J Korean Orthop Assoc.  1974 Jun;9(2):183-197. 10.4055/jkoa.1974.9.2.183.

The Proteolytic Enzyme Activity and Hydrocortisone Effect on Protein Degradation in Long Bone of Albino Rats

• Choi CD

• KMID: 2471503
• DOI: http://doi.org/10.4055/jkoa.1974.9.2.183

Abstract

Synthesis of collagen and mucopolysaccharide in the bone is inhibited the glucocorticoid hormone(Layton. 1961: Schiller and Dorfman, 1957; McCluskey and Thomas. 1959; Bernick and Ershoff, 1963), and triiodothyronine inhibits the incorporation of proline C into collagen (Hahne et al. , 1972). Heller-Steinberg (1951) found that osteoclasts produce an enryme which might be involved in the protein degradation of bone matrix. The administration of excess vitamie A increased the protein degradation (Fell and Mellanby. 1652) and also increased the release of acid protease (Fell and Dingle: 1963: Lucy et al., 1961). Ali (1964) suggested that another enzyme may be present in bone matrix for degrading chondroprotein, even though collagenase and hyaluronidase are absent. Pita et al. (1.970) reported that the protein degradation process preceded mineral formation. Recently, Hahne et al. (1972) observed that triiodothyroaine increased the protein degradation in cultured bone. As a whole, these studies were not on proteolytic enzymes, but on degradation phenomena of protein. Only a few proteolytic enzymes in bone matrix have been identified. The purpose of the present study was to investigate pmteolytic enzymes in the long bone of albino rat at different stages of the gmwth and the effect of hydrocortisone on protein degradation. MATERIAL AND METHOD: a. Adult rats:-healthy, female adult rats. weighing around 200g, were used for obtaining fetuses and newborns. The proestrus stage was checked by vaginal smear and next day the conception was confirmed by the presence of the sperms. b. The fetus was obtained on the 18th day of gestativa by laparatomy. c. Rats in growing stages:-rats in the first day and third day of the neonatal period. were obtained from our laboratory. and those weighing 40, 100, 150 and 200g of body weight were purchased from a local market. The culture was carried out according to, the method of Raisz(1965). The bone(tibia) was excised under stereomicroscope in the tissue culture mom and was cultured in microculture slides or dishes containing BGJb media. Culture was performed in the incubator at 37℃ under 50% CO2 for 4 or 6 days, depending on the experimental design, and the media was changed every two days. A preliminary. study was made on the growth pattern, of tibia and the release of Ca45 as a measure of evaluation of this culture system. The sliced bone (from 18th day fetus, 1st day and 3rd day infant rats) or the homogenatd bone marrow (from rats over 40g) of the tibia was used for measuring cathepsin A,B,C and D activity by the method of Misaka and Tappel(1971). The tibiae of 18th day fetuses and 1 day newborns were incubated for 25 hours in BGJb media with 0.012~0.02uCi of glutamate-C14 (specific activity: 14.9mCi/mM) in order to incorporate, the glutamate-C14 into the bony tissues These cultured bones were divided into 2 groups; owe group of-bones boiled in water for 10 minutes and the other group of nonboiled bones. Thereafter,´ bone culture was continued to examine the release of C14 into the medium fmm that incorporated in the bone. After 48 hours of culture, the bones wen homogenized and treated with 106 trichloracetic acid. and the radioactivity of C14 in the insoluble part and in the soluble partwas measured. The radioactivity of C14 in the media was measured without treatment by 10% trichIoracetin acid. Effect of hydrocortisone on protein degradation: The bones wen cultured for 6 days in BGJb media with 0.5mg of hydrocortisone and measured for radioactivity of C14. In the control group, 0.85 % saline was added to the media in place of hydrocortisone. Ten adult male rats weighing around 200g were divided into 2 groups; The control group was injected hypodermically daily for 3 weeks. with 0.5ml of 0.85% saline and the treated group. with 0.5ml contained of 6 mg of hydrocortisone for each rat. At the completion of the treatment, the rats were sacrificed by.gaillotine and measured the activities of cathepsin A,B,C and D in the bone. Also for 3 days prior to sacrifice. nriae was collected and hydroxyproline excretion was determined by the method of Neuman and logan(1950). The radioactivity of C14 and Ca45 was measured by a Packard-Tricab Liquid Scintillation Spectrometer.
RESULTS
A. Protein degradation in the bone. 1. Growth of cultured bone. The length of tibia of the 18th day fetus was 1.98±0.05mm on the average and the growth rate was 0.5mm after 24-hour cultures. 2. Proteolytic enzyme in the bone. The activity of cathepsin A, B and D in the growing stages was generally high. and the maximum-value was observed. in the 100~150g group. Cathepsia A was 36.14±2.82 in 40g. 50.05±16.20 in 100g. 46.66±8.16 in 150g and 35.00±1.06 nmol/min/mg of protein in 200g rats respectively. Cathepsia B was 24.29±11.50 in 40g. 28.57±13.43 in 100g, 26.43±8.29 in 150g and 12.86±6.21 nmol/min/mg of .protein in 200g, rats. Cathepsia C was 4.50±1.16 is 40g.. 6.03±2.22 ins IOOg,, 6.67±1.70 in 150g and 2.51±0.33 nmol/min/mg of protein in 200g rats. Cathepsia D was 13.40±0.13 in 40g, 15.18±1.97 in 100g, 22.55±3.55 in 150g and 14.20±1.O6 nmol/min/mg of prokein. in 200g rats. 3. Release of glutamate-C14 In the 18th day fetus, the radioactivity of C14 in the insoluble part of the bone after 4 days culture was 243±37.71cpm in the boiled bone and 218±30.99cpm is the non-boiled bone. The released radioactivity in the media was 107±48.52cpm is the boiled bone and 201±79.73cpm in the non-boiled bone. The ratio of the radioactivity of the bone and the media was 1:0.44 is the boiled bone ana 1:0.91. in the non-boiled bone. in 1 day old newborn rat, the radioactivity was 1.371 cpm in the bone and 1.556 cpm in the media of boiled group, and 960 cpm in the bone and 1.556 cpm in the media o the non boiled group The ratio of the radioactivity of the bone and media was 1:0.43 in the boiled bone and l:1.62 in the non-boiled bone. Theses data are suggestive of the presence of proteolytic phenomenon in thet bones. 4. Hydroxyproline excretions is 24 hours uricLe was 5 46±0.99. 2.99±0.23. 1.60±0.11 and 0.87±0.17 µg per g of body weight in the 40, 109, 150 and 200g of body weight groups respectively. These ebservations on the cathepsin activities in the bone and the hydroxyproline excretion is the urine suggest that proteolytae phenomenon in the bone may be mediated by a different proteolytic earyme in addition to cathepsin. B. Effect. of hydrocortisone on protein degradation. 1. On proteolytic enzyme is vivo experiment In the hgdrocortisone treated group, the activites, : were: 32.40±3.51, 15.70±1.37, 3.00±0.59 and 17.00±2.38nmol/min/mg of protein for cathepsin A,B,C and D respectively. In the control group, the cathepsin activites of A. B, C and D were 38.80±5.82, 21.10±1.87, 3.00±0.88 and 15.70±2.40 nmol/min/mg of protein respectively. 2. On glutamate-C14 release. In the 18th day fetus. radioactivity was 218±30.99cpm in the bone and 201±79.73cpm in the media of the control group. and it was 253±24.03 cpm in the bone and 214±77.50cpm in ihe media of the hydrocortisone treated group: In 1 day old newborn, it was 960 cpm in the bone and 1.566 cpm in the media of the control group. and 740 cpm in the bone and 1.331 cpm in the media of the treated groin ´The ratio of radioactivity of the bone to the media was 1:1.62 in the control group and 1:1.79 in the treated group. 3. On hydroxyrproline excretion in urine. Twenty four-hour urinary excretion of hydroxyproline was 0.56±0.017ug per g of body weight in the control group and 0.58±0.044ug per g of body weight in the hydrocortisone treated group. This result indicates that the hydrocortisone has no significant effects on protein degradation in bone.
CONCLUSION
In albino rat bone at different stages of growth, protein degradatiea and cathapeia A, B, C and D activity ware stndiad is both cultured bona and in vivo experiments. The results are as follows: 1. Proteolytic enzymes, cathepsin A, B, C and D were present in the 18th day fetus, in the 1 and 3 days newborn, and in 40, 100, 150 and 200g group rats. 2. The activity of cathepsin A, B, C and D was gradually increased with age. The maximum value was noted in the 100~150g group and the value was less in the 200g group. 3. In general, the activity of cathepsin A was higher than that of cathepsin D at each of different growing stages. 4. Twenty four-hour urinary excretion of hydroxyproline per gram of body weight was higher is younger rats than older one. 5. Hydrocortisone failed to exert a significant effect on protein degradation and cathepsin activity in the cultured bone and in vivo experiments. 6. Proteolytic phenomena in the both may be mediated by different proteolytic enzyme in addition to cathepsin.

MeSH Terms

Adult
Animals
Body Weight
Bone Marrow
Bone Matrix
Cathepsin A
Cathepsin D
Cathepsins
Collagen
Collagenases
Female
Fertilization
Fetus
Groin
Humans
Hyaluronoglucosaminidase
Hydrocortisone*
Hydroxyproline
Incubators
Infant
Infant, Newborn
Male
Methods
Miners
Osteoclasts
Peptide Hydrolases
Proestrus
Proline
Proteolysis*
Radioactivity
Rats*
Research Design
Spermatozoa
Tibia
Triiodothyronine
Vaginal Smears
Water
Cathepsin A
Cathepsin D
Cathepsins
Collagen
Collagenases
Hyaluronoglucosaminidase
Hydrocortisone
Hydroxyproline
Peptide Hydrolases
Proline
Triiodothyronine
Water

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