Hormonal Responsiveness of Adenylate Cyclase Activity in Cartilage DAVID M. SMITH,1 LAWRENCE M. ROTH,2 AND C. CONRAD JOHNSTON, JR.1 The department of Medicine and the department Medicine, Indianapolis, Indiana 46202
of Pathology, Indiana University School of
ABSTRACT. Adenylate cyclase activity was measured in a crude particulate fraction of hyaline cartilage obtained from the xiphoid process of the rat. Bovine parathyroid hormone (PTH) at concentrations as low as 1.3 x 10~7M and porcine calcitonin (CT) at concentrations as low as 2.3 x 10~5M significantly increased adenylate cyclase activity. Glucagon, prostaglandin E, (PGE,) and E2 (PGE2), and epinephrine at concen-
trations of 10 5M also increased activity, whereas, no increased activity was seen with the additions of somatotrophin (10 fxg/m\), PGFi a , PGF 2a , or T3 at 10~5M. The combination of doses of PTH and CT, which individually produced maximal responses, was not additive. These data provide evidence that cartilage in growing rats responds directly to PTH and CT. (Endocrinology 98: 242, 1976)
I
N order to understand better the actions of parathyroid hormone (PTH) and calcitonin (CT), one must know the sites or tissues directly affected by them. This laboratory previously studied the effect of these hormones on specific bone cell types to determine the target tissues (1,2). These studies demonstrated that three cell types, periosteum, osteoblasts, and osteocytes had adenylate cyclase systems which were responsive to both PTH and CT, and that the adenylate cyclase system of marrow cells responded only to CT (1,2). The responsiveness of adenylate cyclase systems is taken as evidence for these specific cell types serving as targets for PTH and CT. With these same objectives, the present study was undertaken using another type of skeletal tissue from the growing rat. One of the studies on embryonic tissue by HerrmannErlee et al. (3) indicated that PTH increased the cyclic 3', 5'-adenosine monophosphate (cyclic AMP) content of embryonic mouse epiphyseal cartilage. The effect of CT was not examined in that system. This study was undertaken to determine whether adenylate cyclase of hyaline cartilage from growing rats was responsive to PTH and CT. The hormonal responsiveness of this enzyme Received April 28, 1975. Reprints: Dr. David M. Smith, Department of Medicine, Indiana University School of Medicine, 1100 W. Michigan Street, Indianapolis, Indiana 46202.
would provide some evidence that a) cartilage serves as another direct target tissue for these hormones, and b) hormonal changes could be potentially mediated through cyclic AMP. Materials and Methods Materials. Purified bovine parathyroid hormone (1100-1900 U/mg) was obtained from Wilson Laboratories. Porcine calcitonin (62 M.R.C. U/mg) was supplied through the courtesy of Armour Pharmaceutical Co. Crystalline glucagon was the gift of Eli Lilly and Co. Prostaglandins E,, E2, Fi a , and F 2a were supplied through the courtesy of Dr. John Pike, the Upjohn Company. Sodium L-triiodothyronine (T3) was a gift from Smith, Kline, and French Laboratories. Ethyleneglycolbis-(/3-amino-ethyl ether) N, N'-tetraacetic acid (EGTA), somatotropin, and epinephrine were obtained from Sigma Chemical Company. The hormones were solubilized in 5 mM acetic acid with 0.013% bovine albumin fraction V (Sigma) except that the solvent for the prostaglandins and T3 was 95% ethanol buffered with Na2CO3. Enzymes, nucleotides, and radioisotopes used in the adenylate cyclase assay were obtained and prepared as described by Heersche, Marcus and Aurbach (4). Tissue preparation. Cox-Holtzman male rats weighing 80 to 100 g were anesthetized with ether and killed by cardiac incision. The unmineralized xiphoid process was surgically removed, cleaned of connective tissue, placed in 0.25M sucrose at 5 C, frozen in liquid nitrogen
242
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ADENYLATE CYCLASE IN CARTILAGE
243
and pulverized with a stainless steel mortar and pestle. A crude particulate fraction (2200 x g) was prepared by the method of Heersche et al. (4) which ultilizes dimethyl sulfoxide and EGTA during homogenization of the tissue.
this preparation. No osteoid or bone was present. Samples from much older, 300 g rats contained gross and microscopic evidence of calcification and were not used for biochemical studies.
Assay of adenylate cyclase activity. Adenylate cyclase activity was determined by a modification (1) of the method described by Marcus and Aurbach (5). Protein concentrations were determined by the method of Lowry et al. (6). Incubation time was 15 min and activity is reported as the amount of cyclic AMP formed (pmol/mg protein/15 min). Control samples contained equal volumes of diluent (acid albumin or buffered ethanol).
Adenylate cyclase activity as a function of time and protein coticentrations. Figure 2 illustrates the linearity of adenylate cyclase activity with respect to time and protein concentration under conditions of maximal stimulation with NaF, 10~2M. These linear relationships, which pass through the origin, permit the adjustment of cyclic AMP formed for these two variables in subsequent experiments.
Histology preparations. Representative samples were removed, cleaned, and fixed in neutral buffered formalin. No decalcification was necessary. Tissue was embedded in paraffin and 5 jum sections were cut and stained with hematoxylin and eosin.
Results Identification of tissue. Sections from samples of the xiphoid processes of 80 to 100 g rats showed hyaline cartilage containing chondrocytes covered by a thin layer of perichondrium (Fig. 1). No calcification was observed. Cell types other than cartilage were not found in significant quantities in
Effect of parathyroid hormone and calcitonin on adenylate cyclase activity. The dose-response curve of adenylate cyclase activity plotted as a function of the log-dose of each hormone is shown in Fig. 3. The lowest dose of PTH which significantly (P < .02) increased activity was 0.1 U/70 fi\. (1.3 x 10-7M, assuming a M.W. of 9000). The activity increased with dose concentration and maximum responsiveness was observed at 15 to 25 U/70 /nl. Significantly increased adenylate cyclase activity was observed with CT at 0.3 U/70 yA (2.3 x 10"5M, assuming a mol wt of 3000). Maximum
FIG. 1. A transverse section of hyaline cartilage from the xiphoid process of a 100 g Cox-Holtzman rat illustrates hyaline cartilage consisting of chondrocytes in lacunae covered by a thin portion of adherent perichondrium. No other cell types are seen in significant numbers nor is there evidence of bone formation. Hematoxylin and eosin; x250.
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SMITH, ROTH AND JOHNSTON
244
Endo • 1976 Vol 98 • No 1
-
800 — /
600 — / /
400 —
-
/
/
200 —
/ \ c
120
-
1
1
1
5
1
20
15
10
FIG. 2. Adenylate cyclase as a function of time and protein concentration. Each point is the mean of four determinations. The brackets indicate the standard error of the means. A linear relationship of activity was observed with time and protein concentration. Each sample contained NaF at 10~2M.
TIME (min)
PROTEIN (u«)
responsiveness was observed at 0.5 to 1.0 U/70 y\. These ranges of doses of PTH and CT producing responses are similar to the doses found effective with preparations from separated bone cells (1).
Specificity. Table 1 shows the effect of other hormones on adenylate cyclase activity in preparations from rat cartilage. PGEi and PGE2, glucagon, and epinephrine at 10~5M increased activity. At higher concentra-
^ 120 —
/
aE
/
in
w
/ "I / / J 17
I 100— bL
1
m
o
n 1/
80 —
E 3 Q U
1
S §60 —
f
b,
s
a,
y
S
In 40 —
/ I
1
FIG. 3. Adenylate cyclase activity as a function of log-dose of parathyroid hormone and calcitonin. The symbols are the same as for Fig. 2.
J J
o
20—
n— 0 PARATHYROID HORMONE (Units/70jil)
.1 .3 .5 1.0 CALCITONIN (Units/70/xl)
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245
ADENYLATE CYCLASE IN CARTILAGE TABLE 1. Effect of hormones on adenylate cyclase activity in preparations from hyaline cartilage Cyclic 3', 5'-AMP formed (pmol/mg protein/15 min) ± SEM
Treatment Experiment I Control (acid-albumin diluent) Calcitonin (0.5U or 3.8 x 10" S M)
Somatotrophin (20 fj.glm\) 5
Clucagon (10" M)
Experiment II Control (buffered ethanol diluent) PGE, ( 1 0 - S M ) PGE, ( 1 0 - » M ) PCF1O ( 1 0 - » M )
PGF to (10- 5 M) P C F l o (7.05 x 1 0 " 5 M ) PGF t o (5.25 x 10" 5 M)
Experiment III Control (acid-albumin) PTH (.64 x 10-5M) Epinephrine (10" 5 M) 5
T a (10- M) NaF(lO-'M)
P vs control
29.8 176.3 20.3 63.0
± ± ± ±
4.7 18.8 2.8 4.2
of Pathology, Indiana University School of
ABSTRACT. Adenylate cyclase activity was measured in a crude particulate fraction of hyaline cartilage obtained from the xiphoid process of the rat. Bovine parathyroid hormone (PTH) at concentrations as low as 1.3 x 10~7M and porcine calcitonin (CT) at concentrations as low as 2.3 x 10~5M significantly increased adenylate cyclase activity. Glucagon, prostaglandin E, (PGE,) and E2 (PGE2), and epinephrine at concen-
trations of 10 5M also increased activity, whereas, no increased activity was seen with the additions of somatotrophin (10 fxg/m\), PGFi a , PGF 2a , or T3 at 10~5M. The combination of doses of PTH and CT, which individually produced maximal responses, was not additive. These data provide evidence that cartilage in growing rats responds directly to PTH and CT. (Endocrinology 98: 242, 1976)
I
N order to understand better the actions of parathyroid hormone (PTH) and calcitonin (CT), one must know the sites or tissues directly affected by them. This laboratory previously studied the effect of these hormones on specific bone cell types to determine the target tissues (1,2). These studies demonstrated that three cell types, periosteum, osteoblasts, and osteocytes had adenylate cyclase systems which were responsive to both PTH and CT, and that the adenylate cyclase system of marrow cells responded only to CT (1,2). The responsiveness of adenylate cyclase systems is taken as evidence for these specific cell types serving as targets for PTH and CT. With these same objectives, the present study was undertaken using another type of skeletal tissue from the growing rat. One of the studies on embryonic tissue by HerrmannErlee et al. (3) indicated that PTH increased the cyclic 3', 5'-adenosine monophosphate (cyclic AMP) content of embryonic mouse epiphyseal cartilage. The effect of CT was not examined in that system. This study was undertaken to determine whether adenylate cyclase of hyaline cartilage from growing rats was responsive to PTH and CT. The hormonal responsiveness of this enzyme Received April 28, 1975. Reprints: Dr. David M. Smith, Department of Medicine, Indiana University School of Medicine, 1100 W. Michigan Street, Indianapolis, Indiana 46202.
would provide some evidence that a) cartilage serves as another direct target tissue for these hormones, and b) hormonal changes could be potentially mediated through cyclic AMP. Materials and Methods Materials. Purified bovine parathyroid hormone (1100-1900 U/mg) was obtained from Wilson Laboratories. Porcine calcitonin (62 M.R.C. U/mg) was supplied through the courtesy of Armour Pharmaceutical Co. Crystalline glucagon was the gift of Eli Lilly and Co. Prostaglandins E,, E2, Fi a , and F 2a were supplied through the courtesy of Dr. John Pike, the Upjohn Company. Sodium L-triiodothyronine (T3) was a gift from Smith, Kline, and French Laboratories. Ethyleneglycolbis-(/3-amino-ethyl ether) N, N'-tetraacetic acid (EGTA), somatotropin, and epinephrine were obtained from Sigma Chemical Company. The hormones were solubilized in 5 mM acetic acid with 0.013% bovine albumin fraction V (Sigma) except that the solvent for the prostaglandins and T3 was 95% ethanol buffered with Na2CO3. Enzymes, nucleotides, and radioisotopes used in the adenylate cyclase assay were obtained and prepared as described by Heersche, Marcus and Aurbach (4). Tissue preparation. Cox-Holtzman male rats weighing 80 to 100 g were anesthetized with ether and killed by cardiac incision. The unmineralized xiphoid process was surgically removed, cleaned of connective tissue, placed in 0.25M sucrose at 5 C, frozen in liquid nitrogen
242
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ADENYLATE CYCLASE IN CARTILAGE
243
and pulverized with a stainless steel mortar and pestle. A crude particulate fraction (2200 x g) was prepared by the method of Heersche et al. (4) which ultilizes dimethyl sulfoxide and EGTA during homogenization of the tissue.
this preparation. No osteoid or bone was present. Samples from much older, 300 g rats contained gross and microscopic evidence of calcification and were not used for biochemical studies.
Assay of adenylate cyclase activity. Adenylate cyclase activity was determined by a modification (1) of the method described by Marcus and Aurbach (5). Protein concentrations were determined by the method of Lowry et al. (6). Incubation time was 15 min and activity is reported as the amount of cyclic AMP formed (pmol/mg protein/15 min). Control samples contained equal volumes of diluent (acid albumin or buffered ethanol).
Adenylate cyclase activity as a function of time and protein coticentrations. Figure 2 illustrates the linearity of adenylate cyclase activity with respect to time and protein concentration under conditions of maximal stimulation with NaF, 10~2M. These linear relationships, which pass through the origin, permit the adjustment of cyclic AMP formed for these two variables in subsequent experiments.
Histology preparations. Representative samples were removed, cleaned, and fixed in neutral buffered formalin. No decalcification was necessary. Tissue was embedded in paraffin and 5 jum sections were cut and stained with hematoxylin and eosin.
Results Identification of tissue. Sections from samples of the xiphoid processes of 80 to 100 g rats showed hyaline cartilage containing chondrocytes covered by a thin layer of perichondrium (Fig. 1). No calcification was observed. Cell types other than cartilage were not found in significant quantities in
Effect of parathyroid hormone and calcitonin on adenylate cyclase activity. The dose-response curve of adenylate cyclase activity plotted as a function of the log-dose of each hormone is shown in Fig. 3. The lowest dose of PTH which significantly (P < .02) increased activity was 0.1 U/70 fi\. (1.3 x 10-7M, assuming a M.W. of 9000). The activity increased with dose concentration and maximum responsiveness was observed at 15 to 25 U/70 /nl. Significantly increased adenylate cyclase activity was observed with CT at 0.3 U/70 yA (2.3 x 10"5M, assuming a mol wt of 3000). Maximum
FIG. 1. A transverse section of hyaline cartilage from the xiphoid process of a 100 g Cox-Holtzman rat illustrates hyaline cartilage consisting of chondrocytes in lacunae covered by a thin portion of adherent perichondrium. No other cell types are seen in significant numbers nor is there evidence of bone formation. Hematoxylin and eosin; x250.
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SMITH, ROTH AND JOHNSTON
244
Endo • 1976 Vol 98 • No 1
-
800 — /
600 — / /
400 —
-
/
/
200 —
/ \ c
120
-
1
1
1
5
1
20
15
10
FIG. 2. Adenylate cyclase as a function of time and protein concentration. Each point is the mean of four determinations. The brackets indicate the standard error of the means. A linear relationship of activity was observed with time and protein concentration. Each sample contained NaF at 10~2M.
TIME (min)
PROTEIN (u«)
responsiveness was observed at 0.5 to 1.0 U/70 y\. These ranges of doses of PTH and CT producing responses are similar to the doses found effective with preparations from separated bone cells (1).
Specificity. Table 1 shows the effect of other hormones on adenylate cyclase activity in preparations from rat cartilage. PGEi and PGE2, glucagon, and epinephrine at 10~5M increased activity. At higher concentra-
^ 120 —
/
aE
/
in
w
/ "I / / J 17
I 100— bL
1
m
o
n 1/
80 —
E 3 Q U
1
S §60 —
f
b,
s
a,
y
S
In 40 —
/ I
1
FIG. 3. Adenylate cyclase activity as a function of log-dose of parathyroid hormone and calcitonin. The symbols are the same as for Fig. 2.
J J
o
20—
n— 0 PARATHYROID HORMONE (Units/70jil)
.1 .3 .5 1.0 CALCITONIN (Units/70/xl)
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245
ADENYLATE CYCLASE IN CARTILAGE TABLE 1. Effect of hormones on adenylate cyclase activity in preparations from hyaline cartilage Cyclic 3', 5'-AMP formed (pmol/mg protein/15 min) ± SEM
Treatment Experiment I Control (acid-albumin diluent) Calcitonin (0.5U or 3.8 x 10" S M)
Somatotrophin (20 fj.glm\) 5
Clucagon (10" M)
Experiment II Control (buffered ethanol diluent) PGE, ( 1 0 - S M ) PGE, ( 1 0 - » M ) PCF1O ( 1 0 - » M )
PGF to (10- 5 M) P C F l o (7.05 x 1 0 " 5 M ) PGF t o (5.25 x 10" 5 M)
Experiment III Control (acid-albumin) PTH (.64 x 10-5M) Epinephrine (10" 5 M) 5
T a (10- M) NaF(lO-'M)
P vs control
29.8 176.3 20.3 63.0
± ± ± ±
4.7 18.8 2.8 4.2
