0.4) of 16 regenerative nodules, and lower (P
Posters / International Hepatology Communications 3 Suppl. (1995) $37-S169
P-237 NEUTROPHIL-MED1ATED SINUSOIDAL CELL INJURY AFTER EXTENDED HEPATECTOMY IN CHOLESTATIC RATS FOLLOWED BY EXTERNAL DRAINAGE M.Ohtsuka, M.Miyazaki, H.Itoh, T.Kaiho,
S.Ambiru,
A.Togawa,
K.Sasada, M.Shiobara, Y.Shimizu, S.Yoshioka, H.Yoshidome, H.Omoto, A.Katoh, S.Nakamura, A.Okuno, Y.Nukui, S.Nozawa, H.Yoshitomi, N.Nakajima, Y.Kondo* 1st Dept. of Surgery and 2nd Dept. of Pathology*, Chiba Univ. School of Medicine, Chiba, Japan Liver failure after extended hepatectomy occurs more frequently in the patients with obstructive jaundice. We hypothesized that such a pathophysiological condition might be mediated by polymorphonuclear neutrophils (PMNs). Serum concentration of cytokine-induced neutrophil chemoattractant (CINC), which is one of the rat interleukin-8 family and a major mediator of PMN infiltration and activation in rats, and PMN accumulation in the hepatic sinusoids increased in 2-week-cholestatic rats. These changes were improved, but still persisted, when cholestasis was relieved by external drainage for 5 days, At this phase, serum alanine aminotransferase
(ALT) and purine nucleoside phosphorylase (PNP)
activities, however, remained almost unchanged. Following about 80% hepatectomy, CINC concentration surged much higher at 12 hours after hepatectomy, along with PMN accumulation. In addition, PNP activities tended to be raised and PNP/ALT ratio was significantly greater in cholestatic rats at 48 hours after hepatectomy. These results suggest that activated PMNs might contribute to the occurrence of sinusoidal cell injury after extended hepatectomy in cholestatic rats followed by external drainage.
P-239 XNTE~CTION OF THE MICROTUBULE CYTOSKELETON WITH ENDOCYTIC VESICLES AND CYTOPLASMIC DYNEIN IN CULTURED RAT HEPATOCYTES H. Oda*,+, R. J. Stockert+, P. M. Novikoff+, P. Satir+, S. Kuroki*, K. Chijiiwa*, M. Tanaka*, and A. W. Wolkoff+ *Dept. of Surgery I, Kyushu Univ., Fukuoka, Japan; +Liver Research Center, Albert Einstein Coll. of Med., Bronx, U.S.A. A requirement of mierotubules (MTs) in receptor-medaated endocytosis (RME) has been well known, but the molecular basis for this requirement is unclear. To elucidate this question, we quantified endocytic vesicles and the MT motors, cytoplasmic dynein and kinesin, which bind to MTs during RME of ASOR by overnight cultured rat hepatocytes. Following a 60 rain single wave of RME of ~=~I-ASOR,hepatocytes were homogenized at 4°C and centrifuged at 40,000 x g. The supematant was incubated at 37°C for 30 min with 20 HM taxol to polymerize endogenous tubulin followed by an additional 30 min incubation at 37°C with or without ATP or other nudeotides. The mixture was centrifuged at 16,000 x g to sedimentMTs and associated endocytic vesicles andproteius. Ligand (L) or receptor (R) -contaimng endocyfic vesicles, dynein, and kinesin were quantified in the MT-pellet by automdiogram or Western blot. In the presence of 5 mM ATP, binding of L-containing vesicles to MTs was reduced to 50.4 % _+ 1.4 % of control (p < 0.001) while that of Rcontaining vesicles was unchanged (97.5 % _+ 1.9 %). For various nudcotides, dynein binding to MTs corrdated significantly with binding of L-containing vesicles (r = 0.77), but not with binding of R-contaiuing vesicles (r = 0.06). Kinesin binding to MTs did not correlate with binding of endocytic vesicles containing either L or R. Analysis of endocytic vesicles that had been released from MTs by ATP revealed that Lcontaining vesicles were immunoprecipitated by monoclonal antibody to dyneia, but not by antibody to kinesin. Confocal microscopy showed that L-contaiuing endosomes colocalized on MTs with dyneia. These results support the view that in RME, L-contaiuing vesicles become bound to MTs by dyaein. While R-containing endosomes remain attached to MTs in an ATP-independeut manner, L-containing domains might be moved away towards pericentrosomal lysosomes by this motor molecules.
P-238
~ ' r r ' r c - r OF BILE ACIDS ON PHAGOCYTIC ACTIVITY ON K U P F F R CELl, M. Ftmaoka, M. Komatsu, T. Kuramitsu, d. Kato, T. ()no, T. Hoshino. T. lshii. I. To2,oshima, O. M a s a m t m e 1st Dept. of Medicine, A k i t a Univ. of Medicine 'Akita, J a p a n We a t t e m p t e d to examine how bile acids effect on phago~,tic activity and movement of Kupffer cell. Cells were isolated from m a l e Spraque-Dawley s t r a i n r a t s by pertizsion of the liver with collagenase a n d pronase-E a n d purified by percoll density gradient centrifugation, a n d seeded on a 24 m m cover slip at a concentration of I x 105 After 48 h culture x~ilh 3 ml GIT ( W A K e ) without sertnn, cells were incubated with taurocholic acid (TCP0, taurodeox),cholic acid (TDCA). taurochenodeoxycholic acid (TCDCA), gtycholitbocholic acid (GLC), or tauroursedeoxTcholic acid (TUDCA) at desired censentration for 3 h. and then 1.'3 × 11lTlatex particles (3pro d i a m e | e r . Polyscience) were added to each medium..M'ter 90 mira the n u m b e r of latex particles incorporated in a Kupffer cell were d e t e r m i n e d by counting 200 cells per cover slip. Phagocytic activity was significantly inhibited at the concentrations over 500 ~tM TDU.A a n d TCDCA ur over 100 IIM GLC. contraly enhanced at the concentralions up to 1.5 mM TCA a n d TUDCA. Ruffling m o v e m e n t s a n d dynamic scene of phagrocytusis of latex parlicles were observed by video- e n h a n c e d differential interterencecontrast (DIC) m i c r o s c o p y . TDCA, TCDCA, and GLC inhibited ruffling m o v e m e n t s a n d i n t r a c e l l u l a r organelle m o v e m e n t s di Kupffer ceils, they were thickened a n d formed filopodia. Contraly TUDCA e n h a n c e d m o v e m e n t s of ruffles a n d intracellular organelle, such as m i t o c h o n t h i a or cytoplasmic vesicles, in conclusion, a d m i n i s t r a t i o n of UDCA m a y play a p a r t in prevention of bacterial inl~ction in cholestasis.
P-240 QUANTITATIVE EVALUATION OF THE ARTERIAL HEMODYNAMICS OF WELL-DIFFERENTIATED HEPATOCELLULAR CARCINOMA BY ENHANCED ULTRASONO-GRAPHY Y.Mlyajimal)2), S.Sakaguchil), K.Tohara, J.Ohara, M.Tanaka, N.Hatono, T.Ueki, S.Nakabayashi, T.Yao and M.Okumura2) Dept. of Gastroenterology, Chikushi Hasp., Fukuoka Univ., Chikushino, Japan 1) and 1st Dept. of Int. Med., Fukuoka univ. , Fukuoka, Japan2) The enhanced ultrasonography(EU) injecting CO2 microbubbles into a hepatic artery showed excellent sensitivity in the detection of the arterial flow in a small hepatocellular carcinoma(HCC). However, well-differentiated HCC(wHCC) with fatty change and/or clear cells were demonstrated hyperechoic on a plain ultrasonogram. In these wHCC, it is difficult to judge whether they were enhanced by CO2 or not. We attempted to make quantitative evaluation of the intensity of the hepatic mass-echoes and their surrounded parenchyma in course of time after injection of CO2 using the Freeland system. Histologically diagnosed 77 hepatic masses less than 2cm in diameter were examined by EU and analyzed the intensity of the echoes of them using Freeland system. We defined Enhancement Index(El) as the slope ratio of the tumor to parenchyma calculated from their time intensity curves by this system. In 36 wHCC, E1(1.3+0.6) was significantly higher (p<0.01) than that(0.5_+0.6) of 6 borderline lesions and that(O.5_+0.4) of 16 regenerative nodules, and lower (P