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 vitamiq A increased the protein degradation (Fell and Mellanby. 1652) and also increased the release of acid pro´´tease (Fell and Dingle: 1963: Lucy et al., 1961). AIi (1964) suggested that another enzyme may be present in bone matrix for degrading chondroproteia, -.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 enrymes, 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 pmteia 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. I00. 150 .and ZOOg of body weight were purchased from alocal market.
The culture was carried out according to, the method of Raisz(196b). 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 37C under 5C% C0: for 4 or 6 days, , depending on the experimental design, sad the media was changed every two days. A preliminary. study was made on the growth pattern ,of tibia and the release of Ca" 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 (fmm 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 cjFay newborns were incubated for 25 hours in BGJb media with 0.012^-0.02G of glutamate-C" (specific activity: 14.9mCi/mM) in order to incorporate, the glutamate-C^14 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 C" into the medium fmm that incorporated in the bona After 48 hours of culture, the bones wen homogenized and treated with 106 trichloracetic acid. and the radioactivity of C´ in the insoluble part and in the soluble partwas measured. The radioactivity of C" 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 C". 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 0.5m1 of 0.85% saline and the treated group. with 0:5m1 con-twined 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 boas Also for 3 days prior to sacrifice. nriae was collected and hydrox3proliae excretion was determined by the method of Neuman and logan(1950). The radioactivity of C" and Ca`S was measured by a Pacl`ard-Tricab Liquid Scint~lation 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 boar 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^-1508 group.
Cathepsia A was 36.14±2.82 in 40g. 50.0516.20 in 100g. 46.66±8.16 in I50g and 35.00±1.06 amol/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 amol/min/mg of .protein in 200g, rats. Cathepsia C was 4.501.16 is 40g.. 6.032.22 ins IOOg,, 6.67 -1.70 in 1508 and 2.510.33 nmol/min/mg of protein in 2008 rats. Cathepsia D was 13.400.13 in 40g. :15.181.97 in.100g, 22. Sli3.55 in. 150g and 14 2p1.O6 nmol/min/mg of prokein. in 200g tats.
3. Release of glutamate-C^14
In the 18th day fetus, the radioactivity of C" in the insoluble part of the bone after 4 d´iys culture was 24337.71cpm in the boiled bone and 21830.99cpm is the non-boiled bone. The. released radioa ctivity is the media was 10´T48.9Qcpm is the boiled bone and 201i9..T3cpm ist thB nvn-boiled bone. The ratio of the radioactivity of the bone and the media was 1:0.44 is the boiled bone ana 1:0.9I. in the non-boiled bone. in 1 day old n~vborn 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=0.48: in. the boiled bone and l:Y, 62 ins the noes boiled hone. 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 0.87e±0.17㎍ per g of body weight in iota 40. 1091 150 aid 200g of body weight groups respectiively.
These ebservations on the eathepsia activities in the bone and´ the hydroxyprol5ne excretion is the urine suggest that proteolytae phenomenon iiv, tl4e bone may be mediated by´ a diff~reat proteolytic earyme in addition to cathepsin.
B. Effect. of hrdcncoctisonn oa protein degradation
1. On proteolytic enzyme is vivo experiment In the hgdrocortisone treated group, the activites, : were: 32.40+_3.51: ~-L~i.Z01.37., 3. G00.59 aid 17.002 38asnol/miu/mg~of protein for cathepsin A. B. ~ and. D respectively. In. the, control group.. the ~cathepsin activites of A. B, C and D were 38.805.82.. 2k 101.82. 3.000.88 and,´ 1Sr7,Q_+2 40`nmol% -min/mg of protein respectively.
2. On glutamate-C^14 release
In the 18th day fetus. radioactivity was`Q1830.99-.epos is the beat a~ 2~tT9..?3 cpeR. is"tba media of the control group. and it was 25324.03 cpm- in the ~ bow aaa "2~4~iT. 59´ cpm, in ihe: medfia "oE tlye hydrocortisone treated group:´ Ih 1 day old newborn, it was 96Q cpm ih the beae aid-?.566 cpm is t1le :media of the control group. and ?40 cpm is the bone and" 1.3~ cpar is the: media ~ 06 the treated groin ´The ratio of radioactivity of tfie bone to the media was 1:152= im. the control group aexk 1r:~Y. T9~ is .the . treated groin. -:
3. On hydroxyrpmliae a:cretioa is urine
Twenty four-hour urinary excretion of hydroxyproline was 0: 560: 01?pg per g of body weight in the .control getup and 0..530.044~eg per g of body weight in -the IIyd´rocortisone treated group.
This result indicates that the hydrocortisone has no significant effects on.protein degradation in bone.
Conclusion
In albino rat bone at different atag+es 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 ZOOg 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 2008 getup.
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 faBed 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.
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