- Email: [email protected]
Comparison of the flow dynamics of erythrocytes in microvessels and glass capillaries
P1-25 COMPARISON OF THE FLOW DYNAMICS OF ERYTHROCYTES IN MICROVESSELS AND GLASS CAPILLARIES M. Soutani, Y. Suzuki, N. Tateishi, N. Maeda Dept. of Physiol., School of Medicine, Ehime Univ., Ehime, Japan Glass capillaries are generally used for the study of flow dynamics of erythrocytes in microvessels. The present study compares the flow dynamics of erythrocytes in glass capillaries and in microvessels. Hand-made glass capillaries and a part of microvascular bed isolated from rabbit mesentery (composed of a few branches of superior mesenteric artery: J. Biomechanics, in press) were used for the perfusion of human erythrocytes suspended in an isotonic phosphate-buffered saline containing 4 g/dl bovine serum albumin. The thickness of cell-free layer forming along the inside wall and the inner diameter of capillaries or microvessels were measured from the digitized video-images using an image processor. The flow velocity of erythrocytes was measured using a dual-spot cross-correlation technique (Circ. Res. 70: 812-819, 1992). (i) The thickness of cell-free layer increased in a saturation manner with an increase of inner diameter of glass capillaries and microvessels. When erythrocyte suspension of 16 % in hematocrit was perfused, the thickness was larger in microvessels than in glass capillaries in inner diameter more than lO ~m. The difference of the thickness was not detected in inner diameter less than I0 pm. (2) With increasing ~he flow velocity of erythrocytes, the thickness of cell-free layer increased. The effect of flow velocity was more clearly detected in glass capillaries. (3) With lowering the hematocrit, the thickness of cell-free layer increased. However, the influence of hematocrit on the thickness was smaller in glass capillaries than in microvessels. (4) A good correlation was obtained in microvessels between the maximum inner diameter for symmetrical (parachute-like) deformation (5 to 8 ~m in length of the deformed cell in the direction of flow axis) and the deformability of erythrocytes (the deformation index, determined by a high shear rheoscope) using diamide-treated cells (Biochim. Biophys. Acta 735: 104-I12, 1983). While, the length of symmetrically deformed cell could be correlated to the deformation index, using glass capillaries. The present results conclude that the flow dynamics of erythrocytes in microvessels is essentially different from those in glass capillaries, and that the symmetrical (parachutelike) deformation of erythrocytes in microvessels gives a quantitative measure of erythrocyte deformability.
P1-26 HIGH-DENSITY LIPOPROTEIN INHIBITS THE PRODUCTION OF PLATELET-ACTIVATING FACTOR ACETYLHYDROLASEBY HEPG2 CELLS K. Satoh, H. Yoshida, M. Koyama, M. Hiramoto, S. Takamatsu Department of Pathological Physiology, Institute of Neurological Disease, Hirosaki University School of Medicine, Hirosaki, J a p a n P l a t e l e t - a c t i v a t i n g f a c t o r (PAF) is a p h o s p h o l i p i d with a wide v a r i e t y o f bioactivities. PAF acetylhydrolase specifically inactivates PAF and may play an important role in the regulation of PAF activity in vivo. Human h e p a t o m a cell line HepG2 secretes PAF acetylhydrolase, which is identical to p l a s m a PAF a c e t y l h y d r o l a s e . PAF a c e t y l h y d r o l a s e is associated w i t h h i g h - d e n s i t y an d low-density lipoproteins (HDL and LDL). We examined the effects of plasma HDL and LDL on the production of PAF acetylhydrolase by HepG2 cells. HDL and LDL were separated by serial ultracentrifugation, and treated with d i i s o p r o p y l f l u o r o p h o s p h a t e to inhibit the endogenous PAF acetylhydrolase. PAF acet y l h y d r o l a s e activity of HepG2-conditioned medium was assayed using [2-acetyl-3H]PAF as the substrate. The activity in the co n t ro l medium conditioned for 24 hours was 37±2.6 p m o l / m g cell p r o t ei n (n=3). DFP-treated HDL inhibited the production in a c o n c e n t r a t i o n - d e p e n d e n t manner, and the inhibition at 100 ~tg/ml was 82%. DFP-treated LDL had a slight inhibitory effect. Metabolic labeling with [35S]methionine disclosed that the enzyme had a molecular weight of about 43 kD, which is consistent with plasma PAF acetylhydrolase. I n the presence of 100 I~g/ml HDL, the labeling of PAF acetylhydrolase was decreased to 42% of the co n t r ol value. DFP-treated HDL did not affect PAF acetylhydrolase activity of conditioned medium when t h e y were mixed immediately before the assay. However, when conditioned medium was incubated for 24 h o ur s with DFP-treated HDL, PAF acetylhydrolase activity was inhibited to 78% of the control. Analysis with gel f i l t r a t i o n c h r o m a t o g r a p h y disclosed that i n c u b a t i o n with HDL resulted in the t r a n s f e r of the LDLassociated e n z y m e to HDL fraction. Since the degradation of PAF by PAF aeetylhydrolase is known to proceed more efficiently in LDL than in HDL, the transfer of the enzyme to addedHDL may have resulted in the a p p a r e n t inhibition of PAF aeetylhydrolase activity. We conclude that plasma HDL inhibits the production of PAF acetylhydrolase by HepG2 cells. In addition, binding of the enzyme to added HDL alters the l i p o p ro te in e n v i r o n m e n t and results in apparent inhibition of the enzyme activity. This is a n o v e l role of HDL, w h i c h may be closely c o n c e r n e d with atherosclerosis. These results suggest t h a t plasma lipoproteins may regulate plasma PAF acetylhydrolase activity by their effect on the liver.
431
P1-26 HIGH-DENSITY LIPOPROTEIN INHIBITS THE PRODUCTION OF PLATELET-ACTIVATING FACTOR ACETYLHYDROLASEBY HEPG2 CELLS K. Satoh, H. Yoshida, M. Koyama, M. Hiramoto, S. Takamatsu Department of Pathological Physiology, Institute of Neurological Disease, Hirosaki University School of Medicine, Hirosaki, J a p a n P l a t e l e t - a c t i v a t i n g f a c t o r (PAF) is a p h o s p h o l i p i d with a wide v a r i e t y o f bioactivities. PAF acetylhydrolase specifically inactivates PAF and may play an important role in the regulation of PAF activity in vivo. Human h e p a t o m a cell line HepG2 secretes PAF acetylhydrolase, which is identical to p l a s m a PAF a c e t y l h y d r o l a s e . PAF a c e t y l h y d r o l a s e is associated w i t h h i g h - d e n s i t y an d low-density lipoproteins (HDL and LDL). We examined the effects of plasma HDL and LDL on the production of PAF acetylhydrolase by HepG2 cells. HDL and LDL were separated by serial ultracentrifugation, and treated with d i i s o p r o p y l f l u o r o p h o s p h a t e to inhibit the endogenous PAF acetylhydrolase. PAF acet y l h y d r o l a s e activity of HepG2-conditioned medium was assayed using [2-acetyl-3H]PAF as the substrate. The activity in the co n t ro l medium conditioned for 24 hours was 37±2.6 p m o l / m g cell p r o t ei n (n=3). DFP-treated HDL inhibited the production in a c o n c e n t r a t i o n - d e p e n d e n t manner, and the inhibition at 100 ~tg/ml was 82%. DFP-treated LDL had a slight inhibitory effect. Metabolic labeling with [35S]methionine disclosed that the enzyme had a molecular weight of about 43 kD, which is consistent with plasma PAF acetylhydrolase. I n the presence of 100 I~g/ml HDL, the labeling of PAF acetylhydrolase was decreased to 42% of the co n t r ol value. DFP-treated HDL did not affect PAF acetylhydrolase activity of conditioned medium when t h e y were mixed immediately before the assay. However, when conditioned medium was incubated for 24 h o ur s with DFP-treated HDL, PAF acetylhydrolase activity was inhibited to 78% of the control. Analysis with gel f i l t r a t i o n c h r o m a t o g r a p h y disclosed that i n c u b a t i o n with HDL resulted in the t r a n s f e r of the LDLassociated e n z y m e to HDL fraction. Since the degradation of PAF by PAF aeetylhydrolase is known to proceed more efficiently in LDL than in HDL, the transfer of the enzyme to addedHDL may have resulted in the a p p a r e n t inhibition of PAF aeetylhydrolase activity. We conclude that plasma HDL inhibits the production of PAF acetylhydrolase by HepG2 cells. In addition, binding of the enzyme to added HDL alters the l i p o p ro te in e n v i r o n m e n t and results in apparent inhibition of the enzyme activity. This is a n o v e l role of HDL, w h i c h may be closely c o n c e r n e d with atherosclerosis. These results suggest t h a t plasma lipoproteins may regulate plasma PAF acetylhydrolase activity by their effect on the liver.
431