O013-7227/78/lO23-0745$02.0O/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 3 Printed in U.S.A
Effect of Somatostatin on Parathyroid Hormone and Calcitonin Secretion* GARY K. HARGIS, GERALD A. WILLIAMS, W. ANN REYNOLDS, BRUCE S. CHERTOW, SUBHASH C. KUKREJA, E. NELSON BOWSER, AND W. J. HENDERSON Section of Endocrinology, Departments of Medicine, Nuclear Medicine, and Anatomy, Veterans Administration West Side Hospital and University of Illinois College of Medicine, Chicago, Illinois 60612 ABSTRACT. This study evaluated the effect of somatostatin on immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) secretion in vivo in rats and monkeys and on iPTH secretion in vitro by normal bovine parathyroid tissue and by a human parathyroid adenoma. Somatostatin infusion promptly (within 0.5 h) suppressed both iPTH and iCT in both species studied in vivo, the suppression being progressive during the infusion period. In in vitro studies, somatostatin caused significant dose-related decreases in basal, low Ca-stimulated, and high Ca-suppressed PTH secretion from normal bovine parathyroid tissue and from basal and low Ca-stimulated PTH secretion from a human
parathyroid adenoma. Therefore, somatostatin 1) suppresses both PTH and CT secretion in vivo; 2) acts directly on the parathyroid cell and presumably directly on the C-cell also; 3) acts upon normal and adenomatous parathyroid tissue; 4) suppresses basal, low Ca-stimulated and high Ca-suppressed PTH secretion; and 5) has a dose-related effect. The possible role of somatostatin in the physiological control of PTH and CT secretion (and therefore in Ca homeostasis), and in the pathogenesis of abnormalities of Ca homeostasis, requires further evaluation. (Endocrinology 102: 745, 1978)
S
OMATOSTATIN was initially discovered in the hypothalamus and characterized for its inhibitory effect on growth hormone (GH) secretion (1). However, it has subsequently been found to exist in several other areas of the body and to have inhibitory effects on secretion of several pituitary (1) and nonpituitary hormones (1, 2), including glucagon (3-5), insulin (4), and gastrin (6). The present in vivo and in vitro studies were undertaken to determine whether somatostatin may modify secretion of the Ca-regulating hormones, parathyroid hormone (PTH), and calcitonin (CT).
for infusion and in the aorta for blood sampling, were fasted for 14-16 h and then placed in restraining cages. Synthetic somatostatin (lot AY-24910, kindly provided by Ayerst Laboratories, Montreal, Quebec) was dissolved in normal saline. Six of the rats received a priming infusion of 25 jug somatostatin in 2 min, then infusion of 109 /ig/kg/h for 1 h, followed by infusion of normal saline for 1 h. Six control rats underwent this same procedure except that normal saline alone was infused throughout. Blood samples were obtained at 0, 0.5, 1,1.5, and 2 h for analysis of serum immunoreactive (i) PTH, plasma iCT, and serum Ca concentrations. Eighteen normal adult Rhesus monkeys [Macaca mulatto) weighing 5.5-7 kg, after fasting for 14-16 h, were tranquillized with thiamylal sodium (Surital; Parke, Davis) and indwelling needles were Materials and Methods placed in an antecubital vein for infusion and a femoral vein for blood sampling. Six of the monkeys In vivo studies received a priming infusion of 18 jug/kg somatoTwelve normal 200-g Sprague-Dawley rats, with statin in 5 min, then infusion of 26 jug/kg/h for 2 previously implanted catheters in the jugular vein h, followed by infusion of normal saline alone for 1 h. Six other animals were similarly infused, but with a lower dose of somatostatin (7 jug/kg in 5 Received February 14, 1977. Requests for reprints should be addressed to: Gary K. min followed by 7 /xg/kg/h for 2h). The remaining Hargis, VA West Side Hospital (M.P. 115), P. 0. Box six animals served as controls and underwent the 8195, Chicago, Illinois 60680. same infusion throughout. Blood samples were ob* VA Research Program MRIS 9420-12 and USPHS tained at 0, 0.5, 1, 2, and 3 h for analysis of serum Grant 1RO1 HD-09373-01. 745
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iPTH, plasma iCT, and serum Ca concentrations. Rat serum iPTH was determined by the method previously reported from this laboratory (7). Monkey serum iPTH was determined by modification of the method of Arnaud et al. (8) by using a guinea pig antiserum to bovine PTH, purified bovine PTH (Wilson Laboratories, lot 147865) for tracer, and dilutions of a pool of human parathyroid tissue culture medium for standards. This antiserum detects both the carboxyl and amino terminals of bovine iPTH, has a high affinity for human iPTH, and detects human and monkey serum iPTH indistinguishably over a 20-fold dilution span. This assay detects as little as 0.5 /xl equivalents of standard human parathyroid tissue culture medium/ml serum (jul Eq/ml). Parathyroidectomized human or monkey serum caused no displacement of the antibody-bound tracer, but normal monkeys responded with appropriate changes in serum iPTH during EDTA or Ca infusions, indicating specificity of the assay. The intraassay and interassay coefficients of variation were 2-6% and 3-8%, respectively, in the utilized portion of the standard curve. The normal range (mean ± 2 SD) for monkey serum iPTH is 4.6-8.3 jul Eq/ml). Rat and monkey plasma iCT was determined by a modification of the method of Sizemore et al. (9) by using a goat antiserum to synthetic hCT and synthetic hCT (N. V. Organon, batch SC 30) for standard and tracer. The antiserum used detects rat, monkey, and human plasma iCT indistinguishably over a 20-fold dilution span. The assay detects as little as 5 pg iCT/ml plasma. Neither polypeptide hormones other than CT, nor thyroidectomized rat, monkey, or human plasma caused any displacement of antibody-bound tracer, but plasma from normal Ca- or EDTA-infused rats, monkeys, and human subjects revealed appropriate changes. The intraassay and interassay coefficients of variation were 1.5-4.5% and 2-7%, respectively, in the utilized portion of the standard curve. The normal range (mean ± 2 SD) for rat plasma iCT is 168-260 pg/ml and for monkey plasma iCT is 242-388 pg/ml. Somatostatin (2 jug/ml) added to serum and plasma and allowed to incubate at room temperature for 2 h had no effect on the assay for iPTH and iCT. Serum Ca concentration was determined with the Technicon AutoAnalyzer by the method of Kessler and Wolfman (10). The normal ranges (mean ± 2 SD) for rat and monkey serum Ca are 8.7-10.9 mg/100 ml and 8.6-10.6 mg/100 ml, respectively. The serum iPTH, plasma, iCT, and serum Ca concentrations were calculated in absolute terms
and also as a percentage of the baseline pre-injection value for each animal. In vitro studies Fresh bovine parathyroid tissue slices were incubated for 4 h in Eagle's Minimal Essential Medium with 10% calf serum by the technique previously described from this laboratory (11). The medium was completely aspirated and replaced by fresh medium hourly. During the first 2 h, the medium in all flasks contained 1.25 mM Ca (considered to approximate the ionized Ca concentration of normal plasma). The first hour of incubation was considered an equilibration period and this medium was discarded. The iPTH in the medium removed at the end of the next hour was considered to represent the control or baseline secretion of the tissue in that flask. The composition of the medium was then modified to contain either a high (3 mM) or a low (0.75 mM) Ca concentration and/or to contain various concentrations of somatostatin, and incubation was continued for an additional 2 h. The iPTH concentration of each hourly medium sample was determined by radioimmunoassay as previously described (11), by using purified bovine PTH for standard and tracer. The concentration of iPTH in pg/wet wt of tissue in the baseline medium sample was designated as 100%. The iPTH concentrations in the media harvested at the end of the next 2 h were then expressed as a percentage of this baseline value for that flask (11). Flasks containing 1.25 mM Ca during the entire incubation period were included to evaluate uniformity of secretion with time. Also, aliquots of media incubated without tissue were assayed to assure that the additives had no nonspecific effects in the immunoassay results. Tissue slices from a parathyroid adenoma removed surgically from a patient with primary hyperparathyroidism were also evaluated by the technique described above. In all studies, the mean and SE for each time period were calculated from the individual percentage values for that time period for each animal (in vivo studies) or each flask (in vitro studies). Statistical tests of significance were carried out with Student's t test.
Results In vivo rat studies The mean baseline values (mean ± SE) for the rat studies were iPTH 5.6 ± 0.25 pg/ml; iCT 224 ± 29.0 pg/ml; Ca 9.3 ± 0.20 mg/100
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SOMATOSTATIN EFFECT ON PTH AND CT ml. Infusion of normal saline alone caused no change in iPTH, iCT, or Ca. However, as shown in Fig. 1, infusion of somatostatin caused a significant decrease in iPTH to 79.4 ± 3.0% of the baseline (P < 0.001) and iCT to 39.2 ± 5.4% of the baseline (P < 0.001) by 30 min, which was the first time evaluated. The decrease in concentration of iPTH was progressive during the 2-h infusion, reaching 53.6 ± 3.3% of the baseline (P < 0.001). The concentration of iCT did not decrease further, being 42.4 ± 2.9% of the baseline (P < 0.001) at the termination of the infusion. The concentration of both hormones had increased to baseline values within 30 min after termination of somatostatin infusion. Serum Ca concentration decreased minimally during somatostatin infusion and the hour thereafter, being significantly decreased at 30 min (P < 0.02) and 90 min (P < 0.01). In vivo monkey studies The mean baseline values for the monkey studies were: iPTH 6.4 ± 0.19 /xl Eq/ml; iCT 297 ± 15.8 pg/ml; Ca 9.6 ± 0.14 mg/100 ml. Infusion of normal saline alone caused no change in iPTH, iCT, or Ca. However, as shown in Fig. 2, infusion of the larger dose of somatostatin caused a significant (P < 0.01) and almost identical decrease in iPTH (to 86.8 ± 5.7% of the baseline) and iCT (to 86.0 ± 3.7% of the baseline) by 30 min, which was the first time evaluated. This very similar
747
•••*
-
5 0 - -Somatostatin( bolus) Somatostatin IV I
Normal Saline 2
3
Time in Hours
FIG. 2. Effect of somatostatin infusion in the monkey on serial serum iPTH, plasma iCT, and serum Ca concentrations during the following 3 h. Values expressed as in Fig. 1. n = 6. Baseline values: iPTH, 6.4 ± 0.19 jtl eq/ml; iCT, 297 ± 15.8 pg/ml; Ca, 9.6 ± 0.14 mg/100 ml.
decrease in concentration of the two hormones was progressive during the 2-h infusion, reaching 57.4 ± 2.4% of the baseline (P < 0.001) for iPTH and 58.9 ± 5.6% of the baseline (P < 0.001) for iCT at the termination of the infusion. The concentration of both hormones had increased toward baseline values by 1 h after termination of somatostatin. Serum Ca concentration did not significantly change during somatostatin infusion or the hour thereafter. Infusion of the lower dose of somatostatin caused significant (P < 0.001) decreases in both iPTH and iCT at all time periods tested, which were of a similar pattern but of lesser degree than those observed with the larger dose of somatostatin. Serum Ca concentration was minimally but significantly decreased at 2 h (P < 0.05) and 3 h (P < 0.02). In vitro studies
Somotostotinl09^q/Kg/h ivl
Normal Saline
I Time in Hours
2
FIG. 1. Effect of somatostatin infusion in the rat on serial serum iPTH, plasma iCT, and serum Ca concentrations during the following 2 h. Values (mean ± SE) at each time period are expressed as percentage of the baseline pre-infusion values (designated as 100%). n = 6. Baseline values: iPTH, 5.6 ± 0.25 pg/ml; iCT, 224 ± 29.0 pg/ml; Ca, 9.3 ± 0.20 mg/100 ml.
Changes in in vitro secretion of iPTH, related to medium changes in Ca ion concentration and/or to addition of somatostatin, are portrayed in Figs. 3-5. Hourly iPTH secretion remained uniform when medium containing 1.25 mM Ca was used during the entire incubation period, and was designated as 100% for each hour. As indicated in Fig. 3, medium containing low (0.75 mM) Ca caused a significant (P < 0.001) increase in iPTH release to 260 ± 24.9% and 348 ± 30.5% of the baseline at the first and second hour of incubation,
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HARGIS ET AL.
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baseline at the first and second hour of incubation, respectively. oO75mM Co + SlOOI/ig/ml) 300In the presence of low Ca stimulation of |f 250iPTH secretion, somatostatin, in concentraS' , * ' ' o 0.75 m M Co**+S(0 05/xg/ml) | S 200tions ranging from 4-0.01 jug/ml, caused sigc "5 yS ,*'£* "1 °O75mM Co** + S(O.I/ig/ml) T ^ 150nificant (P < 0.001-P < 0.05) dose-related -5 0 75mMCa**+S(4/ig/ml) decreases in iPTH at both the first and second I 25mMCa** hour of incubation (Fig. 3). In the presence nM Ca** . _ nM Ca +S(lug/ml) of medium containing 1.25 mM Ca (Fig. 4, " ° 3 0mM Co +S{4ua/ml) note change in ordinate scale) somatostatin in the above series of concentrations also FIG. 3. Effect of Ca concentration and of multiple concentrations of somatostatin (S) in low (0.75 mM) Ca caused significant (P < 0.001-0.05) dose-remedium and high (3.0 mM) Ca medium on hourly iPTH lated decreases in iPTH. The 4 /xg/ml concensecretion in vitro by normal bovine parathyroid tissue. tration of somatostatin in normal (1.25 mM) Medium before zero time contained 1.25 mM Ca and no Ca medium suppressed iPTH to a degree not somatostatin in all flasks. Each value is expressed as a significantly different from that achieved by percentage of the zero time iPTH secretion for that flask. high (3.0 mM) Ca suppression alone. As indiEach point is the mean ± SE for eight flasks. cated in Fig. 3, even in the presence of high l25mM Ca'* (3.0 mM) Ca suppression of iPTH secretion, "r""** *?-~-~ •" l25mM Co**+S(OOI^g/n of somatostatin in a concentration of addition N. ~~^v ^ ^ ~~~-*l25mM Co** + S(OO5/ig/i or 1 jug/ml caused a significant (P < 4 jug/ml ^"1 I 25mM Ca"+S(OI/ig/ml) 0.05) further decrease in medium iPTH. As indicated in Fig. 5, incubation of human § S SO l.25mM Co+SU/ig/ml) Urn adenomatous parathyroid tissue in the presence of 4 jug/ml or 1 jug/ml somatostatin l25mM Co+S(4/ig/ml) caused a decrease in iPTH secretion similar to that observed with normal bovine parathy30; , C roid tissue. O W>
sa=5
V
FIG. 4. Effect of multiple concentrations of somatostatin (S) in normal (1.25 mM) Ca medium on hourly iPTH secretion in vitro by normal bovine parathyroid tissue. Data expressed as in Fig. 3. n = 8.
O75mMCa**
i O75mM
to - c
60-
Si
40-
Co** + Sll/ig/ml) l25mMCa*' 075mM Ca** + S(4/ig/ml) I 25mMCa**+S(l /i g/ml) I 25mM Ca**+S(4pg/ml) 3.0 mM Ca"
Time in Hours
FIG.ft.Effect of Ca concentration and of two concentrations of somatostatin (S) on hourly iPTH secretion in vitro by human parathyroid adenoma. Data expressed as in Fig. 3.
respectively. Medium containing high (3.0 mM) Ca caused a significant (P < 0.001) decrease to 65.3 ± 4.4% and 35.5 ± 5.0% of the
Discussion The in vivo observations indicate that somatostatin can suppress secretion of both PTH and CT to a very similar degree with a prompt onset and dissipation of biologic effect. The in vitro observations indicate that the suppressive effect of somatostatin was directly on the parathyroid cell and was dose-related. The mode of action on the C-cell of the thyroid was not studied, but is inferred to also be direct. However, other possible mechanisms or intermediary steps (such as the known suppressive effect of somatostatin on gastrin secretion, which could possibly lead to a decrease in CT secretion) have not been excluded. In some reported studies, somatostatin has shown its inhibitory effect on secretion of a hormone solely or predominantly under conditions of the stimulated secretory state of
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SOMATOSTATIN EFFECT ON PTH AND CT that hormone (1, 5, 12, 13). However, the observations from both the in vivo and the in vitro components of the present study indicate that somatostatin can suppress basal secretion of both PTH and CT and can even further decrease Ca-suppressed PTH secretion in vitro, as well as decrease the stimulated secretion of PTH. This suggests that somatostatin could have a physiological role in controlling even basal secretion of these two hormones. The findings of these studies are consistent with observations pertaining to somatostatin in relation to other hormones, i.e., a) somatostatin inhibits secretion of multiple polypeptide hormones, and b) the source of somatostatin secretion is topographically closely related to its site of action (1). Two groups of investigators (14, 15) have reported localization of somatostatin in parafollicular cells of the thyroid gland. There are as yet no reported studies of somatostatin localization in parathyroid tissues. Deftos et al. (16) have reported no effect of somatostatin infusion on basal or stimulated PTH secretion in six normal human subjects or in one patient with primary hyperparathyroidism. Metz et al. (17) more recently reported no effect of somatostatin on CT secretion in man. The reasons for the marked difference between our observations and those of these investigators are unexplained. Our studies utilized normal bovine and adenomatous human parathyroid tissue in vitro and normal rat and monkey species in the in vivo studies, whereas those investigators utilized normal and hyperparathyroid human subjects. However, multiple diverse comparative physiological evaluations of PTH and CT secretion in man and monkey in our laboratory have always yielded very similar data. Also, the effect of somatostatin on other hormones has, in general, yielded comparable results in all species tested and in in vitro or in vivo studies. The larger dose of somatostatin in the monkey based on BW was approximately 4-fold greater in the present study than in the study of Deftos et al., but the lower dose was very similar to that used by those investiga-
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tors. The sources of the somatostatin were different, but the potency of both preparations had been demonstrated in other studies. The observation of significant suppression of PTH and CT in vivo in two species, and similar dose-related suppression of basal, stimulated, and suppressed PTH secretion in vitro in two other species lead us to conclude that exogenous somatostatin is effective in the suppression of PTH and CT secretion. The effect and role of endogenous somatostatin in the control of secretion of these two hormones is yet to be established.
Acknowledgments We thank Bertha L. Jackson, Wanda J. Kawahara, and Patricia Johnson for technical assistance.
References 1. Guillemin, R., and J. E. Gerich, Somatostatin: physiological and clinical significance, Ann Rev Med 27: 379, 1976. 2. Copinschi, G., V. Leclercq-Meyer, E. Virasaro, M. L'Hermite, L. Vanhaelst, J. Goldstein, Ft. Leclercq, F. Fery, and C. Robyn, Pituitary and extra-pituitary effects of somatostatin in normal man, Horm Metab Res 8: 226, 1976. 3. Unger, R. H., and L. Orci, The essential role of glucagon in the pathogenesis of diabetes mellitus, Lancet 1: 14, 1975. 4. Mortimer, C. H., D. Carr, T. Lind, S. R. Bloom, C. N. Nallison, A. V. Schally, W. M. G. Tunbridge, L. Yeomans, D. H. Coy, A. Kastin, G. M. Besser, and R. Hall, Effects of growthhormone release-inhibiting hormone on circulating glucagon, insulin, and growth hormone in normal, diabetic, acromegalic and hypopituitary patients, Lancet 1: 697, 1974. 5. Gerich, J. E., M. Lorenzi, V. Schneider, C. W. Kwan, J. H. Karam, R. Guillemin, and P. H. Forsham, Inhibition of pancreatic glucagon responses to arginine by somatostatin in normal man and in insulin dependent diabetics, Diabetes 23: 876, 1974.
6. Bloom, S. R., C. H. Mortimer, M. 0. Thorner, G. M. Besser, R. Hall, A. Gomez-Pan, V. M. Roy, R. C. G. Russell, D. H. Coy, A. J. Kastin, and A. V. Schally, Inhibition of gastrin and gastric acid secretion by growth-hormone release-inhibiting hormone, Lancet 2: 1106, 1974. 7. Hargis, G. K., E. N. Bowser, W. J. Henderson, and G. A. Williams, Radioimmunoassay of rat parathyroid hormone in serum and tissue extracts, Endocrinology 94: 1644, 1974. 8. Arnaud, C. D., H. S. Tsao, and T. Littledike, Radioimmunoassay of human parathyroid hormone in serum, J Clin Invest 50: 21, 1971. 9. Sizemore, G. W., V. L. W. Go, E. L. Kaplan, L. J. Sanzenbacker, K. H. Holtermuller, and C. D. Arnaud, Calcitonin and gastrin in Zollinger-Ellison syndrome and medullary carcinoma of the thyroid, N Engl J. Med 288: 641, 1973. 10. Kessler, G., and M. Wolfman, An automated procedure for the simultaneous determination of calcium and phosphorus, Clin Chem 10: 686, 1964. 11. Williams, G. A., G. K. Hargis, E. N. Bowser, W. J. Henderson, and N. J. Martinez, Evidence for a role of adenosine 3',5'monophosphate on parathyroid hormone release, Endocrinology 92:687, 1973. 12. Lucke, C, B. Hoffken, and A. Von Zur Muhlen, The effect of somatostatin on TSH levels in patients with primary hypothyroidism. J Clin Endocrinol Metab 41: 1082, 1975. 13. Siler, T. M., G. Vanden Borg, S. S. C. Yen, P. Brazeau, W.
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Vale, and R. Guillemin, Inhibition of growth hormone release in humans by somatostatin, J Clin Endocrinol Metab 37: 632, 1973. 14. Hokfelt, T., S. Efendic, C. Hellerstrom, 0. Johansson, R. Luft, and A. Arimura, Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special reference to the Ai-cells of the pancreatic islets and to the hypothalamus, Ada Endocrinol 80(Suppl 200): 5, 1975. 15. Parsons, J. A., S. L. Erlandsen, O. D. Hegre, R. C. McEvoy, and R. P. Elde, Central and peripheral localization of soma-
tostatin: immunoenzyme immunocytochemical studies, J. Histochem Cytochem 24:872, 1976. 16. Deftos, L. J., iCf. Lorenzi, N. Bohanon, E. Tsalakian, V. Schneider, and J. E. Gerich, Somatostatin does not suppress plasma parathyroid hormone, J Clin Endocrinol Metab 43: 205, 1976. 17. Metz, S., L. Deftos, D. Baylink, and R. P. Robertson, Neuroendocrine modulation of calcitonin (CT) and parathyroid hormone (PTH) secretion in normal man, Clin Res 25: 161A, 1977 (Abstract).
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Vol. 102, No. 3 Printed in U.S.A
Effect of Somatostatin on Parathyroid Hormone and Calcitonin Secretion* GARY K. HARGIS, GERALD A. WILLIAMS, W. ANN REYNOLDS, BRUCE S. CHERTOW, SUBHASH C. KUKREJA, E. NELSON BOWSER, AND W. J. HENDERSON Section of Endocrinology, Departments of Medicine, Nuclear Medicine, and Anatomy, Veterans Administration West Side Hospital and University of Illinois College of Medicine, Chicago, Illinois 60612 ABSTRACT. This study evaluated the effect of somatostatin on immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) secretion in vivo in rats and monkeys and on iPTH secretion in vitro by normal bovine parathyroid tissue and by a human parathyroid adenoma. Somatostatin infusion promptly (within 0.5 h) suppressed both iPTH and iCT in both species studied in vivo, the suppression being progressive during the infusion period. In in vitro studies, somatostatin caused significant dose-related decreases in basal, low Ca-stimulated, and high Ca-suppressed PTH secretion from normal bovine parathyroid tissue and from basal and low Ca-stimulated PTH secretion from a human
parathyroid adenoma. Therefore, somatostatin 1) suppresses both PTH and CT secretion in vivo; 2) acts directly on the parathyroid cell and presumably directly on the C-cell also; 3) acts upon normal and adenomatous parathyroid tissue; 4) suppresses basal, low Ca-stimulated and high Ca-suppressed PTH secretion; and 5) has a dose-related effect. The possible role of somatostatin in the physiological control of PTH and CT secretion (and therefore in Ca homeostasis), and in the pathogenesis of abnormalities of Ca homeostasis, requires further evaluation. (Endocrinology 102: 745, 1978)
S
OMATOSTATIN was initially discovered in the hypothalamus and characterized for its inhibitory effect on growth hormone (GH) secretion (1). However, it has subsequently been found to exist in several other areas of the body and to have inhibitory effects on secretion of several pituitary (1) and nonpituitary hormones (1, 2), including glucagon (3-5), insulin (4), and gastrin (6). The present in vivo and in vitro studies were undertaken to determine whether somatostatin may modify secretion of the Ca-regulating hormones, parathyroid hormone (PTH), and calcitonin (CT).
for infusion and in the aorta for blood sampling, were fasted for 14-16 h and then placed in restraining cages. Synthetic somatostatin (lot AY-24910, kindly provided by Ayerst Laboratories, Montreal, Quebec) was dissolved in normal saline. Six of the rats received a priming infusion of 25 jug somatostatin in 2 min, then infusion of 109 /ig/kg/h for 1 h, followed by infusion of normal saline for 1 h. Six control rats underwent this same procedure except that normal saline alone was infused throughout. Blood samples were obtained at 0, 0.5, 1,1.5, and 2 h for analysis of serum immunoreactive (i) PTH, plasma iCT, and serum Ca concentrations. Eighteen normal adult Rhesus monkeys [Macaca mulatto) weighing 5.5-7 kg, after fasting for 14-16 h, were tranquillized with thiamylal sodium (Surital; Parke, Davis) and indwelling needles were Materials and Methods placed in an antecubital vein for infusion and a femoral vein for blood sampling. Six of the monkeys In vivo studies received a priming infusion of 18 jug/kg somatoTwelve normal 200-g Sprague-Dawley rats, with statin in 5 min, then infusion of 26 jug/kg/h for 2 previously implanted catheters in the jugular vein h, followed by infusion of normal saline alone for 1 h. Six other animals were similarly infused, but with a lower dose of somatostatin (7 jug/kg in 5 Received February 14, 1977. Requests for reprints should be addressed to: Gary K. min followed by 7 /xg/kg/h for 2h). The remaining Hargis, VA West Side Hospital (M.P. 115), P. 0. Box six animals served as controls and underwent the 8195, Chicago, Illinois 60680. same infusion throughout. Blood samples were ob* VA Research Program MRIS 9420-12 and USPHS tained at 0, 0.5, 1, 2, and 3 h for analysis of serum Grant 1RO1 HD-09373-01. 745
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iPTH, plasma iCT, and serum Ca concentrations. Rat serum iPTH was determined by the method previously reported from this laboratory (7). Monkey serum iPTH was determined by modification of the method of Arnaud et al. (8) by using a guinea pig antiserum to bovine PTH, purified bovine PTH (Wilson Laboratories, lot 147865) for tracer, and dilutions of a pool of human parathyroid tissue culture medium for standards. This antiserum detects both the carboxyl and amino terminals of bovine iPTH, has a high affinity for human iPTH, and detects human and monkey serum iPTH indistinguishably over a 20-fold dilution span. This assay detects as little as 0.5 /xl equivalents of standard human parathyroid tissue culture medium/ml serum (jul Eq/ml). Parathyroidectomized human or monkey serum caused no displacement of the antibody-bound tracer, but normal monkeys responded with appropriate changes in serum iPTH during EDTA or Ca infusions, indicating specificity of the assay. The intraassay and interassay coefficients of variation were 2-6% and 3-8%, respectively, in the utilized portion of the standard curve. The normal range (mean ± 2 SD) for monkey serum iPTH is 4.6-8.3 jul Eq/ml). Rat and monkey plasma iCT was determined by a modification of the method of Sizemore et al. (9) by using a goat antiserum to synthetic hCT and synthetic hCT (N. V. Organon, batch SC 30) for standard and tracer. The antiserum used detects rat, monkey, and human plasma iCT indistinguishably over a 20-fold dilution span. The assay detects as little as 5 pg iCT/ml plasma. Neither polypeptide hormones other than CT, nor thyroidectomized rat, monkey, or human plasma caused any displacement of antibody-bound tracer, but plasma from normal Ca- or EDTA-infused rats, monkeys, and human subjects revealed appropriate changes. The intraassay and interassay coefficients of variation were 1.5-4.5% and 2-7%, respectively, in the utilized portion of the standard curve. The normal range (mean ± 2 SD) for rat plasma iCT is 168-260 pg/ml and for monkey plasma iCT is 242-388 pg/ml. Somatostatin (2 jug/ml) added to serum and plasma and allowed to incubate at room temperature for 2 h had no effect on the assay for iPTH and iCT. Serum Ca concentration was determined with the Technicon AutoAnalyzer by the method of Kessler and Wolfman (10). The normal ranges (mean ± 2 SD) for rat and monkey serum Ca are 8.7-10.9 mg/100 ml and 8.6-10.6 mg/100 ml, respectively. The serum iPTH, plasma, iCT, and serum Ca concentrations were calculated in absolute terms
and also as a percentage of the baseline pre-injection value for each animal. In vitro studies Fresh bovine parathyroid tissue slices were incubated for 4 h in Eagle's Minimal Essential Medium with 10% calf serum by the technique previously described from this laboratory (11). The medium was completely aspirated and replaced by fresh medium hourly. During the first 2 h, the medium in all flasks contained 1.25 mM Ca (considered to approximate the ionized Ca concentration of normal plasma). The first hour of incubation was considered an equilibration period and this medium was discarded. The iPTH in the medium removed at the end of the next hour was considered to represent the control or baseline secretion of the tissue in that flask. The composition of the medium was then modified to contain either a high (3 mM) or a low (0.75 mM) Ca concentration and/or to contain various concentrations of somatostatin, and incubation was continued for an additional 2 h. The iPTH concentration of each hourly medium sample was determined by radioimmunoassay as previously described (11), by using purified bovine PTH for standard and tracer. The concentration of iPTH in pg/wet wt of tissue in the baseline medium sample was designated as 100%. The iPTH concentrations in the media harvested at the end of the next 2 h were then expressed as a percentage of this baseline value for that flask (11). Flasks containing 1.25 mM Ca during the entire incubation period were included to evaluate uniformity of secretion with time. Also, aliquots of media incubated without tissue were assayed to assure that the additives had no nonspecific effects in the immunoassay results. Tissue slices from a parathyroid adenoma removed surgically from a patient with primary hyperparathyroidism were also evaluated by the technique described above. In all studies, the mean and SE for each time period were calculated from the individual percentage values for that time period for each animal (in vivo studies) or each flask (in vitro studies). Statistical tests of significance were carried out with Student's t test.
Results In vivo rat studies The mean baseline values (mean ± SE) for the rat studies were iPTH 5.6 ± 0.25 pg/ml; iCT 224 ± 29.0 pg/ml; Ca 9.3 ± 0.20 mg/100
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SOMATOSTATIN EFFECT ON PTH AND CT ml. Infusion of normal saline alone caused no change in iPTH, iCT, or Ca. However, as shown in Fig. 1, infusion of somatostatin caused a significant decrease in iPTH to 79.4 ± 3.0% of the baseline (P < 0.001) and iCT to 39.2 ± 5.4% of the baseline (P < 0.001) by 30 min, which was the first time evaluated. The decrease in concentration of iPTH was progressive during the 2-h infusion, reaching 53.6 ± 3.3% of the baseline (P < 0.001). The concentration of iCT did not decrease further, being 42.4 ± 2.9% of the baseline (P < 0.001) at the termination of the infusion. The concentration of both hormones had increased to baseline values within 30 min after termination of somatostatin infusion. Serum Ca concentration decreased minimally during somatostatin infusion and the hour thereafter, being significantly decreased at 30 min (P < 0.02) and 90 min (P < 0.01). In vivo monkey studies The mean baseline values for the monkey studies were: iPTH 6.4 ± 0.19 /xl Eq/ml; iCT 297 ± 15.8 pg/ml; Ca 9.6 ± 0.14 mg/100 ml. Infusion of normal saline alone caused no change in iPTH, iCT, or Ca. However, as shown in Fig. 2, infusion of the larger dose of somatostatin caused a significant (P < 0.01) and almost identical decrease in iPTH (to 86.8 ± 5.7% of the baseline) and iCT (to 86.0 ± 3.7% of the baseline) by 30 min, which was the first time evaluated. This very similar
747
•••*
-
5 0 - -Somatostatin( bolus) Somatostatin IV I
Normal Saline 2
3
Time in Hours
FIG. 2. Effect of somatostatin infusion in the monkey on serial serum iPTH, plasma iCT, and serum Ca concentrations during the following 3 h. Values expressed as in Fig. 1. n = 6. Baseline values: iPTH, 6.4 ± 0.19 jtl eq/ml; iCT, 297 ± 15.8 pg/ml; Ca, 9.6 ± 0.14 mg/100 ml.
decrease in concentration of the two hormones was progressive during the 2-h infusion, reaching 57.4 ± 2.4% of the baseline (P < 0.001) for iPTH and 58.9 ± 5.6% of the baseline (P < 0.001) for iCT at the termination of the infusion. The concentration of both hormones had increased toward baseline values by 1 h after termination of somatostatin. Serum Ca concentration did not significantly change during somatostatin infusion or the hour thereafter. Infusion of the lower dose of somatostatin caused significant (P < 0.001) decreases in both iPTH and iCT at all time periods tested, which were of a similar pattern but of lesser degree than those observed with the larger dose of somatostatin. Serum Ca concentration was minimally but significantly decreased at 2 h (P < 0.05) and 3 h (P < 0.02). In vitro studies
Somotostotinl09^q/Kg/h ivl
Normal Saline
I Time in Hours
2
FIG. 1. Effect of somatostatin infusion in the rat on serial serum iPTH, plasma iCT, and serum Ca concentrations during the following 2 h. Values (mean ± SE) at each time period are expressed as percentage of the baseline pre-infusion values (designated as 100%). n = 6. Baseline values: iPTH, 5.6 ± 0.25 pg/ml; iCT, 224 ± 29.0 pg/ml; Ca, 9.3 ± 0.20 mg/100 ml.
Changes in in vitro secretion of iPTH, related to medium changes in Ca ion concentration and/or to addition of somatostatin, are portrayed in Figs. 3-5. Hourly iPTH secretion remained uniform when medium containing 1.25 mM Ca was used during the entire incubation period, and was designated as 100% for each hour. As indicated in Fig. 3, medium containing low (0.75 mM) Ca caused a significant (P < 0.001) increase in iPTH release to 260 ± 24.9% and 348 ± 30.5% of the baseline at the first and second hour of incubation,
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HARGIS ET AL.
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Endo • 1978 Vol 102 • No 3
baseline at the first and second hour of incubation, respectively. oO75mM Co + SlOOI/ig/ml) 300In the presence of low Ca stimulation of |f 250iPTH secretion, somatostatin, in concentraS' , * ' ' o 0.75 m M Co**+S(0 05/xg/ml) | S 200tions ranging from 4-0.01 jug/ml, caused sigc "5 yS ,*'£* "1 °O75mM Co** + S(O.I/ig/ml) T ^ 150nificant (P < 0.001-P < 0.05) dose-related -5 0 75mMCa**+S(4/ig/ml) decreases in iPTH at both the first and second I 25mMCa** hour of incubation (Fig. 3). In the presence nM Ca** . _ nM Ca +S(lug/ml) of medium containing 1.25 mM Ca (Fig. 4, " ° 3 0mM Co +S{4ua/ml) note change in ordinate scale) somatostatin in the above series of concentrations also FIG. 3. Effect of Ca concentration and of multiple concentrations of somatostatin (S) in low (0.75 mM) Ca caused significant (P < 0.001-0.05) dose-remedium and high (3.0 mM) Ca medium on hourly iPTH lated decreases in iPTH. The 4 /xg/ml concensecretion in vitro by normal bovine parathyroid tissue. tration of somatostatin in normal (1.25 mM) Medium before zero time contained 1.25 mM Ca and no Ca medium suppressed iPTH to a degree not somatostatin in all flasks. Each value is expressed as a significantly different from that achieved by percentage of the zero time iPTH secretion for that flask. high (3.0 mM) Ca suppression alone. As indiEach point is the mean ± SE for eight flasks. cated in Fig. 3, even in the presence of high l25mM Ca'* (3.0 mM) Ca suppression of iPTH secretion, "r""** *?-~-~ •" l25mM Co**+S(OOI^g/n of somatostatin in a concentration of addition N. ~~^v ^ ^ ~~~-*l25mM Co** + S(OO5/ig/i or 1 jug/ml caused a significant (P < 4 jug/ml ^"1 I 25mM Ca"+S(OI/ig/ml) 0.05) further decrease in medium iPTH. As indicated in Fig. 5, incubation of human § S SO l.25mM Co+SU/ig/ml) Urn adenomatous parathyroid tissue in the presence of 4 jug/ml or 1 jug/ml somatostatin l25mM Co+S(4/ig/ml) caused a decrease in iPTH secretion similar to that observed with normal bovine parathy30; , C roid tissue. O W>
sa=5
V
FIG. 4. Effect of multiple concentrations of somatostatin (S) in normal (1.25 mM) Ca medium on hourly iPTH secretion in vitro by normal bovine parathyroid tissue. Data expressed as in Fig. 3. n = 8.
O75mMCa**
i O75mM
to - c
60-
Si
40-
Co** + Sll/ig/ml) l25mMCa*' 075mM Ca** + S(4/ig/ml) I 25mMCa**+S(l /i g/ml) I 25mM Ca**+S(4pg/ml) 3.0 mM Ca"
Time in Hours
FIG.ft.Effect of Ca concentration and of two concentrations of somatostatin (S) on hourly iPTH secretion in vitro by human parathyroid adenoma. Data expressed as in Fig. 3.
respectively. Medium containing high (3.0 mM) Ca caused a significant (P < 0.001) decrease to 65.3 ± 4.4% and 35.5 ± 5.0% of the
Discussion The in vivo observations indicate that somatostatin can suppress secretion of both PTH and CT to a very similar degree with a prompt onset and dissipation of biologic effect. The in vitro observations indicate that the suppressive effect of somatostatin was directly on the parathyroid cell and was dose-related. The mode of action on the C-cell of the thyroid was not studied, but is inferred to also be direct. However, other possible mechanisms or intermediary steps (such as the known suppressive effect of somatostatin on gastrin secretion, which could possibly lead to a decrease in CT secretion) have not been excluded. In some reported studies, somatostatin has shown its inhibitory effect on secretion of a hormone solely or predominantly under conditions of the stimulated secretory state of
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SOMATOSTATIN EFFECT ON PTH AND CT that hormone (1, 5, 12, 13). However, the observations from both the in vivo and the in vitro components of the present study indicate that somatostatin can suppress basal secretion of both PTH and CT and can even further decrease Ca-suppressed PTH secretion in vitro, as well as decrease the stimulated secretion of PTH. This suggests that somatostatin could have a physiological role in controlling even basal secretion of these two hormones. The findings of these studies are consistent with observations pertaining to somatostatin in relation to other hormones, i.e., a) somatostatin inhibits secretion of multiple polypeptide hormones, and b) the source of somatostatin secretion is topographically closely related to its site of action (1). Two groups of investigators (14, 15) have reported localization of somatostatin in parafollicular cells of the thyroid gland. There are as yet no reported studies of somatostatin localization in parathyroid tissues. Deftos et al. (16) have reported no effect of somatostatin infusion on basal or stimulated PTH secretion in six normal human subjects or in one patient with primary hyperparathyroidism. Metz et al. (17) more recently reported no effect of somatostatin on CT secretion in man. The reasons for the marked difference between our observations and those of these investigators are unexplained. Our studies utilized normal bovine and adenomatous human parathyroid tissue in vitro and normal rat and monkey species in the in vivo studies, whereas those investigators utilized normal and hyperparathyroid human subjects. However, multiple diverse comparative physiological evaluations of PTH and CT secretion in man and monkey in our laboratory have always yielded very similar data. Also, the effect of somatostatin on other hormones has, in general, yielded comparable results in all species tested and in in vitro or in vivo studies. The larger dose of somatostatin in the monkey based on BW was approximately 4-fold greater in the present study than in the study of Deftos et al., but the lower dose was very similar to that used by those investiga-
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tors. The sources of the somatostatin were different, but the potency of both preparations had been demonstrated in other studies. The observation of significant suppression of PTH and CT in vivo in two species, and similar dose-related suppression of basal, stimulated, and suppressed PTH secretion in vitro in two other species lead us to conclude that exogenous somatostatin is effective in the suppression of PTH and CT secretion. The effect and role of endogenous somatostatin in the control of secretion of these two hormones is yet to be established.
Acknowledgments We thank Bertha L. Jackson, Wanda J. Kawahara, and Patricia Johnson for technical assistance.
References 1. Guillemin, R., and J. E. Gerich, Somatostatin: physiological and clinical significance, Ann Rev Med 27: 379, 1976. 2. Copinschi, G., V. Leclercq-Meyer, E. Virasaro, M. L'Hermite, L. Vanhaelst, J. Goldstein, Ft. Leclercq, F. Fery, and C. Robyn, Pituitary and extra-pituitary effects of somatostatin in normal man, Horm Metab Res 8: 226, 1976. 3. Unger, R. H., and L. Orci, The essential role of glucagon in the pathogenesis of diabetes mellitus, Lancet 1: 14, 1975. 4. Mortimer, C. H., D. Carr, T. Lind, S. R. Bloom, C. N. Nallison, A. V. Schally, W. M. G. Tunbridge, L. Yeomans, D. H. Coy, A. Kastin, G. M. Besser, and R. Hall, Effects of growthhormone release-inhibiting hormone on circulating glucagon, insulin, and growth hormone in normal, diabetic, acromegalic and hypopituitary patients, Lancet 1: 697, 1974. 5. Gerich, J. E., M. Lorenzi, V. Schneider, C. W. Kwan, J. H. Karam, R. Guillemin, and P. H. Forsham, Inhibition of pancreatic glucagon responses to arginine by somatostatin in normal man and in insulin dependent diabetics, Diabetes 23: 876, 1974.
6. Bloom, S. R., C. H. Mortimer, M. 0. Thorner, G. M. Besser, R. Hall, A. Gomez-Pan, V. M. Roy, R. C. G. Russell, D. H. Coy, A. J. Kastin, and A. V. Schally, Inhibition of gastrin and gastric acid secretion by growth-hormone release-inhibiting hormone, Lancet 2: 1106, 1974. 7. Hargis, G. K., E. N. Bowser, W. J. Henderson, and G. A. Williams, Radioimmunoassay of rat parathyroid hormone in serum and tissue extracts, Endocrinology 94: 1644, 1974. 8. Arnaud, C. D., H. S. Tsao, and T. Littledike, Radioimmunoassay of human parathyroid hormone in serum, J Clin Invest 50: 21, 1971. 9. Sizemore, G. W., V. L. W. Go, E. L. Kaplan, L. J. Sanzenbacker, K. H. Holtermuller, and C. D. Arnaud, Calcitonin and gastrin in Zollinger-Ellison syndrome and medullary carcinoma of the thyroid, N Engl J. Med 288: 641, 1973. 10. Kessler, G., and M. Wolfman, An automated procedure for the simultaneous determination of calcium and phosphorus, Clin Chem 10: 686, 1964. 11. Williams, G. A., G. K. Hargis, E. N. Bowser, W. J. Henderson, and N. J. Martinez, Evidence for a role of adenosine 3',5'monophosphate on parathyroid hormone release, Endocrinology 92:687, 1973. 12. Lucke, C, B. Hoffken, and A. Von Zur Muhlen, The effect of somatostatin on TSH levels in patients with primary hypothyroidism. J Clin Endocrinol Metab 41: 1082, 1975. 13. Siler, T. M., G. Vanden Borg, S. S. C. Yen, P. Brazeau, W.
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Vale, and R. Guillemin, Inhibition of growth hormone release in humans by somatostatin, J Clin Endocrinol Metab 37: 632, 1973. 14. Hokfelt, T., S. Efendic, C. Hellerstrom, 0. Johansson, R. Luft, and A. Arimura, Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special reference to the Ai-cells of the pancreatic islets and to the hypothalamus, Ada Endocrinol 80(Suppl 200): 5, 1975. 15. Parsons, J. A., S. L. Erlandsen, O. D. Hegre, R. C. McEvoy, and R. P. Elde, Central and peripheral localization of soma-
tostatin: immunoenzyme immunocytochemical studies, J. Histochem Cytochem 24:872, 1976. 16. Deftos, L. J., iCf. Lorenzi, N. Bohanon, E. Tsalakian, V. Schneider, and J. E. Gerich, Somatostatin does not suppress plasma parathyroid hormone, J Clin Endocrinol Metab 43: 205, 1976. 17. Metz, S., L. Deftos, D. Baylink, and R. P. Robertson, Neuroendocrine modulation of calcitonin (CT) and parathyroid hormone (PTH) secretion in normal man, Clin Res 25: 161A, 1977 (Abstract).
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