Avian Parathyroid Glands in Organ Culture: Secretion of Parathyroid Hormone and Calcitonin JOEL D. FEINBLATT, LIH-RUEY TAI, AND ALEXANDER D. KENNY1 Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, and Department of Biochemistry, Dalton Research Center, University of Missouri 65201 ABSTRACT. Parathyroid (PT) glands from 20-dayold embryonic chicks cultured in a chemically defined medium secreted a stimulator of in vitro bone resorption. This stimulator was presumed to be parathyroid hormone (PTH) because: 1) the in vitro dose response curve was parallel to that obtained with bovine PTH; 2) the activity was eluted on Sephadex G-100 chromatography at a position similar to that for PTH; and 3) the material produced hypercalcemia in vivo in chicks. The amount of PTH-activity secreted was inversely proportional to the calcium concentration of the medium over the range of 0.75-2.25 mM. The chick PT glands also secreted an inhibitor of PTH-stimulated bone resorption in vitro. This inhibitor was presumed to
T
HERE is an extensive literature on the calcium metabolism of birds and the response of birds to the removal of their parathyroid glands or the administration of exogenous parathyroid hormone (1-3). However, there have been few direct studies on the secretion of endogenous parathyroid hormone (PTH) by the avian parathyroid glands since Gaillard demonstrated that embryonic chick parathyroids secreted a factor in vitro, presumably PTH, which caused resorption of adjacent bone explants (4). It has been concluded from several lines of indirect evidence that the secretion of the avian hormone is similar to that of mammalian PTH with an inverse relationship between the ambient calcium concentration and the rate of secretion (5). We have examined this possibility directly by studying the ability of the embryonic chick parathyroid gland to secrete PTH in organ culture.
be calcitonin (CT) because: 1) the in vitro doseresponse curve was parallel to that obtained with synthetic salmon CT; 2) the activity was eluted on Sephadex G-50 chromatography at a position similar to that for salmon CT; and 3) the material produced hypocalcemia in vivo in rats. In contrast to what would be expected for CT secretion, the CT-activity was secreted by the PT glands in response to a low, not high calcium concentration. The data suggest that the secretion of avian PTH is similar to that of the mammalian hormone, and that the ultimobranchialectomized chick with an intact parathyroid gland may not be deficient in CT. (Endocrinology 96: 282, 1975)
Furthermore, it has been thought that the ultimobranchial (UB) glands are the source of calcitonin (CT) in the chick (1) and that removal of these glands would permit the study of the role of CT in the regulation of calcium metabolism (6,7). CT "deficiency" produced by removal of the ultimobranchials in these studies produced no deficiencies in plasma calcium or in skeletal composition. However, more recent reports have established that the parathyroid gland may also be a source of CT in the chick (8). In the above studies on CT (6,7), the parathyroids were left intact. If these glands were capable of secreting CT, then it must be questioned whether the chickens were really deficient in the hormone. Our initial studies indicated that embryonic chick parathyroid glands are indeed capable of secreting CT-like- activity in vitro (9), and this phenomenon has been studied further. Materials and Methods
Received May 2, 1974. Supported in part by a PMA Foundation Research Starter Grant and U.S.P.H.S. Grants AM-12184 and AM-16550. 1 Present address: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550.
Gland cultures Fertile white Leghorn chicken eggs were obtained from a commercial supplier (Spafas, Inc., Norwich, Connecticut) and incubated in a forced air cabinet with controlled humidity and
282
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AVIAN PARATHYROIDS IN VITRO temperature. Parathyroid (PT) glands were dissected from 20-day-old chick embryos. The glands were found in close association with the thyroid and ultimobranchial (UB) glands (10), but were easily identified and clearly separated from these glands under the dissecting microscope. The PT glands were cultured on pieces of Millipore filter on metal screens in Falcon organ culture dishes (at 20 glands per ml) in a chemically defined medium, modified BGJ2 (11). The glands were incubated at 37 C under 5% CO 2 -40% O 2 -55% N2 and were transferred to fresh medium every 24 or 48 hr. The calcium concentration was varied by adding CaCl2 to the medium. In vitro bioassays The parathyroid culture media were diluted and assayed in vitro for both PTH-like and CTlike activities (10-13). Pregnant rats were injected on the 18th day of gestation with 45 Ca (125 /uCi/rat) and on the 19th day the labeled fetal radii and ulnae were removed and cultured in pairs in control BGJ medium for 24 hr to decrease the release of 45Ca due to physicochemical exchange during the assay period (11). For the assay of PTH-like activity, one member of each pair was then incubated for 48 hr in media obtained from the parathyroid cultures and the other in control BGJ medium containing the same calcium concentration. At the end of this incubation, 0.1 ml aliquots of the media were mixed with 10 ml of PCS Solubilizer (Amersham/Searle) and counted for 45Ca. Background counts were substracted from all sample counts. The results were expressed as the treated/ control (T/C) ratio for 45Ca release with ratios greater than 1.0 representing increased resorption due to PTH-like activity. The in vitro bioassay for PTH activity used in these experiments is a sensitive one, but its major disadvantage is that it is influenced by the presence of CT, and valid potency comparisons for PTH activity may not be easily made when the antagonist is present. Higher levels of PTH may be necessary to produce a given response. These considerations are important 2
Modified BGJ was made using a 10X concentrate of amino acids and vitamins (Grand Island Biological Co.), to which salts and bovine serum albumin Fraction V (1 mg/ml) were added. The complete medium was sterilized by Millipore filtration.
283
because there is CT-like activity in the parathyroid gland media (see below). We have attempted to minimize the effect of CT in the biosassy for PTH by selecting out and reporting only those experiments in which no CT activity was detectable at the doses used to study PTH activity. Although we have not detected CT at the doses used, this does not exclude the possibility of CT being present in the media. The levels of PTH activity may, therefore, represent underestimations of the true levels. Unlike the situation in the in vitro PTH assay where the presence of CT interferes with the response to PTH, the assay for CT is not influenced by the presence of PTH-activity in the PT gland medium. Using the same system, Friedman and coworkers (13) showed that when enough PTH was used to produce a maximal increase in 45Ca release, a given dose of CT caused a similar percentage inhibition of 45Ca release over a 30-fold range of PTH concentration. Therefore, when the media were assayed for CT-like activity, a high dose of PTE was added to the bone cultures to produce a maximal increase in the release of 45Ca. The presence of more PTH in the gland media would not increase this response further and would therefore not affect the response to the CT activity. CT-like activity was measured as: 1) the treated/control ratio for 45Ca release (T/C) for the member of the pair of bones treated with PT gland medium plus PTH compared to its PTH treated mate with ratios less than 1.0 representing decreased resorption due to CT-like activity, or 2) the percent inhibition of the PTH-stimulated release of 45CA (1.00 - T/C x 100). Differences between means and the significance of the T/C ratio compared to 1.00 were evaluated by the t test (14). Dose response curves were first computed by the method of least squares and then standard statistical procedures were used to evaluate parallelism between curves (14). In vivo bioassays PT gland culture media were assayed directly, for hypercalcemic activity in the chick by the method of Parsons and coworkers (2). CT does not interfere with the response to PTH in this assay (2,3). Ten-day old white leghorn chicks were fasted overnight and injected SC with either bovine parathyroid extract (Lilly PTE) or the test preparation. Sufficient calcium chloride was added to the injection vehicle to give a dose of
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 16:34 For personal use only. No other uses without permission. . All rights reserved.
Endo I 1975 Vol 96 , No 2
FEINBLATT, TAI AND KENNY
284
20 ftmoles in an injection volume of 0.4 ml (50 mM CaCl2). The chicks were anesthetized with ether and bled by cardiac puncture 1 hour after injection. Serum calcium concentrations were determined with the use of an automatic calcium titrator (Fiske Associates Inc.) using Calcein as an indicator (15). PT gland culture media were also assayed directly for hypocalcemic activity in the rat by the method of Sturtridge and Kumar, as modified by Kenny (16). Rats weighing 40-60 g were placed on a low calcium diet (General Biochemicals, cat. 170120) for 18 hr and then injected iv with standard porcine CT or unknown preparations in a volume of 0.6 ml/rat, and bled 30 min later. Column chromatography Four ml aliquots of PT culture medium were acidified with one drop of glacial acetic acid and chromatographed on a Sephadex G-100 column (2.5 x 90 cm) at 4 C using a 0.2M ammonium acetate buffer (pH 4.7). Six ml fractions were collected and assayed for protein content by UV absorption at 280 m/x. The fractions were pooled, lyophilized, redissolved in 0.01N acetic acid and then diluted in culture medium for bioassay. Culture medium were also filtered through a Sephadex G-50 column (2.5 x 90 cm) using a 0.1M formic acid buffer according to the method described by Kenny (16). Twelve ml fractions were collected, lyophilized and redissolved in 0.073M sodium acetate buffer (pH 3.0) containing 0.1% bovine serum albumin and assayed for CT using the rat bioassay method.
i
003
0.1 FfcRATHYROID
EXTRACT
"I
U/ml
0.3 »-•
5 ftVRATHYROID MEDIA
glond/ml
6 O-O
FIG. 1. Log-dose response stimulation of 45Ca release by media obtained from embryonic chick parathyroid glands cultured in 0.75 mM calcium (open circles), or Lilly PTE (closed circles). N = 16 pairs of bones. Vertical lines represent ±SE.
TABLE
Material" 1. 2. 3. 4.
Control PTE, 10 U PTE, 20 U PT gland media 8 glds/chick
1. Preliminary experience with the chick PTH assay No. of birds
Plasma calcium1* mg/100 ml
Significance0
5 5 5
9.04 ± 0.22 9.83 ± 0.29 10.88 ± 0.39
p < 0.01
5
9.91 ±0.16
p < 0.05
NS
"All test materials were dissolved in 50 HIM CaCls and injected subcutaneously in a volume of 0.4 ml (20 /imoles CaClj per chick). " Samples obtained by cardiac puncture 1 hr after injection. 0 Compared to control group (1). NS: not significant (p > 0.05).
The synthetic salmon CT (5028 MRC U/mg) used for the in vitro assays was kindly provided by the Armour Pharmaceutical Company (courtesy of Dr. J. W. Bastian).
Results
Parathyroid hormone activity The embryonic chick parathyroid glands secreted into the culture media a stimulator of in vitro bone resorption. The media could be diluted and assayed to obtain the log-dose response curve in Fig. 1, which was parallel to that obtained with bovine parathyroid extract. In the chick bioassay, Lilly PTE at 10 and 20 U/chick produced a dose related increase in plasma calcium (Table 1.). Chick parathyroid gland media also produced a significant increase in the calcium concentration compared to control birds. Estimates made of the relative potency of the parathyroid gland material to Lilly PTE using the in vitro and in vivo assays give discrepant results. The in vitro assay indicates that six glands produce a response comparable to 0.2 U (Fig. 1). While the in vivo assay indicates that eight glands produce a response comparable to 10 U (Table 1). We do not feel that such estimates of "true" potency and direct quantitative comparisons are meaningful for the following reasons: 1) the data reported for the different assays are not for the same preparations and may reflect different activities per se for each individual experiment; 2) dif-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 16:34 For personal use only. No other uses without permission. . All rights reserved.
285
AVIAN PARATHYROIDS IN VITRO ferences in magnitude may also reflect age and treatment of the media before assay. The in vitro assays were done within 3-5 days of incubation whereas the in vivo experiments were performed several weeks later and no measure of degradation of the material can be made. We note that Tregear and associates (17) have reported that the 2-34 fragment of bovine parathyroid hormone appears to be more active in vivo than in vitro for reasons as yet unknown; 3) both PTH and CT may be present in the media and, as previously indicated, the level of PTH activity measured by the in vitro assay may be an underestimation; and 4) Species differences and differences in the precision of the assays. Column
chrojnatography
When medium obtained from PT glands incubated at 0.75 mM calcium for 48 hours was applied to a Sephadex G-100 column, the PTH-like activity, as measured by the
260
J22O-
J
J
180) 140-
100
075
1.50 CALCIUM
2.25
30
FIG. 3. Relationship between the concentration of calcium and the release of PTH-like activity; glands incubated for 48 hr at the indicated calcium concentrations and assayed at 6 glands/ml; N = 8 pairs of bones. Linear relationship derived from treating data as a simple regression by the least squares method (14). Vertical lines represent ±SE.
in vitro bioassay, migrated as shown in Fig. 2. We did not have a purified preparation of PTH available for comparison, but the IQ value for the chick material was approximately 0.40 which is comparable to the migration reported for chick (18) and other species of PTH (19,20) on Sephadex G-100 (Ka = 0.34-0.54). Effect of varying the calcium concentration
•1.00 30
40
50 TUBE
60 NO.
70
80
FIG. 2. Elution pattern of media obtained from parathyroid glands incubated at 0.75 mM calcium for 48 hr. Four ml of media applied to a Sephadex G-100 column (2.5 x 90 cm), and eluted with 0.2M ammonium acetate buffer, pH 4.7; 6 ml fractions collected. Pools of 5 fractions from tubes 30-80 were lyophilized and assayed for PTH-like activity. Only those fractions with significant activity are included (N = 4 pairs of bones. Vertical lines represent ±SE). Void volume (Vo) was determined by the peak of optical density at 280nm after blue dextran. Vt was taken as the peak representing amino acids and other low molecular weight components of the medium.
When parathyroid glands were incubated for 48 hr with increasing calcium concentrations from 0.75 to 3.0 mM (Fig. 3), there was a decrease in the T/C ratio indicating a reduced secretion of PTH with a significant inverse relationship over the range 0.75-2.25 mM calcium. In the chemically defined medium most of the calcium is presumably ionized and the normal value is approximately 1.2 to 1.4 mM. Calcitonin activity The embryonic chick PT glands secreted an inhibitor of in vitro bone resorption into the culture medium. The media could be diluted and assayed to obtain the log-dose
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 16:34 For personal use only. No other uses without permission. . All rights reserved.
FEINBLATT, TAI AND KENNY
286
Endo • 1975 Vol 96 • No 2
tion. Interestingly, in contrast to what would be expected for CT (5,10), the CT-like activity was also secreted by the PT glands in response to the low but not the high calcium concentration.
7060-
E3CO J i
Discussion
x i ^ a o H
20-
1.0
2.0 SALMON CT mil/ml
3
3.0
The present study demonstrates that the 20-day-old embryonic chick PT gland is capable of secreting a PTH-like substance in organ culture. The evidence that the material is in fact PTH is: 1) it is a stimulator of bone resorption in tissue culture (12) and the dose response curve obtained in the
6
PARATHYROID MEDIA glands/ml
1.5
O-O
FIG. 4. Log-dose response inhibition of PTH-stimulated Ca release by media obtained from embryonic chick parathyroid glands culture in 0.75 mM calcium (open circles), or synthetic salmon CT (closed circles). N = 8-12 pairs of bones. Vertical lines represent ±SE.
45
E O
2
response curve in Fig. 4 which was parallel to that obtained using synthetic salmon CT. The CT-like material was assayed directly in vivo in rats (16) without prior concentration and was found to have significant hypocalcemic activity. Column chromatography When medium obtained from PT glands incubated at 0.75 mM calcium for 48 hours was applied to a Sephadex G-50 column, the CT-like activity, as measured by the in vivo bioassay, migrated as shown in Fig. 5. Also included for comparison is the pattern obtained with salmon CT. Effect of varying the calcium concentration Table 2 contains the pooled data for several sets of experiments in which parathyroid glands were incubated for 48 hr at calcium concentrations of either 0.75 or 3.0 mM and the media was assayed for both PTH-like and CT-like activities. As observed in the previous experiments, PTHlike activity (A) was secreted in response to the low but not the high calcium concentra-
Co
1.0
0.5
CHICK PT MEDIUM
a* E
3 U
T
HERE is an extensive literature on the calcium metabolism of birds and the response of birds to the removal of their parathyroid glands or the administration of exogenous parathyroid hormone (1-3). However, there have been few direct studies on the secretion of endogenous parathyroid hormone (PTH) by the avian parathyroid glands since Gaillard demonstrated that embryonic chick parathyroids secreted a factor in vitro, presumably PTH, which caused resorption of adjacent bone explants (4). It has been concluded from several lines of indirect evidence that the secretion of the avian hormone is similar to that of mammalian PTH with an inverse relationship between the ambient calcium concentration and the rate of secretion (5). We have examined this possibility directly by studying the ability of the embryonic chick parathyroid gland to secrete PTH in organ culture.
be calcitonin (CT) because: 1) the in vitro doseresponse curve was parallel to that obtained with synthetic salmon CT; 2) the activity was eluted on Sephadex G-50 chromatography at a position similar to that for salmon CT; and 3) the material produced hypocalcemia in vivo in rats. In contrast to what would be expected for CT secretion, the CT-activity was secreted by the PT glands in response to a low, not high calcium concentration. The data suggest that the secretion of avian PTH is similar to that of the mammalian hormone, and that the ultimobranchialectomized chick with an intact parathyroid gland may not be deficient in CT. (Endocrinology 96: 282, 1975)
Furthermore, it has been thought that the ultimobranchial (UB) glands are the source of calcitonin (CT) in the chick (1) and that removal of these glands would permit the study of the role of CT in the regulation of calcium metabolism (6,7). CT "deficiency" produced by removal of the ultimobranchials in these studies produced no deficiencies in plasma calcium or in skeletal composition. However, more recent reports have established that the parathyroid gland may also be a source of CT in the chick (8). In the above studies on CT (6,7), the parathyroids were left intact. If these glands were capable of secreting CT, then it must be questioned whether the chickens were really deficient in the hormone. Our initial studies indicated that embryonic chick parathyroid glands are indeed capable of secreting CT-like- activity in vitro (9), and this phenomenon has been studied further. Materials and Methods
Received May 2, 1974. Supported in part by a PMA Foundation Research Starter Grant and U.S.P.H.S. Grants AM-12184 and AM-16550. 1 Present address: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550.
Gland cultures Fertile white Leghorn chicken eggs were obtained from a commercial supplier (Spafas, Inc., Norwich, Connecticut) and incubated in a forced air cabinet with controlled humidity and
282
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 16:34 For personal use only. No other uses without permission. . All rights reserved.
AVIAN PARATHYROIDS IN VITRO temperature. Parathyroid (PT) glands were dissected from 20-day-old chick embryos. The glands were found in close association with the thyroid and ultimobranchial (UB) glands (10), but were easily identified and clearly separated from these glands under the dissecting microscope. The PT glands were cultured on pieces of Millipore filter on metal screens in Falcon organ culture dishes (at 20 glands per ml) in a chemically defined medium, modified BGJ2 (11). The glands were incubated at 37 C under 5% CO 2 -40% O 2 -55% N2 and were transferred to fresh medium every 24 or 48 hr. The calcium concentration was varied by adding CaCl2 to the medium. In vitro bioassays The parathyroid culture media were diluted and assayed in vitro for both PTH-like and CTlike activities (10-13). Pregnant rats were injected on the 18th day of gestation with 45 Ca (125 /uCi/rat) and on the 19th day the labeled fetal radii and ulnae were removed and cultured in pairs in control BGJ medium for 24 hr to decrease the release of 45Ca due to physicochemical exchange during the assay period (11). For the assay of PTH-like activity, one member of each pair was then incubated for 48 hr in media obtained from the parathyroid cultures and the other in control BGJ medium containing the same calcium concentration. At the end of this incubation, 0.1 ml aliquots of the media were mixed with 10 ml of PCS Solubilizer (Amersham/Searle) and counted for 45Ca. Background counts were substracted from all sample counts. The results were expressed as the treated/ control (T/C) ratio for 45Ca release with ratios greater than 1.0 representing increased resorption due to PTH-like activity. The in vitro bioassay for PTH activity used in these experiments is a sensitive one, but its major disadvantage is that it is influenced by the presence of CT, and valid potency comparisons for PTH activity may not be easily made when the antagonist is present. Higher levels of PTH may be necessary to produce a given response. These considerations are important 2
Modified BGJ was made using a 10X concentrate of amino acids and vitamins (Grand Island Biological Co.), to which salts and bovine serum albumin Fraction V (1 mg/ml) were added. The complete medium was sterilized by Millipore filtration.
283
because there is CT-like activity in the parathyroid gland media (see below). We have attempted to minimize the effect of CT in the biosassy for PTH by selecting out and reporting only those experiments in which no CT activity was detectable at the doses used to study PTH activity. Although we have not detected CT at the doses used, this does not exclude the possibility of CT being present in the media. The levels of PTH activity may, therefore, represent underestimations of the true levels. Unlike the situation in the in vitro PTH assay where the presence of CT interferes with the response to PTH, the assay for CT is not influenced by the presence of PTH-activity in the PT gland medium. Using the same system, Friedman and coworkers (13) showed that when enough PTH was used to produce a maximal increase in 45Ca release, a given dose of CT caused a similar percentage inhibition of 45Ca release over a 30-fold range of PTH concentration. Therefore, when the media were assayed for CT-like activity, a high dose of PTE was added to the bone cultures to produce a maximal increase in the release of 45Ca. The presence of more PTH in the gland media would not increase this response further and would therefore not affect the response to the CT activity. CT-like activity was measured as: 1) the treated/control ratio for 45Ca release (T/C) for the member of the pair of bones treated with PT gland medium plus PTH compared to its PTH treated mate with ratios less than 1.0 representing decreased resorption due to CT-like activity, or 2) the percent inhibition of the PTH-stimulated release of 45CA (1.00 - T/C x 100). Differences between means and the significance of the T/C ratio compared to 1.00 were evaluated by the t test (14). Dose response curves were first computed by the method of least squares and then standard statistical procedures were used to evaluate parallelism between curves (14). In vivo bioassays PT gland culture media were assayed directly, for hypercalcemic activity in the chick by the method of Parsons and coworkers (2). CT does not interfere with the response to PTH in this assay (2,3). Ten-day old white leghorn chicks were fasted overnight and injected SC with either bovine parathyroid extract (Lilly PTE) or the test preparation. Sufficient calcium chloride was added to the injection vehicle to give a dose of
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 16:34 For personal use only. No other uses without permission. . All rights reserved.
Endo I 1975 Vol 96 , No 2
FEINBLATT, TAI AND KENNY
284
20 ftmoles in an injection volume of 0.4 ml (50 mM CaCl2). The chicks were anesthetized with ether and bled by cardiac puncture 1 hour after injection. Serum calcium concentrations were determined with the use of an automatic calcium titrator (Fiske Associates Inc.) using Calcein as an indicator (15). PT gland culture media were also assayed directly for hypocalcemic activity in the rat by the method of Sturtridge and Kumar, as modified by Kenny (16). Rats weighing 40-60 g were placed on a low calcium diet (General Biochemicals, cat. 170120) for 18 hr and then injected iv with standard porcine CT or unknown preparations in a volume of 0.6 ml/rat, and bled 30 min later. Column chromatography Four ml aliquots of PT culture medium were acidified with one drop of glacial acetic acid and chromatographed on a Sephadex G-100 column (2.5 x 90 cm) at 4 C using a 0.2M ammonium acetate buffer (pH 4.7). Six ml fractions were collected and assayed for protein content by UV absorption at 280 m/x. The fractions were pooled, lyophilized, redissolved in 0.01N acetic acid and then diluted in culture medium for bioassay. Culture medium were also filtered through a Sephadex G-50 column (2.5 x 90 cm) using a 0.1M formic acid buffer according to the method described by Kenny (16). Twelve ml fractions were collected, lyophilized and redissolved in 0.073M sodium acetate buffer (pH 3.0) containing 0.1% bovine serum albumin and assayed for CT using the rat bioassay method.
i
003
0.1 FfcRATHYROID
EXTRACT
"I
U/ml
0.3 »-•
5 ftVRATHYROID MEDIA
glond/ml
6 O-O
FIG. 1. Log-dose response stimulation of 45Ca release by media obtained from embryonic chick parathyroid glands cultured in 0.75 mM calcium (open circles), or Lilly PTE (closed circles). N = 16 pairs of bones. Vertical lines represent ±SE.
TABLE
Material" 1. 2. 3. 4.
Control PTE, 10 U PTE, 20 U PT gland media 8 glds/chick
1. Preliminary experience with the chick PTH assay No. of birds
Plasma calcium1* mg/100 ml
Significance0
5 5 5
9.04 ± 0.22 9.83 ± 0.29 10.88 ± 0.39
p < 0.01
5
9.91 ±0.16
p < 0.05
NS
"All test materials were dissolved in 50 HIM CaCls and injected subcutaneously in a volume of 0.4 ml (20 /imoles CaClj per chick). " Samples obtained by cardiac puncture 1 hr after injection. 0 Compared to control group (1). NS: not significant (p > 0.05).
The synthetic salmon CT (5028 MRC U/mg) used for the in vitro assays was kindly provided by the Armour Pharmaceutical Company (courtesy of Dr. J. W. Bastian).
Results
Parathyroid hormone activity The embryonic chick parathyroid glands secreted into the culture media a stimulator of in vitro bone resorption. The media could be diluted and assayed to obtain the log-dose response curve in Fig. 1, which was parallel to that obtained with bovine parathyroid extract. In the chick bioassay, Lilly PTE at 10 and 20 U/chick produced a dose related increase in plasma calcium (Table 1.). Chick parathyroid gland media also produced a significant increase in the calcium concentration compared to control birds. Estimates made of the relative potency of the parathyroid gland material to Lilly PTE using the in vitro and in vivo assays give discrepant results. The in vitro assay indicates that six glands produce a response comparable to 0.2 U (Fig. 1). While the in vivo assay indicates that eight glands produce a response comparable to 10 U (Table 1). We do not feel that such estimates of "true" potency and direct quantitative comparisons are meaningful for the following reasons: 1) the data reported for the different assays are not for the same preparations and may reflect different activities per se for each individual experiment; 2) dif-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 16:34 For personal use only. No other uses without permission. . All rights reserved.
285
AVIAN PARATHYROIDS IN VITRO ferences in magnitude may also reflect age and treatment of the media before assay. The in vitro assays were done within 3-5 days of incubation whereas the in vivo experiments were performed several weeks later and no measure of degradation of the material can be made. We note that Tregear and associates (17) have reported that the 2-34 fragment of bovine parathyroid hormone appears to be more active in vivo than in vitro for reasons as yet unknown; 3) both PTH and CT may be present in the media and, as previously indicated, the level of PTH activity measured by the in vitro assay may be an underestimation; and 4) Species differences and differences in the precision of the assays. Column
chrojnatography
When medium obtained from PT glands incubated at 0.75 mM calcium for 48 hours was applied to a Sephadex G-100 column, the PTH-like activity, as measured by the
260
J22O-
J
J
180) 140-
100
075
1.50 CALCIUM
2.25
30
FIG. 3. Relationship between the concentration of calcium and the release of PTH-like activity; glands incubated for 48 hr at the indicated calcium concentrations and assayed at 6 glands/ml; N = 8 pairs of bones. Linear relationship derived from treating data as a simple regression by the least squares method (14). Vertical lines represent ±SE.
in vitro bioassay, migrated as shown in Fig. 2. We did not have a purified preparation of PTH available for comparison, but the IQ value for the chick material was approximately 0.40 which is comparable to the migration reported for chick (18) and other species of PTH (19,20) on Sephadex G-100 (Ka = 0.34-0.54). Effect of varying the calcium concentration
•1.00 30
40
50 TUBE
60 NO.
70
80
FIG. 2. Elution pattern of media obtained from parathyroid glands incubated at 0.75 mM calcium for 48 hr. Four ml of media applied to a Sephadex G-100 column (2.5 x 90 cm), and eluted with 0.2M ammonium acetate buffer, pH 4.7; 6 ml fractions collected. Pools of 5 fractions from tubes 30-80 were lyophilized and assayed for PTH-like activity. Only those fractions with significant activity are included (N = 4 pairs of bones. Vertical lines represent ±SE). Void volume (Vo) was determined by the peak of optical density at 280nm after blue dextran. Vt was taken as the peak representing amino acids and other low molecular weight components of the medium.
When parathyroid glands were incubated for 48 hr with increasing calcium concentrations from 0.75 to 3.0 mM (Fig. 3), there was a decrease in the T/C ratio indicating a reduced secretion of PTH with a significant inverse relationship over the range 0.75-2.25 mM calcium. In the chemically defined medium most of the calcium is presumably ionized and the normal value is approximately 1.2 to 1.4 mM. Calcitonin activity The embryonic chick PT glands secreted an inhibitor of in vitro bone resorption into the culture medium. The media could be diluted and assayed to obtain the log-dose
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 16:34 For personal use only. No other uses without permission. . All rights reserved.
FEINBLATT, TAI AND KENNY
286
Endo • 1975 Vol 96 • No 2
tion. Interestingly, in contrast to what would be expected for CT (5,10), the CT-like activity was also secreted by the PT glands in response to the low but not the high calcium concentration.
7060-
E3CO J i
Discussion
x i ^ a o H
20-
1.0
2.0 SALMON CT mil/ml
3
3.0
The present study demonstrates that the 20-day-old embryonic chick PT gland is capable of secreting a PTH-like substance in organ culture. The evidence that the material is in fact PTH is: 1) it is a stimulator of bone resorption in tissue culture (12) and the dose response curve obtained in the
6
PARATHYROID MEDIA glands/ml
1.5
O-O
FIG. 4. Log-dose response inhibition of PTH-stimulated Ca release by media obtained from embryonic chick parathyroid glands culture in 0.75 mM calcium (open circles), or synthetic salmon CT (closed circles). N = 8-12 pairs of bones. Vertical lines represent ±SE.
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2
response curve in Fig. 4 which was parallel to that obtained using synthetic salmon CT. The CT-like material was assayed directly in vivo in rats (16) without prior concentration and was found to have significant hypocalcemic activity. Column chromatography When medium obtained from PT glands incubated at 0.75 mM calcium for 48 hours was applied to a Sephadex G-50 column, the CT-like activity, as measured by the in vivo bioassay, migrated as shown in Fig. 5. Also included for comparison is the pattern obtained with salmon CT. Effect of varying the calcium concentration Table 2 contains the pooled data for several sets of experiments in which parathyroid glands were incubated for 48 hr at calcium concentrations of either 0.75 or 3.0 mM and the media was assayed for both PTH-like and CT-like activities. As observed in the previous experiments, PTHlike activity (A) was secreted in response to the low but not the high calcium concentra-
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1.0
0.5
CHICK PT MEDIUM
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