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IV. Peroxidase technique
V I. P E R O X I D A S E
TECHNIQUE
This technique, originated by Straus (90) and Daems et al. (23), and much improved by modifications of the substrate (Graham and Karnovsky, 40), gives a black reaction product of high contrast and of a clumpy nature. The terms reaction product (RP), activity, staining, and peroxidase will be used interchangeably.
Controls a. Noninjected animals', nonincubated. Droplets of the same electron density as the reaction product are found in pericanalicular dense bodies of the hepatocytes and in the residual bodies of Kupffer cells. Fat droplets and osmiophilic droplets in the Golgi apparatus are homogeneous and exhibit a sharp outline. Glycogen is recognized by its particulate nature. b. Noninjected animals, incubated. With an incubation time of 15-30 min, reaction product is found in three different types: on the whole surface of erythrocytes, in the granules of eosinophilic leukocytes, and in the pericanalicular dense bodies (Figs. 44 and 45). In the pericanalicular dense bodies, it is very difficult to distinguish the reaction product from the droplets found in the dense bodies in noninjected and nonincubated controls. At concentrations as high as 0.1%, this reaction is much more pronounced (Fig. 46). It is remarkable to find reaction product in autophagosomes. It may represent catalase, exhibiting peroxidatic activity (Figs. 38 and 46). Microbodies show no activity with our incubation time schedules. Without hydrogen peroxide, these reactions do not occur. There are no difference from the nonincubated liver. c. Noninjected animals, incubated with reaction product. The purpose is to examine whether floating reaction product deposits in sites other than in the injected enzyme. Before incubation, drops of a solution of 1 mg of peroxidase in 5 ml of water were added to incubation medium until the latter became slightly brownish, and, in a second case, dark brown. No deposits of the reaction product are ever found on cell surfaces or in interstitial spaces, in contrast to findings in peroxidase-injected animals. They are not seen even on the surface of the tissue block. d. Injected animals, nonincubated or incubated without hydrogen peroxide. Neither of these controls shows differences from the noninjected, nonincubated controls. e. Other effects" of the incubation medium on noninjected, glutaraldehyde-perfused liver include a partial loss of glycogen and a partial loss of its stainability; the fine structure of the liver remains unaltered.
38
MATTER ET AL.
Intravenously injected peroxidase Reaction product can be found with fixation beginning (either perfusion or immersion) 60 secs after the end of the injection, which lasted for 20 to 30 secs. Within this lapse of time, practically all extracellular spaces, including fine ramifications of the interstitial space, are homogeneously blackened by the reaction product (Figs. 32 and 33). The endothelial cytomembrane shows a layer of RP, the microvilli of the space of Disse are covered with it (Fig. 31), and the interhepatocytic cleft is drawn out as a fine dark line. These lines stop at each side of the bile canaliculus, at a distance of 0.5 to 1 # from the lumen (Figs. 34-36). This pattern of the filling of the extracellular space stays constant for about 10-15 minutes after peroxidase injection. Then the extracellular activity decreases slowly (disappearing within 1 to 2 hours), concomitant with the increasing intracellular activity. As early as 1 rain after injection, numerous little black spheres of the magnitude of micropinocytotic vesicles can be found, 10-20 on one section in one hepatocyte, mostly along the cell surface. These spheres are membrane bound and possess usually a coating. Beyond these small vesicles, there exist larger ones, 0.5 to 1 # in diameter, homogeneously filled with reaction product and distributed throughout the cell with a distinct accumulation around the bile canaliculus (Figs. 39-43). Then, within 15 to 30 minutes, the peroxidase found intracellularly occurs in three different forms: (a) in smooth or coated micropinocytotic vesicles lining the cell membrane, (b) in small branched tubules scattered throughout the cytoplasm, and (c) in Golgi cisternae and Golgi vesicles, the latter fusing sometimes with larger vacuoles (Figs. 39 and 40).~Finally, after 30 rain the peroxidase begins to appear on microvilli of the bile canaliculi building a layer of reaction product on the wall of the bile canaliculi (Figs. 42 and 43). Sometimes, a single microvillus shows reaction product inside its plasma membrane (Fig. 41). Though there is an overlapping in time course in these two processes of filling of the interstitial space and of filling of the bile canaliculi, it is noteworthy that peroxidase is not found in the region of the junctional complex at any time. It could not be decided whether lysosomes, containing droplets with the density of reaction product, showed exogenous or endogenous peroxidatic activity (see
28-30. Rat livers fixed by immersion 5 min after intravenous injection of horseradish peroxidase. Reaction product is seen in the sinusoids, on the red blood cells, in the interhepatocytic cleft, and in little vacuoles, often surrounding the bile canaliculus, but not yet in the lumen of the bile canaliculus itself. Fig. 28: x 3000; Fig. 29: x 2000; Fig. 30: × 1800. FIG. 31. A sinusoid with two endothelial cells, a Kupffer cell, bordered on each side by hepatocytes, fixed by immersion 5 rain after intravenous injection of peroxidase. The space of Disse is completely filled with reaction product. Intracellularly one can see in the Kupffer cell a very high activity in the micropinocytotic system and large vacuoles filled with reaction product. Some micropinocytotic vesicles and tubules can also be seen in the hepatocytes and occasionally in the endothelial cells. × 24,000. FIGs.
~iiii
MORPHOLOGICAL ASPECTS OF BILE FORMATION
41
FIGS. 32 and 33. Two examples of filling of the extracellular space with reaction product 1 min after injection. The filling stops at the junctional complexes of the bile canaliculi. Some activity appears already inside the cell: in coated and smooth vesicles, and tubules, probably belonging to the smooth endoplasmic reticulum. Fig. 32: x 17,250; Fig. 33: x 22,000.
42
MATTER ET AL.
control
group
b).
The
vacuoles containing
peroxidase
are n o t
homogeneously
b l a c k , b u t suggest a v e s i c u l a r s u b s t r u c t u r e r e m i n i s c e n t of m u l t i v e s i c u l a r bodies. T h i s f e a t u r e is b e t t e r seen w i t h r e t r o g r a d e i n j e c t i o n of p e r o x i d a s e (see b e l o w ) .
Peroxidase injected retrogradely into the common bile duct T h e s e e x p e r i m e n t s yield, as a w h o l e , the i n v e r s e process. I m m e d i a t e l y after the i n j e c t i o n , r e a c t i o n p r o d u c t c a n b e f o u n d filling p r a c t i c a l l y the w h o l e l u m e n of the bile c a n a l i c u l i , b u t n e v e r i n f i l t r a t i n g t h e cell j u n c t i o n s (Figs. 47, 48 a n d 50-53). T h i s filling d e c r e a s e s slowly after 30 m i n a n d d i s a p p e a r s w i t h i n 1 h o u r . R a t h e r often, t h e l u m e n of the bile c a n a l i c u l i s e e m s to b e w i d e n e d w i t h a d i s a p p e a r a n c e of the m i c r o villi. W i t h i n 15 rain, activity is f o u n d in d i f f e r e n t f o r m s a r o u n d t h e bile c a n a t i c u l u s (Figs. 55-62): a. little, s m o o t h ,
m i c r o p i n o c y t o t i c vesicles, in close p r o x i m i t y of t h e bile c a n a -
liculus; b. s m a l l t u b u l a r profiles, m o s t l y in t h e v i c i n i t y of t h e bile c a n a l i c u l u s o r the space of D i s s e ;
FIGs. 34-36. Three bile canaliculi in livers fixed by immersion 5 min after the intravenous injection of peroxidase. The tight junction ( - region of the close-contact-points) is nowhere penetrated by the peroxidase. In Fig. 34 there are already some vacuoles filled with reaction product; the dense body in Fig. 36 does not necessarily contain peroxidase, as such dense bodies are also found in noninjected, incubated livers. The arrows indicate the tight junction. Fig. 34: × 39,000; Figs. 35 and 36: × 40,500. FIG. 37. A bile canaliculus 30 min after intravenous injection of peroxidase. The extracellular space is practically empty, reaction product is accumulated in vacuoles lining the cell border and in vesicles, tubules, and vacuoles which are associated with the Golgi region. The arrows point to osmiophilic droplets, which are also found in noninjected, nonincubated livers. Also the black material inside the bile canaliculus can be found in controls (see Fig. 45). x 37,500. FIG. 38. Portions of three hepatocytes 3 min after injection of peroxidase. The peroxidase has infiltrated a nexus and appears intracellularly in coated vesicles and little vacuoles. The small arrows point to activity (presumably autophagosomes), which also can be found in controls (see Fig. 46) The big arrows point to osmiophilic droplets, x 24,000. FIGS. 39 and 40. Two examples of pericanalicular regions 30 rain after intravenous injection of peroxidase. Reaction product is present in vacuoles, associated with the Golgi complex, and in numerous vesicles and tubules. Vesicles fuse with larger vacuoles in a number of cases (arrows); these present a porous distribution of reaction product reminiscent of multivesicular bodies. The large arrow in Fig. 40 points to osmiophilic droplets in a Golgi vacuole. Fig. 39: × 26,000; Fig. 40: x 34,000. FIO. 41. Bile canaliculus 15 min after intravenous injection of peroxidase. Reaction product is present in the intercellular cleft, in vacuoles, tubules, and vesicles, situated around the bile canaliculus and within a microvillus. × 31,500. FIGS. 42 and 43. Bile canaliculi 1 hour after intravenous injection of peroxidase. Accumulation of reaction product around the bile canaliculus in organelles of the Golgi complex: vesicles, vacuoles, tubules, lysosomes. Reaction product is now found inside the canaliculi, without penetrating the tight junction. Fig. 36: × 22,500; Fig. 37: × 18,000. Fins. 44 and 45. Control livers without injection of peroxidase, incubated with the usual incubation medium. Black material which is very similar to reaction product appears in dense bodies around the bile canaliculus. Osmiophilic droplets, present in Golgi vacuoles can be differentiated from reaction product by their less dense, grayish, round appearance. Incubation medium: 0.05 % 3,3'-diaminobenzidine, 0.01% H202, in Trisbuffer, pH 7.6. Fig. 44: x 31,000; Fig. 45: × 24,000.
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48
MATTER ET AL.
c. r o u n d , m e m b r a n e - l i m i t e d vacuoles with a diameter of a b o u t 1/~ c o n t a i n i n g reaction product, n o t homogeneous, b u t porous, full of holes with the diameter of micropinocytotic vesicles, reminiscent of multivesicular bodies. These bodies fuse very often with smooth, peroxidase-filled vesicles (a), and in some cases with small, coated vesicles, which do not c o n t a i n peroxidase (Fig. 59); d. As in the intravenously injected animals, sometimes we find reaction p r o d u c t inside the cell m e m b r a n e of a microvillus (Figs. 59 a n d 60). We c a n n o t decide whether the dense bodies with one or several black droplets c o n t a i n exogenous peroxidase, because similar organelles occur in the controls (see control group b). Beyond this activity, situated mainly a r o u n d the bile canaliculus, we notice vacuoles of 1-2 # in diameter that are scattered at r a n d o m i n the cytoplasm a n d whose walls are often covered with reaction product. W e see also vacuoles f o r m i n g from outbulging bile canaliculi a n d others fusing with the cell m e m b r a n e (Figs. 63-67). Outside the hepatocytes, reaction p r o d u c t m a y appear as a layer on the surface of both hepatocytes a n d endothelial cells, as well as in micropinocytotic vesicles and vacuoles of endothelial and K u p f f e r cells.
Experiments concerning the leakage problem F o r all experiments with diffusion tracers, the leakage p r o b l e m is a very serious one. Either r u p t u r e of barriers before fixation or diffusible reaction p r o d u c t plus rupture FIG. 46. Control liver without injection of peroxidase, incubated with a medium containing 0.5 % 3,3'-diaminobenzidine, 0.1% H~O2 in Tris buffer, at pH 7.6. The occurrence of dense, black precipitates in dense bodies, in multivesicular bodies, and in autophagosomes is remarkable, x 27,000. FIGS. 47 and 48. Portions of livers, fixed by perfusion, 5 rain after retrograde injection of peroxidase. The extended bile canaliculi and the sinusoids show strong deposits of reaction product. × 1920. FIG. 49. Portion of a liver fixed by perfusion 15 min after retrograde injection of peroxidase. The bile canaliculi are empty; a Kupffer cell in the sinusoid is heavily charged with peroxidase, x 2100. FIGS. 50 53. Four bile canaliculi, fixed by perfusion, 5 min after retrograde injection of peroxidase. The tight junction is nowhere penetrated by the reaction product. Fig. 50: x 18,000; Fig. 51 : x 20,000; Fig. 52: x 19,000; Fig. 53: x 22,200. FIG. 54. Bile canaliculus of the same liver but without incubation. × 35,000. FIG. 55. Reaction product present in the bile canaliculus and endothelial cell 15 rain after retrograde injection. Vacuoles, multivesicular bodies, and dense bodies also show activity. × 18,600. FIGS. 56 and 57. Activity present after 15 min in the bile canaliculus, in micropinocytotic vesicles, multivesicular bodies, dense bodies, associated with the Golgi complex. Fig. 56: x 18,600; Fig. 57: × 25,200. FIGS. 58-60. Tubular profiles filled with reaction product were not very frequently observed after retrograde injection. Figs. 59 and 60 show microvilli with reaction product inside their plasma membrane. Fig. 59 shows also the fusion of a small coated vesicle with a multivesicular body, which contains reaction product. This might be the pathway of the development of a phagosome into a secondary lysosome. Fig. 58: x 27,750; Fig. 59: x 26,250; inset: x 56,000; Fig. 60: × 27,000. FIG. 61. Reaction product in vacuoles, multivesicular bodies, dense bodies around a slightly distended bile canaliculus, itself showing no reaction product after 30 min. x 28,000. FIG. 62. The organelles of this hepatocyte, 30 min after injection, are loaded with peroxidase. They may be in the close vicinity of the cytomembrane, but have never been seen to fuse with it. x 18,000.
MORPHOLOGICAL
4 - 691833 Matter
ASPECTS OF BILE FORMATION
49
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62
56
MATTER ET AL.
Fro. 63. Bile canaliculus with reaction product, 2 rain after injection, surrounded by several vacuoles, which themselves also show reaction product on their walls, x 18,500.
of barriers during or after fixation could simulate pathways which in fact do not exist. Having rejected the diffusibility of reaction p r o d u c t in control group c, we tried to explain the cause of the ruptured junctional complexes, that we encountered regularly, especially at the border of the tissue block. We could exclude that the histamine and serotonin release (Cotran and Karnovsky, 19), induced by peroxidase in the rat, was the causative agent, because two rats, treated with pyrilamine maleate and B O L 148, showed no differences in the n u m b e r of ruptured junctional complexes. Noninjected, incubated livers also show ruptured junctional complexes, which disappear when the hydrogen peroxide in the incubation medium is omitted. So it :is very probable, that oxygen bubbles, formed by the catalatic action of the tissue, FIGS. 64 and 65. Fig. 64 should explain the foregoing; the bulging out of the bile canaliculus could lead to vacuoles, which then distribute in the cytoplasm, as seen in Fig. 65. Fig. 64: x 20,200; Fig. 65: × 18,500.
5
691833 M a t t e r
58
MATTER ET AL.
create the majority of these ruptured junctional complexes, at least in intravenously injected rats. For rats which had been injected retrogradely through the common bile duct, we wanted to have more direct evidence for intact junctional complexes. First we reduced the injection pressure to the minimal amount to get 0.2 ml of Ringer containing 5 mg of peroxidase into the biliary system. This pressure is observed to range between 3- and 6-fold the normal bile pressure. Still, the same extracellular activity as before appears already 1 min after injection. As very few ruptured junctional complexes would be sufficient for a marked leakage, we tried in another experiment to decide this point (see Chart 1): a. Two rats received 1 #Ci inulin-l~C intravenously. After an hour, there was less than 1 % of the injected quantity in the common bile duct. b. Two rats received 20 mg of peroxidase i.v. and then 1 #Ci of inulinJ4C i.v. After an hour, less than 2 % of the injected quantity had appeared in the common bile duct. c. Four rats received 5 mg of peroxidase through the common bile duct, and then 1/zCi of inulin-~C i.v. After an hour, less than 2 % of the injected quantity was in the common bile duct.
Discussion Reviewing the time sequence for the intravenously injected peroxidase, we find a peak for the extracellular distribution between 30 sec and 15 min, a plateau for the intracellular distribution between 15 and 60 min, and the beginning of appearance in canaliculi at 30 min. This agrees with biochemical data (Jacques, 50), but it is noteworthy that we find passage a faster process than did Straus (91), who studied this transport with light microscopy. In view of this time sequence and the impermeability of the junctional complex, we think that it is justified to speak of an active, intracellular transport. This transport includes three steps: resorption, transport inside the cell, secretion. The mechanism of resorption is pinocytosis, a process requiring energy (Berger and Karnovsky, 6) and therefore considered "active." There is no indication for other uptake mechanisms such as diffusion or phagocytosis. The second step, transport inside the cell, is supposedly mediated by the smooth endoplasmic reticulum, recognizable as black, tubular, and sometimes branched profiles. It is interesting to note that these two first steps are the same as for lipid absorption, at least in its particulate form (Palay and Karlin, 67). The third step, secretion, involves the Golgi apparatus, the lysosomes, and the bile canaliculus and
FIGS. 66 and 67. Other vacuoles are seen close to the space of Disse, sometimes fusing with it. These figures and the Figs. 63-65 suggest a diacytotic process, the pathway of regurgitation, the adaptation of the hepatocyte to acutely increased bile pressure. Fig. 66: x 18,000; Fig. 67: x 19,200.
60
MATTER ET AL. 8~
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od A
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t
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2-
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30'
45'
60'
CHART I. Radioactivity in the common bile duct was followed during an hour in 8 rats (a-h). Two rats (a and b) received 1 #Ci of inulin-14C intravenously. Within 1 hour less than 1% of the injected quantity appeared in the common bile duct. Two rats (c and d) received 20 mg of horseradish peroxidase intravenously, then 1 ffCi of inulin-14C intravenously. Within 1 hour less than 2 % of the injected quantity appeared in the common bile duct. Four rats (e-h) received 5 mg of horseradish peroxidase through the common bile duct and then 1 ffCi of inulin-l~C intravenously. Less than 2 % of the injected quantity appeared in the common bile duct within 1 hour.
is more difficult to understand. We might try to explain our results in terms of the concept of de Duve and Wattiaux (25), correlating them with the recent work done by Smith and Farquhar (85), Friend and Farquhar (37), Lane (56), and Orci et al. (65). We might then suppose that peroxidase, either by means of micropinocytotic vesicles or mediated by smooth endoplasmic reticulum, Golgi cisternae, and Golgi vesicles, is concentrated in larger vacuoles around the bile canaliculus. These vacuoles would develop into multivesicular bodies by fusion with small coated Golgi vesicles and then become dense bodies. If the peroxidase-charged Golgi vesicles do not undergo fusion with vacuoles, they may enter the secretory pathway. A point of doubt rests with the arrival of the peroxidase at the lumen of the bile canaliculus. We have never found vacuoles or tubules opening directly into the lumen, but only a blackening of the microvilli themselves. It is not clear how the peroxidase, a protein with the molecular weight of 40,000, can pass through the membrane of the microvilli. There exist two possibilities at least: (a) The peroxidase leaves the vesicles and goes
61
M O R P H O L O G I C A L ASPECTS OF BILE F O R M A T I O N
.'."
• . .
;
.~
o
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CHART 2. Summary of our interpretation of the results with intravenously injected horseradish peroxidase: uptake, intracellular transport, digestion, and secretion.
into the microvilli in a diffuse form; there it would cross the cell membrane. This would fit well with the histochemical demonstration of phosphatases, especially adenosine phosphatase at the membrane, as described by Wachstein and Meisel (93). (b) The fusion of a vesicle with the cytoplasmic membrane, a thermodynamic chance process depending on Brownian motion (Shea and Karnovsky, 83) could be so quick as to be practically undetectable. We are inclined to believe rather in the first possibility, because the same blackening of the microvilli inside their plasma membrane has been observed in the intestine (Hugon and Borgers, 49) and in the kidney (Orci et al. 66). We have no indication of the function of the external coating of the microvilli, the glycocalix of Bennett (5), either in the secretion or in the resorption of peroxidase. The last traces of retrogradely injected peroxidase are seen at the surface of the bile
62
MATTER ET AL.
5
3
CHART 3. Summary of our interpretation of the results with retrogradely injected horseradish peroxidase: uptake and digestion, bypassed by a fast diacytotic process.
canaliculi after about 30 min. The retrograde injection of peroxidase yields much activity in the sinusoids already 1 min after injection. Given the impermeability of the junctional complexes for peroxidase in both directions, we had to ascertain that all the junctional complexes were intact after injection. Inulin with a molecular weight of 5000-6000 is just at the limit for passing into the bile (Chenderovitch et al., 15). As the permeability of the liver epithelium was not altered for this molecule after intravenous or retrograde injection of peroxidase, we concluded that peroxidase had been transported intracellularly from the bile canaliculus to the sinusoids. It remained to explain by which vehicle it had been transported. Indeed, we see vesicles, multivesicular bodies and dense bodies, charged with peroxidase, very similar to those found after intravenous injection, but these organelles stay in the vicinity of the bile canaliculus and do not fuse with the sinusoidal cell membrane, So, a lysosomal
M O R P H O L O G I C A L ASPECTS OF BILE F O R M A T I O N
63
transport, as claimed by Essner and Novikoff (32), seems very unlikely. A diacytotic process, however, with vacuoles pinching off from the bile canaliculus and then fusing with the sinusoidal cell border, would have large transport capacities, sufficient for the total amount found in the sinusoids. This diacytosis may well be caused by the increased pressure, exerted during injection. The results with peroxidase are summarized in Charts 2 and 3.
TECHNIQUE
This technique, originated by Straus (90) and Daems et al. (23), and much improved by modifications of the substrate (Graham and Karnovsky, 40), gives a black reaction product of high contrast and of a clumpy nature. The terms reaction product (RP), activity, staining, and peroxidase will be used interchangeably.
Controls a. Noninjected animals', nonincubated. Droplets of the same electron density as the reaction product are found in pericanalicular dense bodies of the hepatocytes and in the residual bodies of Kupffer cells. Fat droplets and osmiophilic droplets in the Golgi apparatus are homogeneous and exhibit a sharp outline. Glycogen is recognized by its particulate nature. b. Noninjected animals, incubated. With an incubation time of 15-30 min, reaction product is found in three different types: on the whole surface of erythrocytes, in the granules of eosinophilic leukocytes, and in the pericanalicular dense bodies (Figs. 44 and 45). In the pericanalicular dense bodies, it is very difficult to distinguish the reaction product from the droplets found in the dense bodies in noninjected and nonincubated controls. At concentrations as high as 0.1%, this reaction is much more pronounced (Fig. 46). It is remarkable to find reaction product in autophagosomes. It may represent catalase, exhibiting peroxidatic activity (Figs. 38 and 46). Microbodies show no activity with our incubation time schedules. Without hydrogen peroxide, these reactions do not occur. There are no difference from the nonincubated liver. c. Noninjected animals, incubated with reaction product. The purpose is to examine whether floating reaction product deposits in sites other than in the injected enzyme. Before incubation, drops of a solution of 1 mg of peroxidase in 5 ml of water were added to incubation medium until the latter became slightly brownish, and, in a second case, dark brown. No deposits of the reaction product are ever found on cell surfaces or in interstitial spaces, in contrast to findings in peroxidase-injected animals. They are not seen even on the surface of the tissue block. d. Injected animals, nonincubated or incubated without hydrogen peroxide. Neither of these controls shows differences from the noninjected, nonincubated controls. e. Other effects" of the incubation medium on noninjected, glutaraldehyde-perfused liver include a partial loss of glycogen and a partial loss of its stainability; the fine structure of the liver remains unaltered.
38
MATTER ET AL.
Intravenously injected peroxidase Reaction product can be found with fixation beginning (either perfusion or immersion) 60 secs after the end of the injection, which lasted for 20 to 30 secs. Within this lapse of time, practically all extracellular spaces, including fine ramifications of the interstitial space, are homogeneously blackened by the reaction product (Figs. 32 and 33). The endothelial cytomembrane shows a layer of RP, the microvilli of the space of Disse are covered with it (Fig. 31), and the interhepatocytic cleft is drawn out as a fine dark line. These lines stop at each side of the bile canaliculus, at a distance of 0.5 to 1 # from the lumen (Figs. 34-36). This pattern of the filling of the extracellular space stays constant for about 10-15 minutes after peroxidase injection. Then the extracellular activity decreases slowly (disappearing within 1 to 2 hours), concomitant with the increasing intracellular activity. As early as 1 rain after injection, numerous little black spheres of the magnitude of micropinocytotic vesicles can be found, 10-20 on one section in one hepatocyte, mostly along the cell surface. These spheres are membrane bound and possess usually a coating. Beyond these small vesicles, there exist larger ones, 0.5 to 1 # in diameter, homogeneously filled with reaction product and distributed throughout the cell with a distinct accumulation around the bile canaliculus (Figs. 39-43). Then, within 15 to 30 minutes, the peroxidase found intracellularly occurs in three different forms: (a) in smooth or coated micropinocytotic vesicles lining the cell membrane, (b) in small branched tubules scattered throughout the cytoplasm, and (c) in Golgi cisternae and Golgi vesicles, the latter fusing sometimes with larger vacuoles (Figs. 39 and 40).~Finally, after 30 rain the peroxidase begins to appear on microvilli of the bile canaliculi building a layer of reaction product on the wall of the bile canaliculi (Figs. 42 and 43). Sometimes, a single microvillus shows reaction product inside its plasma membrane (Fig. 41). Though there is an overlapping in time course in these two processes of filling of the interstitial space and of filling of the bile canaliculi, it is noteworthy that peroxidase is not found in the region of the junctional complex at any time. It could not be decided whether lysosomes, containing droplets with the density of reaction product, showed exogenous or endogenous peroxidatic activity (see
28-30. Rat livers fixed by immersion 5 min after intravenous injection of horseradish peroxidase. Reaction product is seen in the sinusoids, on the red blood cells, in the interhepatocytic cleft, and in little vacuoles, often surrounding the bile canaliculus, but not yet in the lumen of the bile canaliculus itself. Fig. 28: x 3000; Fig. 29: x 2000; Fig. 30: × 1800. FIG. 31. A sinusoid with two endothelial cells, a Kupffer cell, bordered on each side by hepatocytes, fixed by immersion 5 rain after intravenous injection of peroxidase. The space of Disse is completely filled with reaction product. Intracellularly one can see in the Kupffer cell a very high activity in the micropinocytotic system and large vacuoles filled with reaction product. Some micropinocytotic vesicles and tubules can also be seen in the hepatocytes and occasionally in the endothelial cells. × 24,000. FIGs.
~iiii
MORPHOLOGICAL ASPECTS OF BILE FORMATION
41
FIGS. 32 and 33. Two examples of filling of the extracellular space with reaction product 1 min after injection. The filling stops at the junctional complexes of the bile canaliculi. Some activity appears already inside the cell: in coated and smooth vesicles, and tubules, probably belonging to the smooth endoplasmic reticulum. Fig. 32: x 17,250; Fig. 33: x 22,000.
42
MATTER ET AL.
control
group
b).
The
vacuoles containing
peroxidase
are n o t
homogeneously
b l a c k , b u t suggest a v e s i c u l a r s u b s t r u c t u r e r e m i n i s c e n t of m u l t i v e s i c u l a r bodies. T h i s f e a t u r e is b e t t e r seen w i t h r e t r o g r a d e i n j e c t i o n of p e r o x i d a s e (see b e l o w ) .
Peroxidase injected retrogradely into the common bile duct T h e s e e x p e r i m e n t s yield, as a w h o l e , the i n v e r s e process. I m m e d i a t e l y after the i n j e c t i o n , r e a c t i o n p r o d u c t c a n b e f o u n d filling p r a c t i c a l l y the w h o l e l u m e n of the bile c a n a l i c u l i , b u t n e v e r i n f i l t r a t i n g t h e cell j u n c t i o n s (Figs. 47, 48 a n d 50-53). T h i s filling d e c r e a s e s slowly after 30 m i n a n d d i s a p p e a r s w i t h i n 1 h o u r . R a t h e r often, t h e l u m e n of the bile c a n a l i c u l i s e e m s to b e w i d e n e d w i t h a d i s a p p e a r a n c e of the m i c r o villi. W i t h i n 15 rain, activity is f o u n d in d i f f e r e n t f o r m s a r o u n d t h e bile c a n a t i c u l u s (Figs. 55-62): a. little, s m o o t h ,
m i c r o p i n o c y t o t i c vesicles, in close p r o x i m i t y of t h e bile c a n a -
liculus; b. s m a l l t u b u l a r profiles, m o s t l y in t h e v i c i n i t y of t h e bile c a n a l i c u l u s o r the space of D i s s e ;
FIGs. 34-36. Three bile canaliculi in livers fixed by immersion 5 min after the intravenous injection of peroxidase. The tight junction ( - region of the close-contact-points) is nowhere penetrated by the peroxidase. In Fig. 34 there are already some vacuoles filled with reaction product; the dense body in Fig. 36 does not necessarily contain peroxidase, as such dense bodies are also found in noninjected, incubated livers. The arrows indicate the tight junction. Fig. 34: × 39,000; Figs. 35 and 36: × 40,500. FIG. 37. A bile canaliculus 30 min after intravenous injection of peroxidase. The extracellular space is practically empty, reaction product is accumulated in vacuoles lining the cell border and in vesicles, tubules, and vacuoles which are associated with the Golgi region. The arrows point to osmiophilic droplets, which are also found in noninjected, nonincubated livers. Also the black material inside the bile canaliculus can be found in controls (see Fig. 45). x 37,500. FIG. 38. Portions of three hepatocytes 3 min after injection of peroxidase. The peroxidase has infiltrated a nexus and appears intracellularly in coated vesicles and little vacuoles. The small arrows point to activity (presumably autophagosomes), which also can be found in controls (see Fig. 46) The big arrows point to osmiophilic droplets, x 24,000. FIGS. 39 and 40. Two examples of pericanalicular regions 30 rain after intravenous injection of peroxidase. Reaction product is present in vacuoles, associated with the Golgi complex, and in numerous vesicles and tubules. Vesicles fuse with larger vacuoles in a number of cases (arrows); these present a porous distribution of reaction product reminiscent of multivesicular bodies. The large arrow in Fig. 40 points to osmiophilic droplets in a Golgi vacuole. Fig. 39: × 26,000; Fig. 40: x 34,000. FIO. 41. Bile canaliculus 15 min after intravenous injection of peroxidase. Reaction product is present in the intercellular cleft, in vacuoles, tubules, and vesicles, situated around the bile canaliculus and within a microvillus. × 31,500. FIGS. 42 and 43. Bile canaliculi 1 hour after intravenous injection of peroxidase. Accumulation of reaction product around the bile canaliculus in organelles of the Golgi complex: vesicles, vacuoles, tubules, lysosomes. Reaction product is now found inside the canaliculi, without penetrating the tight junction. Fig. 36: × 22,500; Fig. 37: × 18,000. Fins. 44 and 45. Control livers without injection of peroxidase, incubated with the usual incubation medium. Black material which is very similar to reaction product appears in dense bodies around the bile canaliculus. Osmiophilic droplets, present in Golgi vacuoles can be differentiated from reaction product by their less dense, grayish, round appearance. Incubation medium: 0.05 % 3,3'-diaminobenzidine, 0.01% H202, in Trisbuffer, pH 7.6. Fig. 44: x 31,000; Fig. 45: × 24,000.
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c. r o u n d , m e m b r a n e - l i m i t e d vacuoles with a diameter of a b o u t 1/~ c o n t a i n i n g reaction product, n o t homogeneous, b u t porous, full of holes with the diameter of micropinocytotic vesicles, reminiscent of multivesicular bodies. These bodies fuse very often with smooth, peroxidase-filled vesicles (a), and in some cases with small, coated vesicles, which do not c o n t a i n peroxidase (Fig. 59); d. As in the intravenously injected animals, sometimes we find reaction p r o d u c t inside the cell m e m b r a n e of a microvillus (Figs. 59 a n d 60). We c a n n o t decide whether the dense bodies with one or several black droplets c o n t a i n exogenous peroxidase, because similar organelles occur in the controls (see control group b). Beyond this activity, situated mainly a r o u n d the bile canaliculus, we notice vacuoles of 1-2 # in diameter that are scattered at r a n d o m i n the cytoplasm a n d whose walls are often covered with reaction product. W e see also vacuoles f o r m i n g from outbulging bile canaliculi a n d others fusing with the cell m e m b r a n e (Figs. 63-67). Outside the hepatocytes, reaction p r o d u c t m a y appear as a layer on the surface of both hepatocytes a n d endothelial cells, as well as in micropinocytotic vesicles and vacuoles of endothelial and K u p f f e r cells.
Experiments concerning the leakage problem F o r all experiments with diffusion tracers, the leakage p r o b l e m is a very serious one. Either r u p t u r e of barriers before fixation or diffusible reaction p r o d u c t plus rupture FIG. 46. Control liver without injection of peroxidase, incubated with a medium containing 0.5 % 3,3'-diaminobenzidine, 0.1% H~O2 in Tris buffer, at pH 7.6. The occurrence of dense, black precipitates in dense bodies, in multivesicular bodies, and in autophagosomes is remarkable, x 27,000. FIGS. 47 and 48. Portions of livers, fixed by perfusion, 5 rain after retrograde injection of peroxidase. The extended bile canaliculi and the sinusoids show strong deposits of reaction product. × 1920. FIG. 49. Portion of a liver fixed by perfusion 15 min after retrograde injection of peroxidase. The bile canaliculi are empty; a Kupffer cell in the sinusoid is heavily charged with peroxidase, x 2100. FIGS. 50 53. Four bile canaliculi, fixed by perfusion, 5 min after retrograde injection of peroxidase. The tight junction is nowhere penetrated by the reaction product. Fig. 50: x 18,000; Fig. 51 : x 20,000; Fig. 52: x 19,000; Fig. 53: x 22,200. FIG. 54. Bile canaliculus of the same liver but without incubation. × 35,000. FIG. 55. Reaction product present in the bile canaliculus and endothelial cell 15 rain after retrograde injection. Vacuoles, multivesicular bodies, and dense bodies also show activity. × 18,600. FIGS. 56 and 57. Activity present after 15 min in the bile canaliculus, in micropinocytotic vesicles, multivesicular bodies, dense bodies, associated with the Golgi complex. Fig. 56: x 18,600; Fig. 57: × 25,200. FIGS. 58-60. Tubular profiles filled with reaction product were not very frequently observed after retrograde injection. Figs. 59 and 60 show microvilli with reaction product inside their plasma membrane. Fig. 59 shows also the fusion of a small coated vesicle with a multivesicular body, which contains reaction product. This might be the pathway of the development of a phagosome into a secondary lysosome. Fig. 58: x 27,750; Fig. 59: x 26,250; inset: x 56,000; Fig. 60: × 27,000. FIG. 61. Reaction product in vacuoles, multivesicular bodies, dense bodies around a slightly distended bile canaliculus, itself showing no reaction product after 30 min. x 28,000. FIG. 62. The organelles of this hepatocyte, 30 min after injection, are loaded with peroxidase. They may be in the close vicinity of the cytomembrane, but have never been seen to fuse with it. x 18,000.
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Fro. 63. Bile canaliculus with reaction product, 2 rain after injection, surrounded by several vacuoles, which themselves also show reaction product on their walls, x 18,500.
of barriers during or after fixation could simulate pathways which in fact do not exist. Having rejected the diffusibility of reaction p r o d u c t in control group c, we tried to explain the cause of the ruptured junctional complexes, that we encountered regularly, especially at the border of the tissue block. We could exclude that the histamine and serotonin release (Cotran and Karnovsky, 19), induced by peroxidase in the rat, was the causative agent, because two rats, treated with pyrilamine maleate and B O L 148, showed no differences in the n u m b e r of ruptured junctional complexes. Noninjected, incubated livers also show ruptured junctional complexes, which disappear when the hydrogen peroxide in the incubation medium is omitted. So it :is very probable, that oxygen bubbles, formed by the catalatic action of the tissue, FIGS. 64 and 65. Fig. 64 should explain the foregoing; the bulging out of the bile canaliculus could lead to vacuoles, which then distribute in the cytoplasm, as seen in Fig. 65. Fig. 64: x 20,200; Fig. 65: × 18,500.
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MATTER ET AL.
create the majority of these ruptured junctional complexes, at least in intravenously injected rats. For rats which had been injected retrogradely through the common bile duct, we wanted to have more direct evidence for intact junctional complexes. First we reduced the injection pressure to the minimal amount to get 0.2 ml of Ringer containing 5 mg of peroxidase into the biliary system. This pressure is observed to range between 3- and 6-fold the normal bile pressure. Still, the same extracellular activity as before appears already 1 min after injection. As very few ruptured junctional complexes would be sufficient for a marked leakage, we tried in another experiment to decide this point (see Chart 1): a. Two rats received 1 #Ci inulin-l~C intravenously. After an hour, there was less than 1 % of the injected quantity in the common bile duct. b. Two rats received 20 mg of peroxidase i.v. and then 1 #Ci of inulinJ4C i.v. After an hour, less than 2 % of the injected quantity had appeared in the common bile duct. c. Four rats received 5 mg of peroxidase through the common bile duct, and then 1/zCi of inulin-~C i.v. After an hour, less than 2 % of the injected quantity was in the common bile duct.
Discussion Reviewing the time sequence for the intravenously injected peroxidase, we find a peak for the extracellular distribution between 30 sec and 15 min, a plateau for the intracellular distribution between 15 and 60 min, and the beginning of appearance in canaliculi at 30 min. This agrees with biochemical data (Jacques, 50), but it is noteworthy that we find passage a faster process than did Straus (91), who studied this transport with light microscopy. In view of this time sequence and the impermeability of the junctional complex, we think that it is justified to speak of an active, intracellular transport. This transport includes three steps: resorption, transport inside the cell, secretion. The mechanism of resorption is pinocytosis, a process requiring energy (Berger and Karnovsky, 6) and therefore considered "active." There is no indication for other uptake mechanisms such as diffusion or phagocytosis. The second step, transport inside the cell, is supposedly mediated by the smooth endoplasmic reticulum, recognizable as black, tubular, and sometimes branched profiles. It is interesting to note that these two first steps are the same as for lipid absorption, at least in its particulate form (Palay and Karlin, 67). The third step, secretion, involves the Golgi apparatus, the lysosomes, and the bile canaliculus and
FIGS. 66 and 67. Other vacuoles are seen close to the space of Disse, sometimes fusing with it. These figures and the Figs. 63-65 suggest a diacytotic process, the pathway of regurgitation, the adaptation of the hepatocyte to acutely increased bile pressure. Fig. 66: x 18,000; Fig. 67: x 19,200.
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CHART I. Radioactivity in the common bile duct was followed during an hour in 8 rats (a-h). Two rats (a and b) received 1 #Ci of inulin-14C intravenously. Within 1 hour less than 1% of the injected quantity appeared in the common bile duct. Two rats (c and d) received 20 mg of horseradish peroxidase intravenously, then 1 ffCi of inulin-14C intravenously. Within 1 hour less than 2 % of the injected quantity appeared in the common bile duct. Four rats (e-h) received 5 mg of horseradish peroxidase through the common bile duct and then 1 ffCi of inulin-l~C intravenously. Less than 2 % of the injected quantity appeared in the common bile duct within 1 hour.
is more difficult to understand. We might try to explain our results in terms of the concept of de Duve and Wattiaux (25), correlating them with the recent work done by Smith and Farquhar (85), Friend and Farquhar (37), Lane (56), and Orci et al. (65). We might then suppose that peroxidase, either by means of micropinocytotic vesicles or mediated by smooth endoplasmic reticulum, Golgi cisternae, and Golgi vesicles, is concentrated in larger vacuoles around the bile canaliculus. These vacuoles would develop into multivesicular bodies by fusion with small coated Golgi vesicles and then become dense bodies. If the peroxidase-charged Golgi vesicles do not undergo fusion with vacuoles, they may enter the secretory pathway. A point of doubt rests with the arrival of the peroxidase at the lumen of the bile canaliculus. We have never found vacuoles or tubules opening directly into the lumen, but only a blackening of the microvilli themselves. It is not clear how the peroxidase, a protein with the molecular weight of 40,000, can pass through the membrane of the microvilli. There exist two possibilities at least: (a) The peroxidase leaves the vesicles and goes
61
M O R P H O L O G I C A L ASPECTS OF BILE F O R M A T I O N
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CHART 2. Summary of our interpretation of the results with intravenously injected horseradish peroxidase: uptake, intracellular transport, digestion, and secretion.
into the microvilli in a diffuse form; there it would cross the cell membrane. This would fit well with the histochemical demonstration of phosphatases, especially adenosine phosphatase at the membrane, as described by Wachstein and Meisel (93). (b) The fusion of a vesicle with the cytoplasmic membrane, a thermodynamic chance process depending on Brownian motion (Shea and Karnovsky, 83) could be so quick as to be practically undetectable. We are inclined to believe rather in the first possibility, because the same blackening of the microvilli inside their plasma membrane has been observed in the intestine (Hugon and Borgers, 49) and in the kidney (Orci et al. 66). We have no indication of the function of the external coating of the microvilli, the glycocalix of Bennett (5), either in the secretion or in the resorption of peroxidase. The last traces of retrogradely injected peroxidase are seen at the surface of the bile
62
MATTER ET AL.
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CHART 3. Summary of our interpretation of the results with retrogradely injected horseradish peroxidase: uptake and digestion, bypassed by a fast diacytotic process.
canaliculi after about 30 min. The retrograde injection of peroxidase yields much activity in the sinusoids already 1 min after injection. Given the impermeability of the junctional complexes for peroxidase in both directions, we had to ascertain that all the junctional complexes were intact after injection. Inulin with a molecular weight of 5000-6000 is just at the limit for passing into the bile (Chenderovitch et al., 15). As the permeability of the liver epithelium was not altered for this molecule after intravenous or retrograde injection of peroxidase, we concluded that peroxidase had been transported intracellularly from the bile canaliculus to the sinusoids. It remained to explain by which vehicle it had been transported. Indeed, we see vesicles, multivesicular bodies and dense bodies, charged with peroxidase, very similar to those found after intravenous injection, but these organelles stay in the vicinity of the bile canaliculus and do not fuse with the sinusoidal cell membrane, So, a lysosomal
M O R P H O L O G I C A L ASPECTS OF BILE F O R M A T I O N
63
transport, as claimed by Essner and Novikoff (32), seems very unlikely. A diacytotic process, however, with vacuoles pinching off from the bile canaliculus and then fusing with the sinusoidal cell border, would have large transport capacities, sufficient for the total amount found in the sinusoids. This diacytosis may well be caused by the increased pressure, exerted during injection. The results with peroxidase are summarized in Charts 2 and 3.