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The fine structure of endothelial cells in freeze-fracture preparations
JOURNAL OF ULTRASTRUCTURE RESEARCH 54, 22-28 (1976)
The Fine Structure of Endothelial Cells in Freeze-Fracture Preparations K o J I YAMAMOTO, SUNAO FUJIMOTO, AND YOSHIO TAKESHIGE
Department of Anatomy, Kurume University, School of Medicine, Kurume, Japan Received March 25, 1975 Endothelial cells of small blood vessels and capillaries of rabbits have been examined in freeze-fracture preparations. In small arteries of the penis and in myocardial capillaries, the cells contain a variable distribution and number of pinocytotic caveoles. The greatest concentrations were observed at the tissue front adjacent to the lumen. In kidney tissue, pores of fenestrated capillaries were usually seen on the endothelial surface, where they may be clustered together or grouped into chainlike arrangements. However, they have not been found in the vicinity of the cell boundaries. Cleaved images between adjacent endothelial cells have been observed in capillaries of both brain and myocardium. The extracellular surfaces of tight junctions are characterized by a network of ridgelike particles about 100 ~ in diameter.
The fine structure of endothelial cells from various blood vessels has been widely studied with T E M and utilized for interpretation of physiological findings on vascular permeability. Although the recent application of freeze-cleaving techniques has revealed three-dimensional features of various cells and tissues (4, 8, 14, I5), such studies, with respect to vascular endothelia, have thus far been very limited. Leak (9) and Simionescu et al. (I8), for example, have studied the distribution of pinocytotic caveoles of capillary endothelial cells, and Friederici (2, 3) and Maul (11) have shown that fenestrated capillaries contain numerous pores. Further studies of freeze-fractured endothelia might contribute additional information on vascular permeability. The purpose of this study is to examine the three-dimensional structure of endothelial cells from various vessels with respect to obtaining detailed information on: (1) the distribution of pinocytotic caveoles and pores, and (2) the endothelial cell boundaries, especially regions of tight junctions.
M cacodylate buffer (pH 7.4) was then perfused through each animal in the same manner for 5 min. The brain, kidney, myocardium, and corpus cavernosum were then dissected out and immersed in the fixative for 3 hr, rinsed 30 min in 0.1 M cacodylate buffer, and then soaked in 40% cacodylate buffered glycerol solution for more than 2 hr. The tissues were cut into small pieces (approximately 1 x 1 x 3 mm), frozen in liquid nitrogen, and fractured by a Hitachi freeze-etch device (HFZ-1 type) at a pressure of 5 x 10 -8 Torr. Carbon-platinum film replicas of the fractured surfaces were made and the cleaned films were observed with a Hitachi HU-12AS electron microscope. RESULTS
Pinocytotic caveoles of the endothelial plasmalemma. Pinocytotic caveoles of the endothelial plasma m e m b r a n e were observed from small vessels of the corpus cavernosum and myocardium. Four different faces of the plasma m e m b r a n e can be seen in fractures of endothelial cells: (1) a cytoplasmic (A) face on the luminal front (AL), (2) an A face on the tissue front (AT), (3) an extracellular (B) face on the luminal front (BL), and (4) a B face on the tissue front (BT). The caveoles on both the luminal and tissue fronts have a craterlike appearance on the B face and a complem e n t a l appearance on the A face. However, a few caveoles on the B face are not craterlike, b u t appear elevated. These fea-
MATERIALS AND METHODS Six adult rabbits were perfused by heart puncture with physiological saline until the venous effluent was free of blood. A solution of 2.5% glutaraldehyde in 0.1 22 Copyright © 1976by Academic Press, Inc. All rights of reproduction in any form reserved.
ENDOTHELIAL CELLS IN FREEZE-FRACTURE
23
tures suggest the existence of many thin some pores lack a diaphragm. This may projections on the extracellular surface of argue for the existence of a "large pore" proposed by Clementi and Palade (1). A the caveolar membranes (Fig. 1). The luminal and tissue fronts of endo- considerable number of pores appear to thelial plasma membranes from a small lack the central knob of the diaphragm (see artery of the corpus cavernosum are shown arrows of Fig. 4). There is no remarkable in Fig. 2. The pinocytotic caveoles are difference in size between pores with or irregularly distributed on both fronts and without the central knob. The central knob show a distinct density difference on the is variable in diameter, larger ones measurtwo faces. In this example, there are twice ing about 470 ~,, smaller ones measuring as many on the tissue front as on the about 130 A. Division of the tips is evident luminal front. Fig. 3 shows a fractured in a few cases. image of two adjacent endothelial cells of a Endothelial cell junctions. Cleaved prepmyocardial capillary. Four faces of the arations of endothelial cells also revealed plasma membrane are represented: (1) the various aspects of the cell boundary region. B face of the tissue front (BT), (2) the A In nonfenestrated capillary of the brain, face of t h e luminal front (AL) of the cell on the endothelial cells are intricately apthe right side, (3) the A face of the luminal posed to one another (Fig. 6). The cleaved front (AL), and (4) the B face of the tissue image of a tight junction is shown in the front (BT) of the left cell. Although the lower right of the micrograph. The cleaved number of pinocytotic caveoles of the right planes of the endothelial tight junctions cell appears to be about the same as that of usually appear either as linear and anasthe luminal and tissue fronts, they are tomosing ridges on the A face, or as correquite different in number between both sponding grooves on the B face. At higher faces of the left cell. Pinocytotic caveoles magnifications, the A face of nonfeneshave consistently been found to vary in trated brain capillaries appears as an exnumber from cell to cell. However, the tensive continuous network of small elefrequency of pinocytotic caveoles on the vated ridges (100 A in diameter, Fig. 7). tissue front is always the same as or greater The extension of such networks is very than that of the luminal front. limited in the case of myocardial capilPores of fenestrated capillaries. Endo- laries (Fig. 8). Since this micrograph shows thelial pores of fenestrated capillaries were only the B face of the cleaved plane, the observed in the proximal tubule of the extent of the network is quite limited and kidney. In fractured preparations, they appears to be discontinuous. The cleaved appear to be craterlike on the B face and plane of a myocardial capillary tight junccomplementary to this appearance on the tion appears to consist of several localized A face, similar to the pinocytotic caveoles. network regions that are discontinuous to The cleaved images of each pore are almost one another. The cleaved images of termithe same dimension as that calculated nal bars in the epithelium of small intesfrom sectioned specimens (X = 640 A ; R = tine also appear as continuous networks, 530-660 •, Fig. 4). They are clustered or and it is apparent that each network is organized in the form of distinct chains much larger (4). over the endothelial surface. However, they DISCUSSION never exist near the region of the endothelial cell boundary (Fig. 5). Leak (9) reported that nearly all endoThree-dimensional features of the dia- thelial cells of mouse myocardial capilphragm and its central knob also were laries have a similar density of pinocytotic examined. Our micrographs indicate that caveoles between the luminal and the tis-
24
YAMAMOTO, FUJIMOTO AND TAKESHIGE
FI(~. 1. S m a l l artery of t h e corpus c a v e r n o s u m . T h e fracture exposes t h e B face of the p l a s m a l e m m a on t h e tissue front. T h e direction of s h a d o w i n g is i n d i c a t e d by t h e arrow, x 44 000. Fro. 2. E n d o t h e l i a l cell of a s m a l l artery in t h e corpus c a v e r n o s u m . Diagonally cleaved i m a g e of t h e cells (E) shows b o t h t h e l u m i n a l (AL) a n d t h e tissue front (BT). x 40 000.
ENDOTHELIAL CELLS IN FREEZE-FRACTURE
25
FIG. 3. Myocardial capillary. The fracture followed the cleaved plane of the B face of the tissue front (BT), the A face of the luminal front (AL) of the endothelial cell (E) on the right side, and then followed the A face of the luminal front (AL), and the B face of the tissue front (BT) on the left side. L; lumen, x 18 000. FIG. 4. Fenestrated capillary of a kidney proximal tubule. Central knobs (CK) are seen oll the diaphragm, but are lacking in some pores (arrows). x 89 000. sue fronts. T h e s e d a t a argue a g a i n s t our findings from t h e s m a l l a r t e r y of t h e penis a n d the m y o c a r d i a l c a p i l l a r y . A l t h o u g h we o b s e r v e d a few e n d o t h e l i a l cells w i t h similar d e n s i t i e s b e t w e e n b o t h fronts, our obs e r v a t i o n s i n d i c a t e a h i g h e r d e n s i t y at the tissue front. W e have not o b s e r v e d endot h e l i a l cells w i t h a g r e a t e r d e n s i t y of caveoles on t h e l u m i n a l front. Our o b s e r v a tions are c o n s i s t e n t w i t h t h e d a t a of S i m ionescu et al. (18), which show t h a t the d e n s i t y of caveoles in r a t m u s c l e c a p i l l a r y is higher (by 20-40%) on t h e tissue front. It
is possible t h a t the higher d e n s i t y of caveoles on the tissue front is c h a r a c t e r i s t i c of e n d o t h e l i a l cells of m a n y vessels. Since p i n o c y t o t i c caveoles or vesicles are involved in e n d o t h e l i a l cell p e r m e a b i l i t y (6, I0, 13, 16, 19), it is r e a s o n a b l e to a s s u m e t h a t a higher d e n s i t y on the tissue front is r e l a t e d to h i g h e r m o l e c u l a r pass a c t i v i t y for a b s o r p t i o n a n d secretion. Regional differences in t h e p e r m e a b i l i t y of large molecules in various c a p i l l a r i e s h a v e been e x p l a i n e d in p h y s i o l o g i c a l s t u d ies by a s s u m i n g t h a t t h e r e are two sets of
26
YAMAMOTO, FUJIMOTO AND TAKESHIGE
FIG. 5. Fenestrated capillary of a kidney proximal tubule. Surface image of the endothelial cells show the existence of abundant pores (P). CB, ceil boundary region; L, lumen, x 14 000.
FIG. 6. A face of a tight j u n c t i o n (TJ) from a b r a i n capillary. Craters of pinocytotic caveoles, as seen in Fig. 2, are not seen on the surface of the endothelial cell (E). L, lumen. × 27 000. Fro. 7. B r a i n capillary. A higher m a g n i f i c a t i o n of A face of the tight j u n c t i o n s (TJ). x 78 000. FIG. 8. Myocardial capillary. B face of the tight j u n c t i o n s (TJ) in an endothelial cell is shown. L, lumen; M, cardiac muscle, x 57 000. 27
28
YAMAMOTO, FUJIMOTO AND TAKESHIGE
pores (5, I2). The fenestrations of certain capillary endothelial cells (e.g., intestinal mucosa and kidney) observed in electron micrographs may be the structural equivalent of "pores" supposed by previous physiologists. Recently, Clementi and Palade (1) proposed from TEM observations, that the "large pore" system proposed by physiological investigations, corresponds to endothelial pores lacking a diaphragm, and the "small pore" system to those possessing a diaphragm. The distribution of endothelial cell pores of fenestrated capillaries is apparent in our freeze fracture preparations. Such studies may be suitable for examination of the ratios of pores with diaphragms to those without. In endothelial cells of kidney capillaries, a ratio of 12 to 1 was observed. If we accept the proposal by Clementi and Palade (1) that the "small pore" system is the structural equivalent of pores possessing a diaphragm in fenestrated capillaries, then our data conflict with the ratios determined from physiological calculations (5), where the ratio of small to large pores is 340:1. Cleaved images of endothelial cell junctions, especially tight junctions, were observed in brain and myocardial capillaries. Several workers (e.g. 7, i7) have classified two types of junctions: the zonula occuludens and the macula occuludens. In the latter study, horseradish peroxidase was observed to pass through the endothelial junctions. Karnovsky (7) observed from serial observations that adjacent macula occuludens are separated from each other by 40 A. The cleaved surface of endothelial tight junctions from brain capillaries is characterized by an extensive continuous network, consisting either of chains of elevated ridgelike particles on the A face or the corresponding grooves on the B face. Although a similar network is also characteristic of the tight junction of myo-
.cardial capillaries, they are more limited in extent and are discontinuous. Thus, it seems likely that molecules may pass across the endothelial cells between the regions of discontinuity. Furthermore, it is interesting that the networks of endothelial tight junctions are much coarser than those of the intestinal terminal bar (4). More detailed observations on fractured images of tight junctions, particularly with reference to endothelial permeability, are now in progress in our laboratory. The authors wish to express their thanks to Dr. D. R. Stokes, Emory University, Atlanta, Georgia, for correcting the manuscript. REFERENCES 1. CLEMENTI,F., ANDPALADE,G. E., J. Cell Biol. 41, 33 (1969). 2. FRIEDERICI~H. H. R., J. Ultrastruct. Res. 23, 444 (1968). 3. FRIEDERICI,H. H. R., J. Ultrastruct. Res. 27, 373 (1969). 4. FRIEND,D. S., ANDGILULA,N. B., J. Cell Biol. 53, 758 (1972). 5. GROTTE, G., Acta Chir. Scand. Suppl. 211, 1 (1956). 6. JENNING, M. A., MARCHESI~ V. T., AND FLOREY, H., Proc. Roy. Soc. (London) Ser. B. 156, 14 (1962). 7. KARNOVSKY,M. J., J. Cell Biol. 35, 213, {1967). 8. LEAK, L. V., J. Ultrastruct. Res. 25, 253 (1968). 9. LEAK, L. V., J. Ultrastruct. Res. 35, 127 (1971). 10. MARCHESI, V. T., AND BARRNETT, R. J., J. Cell Biol. 17, 547 (1963). 11. MAUL, G. G., J. Ultrastruct. Res. 36:768 (1971). 12. MAYERSON,H. S., WOLFRAM, C. G., SHIRLEY, H. H., JR., ANDWASSERMAN,K., Amer. J. Physiol. 198, 155 (1960). 13. MOORE, D. H., AND RUSKA, M., J. Biophysic. Biochem. Cytol. 3, 457 (1957). 14. ORCl, L., MATTER, A., AND ROUILLER, CH., J. Ultrastruct. Res. 35, 1 (1971). 15. ORWIN,D. F. G., THOMSON,R. W., ANDFLOWER,N.
E., J. Ultrastruct. Res. 45, 30 (1973). i6. PALADE,G. E., g. Appl. Phys. 24, 1424 (1953). 17. REESE, W. S., ANDKARNOVSKY,M. J., J. Cell Biol. 34, 207 (1967). 18. SIMIONESCU,M., SIMIONESCU,N., AND PALADE, G. E., J. Cell Biol. 60, 128 (1974).. 19. Wmsm, S. L., Anat. Rec. 130, 467 (1958).
The Fine Structure of Endothelial Cells in Freeze-Fracture Preparations K o J I YAMAMOTO, SUNAO FUJIMOTO, AND YOSHIO TAKESHIGE
Department of Anatomy, Kurume University, School of Medicine, Kurume, Japan Received March 25, 1975 Endothelial cells of small blood vessels and capillaries of rabbits have been examined in freeze-fracture preparations. In small arteries of the penis and in myocardial capillaries, the cells contain a variable distribution and number of pinocytotic caveoles. The greatest concentrations were observed at the tissue front adjacent to the lumen. In kidney tissue, pores of fenestrated capillaries were usually seen on the endothelial surface, where they may be clustered together or grouped into chainlike arrangements. However, they have not been found in the vicinity of the cell boundaries. Cleaved images between adjacent endothelial cells have been observed in capillaries of both brain and myocardium. The extracellular surfaces of tight junctions are characterized by a network of ridgelike particles about 100 ~ in diameter.
The fine structure of endothelial cells from various blood vessels has been widely studied with T E M and utilized for interpretation of physiological findings on vascular permeability. Although the recent application of freeze-cleaving techniques has revealed three-dimensional features of various cells and tissues (4, 8, 14, I5), such studies, with respect to vascular endothelia, have thus far been very limited. Leak (9) and Simionescu et al. (I8), for example, have studied the distribution of pinocytotic caveoles of capillary endothelial cells, and Friederici (2, 3) and Maul (11) have shown that fenestrated capillaries contain numerous pores. Further studies of freeze-fractured endothelia might contribute additional information on vascular permeability. The purpose of this study is to examine the three-dimensional structure of endothelial cells from various vessels with respect to obtaining detailed information on: (1) the distribution of pinocytotic caveoles and pores, and (2) the endothelial cell boundaries, especially regions of tight junctions.
M cacodylate buffer (pH 7.4) was then perfused through each animal in the same manner for 5 min. The brain, kidney, myocardium, and corpus cavernosum were then dissected out and immersed in the fixative for 3 hr, rinsed 30 min in 0.1 M cacodylate buffer, and then soaked in 40% cacodylate buffered glycerol solution for more than 2 hr. The tissues were cut into small pieces (approximately 1 x 1 x 3 mm), frozen in liquid nitrogen, and fractured by a Hitachi freeze-etch device (HFZ-1 type) at a pressure of 5 x 10 -8 Torr. Carbon-platinum film replicas of the fractured surfaces were made and the cleaned films were observed with a Hitachi HU-12AS electron microscope. RESULTS
Pinocytotic caveoles of the endothelial plasmalemma. Pinocytotic caveoles of the endothelial plasma m e m b r a n e were observed from small vessels of the corpus cavernosum and myocardium. Four different faces of the plasma m e m b r a n e can be seen in fractures of endothelial cells: (1) a cytoplasmic (A) face on the luminal front (AL), (2) an A face on the tissue front (AT), (3) an extracellular (B) face on the luminal front (BL), and (4) a B face on the tissue front (BT). The caveoles on both the luminal and tissue fronts have a craterlike appearance on the B face and a complem e n t a l appearance on the A face. However, a few caveoles on the B face are not craterlike, b u t appear elevated. These fea-
MATERIALS AND METHODS Six adult rabbits were perfused by heart puncture with physiological saline until the venous effluent was free of blood. A solution of 2.5% glutaraldehyde in 0.1 22 Copyright © 1976by Academic Press, Inc. All rights of reproduction in any form reserved.
ENDOTHELIAL CELLS IN FREEZE-FRACTURE
23
tures suggest the existence of many thin some pores lack a diaphragm. This may projections on the extracellular surface of argue for the existence of a "large pore" proposed by Clementi and Palade (1). A the caveolar membranes (Fig. 1). The luminal and tissue fronts of endo- considerable number of pores appear to thelial plasma membranes from a small lack the central knob of the diaphragm (see artery of the corpus cavernosum are shown arrows of Fig. 4). There is no remarkable in Fig. 2. The pinocytotic caveoles are difference in size between pores with or irregularly distributed on both fronts and without the central knob. The central knob show a distinct density difference on the is variable in diameter, larger ones measurtwo faces. In this example, there are twice ing about 470 ~,, smaller ones measuring as many on the tissue front as on the about 130 A. Division of the tips is evident luminal front. Fig. 3 shows a fractured in a few cases. image of two adjacent endothelial cells of a Endothelial cell junctions. Cleaved prepmyocardial capillary. Four faces of the arations of endothelial cells also revealed plasma membrane are represented: (1) the various aspects of the cell boundary region. B face of the tissue front (BT), (2) the A In nonfenestrated capillary of the brain, face of t h e luminal front (AL) of the cell on the endothelial cells are intricately apthe right side, (3) the A face of the luminal posed to one another (Fig. 6). The cleaved front (AL), and (4) the B face of the tissue image of a tight junction is shown in the front (BT) of the left cell. Although the lower right of the micrograph. The cleaved number of pinocytotic caveoles of the right planes of the endothelial tight junctions cell appears to be about the same as that of usually appear either as linear and anasthe luminal and tissue fronts, they are tomosing ridges on the A face, or as correquite different in number between both sponding grooves on the B face. At higher faces of the left cell. Pinocytotic caveoles magnifications, the A face of nonfeneshave consistently been found to vary in trated brain capillaries appears as an exnumber from cell to cell. However, the tensive continuous network of small elefrequency of pinocytotic caveoles on the vated ridges (100 A in diameter, Fig. 7). tissue front is always the same as or greater The extension of such networks is very than that of the luminal front. limited in the case of myocardial capilPores of fenestrated capillaries. Endo- laries (Fig. 8). Since this micrograph shows thelial pores of fenestrated capillaries were only the B face of the cleaved plane, the observed in the proximal tubule of the extent of the network is quite limited and kidney. In fractured preparations, they appears to be discontinuous. The cleaved appear to be craterlike on the B face and plane of a myocardial capillary tight junccomplementary to this appearance on the tion appears to consist of several localized A face, similar to the pinocytotic caveoles. network regions that are discontinuous to The cleaved images of each pore are almost one another. The cleaved images of termithe same dimension as that calculated nal bars in the epithelium of small intesfrom sectioned specimens (X = 640 A ; R = tine also appear as continuous networks, 530-660 •, Fig. 4). They are clustered or and it is apparent that each network is organized in the form of distinct chains much larger (4). over the endothelial surface. However, they DISCUSSION never exist near the region of the endothelial cell boundary (Fig. 5). Leak (9) reported that nearly all endoThree-dimensional features of the dia- thelial cells of mouse myocardial capilphragm and its central knob also were laries have a similar density of pinocytotic examined. Our micrographs indicate that caveoles between the luminal and the tis-
24
YAMAMOTO, FUJIMOTO AND TAKESHIGE
FI(~. 1. S m a l l artery of t h e corpus c a v e r n o s u m . T h e fracture exposes t h e B face of the p l a s m a l e m m a on t h e tissue front. T h e direction of s h a d o w i n g is i n d i c a t e d by t h e arrow, x 44 000. Fro. 2. E n d o t h e l i a l cell of a s m a l l artery in t h e corpus c a v e r n o s u m . Diagonally cleaved i m a g e of t h e cells (E) shows b o t h t h e l u m i n a l (AL) a n d t h e tissue front (BT). x 40 000.
ENDOTHELIAL CELLS IN FREEZE-FRACTURE
25
FIG. 3. Myocardial capillary. The fracture followed the cleaved plane of the B face of the tissue front (BT), the A face of the luminal front (AL) of the endothelial cell (E) on the right side, and then followed the A face of the luminal front (AL), and the B face of the tissue front (BT) on the left side. L; lumen, x 18 000. FIG. 4. Fenestrated capillary of a kidney proximal tubule. Central knobs (CK) are seen oll the diaphragm, but are lacking in some pores (arrows). x 89 000. sue fronts. T h e s e d a t a argue a g a i n s t our findings from t h e s m a l l a r t e r y of t h e penis a n d the m y o c a r d i a l c a p i l l a r y . A l t h o u g h we o b s e r v e d a few e n d o t h e l i a l cells w i t h similar d e n s i t i e s b e t w e e n b o t h fronts, our obs e r v a t i o n s i n d i c a t e a h i g h e r d e n s i t y at the tissue front. W e have not o b s e r v e d endot h e l i a l cells w i t h a g r e a t e r d e n s i t y of caveoles on t h e l u m i n a l front. Our o b s e r v a tions are c o n s i s t e n t w i t h t h e d a t a of S i m ionescu et al. (18), which show t h a t the d e n s i t y of caveoles in r a t m u s c l e c a p i l l a r y is higher (by 20-40%) on t h e tissue front. It
is possible t h a t the higher d e n s i t y of caveoles on the tissue front is c h a r a c t e r i s t i c of e n d o t h e l i a l cells of m a n y vessels. Since p i n o c y t o t i c caveoles or vesicles are involved in e n d o t h e l i a l cell p e r m e a b i l i t y (6, I0, 13, 16, 19), it is r e a s o n a b l e to a s s u m e t h a t a higher d e n s i t y on the tissue front is r e l a t e d to h i g h e r m o l e c u l a r pass a c t i v i t y for a b s o r p t i o n a n d secretion. Regional differences in t h e p e r m e a b i l i t y of large molecules in various c a p i l l a r i e s h a v e been e x p l a i n e d in p h y s i o l o g i c a l s t u d ies by a s s u m i n g t h a t t h e r e are two sets of
26
YAMAMOTO, FUJIMOTO AND TAKESHIGE
FIG. 5. Fenestrated capillary of a kidney proximal tubule. Surface image of the endothelial cells show the existence of abundant pores (P). CB, ceil boundary region; L, lumen, x 14 000.
FIG. 6. A face of a tight j u n c t i o n (TJ) from a b r a i n capillary. Craters of pinocytotic caveoles, as seen in Fig. 2, are not seen on the surface of the endothelial cell (E). L, lumen. × 27 000. Fro. 7. B r a i n capillary. A higher m a g n i f i c a t i o n of A face of the tight j u n c t i o n s (TJ). x 78 000. FIG. 8. Myocardial capillary. B face of the tight j u n c t i o n s (TJ) in an endothelial cell is shown. L, lumen; M, cardiac muscle, x 57 000. 27
28
YAMAMOTO, FUJIMOTO AND TAKESHIGE
pores (5, I2). The fenestrations of certain capillary endothelial cells (e.g., intestinal mucosa and kidney) observed in electron micrographs may be the structural equivalent of "pores" supposed by previous physiologists. Recently, Clementi and Palade (1) proposed from TEM observations, that the "large pore" system proposed by physiological investigations, corresponds to endothelial pores lacking a diaphragm, and the "small pore" system to those possessing a diaphragm. The distribution of endothelial cell pores of fenestrated capillaries is apparent in our freeze fracture preparations. Such studies may be suitable for examination of the ratios of pores with diaphragms to those without. In endothelial cells of kidney capillaries, a ratio of 12 to 1 was observed. If we accept the proposal by Clementi and Palade (1) that the "small pore" system is the structural equivalent of pores possessing a diaphragm in fenestrated capillaries, then our data conflict with the ratios determined from physiological calculations (5), where the ratio of small to large pores is 340:1. Cleaved images of endothelial cell junctions, especially tight junctions, were observed in brain and myocardial capillaries. Several workers (e.g. 7, i7) have classified two types of junctions: the zonula occuludens and the macula occuludens. In the latter study, horseradish peroxidase was observed to pass through the endothelial junctions. Karnovsky (7) observed from serial observations that adjacent macula occuludens are separated from each other by 40 A. The cleaved surface of endothelial tight junctions from brain capillaries is characterized by an extensive continuous network, consisting either of chains of elevated ridgelike particles on the A face or the corresponding grooves on the B face. Although a similar network is also characteristic of the tight junction of myo-
.cardial capillaries, they are more limited in extent and are discontinuous. Thus, it seems likely that molecules may pass across the endothelial cells between the regions of discontinuity. Furthermore, it is interesting that the networks of endothelial tight junctions are much coarser than those of the intestinal terminal bar (4). More detailed observations on fractured images of tight junctions, particularly with reference to endothelial permeability, are now in progress in our laboratory. The authors wish to express their thanks to Dr. D. R. Stokes, Emory University, Atlanta, Georgia, for correcting the manuscript. REFERENCES 1. CLEMENTI,F., ANDPALADE,G. E., J. Cell Biol. 41, 33 (1969). 2. FRIEDERICI~H. H. R., J. Ultrastruct. Res. 23, 444 (1968). 3. FRIEDERICI,H. H. R., J. Ultrastruct. Res. 27, 373 (1969). 4. FRIEND,D. S., ANDGILULA,N. B., J. Cell Biol. 53, 758 (1972). 5. GROTTE, G., Acta Chir. Scand. Suppl. 211, 1 (1956). 6. JENNING, M. A., MARCHESI~ V. T., AND FLOREY, H., Proc. Roy. Soc. (London) Ser. B. 156, 14 (1962). 7. KARNOVSKY,M. J., J. Cell Biol. 35, 213, {1967). 8. LEAK, L. V., J. Ultrastruct. Res. 25, 253 (1968). 9. LEAK, L. V., J. Ultrastruct. Res. 35, 127 (1971). 10. MARCHESI, V. T., AND BARRNETT, R. J., J. Cell Biol. 17, 547 (1963). 11. MAUL, G. G., J. Ultrastruct. Res. 36:768 (1971). 12. MAYERSON,H. S., WOLFRAM, C. G., SHIRLEY, H. H., JR., ANDWASSERMAN,K., Amer. J. Physiol. 198, 155 (1960). 13. MOORE, D. H., AND RUSKA, M., J. Biophysic. Biochem. Cytol. 3, 457 (1957). 14. ORCl, L., MATTER, A., AND ROUILLER, CH., J. Ultrastruct. Res. 35, 1 (1971). 15. ORWIN,D. F. G., THOMSON,R. W., ANDFLOWER,N.
E., J. Ultrastruct. Res. 45, 30 (1973). i6. PALADE,G. E., g. Appl. Phys. 24, 1424 (1953). 17. REESE, W. S., ANDKARNOVSKY,M. J., J. Cell Biol. 34, 207 (1967). 18. SIMIONESCU,M., SIMIONESCU,N., AND PALADE, G. E., J. Cell Biol. 60, 128 (1974).. 19. Wmsm, S. L., Anat. Rec. 130, 467 (1958).