Comparison of Somatomedin Activity in Rat Serum and Lymph1 KENNETH L. COHEN AND S. PETER NISSLEY Metabolism Branch, National Maryland 20014
Cancer Institute,
National
ABSTRACT. A chick embryo fibroblast (CEF) assay has been used to measure the somatomedin activity in serum and lymph from normal rats. Lymph contains about one-half the SM activity found in serum. The somatomedin activities in serum and lymph have identical elution volumes on Sephadex G-50 in I N
S
OMATOMEDIN (SM) activity is associated with a family of low molecular weight (MW 4,500-10,000) heat stable polypeptides. In normal serum, SM is bound to one or several larger proteins from which it dissociates at low pH (1,2). The influence of protein binding on the distribution of SM between serum and interstitial fluid has not been reported. Utilizing a recently described chick embryo fibroblast (CEF) assay (3), we have compared the concentration of SM activity in serum and lymph from normal rats. The level of SM activity in lymph is one-half that of serum. The active material in lymph is similar to that in serum in that it is heat stable, it is associated with large proteins at neutral pH, and on Sephadex chromatography under acid (dissociating) conditions it has the same elution volume as the SM activity in rat serum. Thus, the SM peptides appear not to be freely diffusible into the interstitial fluid. Materials and Methods Media, chemicals and hormones Temin's modified Eagle's minimum essential medium (E1) in powder form, and [3H-methyl]thymidine, 13-21 Ci/mmol, were obtained from Schwarz-Mann. Tryptose phosphate broth Received February 21, 1975. 1 Abbreviations used are: SM, somatomedin; CEF, chicken embryo fibroblasts; E, Temin's modified Eagle's minimum essential medium; GH, growth hormone; hypox., hypophysectomized.
Institutes
of Health,
Bethesda,
acetic acid. Chromatography at neutral pH demonstrates that somatomedin peptides are bound to large proteins in both normal rat serum and lymph. The relative restriction of somatomedin to the intravascular space may be due to this protein binding. (Endocrinology 97: 654, 1975)
was obtained from Difco. Fetal calf serum was purchased from GIBCO. Bovine growth hormone (GH) 0.92 IU/mg, NIH-GH-B17 was generously supplied by the Hormone Distribution Officer,
NIAMDD.NIH. Tissue culture and [3H]thymidine incorporation into DNA Tertiary cultures of CEF's were plated in E media containing 20% (vol/vol) tryptose phosphate broth and 0.4% (vol/vol)'fetal calf serum at a density of 1 x 106 cells/60 mm dish (Falcon Plastics). [3H]Thymidine incorporation into DNA was measured using the procedure described by Temin (4). Briefly, after 3 - 5 days the 0.4% medium was replaced with 3 ml of E medium containing the serum or lymph to be assayed. After 12 h incubation at 37 C, 5% CO2 atmosphere, the test medium was replaced with 2 ml Eagle's medium containing 0.2 /xCi/ml [3H]thymidine and incubated for 1 h. The radioactive medium was removed and the cultures washed twice with 3 ml cold Dulbecco's phosphate buffered saline (without Ca or Mg), once with 3 ml cold 10% trichloroacetic acid and once with 3 ml of ethenol:ether (3:1). The dried, fixed cells were dissolved in 0.2N NaOH, an aliquot added to Aquasol (New England Nuclear) and radioactivity measured in a Mark II (Nuclear Chicago) scintillation counter. Cell multiplication Cell multiplication experiments also utilized tertiary cultures of CEF's plated at 1 x 10%0 mm dish; however, the E media contain ing boiled rat serum or lymph were added approximately 4 hours after plating in 0.4% medium. Cell counts were performed on a Particle Data cell counter
654
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SOMATOMEDIN IN SERUM AND LYMPH before the initial addition of media and at 3 days of growth. Rat serum and lymph
655
the column. Six ml fractions were collected from which 2 ml samples were lyophilized, reconstituted in media, and tested on duplicate CEF assay dishes.
Hypophysectomized rats were obtained from Rat cartilage assay Zivic-Miller. The operation was performed on The column fractions obtained from boiled 90-100 g male Sprague-Dawley rats and the rats were maintained for 10 days prior to use. The GH normal serum were pooled for assay in a rat carti3 treatment schedule was 3 ip injections of 200 lage system. [ H]Thymidine uptake into costal ^g of bGH (NIH-GH-B17) over a 24-h period. cartilage from hypophysectomized rats was measSerum was obtained by cardiac puncture 4 h ured by the method of Daughaday and Reeder (5). after the last injection, allowed to clot at 4 C, The cartilages (4 per flask) were incubated for centrifuged at 3,000 rpm in an IEC PR 6000 22 h with 6 ml of medium alone or 1:4 dilutions centrifuge, and the serum stored at —15 C. of the pooled column fractions (1.5 ml aliquots Lymph was obtained from normal Sprague Daw- lyophilized and reconstituted in 6 ml of medium). ley rats weighing 125-150 g. Under ether anes- Incubations were continued for 4 h in labelled thesia, a polyethylene catheter (Intramedic, PE medium. Cartilage pieces were dissolved in con7400) was inserted into the cysterna chyli and centrated formic acid, mixed with Aquasol, and sutured in place. The animals were then main- counted in the liquid scintillation counter. tained for 24 h in restraining cages, while lymph was collected in a sterile iced tube. The lymph Results was then centrifuged and stored at - 1 5 C. Boiled serum and lymph were obtained by diluting with physiologic saline (3 parts saline, 1 part serum or 1) Activity in whole and heat-treated lymph lymph), adjusting the pH to 5.5 with HC1 and Pooled rat lymph (n = 5) and pooled norplacing the serum or lymph in a boiling water mal rat serum (n = 25) were assayed at 1%, bath for 15 min. After removal of precipitate by 5% and 10% concentrations (Fig. 1). The centrifugation, dilutions were made with E greatest activity was noted at 1% with promedium prior to use. The final percent value gressive inhibition at higher concentrations. assigned to the media containing boiled serum Heat treatment of the specimens under acid or lymph was based on the volume of serum or conditions resulted in more linear dose relymph used prior to dilution and boiling. sponse curves, thus allowing a more accurate comparison of the relative activities. In the Gel filtration assay shown in Fig. 1 lymph had 47% of A G50 (fine) Sephadex column (1.5 x 90 cm) the activity of serum at a 5% concentration. equilibrated with Dulbecco's phosphate buf- Five paired specimens (lymph and serum fered saline (without Ca ++ or Mg ++ , pH 7.4) and from the same animal) were also heat treated run at 4 C was used for the experiment shown and assayed at a 5% concentration. Combinin Fig. 2. The column was calibrated with blue ing the data from both pooled and paired dextran 2000 (Pharmacia) and phenol red markspecimens, rat lymph contains 50% ± 8 ers. One ml aliquots of serum or lymph were (SEM) of the SM activity present in rat serum. layered on the column. Six ml fractions were collected from which 2 ml samples were tested on dulplicate CEF assay dishes. A G50 (fine) Sephadex column (1.5 x 90 cm) equilibrated with IN acetic acid and run at 25 C was used for the experiments shown in Fig. 3. The column was calibrated with blue dextran 2000, cytochrome C (MW 12,400) and bacitracin (MW 1,450). Two ml aliquots of serum or lymph were boiled under acid conditions, lyophilized, reconstituted with IN acetic acid, and layered on
2) Sephadex chromatography of serum and lymph Aliquots of serum and lymph from normal rats were passed over a G50 Sephadex column equilibrated with phosphate buffered saline (pH 7.4). The active material in both eluted in the void volume in association with larger proteins (Fig. 2). The areas under the
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Enclo • 1975 Vol 97 • No 3
COHEN AND NISSLEY
656
elution curves paralleled the relative activities of unfractionated serum and lymph. Aliquots of serum from normal, hypophysectomized, and GH treated hypophysectomized rats were boiled under acid conditions and then passed over a G50 Sephadex column equilibrated with I N acetic acid. The active material in each of the 3 specimens eluted as a single peak with the same elution volume (Fig. 3A). There was no loss of activity during gel filtration and the areas under the elution curves paralleled the relative activities of unfractionated, heat-treated rat sera. Furthermore, when pooled column fractions were tested in a rat cartilage assay system, activity was found in the same fractions which were active in the CEF assay (Fig. 3B). Heat-treated rat lymph was chromatographed on the same G50 column and the active material eluted as a single major peak (Fig. 3C). The elution volume was identical to that of the GH-dependent material in rat serum. 3) CEF growth experiments
% SERUM OR LYMPH
FIG. 1. Stimulation of [3H]thymidine incorporation into DNA of CEFs by untreated and boiled rat serum and lymph. The details of the [3H]thymidine incorporation assay and preparation of boiled serum and lymph are given in Materials and Methods. Each point represents the results of the assay on a separate dish of CEFs.
In order to demonstrate that the relative activities of serum and lymph in the [3H]thymidine incorporation assay were a measure of DNA synthesis, cell multiplication experiments were performed. Although
VOID VOLUME
10
SEPHADEX
G-50. pH 7.4 SALT
J
I
7
8
9
10
11
l 12
I 13
I 14
I 15
I 16
TUBE NUMBER
I 17
18
19
20
21
I 22
I 23
l 24
FIG. 2. Stimulation of [3H]thymidine incorporation into DNA of CEFs by Sephadex C-50 (pH 7.4) fractions of normal rat senim and lymph. Details of the [ 3 H]-thymidine incorporation assay and Sephadex chromatography are given in Materials and Methods. Each point represents the result of the assay of a separate dish of CEFs.
25
(6 ml fractions)
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657
SOMATOMEDIN IN SERUM AND LYMPH serum-starved CEF's can be shown to accumulate DNA in response to untreated rat serum most of the cells do not undergo cell division (data not shown). However, heat-treated serum and lymph will support cell multiplication. As shown in Table 1, the increase in cell number in 5% lymph is about 40% of that stimulated by 5% serum. Thus, the results of cell multiplication experiments parallel the thymidine incorporation data.
possibility by comparing SM concentrations in rat serum and lymph. Whole rat serum is more active than rat lymph in the CEF assay, but accurate comparison is difficult because of the inhibition noted at higher concentrations. Heat treatment of lymph and serum removes these inhibitors resulting in nearly linear dose response curves (Fig. 1). Rat lymph contains 50% ± 8 (SEM) of the activity of rat serum in A. BOILED RAT SERA SEPHADEX G-50. IN ACETIC ACID
Discussion
BACITRACIN
v.
Somatomedin is the proposed mediator of growth hormone (GH) action on skeletal tissue (6). Rather than being a unique molecular species, SM appears to encompass a family of polypeptides (1). A common property is the association with larger proteins in normal serum (1,2). Although the physiological significance of the binding is unknown, it could have major effects on the distribution of SM in body fluids, the magnitude of fluctuations in serum SM concentrations, and the serum half-life of SM. In this report we have investigated the first
GH TREATED / / HVPOX i '
SALT
. -
V
B. CARTILAGE ASSAY
FlC. 3. A. Stimulation of [3H]thymidine incorporation into DNA of CEFs by Sephadex G-50 (IN acetic acid) fractions of boiled serum from normal, hypox., and bGH-treated hypox. rats. Details of the [3H]thymidine incorporation assay, heat treatment of serum, and Sephadex chromatography are given in Materials and Methods. Vo represents the void volume of the column as determined with blue dextran 2000. The elution peak of the bacitracin marker and the salt front are noted. The cytochrome C marker appeared in the void volume. Each point represents the result of the assay for a separate dish of CEFs. B. Stimulation of [3H]thymidine incorporation into DNA of costal cartilages from hypox. rats by pooled Sephadex G50 fractions of boiled normal rat serum. Aliquots of the fractions shown in Fig. 3A were used. The cartilage assay method is that of Daughaday and Reeder (4). Results are expressed as the percent of baseline uptake in control cartilages. Each bar represents the mean value of four costal cartilages. C. Stimulation of [3H]thymidine incorporation into DNA of CEFs by Sephadex G50 fractions of boiled lymph. The column was the same as that used in Fig. 3A. Each point represents the result of the assay for a separate dish fo CEFs.
C. BOILED RAT LYMPH
10 11 12 13 14 15 16 17 IB 19 20 21 22 23 TUBE NUMBER 16ml fractions)
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TABLE 1. Growth of chick embryo fibroblasts in boiled serum and lymph 106 Cells/dish 1) DavO 2) Day 3 a) Control b) 5% Serum c) 5% Lymph
Enclo • 1975 Vol 97 • No 3
COHEN AND NISSLEY
65S
Mean
0.84,
0.79,
0.78
0.80
0.84, 1.79, 1.17,
0.92, 1.83, 1.27,
0.89 1.85 1.29
0.88 1.82 1.24
The details of the growth experiment are given in Materials and Methods. Each value is for a separate dish of CEFs.
the CEF assay. This difference was confirmed in cell multiplication experiments (Table 1). When heat-treated sera from normal, hypophysectomized, and GH treated hypophysectomized rats were chromatographed on a G50 Sephadex column, equilibrated with L\ acetic acid, the active material in each of the 3 types of sera eluted as a single peak with identical elution volumes (Fig. 3A). Furthermore, the same fractions active in the CEF assay stimulated thymidine uptake in hypophysectomized rat cartilage, a standard SM assay (Fig. 3B). The three types of sera thus serve as a column marker for the heat stable, low molecular weight, GHdependent activity (i.e., SM) in rat serum. The activity in heat-treated rat lymph, chromatographed on the same G50 column, eluted as a single peak with the same elution volume as the serum SM activity (Fig. 3C). Thus, the physical properties of the active material in serum and in lymph are similar. These results indicate that SM is not freely diffusible from serum into lymph. This is in contrast to immunoreactive insulin which has a similar molecular weight but whose interstitial fluid concentration more closely reflects the intravascular levels (7). Insulin behaves as an unbound molecule in normal serum (8). The association of SM with larger serum proteins may explain its relative restriction to the vascular compartment. When normal rat serum and lymph were chromatographed on a G50 sephadex column equilibrated with phosphate buffered saline, pH 7.4, the active material appeared
in the void volume (Fig. 2). Thus, the association with large proteins described for serum SM also applies to extravascular SM activity. The relative concentration of SM activity may, therefore, be governed by the distribution of the large binding protein between tissue compartments. In studies of one of the purified somatomedins from human serum, SM-B, Yalo et al. reported that its distribution space after IV injection in the dog was about twice plasma volume (9). The data are difficult to interpret, however, since there was no significant binding of the human SM-B to the dog plasma proteins. In conclusion, the association of SM with large serum proteins appears to alter its relative distribution between serum and interstitial fluid. Whether this can function as a regulatory mechanism to limit the concentration of growth factors at target sites is unknown. The effect of protein binding on the half-life of SM in serum is currently being investigated. Acknowledgments We wish to thank Patricia Short for her skillful technical assistance and Dr. James Phang for reviewing the manuscript. We also thank the NIAMDD, NIH, for the gift of bGH HIN-GH-B17.
References 1. VanWyk, J. J., L. E. Underwood, R. L. Hintz, S. J. Voina, R. P. Weaver, and D. R. Clemmons, Recent Progr Horm Res 36: 259, 1974. 2. Hintz, R. L., E. M. Orsini, and M. G. VanCamp, 56th Annual Meeting of the Endocrine Society, A-71, 1974 (Abstract). 3. Cohen, K. L., P. A. Short, and S. P. Nissley, Endocrinology 96: 193, 1975. 4. Pierson, R. W., Jr., and H. M. TeminJ Cell Physiol 79: 319, 1972. 5. Daughaday, W. H., and C. ReederJ Lab Clin Med 68: 357, 1966. 6. , K. Hall, M. Raben, W. D. Salmon, Jr., J. L. Van den Brande, and J. J. VanWyk, Nature 235: 107, 1972. 7. Rasio, E. A., C. L. Hampers, J. S. Soeldner, and G. F. Cahill, J r . J Clin Invest 46: 903, 1967. 8. Berson, S. A., and R. S. Yalow, Amy Med 40: 676, 1966. 9. Yalow, R. S., K. Hall, and R. LuftJ Clin Invest 55: 127, 1975.
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Cancer Institute,
National
ABSTRACT. A chick embryo fibroblast (CEF) assay has been used to measure the somatomedin activity in serum and lymph from normal rats. Lymph contains about one-half the SM activity found in serum. The somatomedin activities in serum and lymph have identical elution volumes on Sephadex G-50 in I N
S
OMATOMEDIN (SM) activity is associated with a family of low molecular weight (MW 4,500-10,000) heat stable polypeptides. In normal serum, SM is bound to one or several larger proteins from which it dissociates at low pH (1,2). The influence of protein binding on the distribution of SM between serum and interstitial fluid has not been reported. Utilizing a recently described chick embryo fibroblast (CEF) assay (3), we have compared the concentration of SM activity in serum and lymph from normal rats. The level of SM activity in lymph is one-half that of serum. The active material in lymph is similar to that in serum in that it is heat stable, it is associated with large proteins at neutral pH, and on Sephadex chromatography under acid (dissociating) conditions it has the same elution volume as the SM activity in rat serum. Thus, the SM peptides appear not to be freely diffusible into the interstitial fluid. Materials and Methods Media, chemicals and hormones Temin's modified Eagle's minimum essential medium (E1) in powder form, and [3H-methyl]thymidine, 13-21 Ci/mmol, were obtained from Schwarz-Mann. Tryptose phosphate broth Received February 21, 1975. 1 Abbreviations used are: SM, somatomedin; CEF, chicken embryo fibroblasts; E, Temin's modified Eagle's minimum essential medium; GH, growth hormone; hypox., hypophysectomized.
Institutes
of Health,
Bethesda,
acetic acid. Chromatography at neutral pH demonstrates that somatomedin peptides are bound to large proteins in both normal rat serum and lymph. The relative restriction of somatomedin to the intravascular space may be due to this protein binding. (Endocrinology 97: 654, 1975)
was obtained from Difco. Fetal calf serum was purchased from GIBCO. Bovine growth hormone (GH) 0.92 IU/mg, NIH-GH-B17 was generously supplied by the Hormone Distribution Officer,
NIAMDD.NIH. Tissue culture and [3H]thymidine incorporation into DNA Tertiary cultures of CEF's were plated in E media containing 20% (vol/vol) tryptose phosphate broth and 0.4% (vol/vol)'fetal calf serum at a density of 1 x 106 cells/60 mm dish (Falcon Plastics). [3H]Thymidine incorporation into DNA was measured using the procedure described by Temin (4). Briefly, after 3 - 5 days the 0.4% medium was replaced with 3 ml of E medium containing the serum or lymph to be assayed. After 12 h incubation at 37 C, 5% CO2 atmosphere, the test medium was replaced with 2 ml Eagle's medium containing 0.2 /xCi/ml [3H]thymidine and incubated for 1 h. The radioactive medium was removed and the cultures washed twice with 3 ml cold Dulbecco's phosphate buffered saline (without Ca or Mg), once with 3 ml cold 10% trichloroacetic acid and once with 3 ml of ethenol:ether (3:1). The dried, fixed cells were dissolved in 0.2N NaOH, an aliquot added to Aquasol (New England Nuclear) and radioactivity measured in a Mark II (Nuclear Chicago) scintillation counter. Cell multiplication Cell multiplication experiments also utilized tertiary cultures of CEF's plated at 1 x 10%0 mm dish; however, the E media contain ing boiled rat serum or lymph were added approximately 4 hours after plating in 0.4% medium. Cell counts were performed on a Particle Data cell counter
654
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SOMATOMEDIN IN SERUM AND LYMPH before the initial addition of media and at 3 days of growth. Rat serum and lymph
655
the column. Six ml fractions were collected from which 2 ml samples were lyophilized, reconstituted in media, and tested on duplicate CEF assay dishes.
Hypophysectomized rats were obtained from Rat cartilage assay Zivic-Miller. The operation was performed on The column fractions obtained from boiled 90-100 g male Sprague-Dawley rats and the rats were maintained for 10 days prior to use. The GH normal serum were pooled for assay in a rat carti3 treatment schedule was 3 ip injections of 200 lage system. [ H]Thymidine uptake into costal ^g of bGH (NIH-GH-B17) over a 24-h period. cartilage from hypophysectomized rats was measSerum was obtained by cardiac puncture 4 h ured by the method of Daughaday and Reeder (5). after the last injection, allowed to clot at 4 C, The cartilages (4 per flask) were incubated for centrifuged at 3,000 rpm in an IEC PR 6000 22 h with 6 ml of medium alone or 1:4 dilutions centrifuge, and the serum stored at —15 C. of the pooled column fractions (1.5 ml aliquots Lymph was obtained from normal Sprague Daw- lyophilized and reconstituted in 6 ml of medium). ley rats weighing 125-150 g. Under ether anes- Incubations were continued for 4 h in labelled thesia, a polyethylene catheter (Intramedic, PE medium. Cartilage pieces were dissolved in con7400) was inserted into the cysterna chyli and centrated formic acid, mixed with Aquasol, and sutured in place. The animals were then main- counted in the liquid scintillation counter. tained for 24 h in restraining cages, while lymph was collected in a sterile iced tube. The lymph Results was then centrifuged and stored at - 1 5 C. Boiled serum and lymph were obtained by diluting with physiologic saline (3 parts saline, 1 part serum or 1) Activity in whole and heat-treated lymph lymph), adjusting the pH to 5.5 with HC1 and Pooled rat lymph (n = 5) and pooled norplacing the serum or lymph in a boiling water mal rat serum (n = 25) were assayed at 1%, bath for 15 min. After removal of precipitate by 5% and 10% concentrations (Fig. 1). The centrifugation, dilutions were made with E greatest activity was noted at 1% with promedium prior to use. The final percent value gressive inhibition at higher concentrations. assigned to the media containing boiled serum Heat treatment of the specimens under acid or lymph was based on the volume of serum or conditions resulted in more linear dose relymph used prior to dilution and boiling. sponse curves, thus allowing a more accurate comparison of the relative activities. In the Gel filtration assay shown in Fig. 1 lymph had 47% of A G50 (fine) Sephadex column (1.5 x 90 cm) the activity of serum at a 5% concentration. equilibrated with Dulbecco's phosphate buf- Five paired specimens (lymph and serum fered saline (without Ca ++ or Mg ++ , pH 7.4) and from the same animal) were also heat treated run at 4 C was used for the experiment shown and assayed at a 5% concentration. Combinin Fig. 2. The column was calibrated with blue ing the data from both pooled and paired dextran 2000 (Pharmacia) and phenol red markspecimens, rat lymph contains 50% ± 8 ers. One ml aliquots of serum or lymph were (SEM) of the SM activity present in rat serum. layered on the column. Six ml fractions were collected from which 2 ml samples were tested on dulplicate CEF assay dishes. A G50 (fine) Sephadex column (1.5 x 90 cm) equilibrated with IN acetic acid and run at 25 C was used for the experiments shown in Fig. 3. The column was calibrated with blue dextran 2000, cytochrome C (MW 12,400) and bacitracin (MW 1,450). Two ml aliquots of serum or lymph were boiled under acid conditions, lyophilized, reconstituted with IN acetic acid, and layered on
2) Sephadex chromatography of serum and lymph Aliquots of serum and lymph from normal rats were passed over a G50 Sephadex column equilibrated with phosphate buffered saline (pH 7.4). The active material in both eluted in the void volume in association with larger proteins (Fig. 2). The areas under the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 August 2016. at 03:27 For personal use only. No other uses without permission. . All rights reserved.
Enclo • 1975 Vol 97 • No 3
COHEN AND NISSLEY
656
elution curves paralleled the relative activities of unfractionated serum and lymph. Aliquots of serum from normal, hypophysectomized, and GH treated hypophysectomized rats were boiled under acid conditions and then passed over a G50 Sephadex column equilibrated with I N acetic acid. The active material in each of the 3 specimens eluted as a single peak with the same elution volume (Fig. 3A). There was no loss of activity during gel filtration and the areas under the elution curves paralleled the relative activities of unfractionated, heat-treated rat sera. Furthermore, when pooled column fractions were tested in a rat cartilage assay system, activity was found in the same fractions which were active in the CEF assay (Fig. 3B). Heat-treated rat lymph was chromatographed on the same G50 column and the active material eluted as a single major peak (Fig. 3C). The elution volume was identical to that of the GH-dependent material in rat serum. 3) CEF growth experiments
% SERUM OR LYMPH
FIG. 1. Stimulation of [3H]thymidine incorporation into DNA of CEFs by untreated and boiled rat serum and lymph. The details of the [3H]thymidine incorporation assay and preparation of boiled serum and lymph are given in Materials and Methods. Each point represents the results of the assay on a separate dish of CEFs.
In order to demonstrate that the relative activities of serum and lymph in the [3H]thymidine incorporation assay were a measure of DNA synthesis, cell multiplication experiments were performed. Although
VOID VOLUME
10
SEPHADEX
G-50. pH 7.4 SALT
J
I
7
8
9
10
11
l 12
I 13
I 14
I 15
I 16
TUBE NUMBER
I 17
18
19
20
21
I 22
I 23
l 24
FIG. 2. Stimulation of [3H]thymidine incorporation into DNA of CEFs by Sephadex C-50 (pH 7.4) fractions of normal rat senim and lymph. Details of the [ 3 H]-thymidine incorporation assay and Sephadex chromatography are given in Materials and Methods. Each point represents the result of the assay of a separate dish of CEFs.
25
(6 ml fractions)
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657
SOMATOMEDIN IN SERUM AND LYMPH serum-starved CEF's can be shown to accumulate DNA in response to untreated rat serum most of the cells do not undergo cell division (data not shown). However, heat-treated serum and lymph will support cell multiplication. As shown in Table 1, the increase in cell number in 5% lymph is about 40% of that stimulated by 5% serum. Thus, the results of cell multiplication experiments parallel the thymidine incorporation data.
possibility by comparing SM concentrations in rat serum and lymph. Whole rat serum is more active than rat lymph in the CEF assay, but accurate comparison is difficult because of the inhibition noted at higher concentrations. Heat treatment of lymph and serum removes these inhibitors resulting in nearly linear dose response curves (Fig. 1). Rat lymph contains 50% ± 8 (SEM) of the activity of rat serum in A. BOILED RAT SERA SEPHADEX G-50. IN ACETIC ACID
Discussion
BACITRACIN
v.
Somatomedin is the proposed mediator of growth hormone (GH) action on skeletal tissue (6). Rather than being a unique molecular species, SM appears to encompass a family of polypeptides (1). A common property is the association with larger proteins in normal serum (1,2). Although the physiological significance of the binding is unknown, it could have major effects on the distribution of SM in body fluids, the magnitude of fluctuations in serum SM concentrations, and the serum half-life of SM. In this report we have investigated the first
GH TREATED / / HVPOX i '
SALT
. -
V
B. CARTILAGE ASSAY
FlC. 3. A. Stimulation of [3H]thymidine incorporation into DNA of CEFs by Sephadex G-50 (IN acetic acid) fractions of boiled serum from normal, hypox., and bGH-treated hypox. rats. Details of the [3H]thymidine incorporation assay, heat treatment of serum, and Sephadex chromatography are given in Materials and Methods. Vo represents the void volume of the column as determined with blue dextran 2000. The elution peak of the bacitracin marker and the salt front are noted. The cytochrome C marker appeared in the void volume. Each point represents the result of the assay for a separate dish of CEFs. B. Stimulation of [3H]thymidine incorporation into DNA of costal cartilages from hypox. rats by pooled Sephadex G50 fractions of boiled normal rat serum. Aliquots of the fractions shown in Fig. 3A were used. The cartilage assay method is that of Daughaday and Reeder (4). Results are expressed as the percent of baseline uptake in control cartilages. Each bar represents the mean value of four costal cartilages. C. Stimulation of [3H]thymidine incorporation into DNA of CEFs by Sephadex G50 fractions of boiled lymph. The column was the same as that used in Fig. 3A. Each point represents the result of the assay for a separate dish fo CEFs.
C. BOILED RAT LYMPH
10 11 12 13 14 15 16 17 IB 19 20 21 22 23 TUBE NUMBER 16ml fractions)
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TABLE 1. Growth of chick embryo fibroblasts in boiled serum and lymph 106 Cells/dish 1) DavO 2) Day 3 a) Control b) 5% Serum c) 5% Lymph
Enclo • 1975 Vol 97 • No 3
COHEN AND NISSLEY
65S
Mean
0.84,
0.79,
0.78
0.80
0.84, 1.79, 1.17,
0.92, 1.83, 1.27,
0.89 1.85 1.29
0.88 1.82 1.24
The details of the growth experiment are given in Materials and Methods. Each value is for a separate dish of CEFs.
the CEF assay. This difference was confirmed in cell multiplication experiments (Table 1). When heat-treated sera from normal, hypophysectomized, and GH treated hypophysectomized rats were chromatographed on a G50 Sephadex column, equilibrated with L\ acetic acid, the active material in each of the 3 types of sera eluted as a single peak with identical elution volumes (Fig. 3A). Furthermore, the same fractions active in the CEF assay stimulated thymidine uptake in hypophysectomized rat cartilage, a standard SM assay (Fig. 3B). The three types of sera thus serve as a column marker for the heat stable, low molecular weight, GHdependent activity (i.e., SM) in rat serum. The activity in heat-treated rat lymph, chromatographed on the same G50 column, eluted as a single peak with the same elution volume as the serum SM activity (Fig. 3C). Thus, the physical properties of the active material in serum and in lymph are similar. These results indicate that SM is not freely diffusible from serum into lymph. This is in contrast to immunoreactive insulin which has a similar molecular weight but whose interstitial fluid concentration more closely reflects the intravascular levels (7). Insulin behaves as an unbound molecule in normal serum (8). The association of SM with larger serum proteins may explain its relative restriction to the vascular compartment. When normal rat serum and lymph were chromatographed on a G50 sephadex column equilibrated with phosphate buffered saline, pH 7.4, the active material appeared
in the void volume (Fig. 2). Thus, the association with large proteins described for serum SM also applies to extravascular SM activity. The relative concentration of SM activity may, therefore, be governed by the distribution of the large binding protein between tissue compartments. In studies of one of the purified somatomedins from human serum, SM-B, Yalo et al. reported that its distribution space after IV injection in the dog was about twice plasma volume (9). The data are difficult to interpret, however, since there was no significant binding of the human SM-B to the dog plasma proteins. In conclusion, the association of SM with large serum proteins appears to alter its relative distribution between serum and interstitial fluid. Whether this can function as a regulatory mechanism to limit the concentration of growth factors at target sites is unknown. The effect of protein binding on the half-life of SM in serum is currently being investigated. Acknowledgments We wish to thank Patricia Short for her skillful technical assistance and Dr. James Phang for reviewing the manuscript. We also thank the NIAMDD, NIH, for the gift of bGH HIN-GH-B17.
References 1. VanWyk, J. J., L. E. Underwood, R. L. Hintz, S. J. Voina, R. P. Weaver, and D. R. Clemmons, Recent Progr Horm Res 36: 259, 1974. 2. Hintz, R. L., E. M. Orsini, and M. G. VanCamp, 56th Annual Meeting of the Endocrine Society, A-71, 1974 (Abstract). 3. Cohen, K. L., P. A. Short, and S. P. Nissley, Endocrinology 96: 193, 1975. 4. Pierson, R. W., Jr., and H. M. TeminJ Cell Physiol 79: 319, 1972. 5. Daughaday, W. H., and C. ReederJ Lab Clin Med 68: 357, 1966. 6. , K. Hall, M. Raben, W. D. Salmon, Jr., J. L. Van den Brande, and J. J. VanWyk, Nature 235: 107, 1972. 7. Rasio, E. A., C. L. Hampers, J. S. Soeldner, and G. F. Cahill, J r . J Clin Invest 46: 903, 1967. 8. Berson, S. A., and R. S. Yalow, Amy Med 40: 676, 1966. 9. Yalow, R. S., K. Hall, and R. LuftJ Clin Invest 55: 127, 1975.
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