Bisphosphonates and bone resorption: effects on collagenase and lysosomal enzyme excretion

When added to cultures of parathyroid hormone (PTH)- bones, dichloromethylenebisphosphonate (C12MBP) and 3-amino-1-hydroxypropydilene-1,1-bisphosphonate (AHPrBP) inhibit completely and in a parallel manner the development of resorption lacunae, the loss of calcium by the explants and their PTH-induced excretion of lysosomal hydrolases (β-glucuronidase and N-acetyl-β-glucosaminidase). The loss of collagen (hydroxyproline) by the bones is usually less inhibited than their loss of calcium and their heparin-induced excretion of collagenase is unaffected. To interpret these data, it is proposed that these bisphosphonates act more on the activity of osteoclasts, suppressing simultaneously their excretion of lysosomal enzymes and their erosion of mineralized bone matrix, than on that of other cell types (osteoblasts ?) responsible for collagenase production and the removal of uncalcified collagen.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Effects of cholera toxin on cyclic AMP accumulation and bone resorption in cultured mouse calvaria

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissociation of organic acid secretion from macrophage mediated bone resorption

A prevailing concept in the literature on bone resorption suggests that the removal of calcium crystals from the bone matrix is the result of the secretion of lactic and/or citric acid. In the present study, we have reassessed this concept using an in vitro bone resorption system consisting of thioglycolate elicited rat peritoneal macrophages co-cultured, for up to 96 hours, with devitalized ⁴⁵Ca-labeled bone particles. In these combined cultures, medium lactate concentration increased linearly for the first 48 hours of culture and remained at a plateau thereafter. Medium citrate concentration, on the other hand, remained constant and at very low levels throughout incubation. In contrast to both citrate and lactate, bone resorption, measured as ⁴⁵Ca release, began a few hours after the onset of culture and increased at a constant rate for the balance of the 96-hour culture period. Alteration of resorptive activity by the addition of 10^?6M cortisol (which stimulates ⁴⁵Ca release) or the interposition of a filter between cells and bone (which inhibits resorption) was not paralleled by similar shifts in lactate or citrate concentration. These experiments indicate that mobilization of the bone mineral can occur in the absence of a concurrent, generalized release of lactic and citric acid by sesorbing cells. On the other hand, the data do not exclude a possible role for these compounds under circumstances where they are secreteo into a “closed” compartment at the cell-bone interface or, in the case of lactate, during the initial period of resorptive activity.     

Relationship between bone resorption and lysosomal enzyme release as demonstrated by the effect of 1α-hydroxy-vitamin D-3 in an organ culture system

The effect of 1α-hydroxy-vitamin D-3 on the release of calcium (⁴⁰Ca, ⁴⁵Ca), inorganic phosphate and lysosomal enzymes, on glucose consumption and lactate production was studied in a bone organ culture system using half calvaria from 6–7-day-old mice. 1α-Hydroxy-vitamin D-3 stimulated the mobilization of minerals and increased the release of β-glucuronidase, β-N-acetylglucosaminidase and acid phosphatase, while no effect on the release of lactate dehydrogenase was seen. 1α-Hydroxy-vitamin D-3 also caused a significant increase in the total activities of acid phosphatase in the bones after culture, indicating increased enzyme synthesis. The stimulatory effect of the release of P_i and β-glucuronidase was also obtained after a temporary exposure to 1α-hydroxy-vitamin D-3. The stimulation by 1α-hydroxy-vitamin D-3 on the release of Ca²⁺, P_i and β-glucuronidase was suppressed by a protein synthesis inhibitor cycloheximide. No effect by 1α-hydroxy-vitamin D-3 on glucose consumption and lactate production was registered, suggesting that increased mineral mobilization does not require increased lactate production. It is concluded that although the data in the present paper do not prove a cause-and-effect relationship between lysosomal enzyme release and bone resorption, they give further support to the concept that the processes are intimately associated.     

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes.

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P20,2) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid beta-glycerophosphatase (EC 3.1.3.2) and acid p-nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p-nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1, 1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1, 1-diphosphonate had any effect on beta-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1, 1-diphosphonic acid inhibited acid p-nitrophenyl phosphatase strongly, the polyphosphate (P20, I) had little effect. Acid p-nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1, 1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.     

A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture

A specific and potent synthetic inhibitor of mammalian tissue collagenase and related metallo-proteinases inhibits the collagen matrix resorption induced by parathyroid hormone (PTH) in cultured embryonic mouse calvaria. The inhibition is reversible, dose-dependent and virtually complete at 50 microM inhibitor concentration whereas that due to a less potent stereoisomer is much weaker. The PTH-enhanced secretion of calvarial lysosomal enzymes and the small spontaneous leakage of lactate dehydrogenase are not affected by the inhibitor. These results suggest that collagenase plays a critical role in bone resorption. Its role is discussed in relation to that of cysteine-proteinases that have also been implicated in this process.     

Blood coagulation and bone metabolism: some characteristics of the bone resorptive effect of thrombin in mouse calvarial bones in vitro

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1-7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic acid and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal disease and rheumatoid arthritis.     

Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages.

Macrophages isolated from the peritoneal cavity of untreated mice and maintained in tissue culture synthesize and release prostaglandins when challenged with zymosan. These cells also selectively release lysosomal acid hydrolases under the same conditions. The major prostaglandins released into the media are found to be prostaglandins E1, E2 and 6-oxoprostaglandin F1a, whereas prostaglandin F2a is not detected. Macrophages isolated from mice that have received an intraperitoneal injection of thioglycollate broth are far less responsive to zymosan challenge. These cells require 300 microgram of zymosan to synthesize and release one-third the amount of prostaglandins released from non-stimulated macrophages exposed to 50 microgram of zymosan. In addition, thioglycollate-stimulated macrophages release less than 10% of their lysosomal acid hydrolases when exposed to 300 microgram of zymosan whereas non-stimulated cells release approximately 50% of these enzymes after treatment with 50 microgram of zymosan. The zymosan-stimulated synthesis and release of prostaglandins are completely inhibited by indomethacin, whereas the increased selective release of lysosomal acid hydrolases is not affected. Macrophages, unlike fibroblasts, do not synthesize and release prostaglandins when exposed to serum or to bradykinin.     

Effects of lowering food intake by high phosphorus diet on parathyroid hormone actions and kidney mineral concentration in rats

We investigated whether lowering food intake by high phosphorus (P) diet influenced parathyroid hormone (PTH) actions, bone turnover markers, and kidney mineral concentration in rats. Rats in two of the three groups were respectively given free access to a control diet (C group) and a high P diet (HP group) for 21 days. Rats in another group (PF group) were pair-fed the control diet with the HP group. Compared to the C and PF groups, serum PTH concentration, urinary C-terminal telopeptide of type I collagen excretion, and kidney calcium and P concentrations were significantly higher in the HP group. Urinary excretion of cAMP was significantly lower in the HP group than in the C and PF groups. These results suggested that high P diet decreased PTH action in the kidney and increased bone resorption and kidney mineral concentrations independently of lowering food intake.     

Effects of Lowering Food Intake by High Phosphorus Diet on Parathyroid Hormone Actions and Kidney Mineral Concentration in Rats

We investigated whether lowering food intake by high phosphorus (P) diet influenced parathyroid hormone (PTH) actions, bone turnover markers, and kidney mineral concentration in rats. Rats in two of the three groups were respectively given free access to a control diet (C group) and a high P diet (HP group) for 21 days. Rats in another group (PF group) were pair-fed the control diet with the HP group. Compared to the C and PF groups, serum PTH concentration, urinary C-terminal telopeptide of type I collagen excretion, and kidney calcium and P concentrations were significantly higher in the HP group. Urinary excretion of cAMP was significantly lower in the HP group than in the C and PF groups. These results suggested that high P diet decreased PTH action in the kidney and increased bone resorption and kidney mineral concentrations independently of lowering food intake.     

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.     

Formation and function of the ruffled border in osteoclasts

Osteoclasts are multinucleated hematopoietic cells specialised for bone resorption. Dissolution of the inorganic fraction of the bone matrix is mediated by acidification of the bone surface in contact with the osteoclast whereas secreted lysosomal enzymes digest organic components. Through massive exocytosis, the plasma membrane in contact with the bone surface enlarges into the ruffled border, which has unusual features more similar to endosomal/lysosomal membranes. Maintenance of the ruffled border during resorption is achieved through a balance between exocytosis and endocytosis. Inactivation of proteins necessary for the extracellular acidification or of the proteases involved in matrix degradation leads to osteopetrosis; a disease characterised by dense bones.     

Effect of shellfish calcium on the apparent absorption of calcium and bone metabolism in ovariectomized rats

Fossil shellfish powder (FS) and Ezo giant scallop shell powder (EG) were rendered soluble with lactate and citrate under decompression (FSEx and EGEx, respectively) and we examined the effects of lactate-citrate solubilization of FS and EG on mineral absorption, tissue mineral contents, serum biochemical indices and bone mineral density (BMD) in ovariectomized (OVX) rats. The apparent absorption ratios of minerals tended to be high in the rats fed with the solubilized mineral sources, those in the FSEx group being significantly higher than in the FS group. There was no significant difference in the tibia mineral content among the OVX groups. BMD at the distal femoral diaphysis was significantly increased by FSEx and EGEx feeding. It is suggested that solubilization with lactate and citrate under decompression increased the solubility and bioavailability of calcium from such natural sources of shellfish calcium as FS and EG.     

Selective release of lysosomal hydrolases from phagocytic cells by cytochalasin B

1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.     

Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity. Acid phosphatase activity greatly exceeded arylsulphatase activity, and most of the measured peptidase activity was due to acid peptidases. Optimum pH and apparent Km values were determined for the most abundant hydrolases. Exposure of human umbilical vein endothelial cells to bradykinin, thrombin or interleukin-1 resulted in negligible release of either hexosaminidase or lactate dehydrogenase (LDH), in contrast to phorbol myristate acetate, which caused a parallel, dose-dependent release of both enzymes. Treatment of these cells with calcium ionophore A23187, trypsin or platelet-activating factor, caused less than 10% release of either hexosaminidase or LDH. Agents known to modulate lysosomal enzyme secretion by other phagocytic cells failed to induce selective secretion of lysosomal enzymes by human umbilical vein endothelial cells.     

Studies on the mechanisms by which 2-chloroadenosine stimulates bone resorption in tissue culture

The effect of 2-chloroadenosine on bone resorption was studied in calvarial bones from 6-7-day-old mice in organ culture. 2-Chloroadenosine stimulated the mobilization of minerals (40Ca, 45Ca) and increased the degradation of matrix ([3H]proline) from the bones. The nucleoside also caused an increased release of beta-glucuronidase, a lysosomal enzyme. In doses above 30 microM 2-chloroadenosine was cytotoxic, as evidenced by an increased release of lactate dehydrogenase. 2-Chloroadenosine-stimulated resorption could be inhibited by calcitonin, increased concentration of phosphate in culture medium, cortisone, dexamethasone, indomethacin, naproxen, meclofenamic acid and 5,8,11,14-eicosatetraynoic acid. 2-Chloroadenosine was much more sensitive to inhibition by dexamethasone than was parathyroid hormone. The response to the maximal dose of 2-chloroadenosine could not be enhanced by parathyroid hormone, 1 alpha-hydroxyvitamin D-3 and prostaglandin E2. An exposure to 2-chloroadenosine for 12 h was not sufficient to produce prolonged resorption. The results suggest that 2-chloroadenosine stimulated bone resorption by a process which is dependent on osteoclastic activity. The possibility that the effect of 2-chloroadenosine, either directly or indirectly, is related to formation of prostaglandins is discussed in the light of the above data.     

Inhibition of bone resorption in vitro by serine-esterase inhibitors

The effect of two synthetic serine esterase inhibitors, N-alpha-dansyl(p-guanidino)phenylalaninepiperidine hydrochloride (I 2581) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (D-Phe-Pro-Arg-CH2Cl), on bone resorption in organ cultured mouse calvaria from neonatal mice has been examined. Mineral mobilization was assessed by analyzing the release of 45Ca, stable calcium (Ca2+) and inorganic phosphate (Pi). Organic matrix degradation was studied by analyzing the release of 3H from [3H]proline-labelled bones, and by quantifying the amounts of hydroxyproline in bone after culture. It was found that I 2581, at and above 30 mumol/l, dose-dependently inhibited 45Ca release induced by thrombin, parathyroid hormone (PTH), prostaglandin E2 and 1-alpha-hydroxyvitamin D-3. I 2581 (50 mumol/l) inhibited PTH-stimulated release of 3H from [3H]proline-labelled bones, and this effect was reversible after withdrawal of I 2581. I 2581 (50 mumol/l) inhibited the release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in bones stimulated by PTH and 1-alpha-hydroxyvitamin D-3, without affecting the release of lactate dehydrogenase. In parallel, I 2581 decreased PTH and 1-alpha-hydroxyvitamin D-3 induced reduction of hydroxyproline levels in bones after culture. I 2581 (50 mumol/l) did not affect the basal release of 45Ca, Ca2+, beta-glucuronidase and beta-N-acetylglucosaminidase, nor the basal amounts of hydroxyproline in bones after culture. D-Phe-Pro-Arg-CH2Cl (100 mumol/l) significantly inhibited PTH- and PGE2-induced release of 45Ca without affecting basal release of radioactive calcium. These data indicate that activation of serine proteinase(s) may be a necessary step in the mechanism of action of several stimulators of bone resorption.     

Enzymologic study of the structural organization of the matrix or rat liver peroxisomes]

The effect of ionic strength and pH on the release of some enzymes of the matrix of peroxisomes in rat's liver was studied. Catalase, L ALpha-hydroxy acid oxidase, isocitrate dehydrogenase, glycerophosphate dehydrogenase and lactate dehydrogenase were easily released from the particles during their lysis and treatment with 0.16 M KCl, whereas urate oxidase, NADH cytochrome c reductase and D-amino acid oxidase were not solubilized. After the solubilization of peroxisomal membrane by 0.2% Triton X-100, the remaining core contained about 50% amino acid oxidase activity, and had 1.28--1.30 g/cm3 density. These results suggest that D-amino acid oxidase associates with urate oxidase in the peroxisomal core.