High glucose-6-phosphatase activity in osteoblasts in the metaphysis of femur of growing rats.код для вставкиСкачать
THE ANATOMICAL RECORD 220:252-257 (1988) High GIucose-6-Phosphatase Activity in Osteoblasts in the Metaphysis of Femur of Growing Rats HIROHIKO TOKUNAGA, SHINSUKE KANAMURA, JUN WATANABE, KAZUO KANAI, AND MINORU SAKAIDA Department of Anatomy and Orthopaedic Surgery, Kansai Medical University, Fumizonecho 1, Moriguchi, Osaka, 570 Japan ABSTRACT Glucose-6-phosphatase (G6Pase) activity was examined cytochemically in the metaphysis of femurs of 3- and 7-day-old rats. G6Pase and hexokinase activities were also examined biochemically in the femur and tibia of 3-day-old animals. The reaction product for G6Pase activity was seen in the endoplasmic reticulum and nuclear envelope of all cell types composing the metaphysis. The amount of the reaction product was abundant in osteoblasts, moderate in osteocytes, and moderate to scarce in osteoclasts and capillary endothelial cells. Biochemical G6Pase activity in the bones was higher than that in the brain, submandibular gland, or pancreas of the animals. Hexokinase activity in the bones was not different from that in the submandibular gland, pancreas, or kidney. The activity ratio of G6Pase and hexokinase in the bones (0.603) was greater than that in the submandibular gland, pancreas, or brain and smaller than that in the kidney. Possible physiological significances of the higher G6Pase activity in osteoblasts are discussed. were fixed with 2% glutaraldehyde containing 0.1 M sodium cacodylate (pH 7.4) at 4°C for 1 hr and washed in 0.1 M sodium cacodylate (pH 7.4) containing 0.2 M ethylenediamine tetraacetate (EDTA) and 8% sucrose for 1 h r at 4°C. EDTA was used for decalcification and inhibiting alkaline phosphatase activity. The fixed slices were sectioned at 30 pm with a freezing microtome and washed in the buffer containing 0.2 M EDTA for 1h r at 4°C. The sections were preincubated in 0.25 M sucrose containing 10 mM levamisole for 15 min and incubated in a reaction medium (3.7 mM G6P, 3.6 mM lead nitrate, 30 mM sodium cacodylate, 230 mM sucrose, and 10 mM levamisole, pH 6.7; Kanai et al., 1983, 1986; Shugyo et al., 1986; Watanabe et al., 1983, 1986) a t room temperature for 1h r with a change of the medium. Levamisole was used to inhibit alkaline phosphatase activity (van Belle, 1972). The sections were postfixed in 1%buffered osmium tetroxide (pH 7.4) for 2 h r a t 4"C, dehydrated, and embedded in Spurr. Thin sections were stained with uranyl acetate and lead citrate and examined in a JEM 100-S electron microscope. To ascertain whether the reaction product is due to G6Pase activity, control experiments (Tice and Barrnett, 1962; Kanamura, 1971a,b, 1975b) were carried out as follows: The glutaraldehyde-fixed sections were incubated in the reaction medium lacking G6P; sections were incubated in 0.1 acetate buffer (pH 5.0) for 30 min at 37°C before incubation in the reaction medium; sections were incubated in a reaction medium containing MATERIALS AND METHODS a n equimolar amount of /3-glycerophosphate in place of One hundred seventy-two male Wistar rats, 3 and 7 G6P; or sections were preincubated in 0.25 M sucrose days old, were used. The animals were killed by decapitation. The osteoblast is a functionally active cell bearing well-developed endoplasmic reticulum and participating in the synthesis and secretion of the organic components of the bone matrix. Generally, glucose-6-phosphatase (G6Pase; E.C. 188.8.131.52; D-glucose-6-phosphate phosphohydrolase) activity is shown to be high in functionally active cells, in which the endoplasmic reticulum is well developed, such as hepatocytes (Tice and Barmett, 1962; Ericsson, 1966; Kanamura, 1971a,b; Leskes et al., 1971) and jejunal epithelial cells (Hugon et al., 1970, 1971). The osteoblast is also supposed to provide storage sites for calcium and phosphate, which are used in the calcification process (Matthews and Martin, 1971; Lehninger, 1977; Brighton and Hunt, 1974,1978; Boskey, 1981). In vivo, G6Pase hydrolyzes glucose-6-phosphate (G6P)to produce glucose and phosphate (Arion et al., 1972); the enzyme is related to phosphate production. For these reasons, it is of interest to examine whether G6Pase activity is present in osteoblasts. However, there have been no data in the literature on G6Pase activity in osteoblasts. In the present study, therefore, we examined in the metaphysis of femurs of 3- and 7-day-old rats whether G6Pase activity is high in osteoblasts and whether the high activity in osteoblasts decreases with the development into osteocytes that are not concerned with the bone formation. Further, biochemical G6Pase and hexokinase activities were measured in the femur and tibia. Cytochemical Methods Metaphyses of the distal femurs were quickly removed and sliced at about 1 mm with razor blades. The slices 0 1988 ALAN R. LISS, INC. Received July 23, 1987; accepted August 31, 1987. GGPASEIN OSTEOBLASTS 253 containing 10 mM NaF for 15 min and then incubated nate was mixed with an equal volume of 12N HCl, and in the reaction medium containing an equimolar amount the pellet was dissolved in 2 ml of 12N HC1. Calcium in of NaF. an aliquot (0.1 ml) of the mixture or the solution was measured by the Calcium C-test (Wako Pure Chemical). Biochemical Methods All biochemical data were subjected to statistical analBone shafts of bilateral femurs and tibias of ten ani- ysis (analysis of variance and Student’s t-test). mals were used in one experiment, and five experiments RESULTS were done within an age group. Bone marrows were Cytochemical Results eliminated from bone shafts. The bone shafts were dissected into small pieces and homogenized in 20 mM TrisIn spite of the washing in the buffer containing 0.2 M HC1 buffer (pH 7.6) containing 50 mM KC1,2 mMMgC12, EDTA, the preservation of the fine structures of cells and 0.25 M sucrose in a Potter-Elvehjem homogenizer appeared generally satisfactory. The reaction product at 4,000 revlmin for 2 min at 4°C. The homogenate was €or G6Pase activity was present in the endoplasmic recentrifuged at 3,OOOg for 10 min at 4°C. The 3,OOOg ticulum and nuclear envelope in all cell types composing supernate was used for the measurement of G6Pase and the metaphysis of both 3- and 7-day-old animals: osteohexokinase activities (3-day-oldanimals). blasts, osteocytes, osteoclasts, capillary endothelial cells, Microsomal fractions were prepared according to the and fibroblasts. However, the amount of reaction prodmethod of Hogeboom et al. (1948). The 3,OOOg supernate uct was abundant in osteoblasts (Fig. l), decreased to was saved, and the pellet was again homogenized and moderate with development into the typical osteocyte centrifuged as above. The first and second supernates (Fig. 2), and moderate to scarce in osteoclasts (Fig. 41, were combined and centrifuged at 8,OOOg €or 10 min at fibroblasts, and endothelial cells. 4°C. The supernate was then centrifuged at 12,OOOgfor Deposition of final product was sometimes observed in 15 rnin at 4°C. The supernate was further centrifuged lysosomes and in the Golgi apparatus of osteocytes and at 105,OOOg for 60 rnin at 4°C. The resultant pellet was osteoclasts (Fig. 2); on the plasma membrane facing the washed with 0.15 M KC1, resuspended, and centrifuged free surface and in lysosomes and the Golgi apparatus again at 105,OOOg for 60 rnin at 4°C. The pellet was of osteoblasts (Fig. 1);and on the plasma membrane of suspended in 2 ml of 0.25 M sucrose and was used for endothelial cells and fibroblasts. Mitochondria in all cell assay of G6Pase activity (3-and 7-day-oldanimals). types showed no reaction product. No reaction product For comparing levels of G6Pase and hexokinase activ- was seen in matrix vesicles. ities in the bones with other organs, the 3,OOOg superOmission of G6P from the incubation medium resulted nates were also made from the brain, submandibular in a complete absence of the reaction product. Immergland, pancreas, and kidney of 3-day-oldanimals. sion of the fixed sections in 0.1 M acetate buffer (pH 5.0) G6Pase activity was assayed according to the method before incubation in the reaction medium or use of 0of Leskes et al. (1971). An aliquot (0.1 ml) of the 3,OOOg glycerophosphate in place of G6P in the reaction mesupernate or microsomal fraction was incubated with 1 dium caused a loss of the reaction product except in ml of the medium (30 mM G6P, 30 mM sodium cacodyl- lysosomes and in the Golgi apparatus and on the plasma ate, pH 6.7). Levamisole (5 mM) was added to the reac- membrane (Fig. 3). Preincubation and incubation of the tion medium to inhibit alkaline phosphatase activity. fixed sections with NaF abolished the total reaction, but Incubation was done at 37°C for 30 min. The inorganic the final product was still visible on the plasma memphosphorus released was determined by a Phosphor C- brane and sometimes in the Golgi apparatus. These test (Wako Pure Chemical, Osaka, Japan). Protein was results indicate that the reaction product in the endodetermined according to the method of Lowry et al. plasmic reticulum and nuclear envelope is due to G6Pase (1951).The enzyme activity was expressed as nanomoles activity, but the deposition of final product in lysosomes and in the Golgi apparatus and on the plasma memof G6P used per minute per milligram of protein. Hexokinase activity was assayed according to the brane is probably related to acid or alkaline phosphatase method of Joshi and Jagannathan (1966). An aliquot (0.1 activity. ml) of the 3,OOOg supernate was mixed with 2.8 ml of the medium containing 15 mM glucose, 10 mM MgC12, Biochemical Results 40 mM Tris-HC1 buffer, 1.4 IU glucose-6-phosphate deG6Pase activity in the 3,OOOg supernate from the bones hydrogenase, 0.01 mM EDTA and 0.3 mM 0-nicotinamide adenine dinucleotide phosphate (NADP), pH 7.4. of 3-day-old animals was 9.37 & 1.90 (nmol G6P used Then, 0.1 ml of 3.3 mM adenosine triphosphate (ATP) per min per mg of protein; mean f S.D. for five experiwas added to the mixture, and the formation of NADP- ments), and this value was higher than values of the reduced form was estimated at 30°C from the second to brain, submandibular gland, and pancreas, although it the tenth minute after adding ATP. The enzyme activity was lower than the value of the kidney (Table 1).The was expressed as nanomoles of G6P formed per minute activity in the microsomal fraction was 30.13 k 7.99 in 3-day-oldanimals and 36.73 k 3.17 in 7-day-oldanimals. per milligram of protein. Hexokinase activity (11.3 f 3.77 nmol G6P formed per As an index showing whether an organ is inclined to uptake glucose or to release glucose, the ratio of G6Pase min per mg of protein; mean f S.D. for five experiactivity and hexokinase activity at 37°C was calculated. ments) in the bones of 3-day-old animals was not differHexokinase activity was converted into the value at ent from values in the submandibular gland, pancreas, and kidney, although it was lower than the value in the 37°C by the method of Long (1952). To estimate the contamination of the bone matrix in brain. The activity ratio of G6Pase and hexokinase was 0.603. the 3,OOOg supernate, calcium was assayed in the 3,OOOg supernate and 3,OOOg pellet. One milliliter of the super- This value was greater than that of the submandibular Figs. 1-4. Cytochemical demonstration of GGPase activity in the cells of the metaphyses of femurs of 3- and 7-day-old rats. Sections (30 pm) cut from glutaraldehyde-fixed tissues and washed in the buffer containing 0.2 M EDTA were incubated for 1 hr in a medium modified from that of Wachstein and Meisel (1956). x 16,000. Fig. 1. A osteoblast from a 7-day-old animal. An abundant amount of the reaction product for GGPase activity is seen in the nuclear envelope and well-developed endoplasmic reticulum. The reaction product in lysosomes and on the plasma membrane facing the free surface (the upper side) is probably related to acid or alkaline phosphatase activity. Fig. 2. Two osteocytes from a 3-day-old (a)and a 7-day-old(a) animal. Note the decrease in the amount of reaction product for G6Pase activity in the endoplasmic reticulum. In addition, the endoplasmic reticulum decreases markedly in amount. The reaction product in lysosomes is probably related to acid phosphatase activity. 255 G ~ P A SIN E OSTEOBLASTS TABLE 1. G6Pase and hexokinase activities in 3,OOOg supernates from the bone and other organs of 3-day-old rats* Brain Bone Submandibular gland Pancreas Kidney G6Pase (37°C) Hexokinase (30°C) Hexokinase (37°C)' G6PaseI hexokinase at 37°C 3.6 & 0.72 9.4 & 1.90 3.9 ?C 1.75 21.9 f 4.30 11.3 & 3.77 8.7 f 3.00 30.0 & 5.90 15.5 + 5.17 11.9 f 4.11 0.120 0.603 0.328 4.2 & 0.53 52.4 & 4.64 8.4 1.22 17.4 2 6.04 + 11.5 & 1.67 23.8 8.28 0.368 2.201 + *Values are means f S.D. for five experiments (ten animals/experiment). Activities of G6Pase and hexokinase are nmol G6P usedmidmg protein and nmol G6P formedmidmg protein, respectively. 'The rate of G6P formed at 37°C was calculated by the method of Long (1952). Fig. 3. Portion of an osteoblast from a 7-day-old animal. The fixed section was incubated in the reaction medium containing an equimolar amount of 0-glycerophosphate in place of G6P. Note disappearance of Fig. 4. An osteoclast from a 7-day-old animal. A moderate amount of the reaction product in the endoplasmic reticulum and nuclear envelope. The reaction product in the Golgi apparatus and a lysosome is the reaction product for G6Pase activity is seen in the endoplasmic reticulum and nuclear envelope. probably due to acid phosphatase activity. clear envelope of osteoblasts in the metaphyses of femurs of 3- and 7-day-old rats. Further, biochemical G6Pase activities in the femur and tibia (except bone marrow) were higher than values in the brain, submandibular gland, and pancreas of the animals. The higher G6Pase activity in osteoblasts is probably related to their functions. G6Pase has a wide spectrum of hydrolytic and synthetic activities (Nordlie, 1972). However, hydrolysis of DISCUSSION G6P is probably the sole function of this enzyme in vivo As revealed in the present cytochemical results, an (Arion et al., 1972). The role of the enzyme in the liver abundant amount of reaction product for G6Pase activ- and kidney is to release glucose into blood by hydrolyzity was present in the endoplasmic reticulum and nu- ing G6P produced via gluconeogenesis and glycogeno- gland or pancreas, much greater than that of the brain, and smaller than that of the kidney (Table 1). Calcium content in the bones was 6.10 k 2.48 pglmg of wet tissue (mean _+ S.D. for five experiments). Percentages of calcium recovered in the 3,OOOg pellet and 3,OOOg supernate were 94.7 + 1.7 and 5.3 k 1.7, respectively. Therefore, contamination of the bone matrix in the 3,OOOg supernate was negligible. 256 H. TOKUNAGA ET AL. lysis (Krebs, 1963; Nordlie, 1972). We postulated that the role of relatively high activity in epididymal principal cells or in the seminal epithelium is to supply glucose into the epididymal fluid or fructose into the seminal fluid (Kanai et al., 1981, 1983, 1986) and that the role of the increased activity in skeletal muscle cells of starved mice is to release glucose into the blood (Hirose et al., 1986; Sakaida et al., 1987). However, the role of this enzyme in other various cell types containing low or moderate activities is unknown, although we supposed a role of regulation of G6P concentration in the cells, hydrolyzing if there is any excess (Kanamura, 1975a; Watanabe et al., 1983, 1986). Osteoblasts probably consume a large amount of glucose from the blood for the synthesis of organic components of bone matrix. In the present results, hexokinase activity in the bone was not different from that in the submandibular gland or pancreas, which is considered to be functionally active and to use glucose from the blood abundantly (Martin, 1967; Hokin, 1967). Therefore, it is probable that G6P is steadily produced by the hexokinase activity from the blood glucose in osteoblasts. The higher G6Pase activity possibly hydrolyzes G6P thus produced, if there is any excess. Thus, a role of the higher G6Pase in osteoblasts is possibly to regulate the intracellular concentration of G6P. Phosphate that is thus produced in the well-developed endoplasmic reticulum of osteoblasts may be abundant. Some of such phosphate may be used for new calcification in the bone; the phosphate may diffuse freely to new mineralization sites to increase regional phosphate levels. As to the mechanism of how calcium-phosphate particles in mitochondria of chondrocytes can influence the formation of large amounts of a solid phase of calcium-phosphate in the extracellular tissue spaces, Glimcher (1976) and Landis and Glimcher (1982) postulated as follows: Calcium and phosphate of mitochondrial particles may be released and then dissolved in the extracellular fluid and the resulting increases in ion concentrations produce a more metastable solution of calcium and phosphate, a situation that facilitates the heterogenous nucleation of solid phase calcium-phosphate particles by major connective tissue components such as collagen. A similar mechanism is supposed for the participation of calcium and phosphate in the matrix vesicle for tissue calcification (Anderson, 1976). The phosphate released by G6Pase activity from osteoblasts may also serve for the calcification by a similar mechanism. However, the mechanism is postulated for the initial step of calcification without any existing mineralization front, while considerable mineralization front, which can serve as a nucleator of new crystals (Glimcher, 19761, already exists in the bone used in the present study. Thus, the present results suggest that the endoplasmic reticulum in osteoblasts may play a role of phosphate production for new calcification in the bone. During the process of development into osteocytes, G6Pase activity decreased to a moderate level with a decline in the amount of the endoplasmic reticulum. Thus, GGPase activity decreases with the decline in functional activity of the cell. The production of phosphate also probably becomes lower in osteocytes, which are not concerned with the bone formation, than in osteoblasts. The activity ratio of G6Pase and hexokinase was 0.120 in the brain, and it was 2.201 in the kidney. The values are consistent with the facts that the brain is a n organ consuming much glucose (Sokoloff, 1960) and that the kidney releases glucose into the blood (Nordlie, 1972). The ratios (0.328 and 0.368) of the submandibular gland and pancreas possibly show that these organs neither consume much glucose nor release glucose into the blood. However, the ratio in the bone was 0.603. This suggests that the cells of the bone are more inclined to “release glucose and phosphate” than the cells of the submandibular gland and pancreas. G6Pase activity was moderate in osteocytes and moderate to scarce in osteoclasts and endothelial cells. The role of the activity in these cells is also possibly to regulate the intracellular concentration of G6P, as has been postulated in various cell types containing low or moderate activities (Kanamura, 1975a; Watanabe et al., 1983, 1986). LITERATURE CITED Anderson, H.C. (1976) Matrix vesicles of cartilage and bone. 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