Institute of of
University
Pathology and Department of Nuclear Medicine, Hamburg, Hamburg, Federal Republic of Germany
THE EFFECT OF 1,25-DIHYDROXYCHOLECALCIFEROL ON THE PARATHYROID HORMONE SECRETION OF PORCINE PARATHYROID GLANDS AND HUMAN PARATHYROID ADENOMAS IN VITRO
By E. Alten\l=a"\hr,M. Dietel, G. Dorn and R. Montz
ABSTRACT The effect of 1,25-dihydroxycholecalciferol (1.25-(OH)2-D3) on parathyroid hormone secretion by porcine parathyroid glands and human parathyroid adenoma tissue was investigated by in vitro incubation. The addition of 100 nmoles 1,25-(OH)2-D3 to the medium inhibited significantly the release of immunoreactive parathyroid hormone by 63\p=n-\65%. This suppression was reversible when 1.25-(OH)2-D3 was removed again. The inhibition of parathyroid hormone release observed in human parathyroid adenoma tissue was similar to that in normal porcine parathyroid glands. This indicates that adenoma tissue is sensitive to regulatory influences. As well as calcium, 1,25-(OH)2-D3 may act as another feedback inhibitor of parathyroid hormone secretion.
The significance of vitamin D for parathyroid gland (PTG) function has been the subject of many studies (Au 8c Raisz 1965; Oldham et al. 1974a; Fischer et al. 1973; Lumb 8c Stanbury 1974). Whereas Au 8c Raisz (1965) concluded that both the size and functional activity of rat PTG are inversely related to serum calcium concentration, and do not depend on the presence or absence of vitamin D, evidence is now available for the role of vitamin D and its meta¬ bolites in PTG function (Fischer et al. 1973; Oldham et al. 1974è). As a resuit of their clinical studies Lumb 8c Stanbury (1974) suggested that vitamin D may act on the PTG, conceivably through the formation of calcium binding
protein.
533
1,25-Dihydroxycholecalciferol (l,25-(OH)2-Da) has been established as an important biologically active metabolite of vitamin D (Holick et al. 1971; Wong et al. 1972). In vitamin D-deficient chicks in which l,25-(OH)2-D3 was administered orally Henry 8c Norman (1975) have reported an accumulation of this steroid in the PTG to a concentration four times that in the blood. This concentration is equivalent to the levels observed in the target intestine. Other metabolites of vitamin D have been found to decrease the secretion of parathyroid hormone (PTH), e. g. 25-hydroxycholecalciferol in vitamin D-deficient puppies (Oldham et al. 1973) and 24,25-dihydroxycholecalciferol as well as 25,26-dihydroxycholecalciferol in isolated perfused superior PTGs of the goat (Care et al. 1976). With regard to the effect of l,25-(OH)2-D3 on PTH secretion, the few findings reported up to the present are apparently somewhat contradictory. Chertow et al. (1975) found a decrease in serum immunoreactive PTH in rats injected with l,25-(OH)2-D3 as well as an inhibition of PTH secretion into the medium by bovine PTG tissue incubated with 1 nmol and with 100 nmoles l,25-(OH)o-D.¡). Inhibition has also been observed by Okano et al. (1976) in bovine PTG monolayer cell cultures. In contrast to this, Care et al. (1976) observed a stimulation of PTH secretion of isolated goat PTGs in two of five experiments using l,25-(OH)g-Dg at concentrations of 50-125 pg/ml; a higher concentration (25 ng/ml) was without effect. In the present study we examined the effect of l,25-(OH)2-Da on the secre¬ tion of immunoreactive PTH by porcine PTG tissue and by human parathyroid adenoma (PTG-Ad) tissue using in vitro incubation.
MATERIAL AND METHODS Porcine PTGs were dissected from 8 normal pigs immediately after killing at the slaughter-house. The human PTG-Ad were surgically removed from patients with primary hyperparathyroidism. The diagnosis was based on the macroscopic presence of a solitary parathyroid tumour together with histological evidence. An enlargement of the other parathyroid glands was excluded. In each case surgical removal of the PTG-Ad resulted in a sudden decrease of serum calcium levels. The porcine PTGs and the human PTG-Ads were put in a cold sterile culture medium immediately after their removal and transported to the laboratory. After removing the connective tissue the parathyroid tissue was cut into slices 1 mm thick using a hand slicer (A. H. Thomas, Philadelphia) and subsequently reduced to pieces with a size of about 1 mm5. Five tissue specimens were placed on a stainless steel grid and put into one culture dish (Falcon Plastic). For culture Hams F 10 (modified) medium was used. Per 100 ml medium there was added 10 ml foetal bovine serum and 0.5 ml glutamine (both obtained from Flow Laboratories). The calcium concentration of the medium was adjusted to 1.2 mM Ca and checked by atomic absorption spectrophotometry. This concentration corresponds to that of the ionised normal extracellular fluid (Rasmussen 1970). The magnesium
534
concentration was 0.8 mM. The specimens were incubated at a temperature of 37°C in an atmosphere of 95 °/o air and 5 %> CO.?. The porcine PTG tissue and the human PTG-Ad tissue of all patients were tested in the same way. The test period covered 6 h during which the medium was changed hourly. During the 1st and 2nd h the parathyroid tissue was incubated in the above described medium. During h 3 and 4 100 nmoles l,25(OH)2-D3 was added to the medium in the test dishes, and during h 5 and 6 the medium was used again without addition of this steroid. To ensure the constancy of the PTH secretion throughout the test period, control samples of porcine PTG tissue and of each PTG-Ad were incubated in this medium for 6 h without addition of l,25(OH)2-D3. After each hour the immunoreactive PTH released into the medium was determined by radioimmunoassay using the method of Arnaud et al. (1971) with slight modifica¬ tions. The assay was done without pre-incubation. The materials used were highly purified bovine parathyroid hormone (Nordmeyer et al. 1976) for standards and for radioiodination (Hunter Se Greenwood 1962) with 125I 10 Ci/mg (Behring Werke. Marburg) and an anti-porcine PTH antiserum from rabbit in a final concentration of 1:10 000. With this antiserum cross-reaction and superimposable dilution curves were found with bovine PTG highly purified, 1450 MRC units/mg protein (Nordmeyer et cd. 1976) total medium from human parathyroid adenoma tissue culture and serum of a patient with secondary renal hyperparathyroidism. There was no reaction with the human 1-34 synthetic fragment (Beckman, Palo Alto) up to a concentration of 100 ng/tube, suggesting specificity of binding to the COOH-terminal region of the PTH molecule. The non-specific binding effect was measured with the culture medium batches from each experiment. The range of the standard curves was 0.2-3.0 ng bPTH/100 ml medium. As in previous studies (Dietel et al. 1977) the PTH release during the first hour into the original medium (i. e. without l,25-(OH)9-D3) was used as a reference value corresponding to 100 %>, and the release during the following test hours was expressed as a percentage of this initial value. Statistical tests of the significance of the results from porcine PTGs and human PTG-Ad were carried out with Student's ¿-test.
RESULTS
The values of immunoreactive PTH secreted into the medium during 1 h were generally between 20-300 ng/ml. Only very few samples were outside this range.
When incubated in the control medium during the 6 h period the PTH release per hour into the medium was rather constant. This applies to the porcine PTG tissue as well as to the human PTG-Ad tissue. Related to the release during the first hour (100 %) the mean value of PTG release decreased only non-significantly to 95-98 %. During the 3rd and 4th h of incubation we added 100 nmoles l,25-(OH)2-D3 to the medium which resulted in a significant decrease of PTH release. The secretion into the medium during the 4th h amounted to only 63 °/o in porcine PTG tissue and 65% in human PTG-Ad tissue (P < 0.005) relative to the first hour value. 535
*---J
ioo%-
J
j
/
\
porcine PTG
o
a
t 50%;_
X
«
Ô
-4_í_I
~T**
v
•S ioo%-
S
SS
T
T
•——._
-I
T—Hh~—T— i-1
*
\
-
human PTG-Ad
*
\
\A
J/ Jf
T
-
ft
T
y"^
1
50%; _
-I-1-i-I-1-1 3
12
4
5
6
hours
Fig. 1. PTH release of porcine PTG tissue incubated without l,25-(OH)o-D3, D-D porcine PTG tissue incubated with l,25-(OH)2-D3, •-• human PTG adenoma tissue incubated without l,25-(OH).2-D3, O-O human PTG adenoma tissue incubated with l,25-(OH)2-D3. Number of porcine PTG N 8, number of human PTG adenomas N 8. Significant differences between 2nd and 4th value after incuba¬ tion with l,25-(OH)2-D3 identically for porcine and human material (P < 0.01) and between 4th and 6th h value when incubated without l,25-(OH)2-D3 again (P < 0.01). The values are expressed as percentage of the 1st h value. Each point represents Hourly
=
=
mean
+ sd.
The PTH secretion increased again, when incubation was done without l,25-(OH)o-D3 during the 5th and 6th h. There was only a minor difference between the porcine PTG tissue and the human PTG-Ad tissue with regard to the time course of changes in PTH release. In the tests with human PTG-Ad tissue the decrease of PTH secretion in the presence of the steroid was faster while the increase following its removal was slower (and incomplete within the experimental period) than in the tests with porcine PTG tissue (Fig. 1). DISCUSSION
The inhibitory effect of l,25-(OH)2-D3 on PTH release by porcine PTG tissue or human PTG-Ad tissue in vitro demonstrated in this study is consistent with the results of Chertow et al. (1975) in rats in vivo and in bovine PTG in vitro and also with the results of Okano et al. (1976) in bovine PTG monolayer culture. The reversibility of the inhibitory effect when the steroid was sub¬ sequently removed again and the constant secretion of tissue incubated all 536
the time in control medium provide further evidence that the decrease of PTH is a specific effect of l,25-(OH)o-D3 in our in vitro system. This effect and the accumulation of l,25-(OH)2-D3 in parathyroid glands of vitamin D-deficient chicks (Henry 8c Norman 1975) speak in favour of the PTG being a target organ for this vitamin D metabolite. As in the other target organs small intestine (Wasserman et al. 1968; Emtage et al. 1973) and kidney (Taylor 8c Wasserman 1972), it may stimulate the synthesis of a calcium binding protein, which has been found to occur in porcine PTG (Oldham et al. 19746). Furthermore Oldham et al. (1973, 19746) have reported that an in¬ creased calcium binding activity in the PTG was associated with a decreased PTH secretion. However, recent experiments by Oldham et al. (1976) have demonstrated that the investigators were unable to confirm their own results regarding to relationships between the PTH secretion and parathyroid calcium binding proteins. From the sequence of events it seems that calcium ions inhibit the activity of parathyroid adenylcyclase (Dufresne et al. 1971; Matsuzaki 8c Durnont 1972) and mediate via cyclic AMP the PTH secretion (Abe 8c Sherwood 1972; Williams et al. 1973; Dietel et al. 1977). Since PTH stimulates the renal synthesis of l,25-(OH)¿-D3 (Fraser 8c Kodicek 1973) this steroid may act in addition to calcium as another feed-back inhibitor of PTH secretion. The inhibition of PTH secretion by l,25-(OH)o-D3 and its subsequent re¬ stitution was observed in a very similar way in normal porcine PTG tissue and in human PTG-Ad tissue. These findings suggest that adenoma tissue is sensitive to the same regulatory agents as normal PTGs. as has previously also been shown for calcium concentration and dibutyryl-cyclic AMP in vitro (Dietel et al. 1977). However, it must be stressed that the l,25-(OH)2-D3 con¬ centration used in these in vitro experiments is not a physiological one. Quan¬ titative differences between parathyroid adenoma tissue and normal para¬ thyroid glands may therefore exist in the sensitivity to different regulatory influences at the physiological level.
ACKNOWLEDGMENTS
l,25-(OH)2"D3
synthesized by Dr. A. W. Norman (University of California) and disposal by Prof. Dr. F. Kuhlencordt (University of Hamburg). and surgery of the patients with primary hyperparathyroidism we are indebted to Prof. Dr. F. Kuhlencordt and Prof. E. Farthmann (both University of Hamburg), Dr. M. Bressel and Dr. H. Hüsselmann (both General Hospital, HamburgHarburg). The excellent technical assistance of Mrs. E. Lehmann and Mrs. B. Sehringer is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 34, Endokrinologie, Hamburg.
kindly placed For diagnosis
was
at
our
537
REFERENCES Abe M. Se Sherwood L. M.: Biochem. biophys. Res. Commun. 48 (1972) 396. Arnaud C. D., Tsao H. S. Se Littledyke T.: J. clin. Invest. 50 (1971) 20. Au W. Y. W. Se Raisz L. G.: Amer. J. Physiol. 20.9 (1965) 637. Care A. D., Bates R. F. L., Pickard D. W., Peacock M., Tomlinson S., O'Riordan J. L. H., Mawer E. B., Taylor C. M., DeLuca H. F. Se Norman A. W.: Calcif. Tiss. Res. Suppl.
21 (1976) 142. Chertow B. S., Baylink D. J., Wergedahl J. E., Su M. H. H. Se Norman A. W.: J. clin. Invest. 56 (1975) 668. Dietel M., Dorn G., Montz R. Se Altenähr E.: Acta endocr. (Kbh.) 85 (1977) 541. Dufresne L. R., Andersen R. Se Gitelman H. J.: Clin. Res. 19 (1971) 529. Emtage J. S., Lawson D. E. M. 8c Kodicek E.: Nature (Lond.) 246 (1973) 100. Fischer J. A., Binswanger U., Fanconi A., Jelig R., Baerlocher K. 8c Prader A.: Horm. Metab. Res. 5 (1973) 381. Fraser D. R. Se Kodicek E.: Nature New Biol. 241 (1973) 163. Henry H. L. 8e Norman A. W.: Biochem. biophys. Res. Commun. 62 (1975) 781. Holick M. F., Schnoes H. K. Se DeLuca H. F.: Proc. nat. Acad. Sei. (USA) 68
(1971)
803.
Hunter W. M. Se Greenwood F. C: Nature (Lond.) 194 (1962) 494. Lumb G. A. Se Stanbury S. W.: Amer. J. Med. 56 (1974) 833. Matsuzaki S. Se Dumont J. E.: Biochim. biophys. Acta (Amst.) 284 (1972) 227. Nordmeyer J. P., Dorn G. Se Mayer H.: Hoppe-Seylers Z. physiol. Chem. 357 (1976) 893. Okano K., Nakai R., Fujita T. Se Yoshikawa M.: Vth International Congress of Endo¬ crinology, Hamburg. Abstr. No. 711 (1976) p. 292. Oldham S. B., Arnaud C. D. Se Jowsey J.: Clin. Res. 21 (1973) 254. Oldham S. B., Arnaud C. D. 8c Jowsey J.: Endocrinology 1973. W. Heinemann, London
(1974a)
p. 261.
Oldham S. B., Fischer J. A., Shen L. H. & Arnaud C. D.: Biochemistry 13 (1974a) 4790. Oldham S. B., Smith R., Hartenbower D., Henry H., Norman A. W. & Coburn J.: Clin. Res. 24 (1976) 118. Rasmussen H.: Science 170 (1970) 404. Taylor A. N. Se Wasserman R. H.: Amer. J. Physiol. 223 (1972) 110. Wasserman R. H., Corradino R. A. Se Taylor A. N.: J. biol. Chem. 243 (1968) 3978. Williams G. A., Hargis G. K., Bowser E. N., Henderson W. J. 8c Martinez N. J.:
Endocrinology 92 (1973) 687. Wong R. G., Myrtle J. F., Tsai H. Received
on
C. 8c Norman A. W.:
March 4th, 1977.
538
J.
biol. Chem. 247
(1972)
5728.
University
Pathology and Department of Nuclear Medicine, Hamburg, Hamburg, Federal Republic of Germany
THE EFFECT OF 1,25-DIHYDROXYCHOLECALCIFEROL ON THE PARATHYROID HORMONE SECRETION OF PORCINE PARATHYROID GLANDS AND HUMAN PARATHYROID ADENOMAS IN VITRO
By E. Alten\l=a"\hr,M. Dietel, G. Dorn and R. Montz
ABSTRACT The effect of 1,25-dihydroxycholecalciferol (1.25-(OH)2-D3) on parathyroid hormone secretion by porcine parathyroid glands and human parathyroid adenoma tissue was investigated by in vitro incubation. The addition of 100 nmoles 1,25-(OH)2-D3 to the medium inhibited significantly the release of immunoreactive parathyroid hormone by 63\p=n-\65%. This suppression was reversible when 1.25-(OH)2-D3 was removed again. The inhibition of parathyroid hormone release observed in human parathyroid adenoma tissue was similar to that in normal porcine parathyroid glands. This indicates that adenoma tissue is sensitive to regulatory influences. As well as calcium, 1,25-(OH)2-D3 may act as another feedback inhibitor of parathyroid hormone secretion.
The significance of vitamin D for parathyroid gland (PTG) function has been the subject of many studies (Au 8c Raisz 1965; Oldham et al. 1974a; Fischer et al. 1973; Lumb 8c Stanbury 1974). Whereas Au 8c Raisz (1965) concluded that both the size and functional activity of rat PTG are inversely related to serum calcium concentration, and do not depend on the presence or absence of vitamin D, evidence is now available for the role of vitamin D and its meta¬ bolites in PTG function (Fischer et al. 1973; Oldham et al. 1974è). As a resuit of their clinical studies Lumb 8c Stanbury (1974) suggested that vitamin D may act on the PTG, conceivably through the formation of calcium binding
protein.
533
1,25-Dihydroxycholecalciferol (l,25-(OH)2-Da) has been established as an important biologically active metabolite of vitamin D (Holick et al. 1971; Wong et al. 1972). In vitamin D-deficient chicks in which l,25-(OH)2-D3 was administered orally Henry 8c Norman (1975) have reported an accumulation of this steroid in the PTG to a concentration four times that in the blood. This concentration is equivalent to the levels observed in the target intestine. Other metabolites of vitamin D have been found to decrease the secretion of parathyroid hormone (PTH), e. g. 25-hydroxycholecalciferol in vitamin D-deficient puppies (Oldham et al. 1973) and 24,25-dihydroxycholecalciferol as well as 25,26-dihydroxycholecalciferol in isolated perfused superior PTGs of the goat (Care et al. 1976). With regard to the effect of l,25-(OH)2-D3 on PTH secretion, the few findings reported up to the present are apparently somewhat contradictory. Chertow et al. (1975) found a decrease in serum immunoreactive PTH in rats injected with l,25-(OH)2-D3 as well as an inhibition of PTH secretion into the medium by bovine PTG tissue incubated with 1 nmol and with 100 nmoles l,25-(OH)o-D.¡). Inhibition has also been observed by Okano et al. (1976) in bovine PTG monolayer cell cultures. In contrast to this, Care et al. (1976) observed a stimulation of PTH secretion of isolated goat PTGs in two of five experiments using l,25-(OH)g-Dg at concentrations of 50-125 pg/ml; a higher concentration (25 ng/ml) was without effect. In the present study we examined the effect of l,25-(OH)2-Da on the secre¬ tion of immunoreactive PTH by porcine PTG tissue and by human parathyroid adenoma (PTG-Ad) tissue using in vitro incubation.
MATERIAL AND METHODS Porcine PTGs were dissected from 8 normal pigs immediately after killing at the slaughter-house. The human PTG-Ad were surgically removed from patients with primary hyperparathyroidism. The diagnosis was based on the macroscopic presence of a solitary parathyroid tumour together with histological evidence. An enlargement of the other parathyroid glands was excluded. In each case surgical removal of the PTG-Ad resulted in a sudden decrease of serum calcium levels. The porcine PTGs and the human PTG-Ads were put in a cold sterile culture medium immediately after their removal and transported to the laboratory. After removing the connective tissue the parathyroid tissue was cut into slices 1 mm thick using a hand slicer (A. H. Thomas, Philadelphia) and subsequently reduced to pieces with a size of about 1 mm5. Five tissue specimens were placed on a stainless steel grid and put into one culture dish (Falcon Plastic). For culture Hams F 10 (modified) medium was used. Per 100 ml medium there was added 10 ml foetal bovine serum and 0.5 ml glutamine (both obtained from Flow Laboratories). The calcium concentration of the medium was adjusted to 1.2 mM Ca and checked by atomic absorption spectrophotometry. This concentration corresponds to that of the ionised normal extracellular fluid (Rasmussen 1970). The magnesium
534
concentration was 0.8 mM. The specimens were incubated at a temperature of 37°C in an atmosphere of 95 °/o air and 5 %> CO.?. The porcine PTG tissue and the human PTG-Ad tissue of all patients were tested in the same way. The test period covered 6 h during which the medium was changed hourly. During the 1st and 2nd h the parathyroid tissue was incubated in the above described medium. During h 3 and 4 100 nmoles l,25(OH)2-D3 was added to the medium in the test dishes, and during h 5 and 6 the medium was used again without addition of this steroid. To ensure the constancy of the PTH secretion throughout the test period, control samples of porcine PTG tissue and of each PTG-Ad were incubated in this medium for 6 h without addition of l,25(OH)2-D3. After each hour the immunoreactive PTH released into the medium was determined by radioimmunoassay using the method of Arnaud et al. (1971) with slight modifica¬ tions. The assay was done without pre-incubation. The materials used were highly purified bovine parathyroid hormone (Nordmeyer et al. 1976) for standards and for radioiodination (Hunter Se Greenwood 1962) with 125I 10 Ci/mg (Behring Werke. Marburg) and an anti-porcine PTH antiserum from rabbit in a final concentration of 1:10 000. With this antiserum cross-reaction and superimposable dilution curves were found with bovine PTG highly purified, 1450 MRC units/mg protein (Nordmeyer et cd. 1976) total medium from human parathyroid adenoma tissue culture and serum of a patient with secondary renal hyperparathyroidism. There was no reaction with the human 1-34 synthetic fragment (Beckman, Palo Alto) up to a concentration of 100 ng/tube, suggesting specificity of binding to the COOH-terminal region of the PTH molecule. The non-specific binding effect was measured with the culture medium batches from each experiment. The range of the standard curves was 0.2-3.0 ng bPTH/100 ml medium. As in previous studies (Dietel et al. 1977) the PTH release during the first hour into the original medium (i. e. without l,25-(OH)9-D3) was used as a reference value corresponding to 100 %>, and the release during the following test hours was expressed as a percentage of this initial value. Statistical tests of the significance of the results from porcine PTGs and human PTG-Ad were carried out with Student's ¿-test.
RESULTS
The values of immunoreactive PTH secreted into the medium during 1 h were generally between 20-300 ng/ml. Only very few samples were outside this range.
When incubated in the control medium during the 6 h period the PTH release per hour into the medium was rather constant. This applies to the porcine PTG tissue as well as to the human PTG-Ad tissue. Related to the release during the first hour (100 %) the mean value of PTG release decreased only non-significantly to 95-98 %. During the 3rd and 4th h of incubation we added 100 nmoles l,25-(OH)2-D3 to the medium which resulted in a significant decrease of PTH release. The secretion into the medium during the 4th h amounted to only 63 °/o in porcine PTG tissue and 65% in human PTG-Ad tissue (P < 0.005) relative to the first hour value. 535
*---J
ioo%-
J
j
/
\
porcine PTG
o
a
t 50%;_
X
«
Ô
-4_í_I
~T**
v
•S ioo%-
S
SS
T
T
•——._
-I
T—Hh~—T— i-1
*
\
-
human PTG-Ad
*
\
\A
J/ Jf
T
-
ft
T
y"^
1
50%; _
-I-1-i-I-1-1 3
12
4
5
6
hours
Fig. 1. PTH release of porcine PTG tissue incubated without l,25-(OH)o-D3, D-D porcine PTG tissue incubated with l,25-(OH)2-D3, •-• human PTG adenoma tissue incubated without l,25-(OH).2-D3, O-O human PTG adenoma tissue incubated with l,25-(OH)2-D3. Number of porcine PTG N 8, number of human PTG adenomas N 8. Significant differences between 2nd and 4th value after incuba¬ tion with l,25-(OH)2-D3 identically for porcine and human material (P < 0.01) and between 4th and 6th h value when incubated without l,25-(OH)2-D3 again (P < 0.01). The values are expressed as percentage of the 1st h value. Each point represents Hourly
=
=
mean
+ sd.
The PTH secretion increased again, when incubation was done without l,25-(OH)o-D3 during the 5th and 6th h. There was only a minor difference between the porcine PTG tissue and the human PTG-Ad tissue with regard to the time course of changes in PTH release. In the tests with human PTG-Ad tissue the decrease of PTH secretion in the presence of the steroid was faster while the increase following its removal was slower (and incomplete within the experimental period) than in the tests with porcine PTG tissue (Fig. 1). DISCUSSION
The inhibitory effect of l,25-(OH)2-D3 on PTH release by porcine PTG tissue or human PTG-Ad tissue in vitro demonstrated in this study is consistent with the results of Chertow et al. (1975) in rats in vivo and in bovine PTG in vitro and also with the results of Okano et al. (1976) in bovine PTG monolayer culture. The reversibility of the inhibitory effect when the steroid was sub¬ sequently removed again and the constant secretion of tissue incubated all 536
the time in control medium provide further evidence that the decrease of PTH is a specific effect of l,25-(OH)o-D3 in our in vitro system. This effect and the accumulation of l,25-(OH)2-D3 in parathyroid glands of vitamin D-deficient chicks (Henry 8c Norman 1975) speak in favour of the PTG being a target organ for this vitamin D metabolite. As in the other target organs small intestine (Wasserman et al. 1968; Emtage et al. 1973) and kidney (Taylor 8c Wasserman 1972), it may stimulate the synthesis of a calcium binding protein, which has been found to occur in porcine PTG (Oldham et al. 19746). Furthermore Oldham et al. (1973, 19746) have reported that an in¬ creased calcium binding activity in the PTG was associated with a decreased PTH secretion. However, recent experiments by Oldham et al. (1976) have demonstrated that the investigators were unable to confirm their own results regarding to relationships between the PTH secretion and parathyroid calcium binding proteins. From the sequence of events it seems that calcium ions inhibit the activity of parathyroid adenylcyclase (Dufresne et al. 1971; Matsuzaki 8c Durnont 1972) and mediate via cyclic AMP the PTH secretion (Abe 8c Sherwood 1972; Williams et al. 1973; Dietel et al. 1977). Since PTH stimulates the renal synthesis of l,25-(OH)¿-D3 (Fraser 8c Kodicek 1973) this steroid may act in addition to calcium as another feed-back inhibitor of PTH secretion. The inhibition of PTH secretion by l,25-(OH)o-D3 and its subsequent re¬ stitution was observed in a very similar way in normal porcine PTG tissue and in human PTG-Ad tissue. These findings suggest that adenoma tissue is sensitive to the same regulatory agents as normal PTGs. as has previously also been shown for calcium concentration and dibutyryl-cyclic AMP in vitro (Dietel et al. 1977). However, it must be stressed that the l,25-(OH)2-D3 con¬ centration used in these in vitro experiments is not a physiological one. Quan¬ titative differences between parathyroid adenoma tissue and normal para¬ thyroid glands may therefore exist in the sensitivity to different regulatory influences at the physiological level.
ACKNOWLEDGMENTS
l,25-(OH)2"D3
synthesized by Dr. A. W. Norman (University of California) and disposal by Prof. Dr. F. Kuhlencordt (University of Hamburg). and surgery of the patients with primary hyperparathyroidism we are indebted to Prof. Dr. F. Kuhlencordt and Prof. E. Farthmann (both University of Hamburg), Dr. M. Bressel and Dr. H. Hüsselmann (both General Hospital, HamburgHarburg). The excellent technical assistance of Mrs. E. Lehmann and Mrs. B. Sehringer is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 34, Endokrinologie, Hamburg.
kindly placed For diagnosis
was
at
our
537
REFERENCES Abe M. Se Sherwood L. M.: Biochem. biophys. Res. Commun. 48 (1972) 396. Arnaud C. D., Tsao H. S. Se Littledyke T.: J. clin. Invest. 50 (1971) 20. Au W. Y. W. Se Raisz L. G.: Amer. J. Physiol. 20.9 (1965) 637. Care A. D., Bates R. F. L., Pickard D. W., Peacock M., Tomlinson S., O'Riordan J. L. H., Mawer E. B., Taylor C. M., DeLuca H. F. Se Norman A. W.: Calcif. Tiss. Res. Suppl.
21 (1976) 142. Chertow B. S., Baylink D. J., Wergedahl J. E., Su M. H. H. Se Norman A. W.: J. clin. Invest. 56 (1975) 668. Dietel M., Dorn G., Montz R. Se Altenähr E.: Acta endocr. (Kbh.) 85 (1977) 541. Dufresne L. R., Andersen R. Se Gitelman H. J.: Clin. Res. 19 (1971) 529. Emtage J. S., Lawson D. E. M. 8c Kodicek E.: Nature (Lond.) 246 (1973) 100. Fischer J. A., Binswanger U., Fanconi A., Jelig R., Baerlocher K. 8c Prader A.: Horm. Metab. Res. 5 (1973) 381. Fraser D. R. Se Kodicek E.: Nature New Biol. 241 (1973) 163. Henry H. L. 8e Norman A. W.: Biochem. biophys. Res. Commun. 62 (1975) 781. Holick M. F., Schnoes H. K. Se DeLuca H. F.: Proc. nat. Acad. Sei. (USA) 68
(1971)
803.
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