Takuma Saito and Kazuo Ogawa. Department of Anatomy, Kansai Medical School Moriguchi, Osaka, Japan

Transcription

1 Okajimas Fol. anat. jap., 44 : 11-27, 1967 Ultracytochemical Changes of the Glucose-6-Phosphatase (D-Glucose-6-Phosphate Phosphohydrolase) Activity in Liver Cells of the Rat treated with Phenobarbital By Takuma Saito and Kazuo Ogawa Department of Anatomy, Kansai Medical School Moriguchi, Osaka, Japan When Tice and B a r r nett (1962) first reported the fine structural localization of the glucose-6-phosphatase (G-6-Pase) activity in the rat liver, it was described that the enzymatic activity was positive in both smooth- and rough-surfaced elements of the endoplasmic reticulum of hepatic cells, including the nuclear envelope. However, since the fresh or hydroxyadipaldehyde-fixed specimens were used in their observation, the preservation of fine structural details were not satisfactorily good and the presence of the enzymatic activity in the smooth-surfaced variant of the endoplasmic reticulum was not confirmative. Recently Ericsson (1966) reported in a brief form the presence of the G-6-Pase activity in the smooth-surfaced type of the endoplasmic reticulum around the glycogen area in the glutaraldehyde-fixed rat liver. However, since an amount of smooth-surfaced endoplasmic reticulum is extremely scanty in the normal liver in usual, the unequivocal demonstration of the enzymatic activity in the smooth-surfaced endoplasmic reticulum awaited further clarification. The aims of the present investigation were to see the ultracytochemical localization of the G-6-Pase activity in the well-preserved liver of the normal rat to confirm the presence of the enzymatic activity in the smooth-surfaced endoplasmic reticulum, and furthermore to see changes of the enzymatic activity, if any, in the liver 1) This investigation has been supported by grants from the Anna Fuller Fund and the Jane Coffin Childs Memorial Fund for Medical Research (Project No.. 196). 2) A part of this work has been presented at the 6th Annual Meeting of the American Society for Cell Biology held in Houston, Texas, on Nov , 1966, in the section of read by title " (S a i t o and O g a w a, 1966a). 11

2 12 Takuma Saito and Kazuo Ogawa of the phenobarbital-treated rats at the level of ultrastructure, since it has been shown that the smooth-surfaced endoplasmic reticulum reveals enormous proliferation in the intoxicated rat liver (R e m m e r and M e r k e r, 1965 ; Jones and F a w c e t t, 1966). The work on the phenobarbital-treated rat liver serves as a part of ancillary works of the enzyme ultracytochemistry on the azo-dye hepatocarcinogenesis being carried out in our laboratory (S a i t o, 1966 ; Saito and Ogaw a, 1966a, b) also. Material and Methods Male Sprague-Dawley rats, weighing approximately 150 g, were used. The experimental animals received daily one intraperitoneal injection of 80 mg of sodium phenobarbital per kg body weight for 9 days. After the 3rd day, daily one rat was sacrificed by a blow on the neck. The liver tissues removed were immersed immediately in 2% glutaraldehyde or 12.5% hydroxyadipaldehyde in 0.1 M cacodylate buffer, ph 7.2, containing 8% sucrose (S abatini et al., 1963), for 30 minutes at 0-4 C. During the fixation tissues were cut in a small needle-like shape to facilitate the penetration of the fixative into tissues. The pieces of fixed liver tissues were then transferred into cold 0.1 M cacodylate buffer, ph 7.2, with 8% sucrose, and washed overnight. Following washing in buffer, frozen sections, approximately 40-60a in thickness, were made. Occasionally thick non-frozen sections were made by an automicrochopper model KO-1 (0 g a w a et al., 1966) or a tissue sectioner model TC-2 (S m i t h and F a r q u h a r, 1965). Thick frozen or non-frozen sections were incubated in the Wachstein-Meisel medium for the G-6-Pase activity (W a c h stein and M e i se 1, 1956) for 15 minutes at 37 C. After incubation specimens were postosmificated in Caulfield's solution (1957) for 1 hour at 0-4 C, dehydrated in graded ethanols and embedded in open (L u f t, 1961). Thin sections were cut by LKB ultrotome using glass knives, mounted directly on 400-mesh copper grids, stained with uranyl acetate and/or lead citrate (R e y n o 1 d s, 1963) and observed with JEM-7 electron microscope. Photographs were taken at original magnification of 5,000 to 15,000. In some cases, in order to select bile duct regions in the hepatic lobule approximately 111 thick sections cut by LKB ultrotome were stained with 1% toluidine blue and examined with light microscope prior to making thin sections from the same block. Specimens were incubated in the substrate-free medium for

3 Ultracytochernical Changes of the Glucose-6-Phosphatase Activity 13 control. In addition to the ultracytochemical observation of the enzymatic activity, the G-6-Pase activity was observed at the light microscopic level also. For the light microscopic observation, specimens incubated in the incubating medium were rinsed in buffer, immersed in 1% yellow ammonium sulfide solution for several minutes at room temperature, washed in the double distilled water and mounted in glycerinejelly. Results Findings in the normal rat liver cell Although the inhibitory effect on the enzymatic activity was slightly stronger in glutaraldehyde-fixed tissues than in hydroxyadipaldehyde-fixed ones, fixation in 2% glutaraldehyde for 30 minutes at 0-4 C was quite satisfactory in terms of the preservation of the enzymatic activity and fine structures. Fixation in glutaraldehyde longer than 1 hour destroyed the G-6-Pase activity almost completely. In the normal rat hepatic parenchymal cells, lead phosphate showing the G-6-Pase activity was found in the endoplasmic reticulum including the nuclear envelope and the transitional element near the Golgi apparatus (Figs. 1-5). In many places reaction product filled the cisternal space. In some areas deposits appeared to be adhered to the cisternal side of the membrane. The matricial side of the membrane, ribosomes and the cytoplasmic matrix revealed entirely no activity. Membranes such as plasma membranes, membranes of bile canaliculi (b), smooth membranes constituting Golgi apparatus (Go), multivesicular bodies (mu), coated vesicles (co), microbodies (mc), mitochondrial membranes (m), and membranes limiting lysosomes or cytolysomes (c) were devoid of activity. No activity was observed in the nucleus (n) itself. Lipid droplets (1i) were also negative. It was of interest to find that the endoplasmic reticulum captured recently in cytolysomes still retained G-6-Pase activity (c in Figs. 1 and 4), while cytolysomes in the later stage revealed no enzymatic activity any more (c1 in Fig. 2). The diffuse and weak deposition of lead phosphate in cytolysomes (c, in Fig. 2) might mean the intermediate stage of degeneration of the endoplasmic reticulum in cytolysomes. The tubular smooth-surfaced endoplasmic reticulum in and around glycogen areas (g) was positive for the G-6-Pase. The G-6-Pase activity was positive only in the hepatic parenchymal cell the endothelial cell (Fig. 5), the connective tissue cells and

4 14 Takuma Saito and Kazuo Ogawa the cuboidal epithelial cells of the bile duct (Fig. 6) were essentially devoid of reaction products. Membranes covering Disse's space (D) were also not positive. Findings in the phenobarbital-treated rat liver cells The prominent findings in the phenobarbital-treated hepatic cells were the loss of glycogen from the glycogen area (g) (Figs. 7 and 8) and the enormous proliferation of the tubular smooth-surfaced endoplasmic reticulum (Fig. 9) which could be seen already on the 3rd day following the onset of injection. The fine structural localization. of the G-6-Pase activity in the experimental hepatic cell was essentially similar to that observed in the normal cell. The G-6-Pase activity was positive in the nuclear envelope and the endoplasmic reticulum (Figs. 7-9). The enzymatic activity was still positive in the tubular smooth-surfaced endoplasmic reticulum in and around the preexisting glycogen area (g) (Fig. 7) and in the proliferated tubular smooth-surfaced endoplasmic reticulum (Fig. 9). The mode of distribution of the reaction product within the enzymatically positive fine structural elements was entirely the same as that seen in the normal hepatic cell ; the G-6-Pase activity was positive in the cisternae or the cisternal side of the membrane. Judging from the over-all light microscopic observation, the enzymatic activity per cell did not seem to reveal any appreciable changes. Discussion It was unequivocally shown in the present study that in addition to the rough-surfaced endoplasmic reticulum and the nuclear envelope (Tice and Barrnett, 1962; Goldfischer et al., 1964; Er i c s s o n, 1966), the smooth-surfaced endoplasmic reticulum, in many cases in the tubular profile, and the transitional element near the Golgi apparatus were positive for the G-6-Pase activity in the normal hepatic parenchymal cell. Furthermore the site of reaction product was in many areas within the cisternae of the system, although in some areas the reaction product appeared to be adhered to the cisternal (inner) side of the membrane. In no places the deposition of lead phosphate was on the membrane per se, although it has been shown by the fractionation studies that the G-6-Pase is bound to microsomal membranes in the liver of various animals (de D u v e et al., 1962 ; Dallner et al., 1966). This discrepancy may be due to differences in methods used. However, an alternative explanation such as follows may not be improbable. The actual site

5 Ultracytochemical Changes of the Glucose-6-Phosphatase Activity 15 of the enzyme molecule may be in membranes, but the site of hydrolysis of glucose-6-phosphate resulting in the formation of glucose is in the cisternal space. The complicated biochemical interaction between the enzyme and the substrate may be taking place at the cisternal surface of the membrane of the endoplasmic reticulum. Phosphates, split from the substrate by the enzyme action and captured in a form of lead phosphate, might easily be transferred from the locale of the enzyme action, which is presumably at the cisternal surface of the membrance, to the nearby cisternal space. There remains, however, still another possibility that the reaction product in the cisternae is showing directly the soluble moiety of G-6-Pase molecule. At any rate, it is evident from the present investigation that the final step of gluconeogenesis, formation of glucose from glucose-6-phosphate, is being carried out in the cisternae of the endoplasmic reticulum, not in the cytoplasmic matrix. This is of great interest in terms of the intracellular topographic chemistry of the glycogen metabolism, since it has been shown that activities of fructose-1, 6-diphosphatase, which is also involved in the gluconeogenesis (S alto and 0 g a w a, 1967), as well as a-glucan phosphorylase (H or i, 1966) are localized in the cytoplasmic matrix, but not in the cisternae of the endoplasmic reticulum. The fine structural topography of the G-6-Pase activity in the phenobarbital-treated rat hepatic parenchymal cell was essentially similar to that observed in the normal rat hepatic cells and the enzymatic activity was positive even in the proliferated tubular smooth-surfaced endoplasmic reticulum. This is in good accordance with findings obtained by Orr e n i u s and Ericsson (1966) recently independently. However, the spurious reaction in the nucleus obtained by Orrenius and Ericsson (Fig. 8 in Orrenius and Eric s s o n, 1966) was not seen in the present study. As to the G-6-Pase activity in the phenobarbital-treated rat liver, R e m m e r and M e r k e r (1965) has reported an increased enzymatic activity. in the treated rat liver, while Or renius et al. (1965) and 0 r r e- n i u s and Er i c s s o n (1966) obtained the decreased activity, and there seems to exist discrepancy among data obtained by biochemical methods. This discrepancy may be due, at least in part, to differences in the way of expression of the biochemical enzymatic activity ; Remmer and M e r k e r (1965) expressed the G-6-Pase activity as mp moles of substrate metabolized in one minute per 1 g liver tissue, while Orr e n i u s and Ericsson (1965) used i moles of inorganic phosphate (Pi) liberated in 20 minutes per mg protein

6 16 Takuma Saito and Kazuo Ogawa as standard. It is difficult to compare data expreseed by different standards. If any changes of the enzymatic activity occurred in the cell of the phenobarbital-treated rats, the question uaturally arises here, whether the proliferated tubular smooth-surfaced endoplasmic reticulum is of the same physicochemical nature as that seen in the normal hepatic cell or not. There seems to be no work so far which is capable of giving a direct answer to this question. However, since it has been shown that the proliferation of the tubular smooth-surfaced endoplasmic reticulum occur transientally during the development of the rodent liver (P e t e r s et al., 1963 ; Rosen et al., 1966) or as a pharmacological response to certain carcinogens in the hepatic cell (P or ter and Brun i, 1959 ; S a i t o, 1966 ; S a i t o and Cigaw a, 1966a, b), it seems not unlikely that the normal cell has the potential capacity to proliferate the smooth-surfaced endoplasmic reticulum having normal physicochemical properties as the cellular metabolic adaptation mechanism. The question of this sort further relates to the problem of the biogenesis of the smooth-surfaced endoplasmic reticulum. With regard to the latter question, D a 11 n e r et al. (1966) maintain that the proteins are synthesized in the rough-surfaced endoplasmic reticulum, then transferred to the smooth-surfaced part of the endoplasmic reticulum. However, the questions pertinent to this problem do not seem to be completely solved by this work only, and further studies on the fine structural changes of the biochemical enzyme activity in the cell under various conditions of induction or suppression may be necessary to find keys to answer these problems Summary Using Wachstein-Meisel medium, the glucose-6-phosphatase (G-6- Paso) activity was demonstrated ultracytochemically in the liver cells of normal and phenobarbital-treated rats. The G-6-Pase activity was positive only in the hepatic parenchymal cell. In the normal hepatic parenchymal cell the enzymatic activity was observed in the rough-surfaced endoplasmic reticulum, the smooth-sufaced endoplasmic reticulum, the transitional element near the Golgi apparatus and the nuclear envelope. The reaction product was localized in the cisternae of the membrane system. In some areas deposits were found to be adhered to the cisternal side of the membrane. The matricial side of the membrane, ribosomes and the cytoplasmic matrix revealed no activity. Smooth membranes constituting Golgi

7 Ultracytochemical Changes of the Glucose-6-Phosphatase Activity 17 apparatus, multivesicular bodies and coated vesicles, mitochondrial membranes, membranes limiting lysosomes or cytolysomes and plasma membranes were entirely devoid of activity. In the phenobarbital-treated rat liver, the loss of glycogen and the proliferation of the tubular smooth-surfaced endoplasmic reticulum was evident. The intracellular topography of the G-6-Pase activity in the phenobarbital-treated rat hepatic parenchymall cell was essentially the same as that seen in the normal rat hepatic parenchymal cell and the enzymatic activity was positive in the proliferated tubular smooth-surfaced endoplasmic reticulum. Acknowledgement The various technical assistance of Mr. Y. F u r u saw a, Miss. H. T a b at a and Miss. N. Kobayashi is gratefully acknowledged. References Ca ul If i e 1 d, J. B.: Effects of varying the vehicle for 0504 in tissue fixation. J. Biophys. Biochem. Cytol., 3: , D a 11 n e r, G., P. Sick e v i t z and G. E. Pa la d e: Biogenesis of endoplasmic reticulum membranes. II. Synthesis of constitutive microsomal enzymes in developing rat hepatocyte. J. Cell Biol., 30, , de D u v e, C., R. W a t t i a u x and P. B a u d h u i n: Distribution of enzymes between subcellular fractions in animal tissues. Advanc. Enzymol., 24 : , Er i c s s o n, J. L. E.: On the fine structural demonstration of glucose-6-phosphatase. J. Histochem. Cytochem., 14: , Goldf ische r, S., E. Essner and A.B. Novikof f: The localization of phosphatase activities at the level of ultrastructure. J. Histochem. Cytochem., , H o r i, S. H.: Fine-structure locations of a-glucan phosphorylase, as shown by lead precipitation and electron microscopy. Stain Tech., 41 : 91-95, J o n e s, A. L. and D. E. Fa w c e t t: Hypertrophy of the agranular endoplasmic reticulum in hamster liver induced by phenobarbital. J. Histochem. Cytochem., 14: , L u f t, J. H. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: , O g a w a, K., T. S a i t o, H. H i r a no and H. Ma y a h a r a: Automicrochopper model KO-1 for making non-frozen sections (In Japanese). 7th Ann. Meet. Jap. Soc. Histo- and Cyto-chem., Chiba, Nov. 7, Orr e n i u s, S., J. L. E. E r i c s s on and L. Er n s t e r: Phenobarbital induced synthesis of the microsomal drug-metabolizing enzyme system and its relationship to the proliferation of endoplasmic membranes. A morphological and biochemical study. J. Cell Biol., 25 : , Orr e n i u s, S. and J. L. E. Ericsson: On the relationship of liver glucose-6-phosphatase to the proliferation of endoplasmic reticulum in phenobarbital induction. J. Cell Biol., 31 : , 1966.

8 18 Takuma Saito and Kazuo Ogawa Peter s, V. B., G. W. Kell y and H. M. Dembit zer: Cytologic changes in fetal and neonatal hepatic cells of the mouse. Ann. N. Y. Acad. Sci., 111 : , Porte r, K. R. and C. Br uni: An electron microscopic study of the early effects of 3'-Me-DAB on rat liver cells. Can. Res., 19: , R e m m e r, H. and H. J. M e r k e r : Effect of drugs on the formation of smooth end. plasmic reticulum and drug-metabolizing enzymes. Ann. N. Y. Acad. Sci., 123: 79-97, Reynold s, E. S. : The use of lead citrate at high ph as an electron opaque stain in electron microscopy. J. Cell Biol., 17 : , R o s e n, S. I., G. W. Kelly and V. B. Peters: Glucose-6-phosphatase in tubular endoplasmic reticulum of hepatocytes. Sci., 152 : , Sabatin i, D. D., K. Bensch and R. J. Barrnett: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17 : 19-57, S a i t o, T.: Fine structural localizatign of several phosphatases in the rat hepatic parenchymal cell. In " Electron Microscopy 1966", ed. by R. U y e d a, vol. II, Maruzen Co., Tokyo, , S a i t o, T. and K. 0 g a w a : The smooth-surfaced endoplasmic reticulum and glucose- 6-phosphatase activity in rat liver cells. J. Cell Biol., 31 : 158A, 1966a. S a i t o, T. and K. 0 g,a w a : Electron cytochemical study of glucose-6-phosphatase activity in rat hepatic parenchymal cells during the early stage of carcinogenesis induced by 3'-MeDAB. 9th Internat. Can. Congr., Tokyo, Oct , 1966b. S a i t o, T. and K. 0 g a w a: Ultracytochemical demonstration of D-fructose-1, 6-diphosphatase (D-fructose-1, 6-diphosphate 1-phosphohydrolase) activity in the rat liver using lead citrate as capture reagent. J. Ultrastr. Res., in press. S m i t h, R. E. and M. G. Far q u h a r: Preparation of non-frozen sections for electron microscope cytochemistry. RCA Scient. Instr. News, 10: 13-18, T i c e, L. W. and R. J. Barr net t: The fine structural localization of glucose-6- phosphatase in rat liver. J. Histochem. Cytochem. 10: , W a c h s t e i n, M. and E. M. M e i s e 1: On the histochemical demonstration of glucose- 6-phosphatase. J. Histochem. Cytochem., 4: 592, Explanation of Figures Fig. 1. A normal rat hepatic parenchymal cell. The glucose-6-phosphatase (G-6-Pase) activity is positive in the endoplasmic reticulum, the nuclear envelope and the transitional element near the Golgi apparatus (Go). No activity is seen in mitochondria (in), the Golgi apparatus (Go) per se, the microbody (mi), multivesicular bodies (mu), coated vesicles (co) and the nucleus (n). The endoplasmic reticulum captured recently in cytolysomes (c) still shows G-6- Pase activity. The limiting membrane surrounding cytolysomes does not show any activity. x 24,000 Fig. 2. Normal rat hepatic parenchymal cells. The enzymatic activity is seen in the intracisternal space of the rough-surfaced endoplasmic reticulum and the transitional element. In some areas the reaction product appears to be adhered to the cisternal side of the membrane. Mitochondria (in), the Golgi apparatus (Go) and plasma membranes have no activity. Some cytolysomes (c2) contain lead phosphate precipitates. Others (c,) do not. Cytolysome c2 may show an intermediate stage of degradation of the endoplasmic reticulum within cytoly.

9 Ultracytochemical Changes of the Glucose-6-Phosphatase Activity 19 somes. x 23,000 Fig. 3. A normal rat hepatic parenchymal cell. Well-developed parallel arrays of the rough-surfaced endoplasmic reticulum can be seen in the center and the glycogen area (g) above it. The G-6-Pase activity is demonstrated in the cisternae of the rough-surfaced endoplasmic reticulum and the smooth-surfaced variant in and around the glycogen area (g). x 17,000 Fig. 4. Normal rat hepatic parenchymal cells. The glycogen area (g) can be observed in the central part and the G-6-Pase activity is positive in the smooth-surfaced endoplasmic reticulum in and around the glycogen area. Reaction products are observed in the intracisternal space. No activity is observed in the plasma membrane and the bile canaliculus (b). Some of final products are found in the degrading endoplasmic reticulum in a cytolysome (c). x 25,000 Fig. 5. An endothelial cell adjacent to a normal hepatic parenchymal cell. The G-6- Pase activity is observed in the endoplasmic reticulum and the nuclear envelope of a parenchymal cell, but no activity is found in an endothelial cell. No activity is observed in the plasma membrane facing to the Disse's space (D) also. Some lipid droplets (1i) without activity can be seen in a parenchymal cell. x 11,000 Fig. 6. Normal rat liver bile duct. The G-6-Pase activity is not positive in epithelia of the bile duct. Mitochondria (m), the Golgi apparatus (Go), free ribosomes, the endoplasmic reticulum (arrows) and the nucleus (n) do not show any G-6-Pase activity. x 23,000 Fig. 7. A hepatic parenchymal cell from the rat treated with phenobarbital for 9 days. The glycogen area (g) is observed in the center, however, no glycogen granules can be seen among the mesh-work of the tubular smooth-surfaced endoplasmic reticulum. The G-6-Pase activity is positive in the rough-surfaced endoplasmic reticulum, the tubular smooth-surfaced endoplasmic reticulum in and around the preexisting glycogen area, the transitional element near the Golgi apparatus (Go) and the nuclear envelope. Reaction products are in the cisternae. x 30,00D Fig. 8. Phenobarbital-treated rat hepatic parenchymal cells. The enzymatic activity is observed in the proliferated tubular smooth-surfaced endoplasmic reticulum near the bile canaliculus (b) in addition to the smooth-surfaced endoplasmic reticulum in and around the preexisting glycogen area. x 29,000 Fig. 9. Phenobarbital-treated rat hepatic parenchymal cells. The proliferated tubular smooth-surfaced endoplasmic reticulum near the Golgi apparatus (Go) is positive for the enzymatic activity. x 20,000

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