Organ culture of human parathyroid glands: Effects of catecholamines

JOURNAL

OF SURGICAL

30, 86-95 (1981)

RESEARCH

Organ Culture of Human Parathyroid Effects of Catecholaminesl FRANTZ DONALD MARK

Glands:

J. DERENONCOURT, M.D.,2 JOHN P. BILEZIKIAN, M.D., CARL R. FEIND, M.D., E. GAMMON, A.B., ELIZABETH SHANE, M.D., SIEGFRIED KRUTZIK, PH.D.,~ A. HARDY, M.D., COLLIN J. WEBER, M.D., AND KEITH REEMTSMA, M.D.

Departments

of Medicine

and Surgery, Columbia University, College of Physicians New York, New York 10032

and Surgeons,

Submitted for publication March 3, 1980 Norma1 and adenomatous human parathyroid glands were studied in organ culture to determine basal secretory rates of cyclic AMP and parathyroid hormone as well as sensitivity to padrenergic catecholamines. Basal cyclic AMP secretion was relatively constant over 4 weeks but parathyroid hormone secretion declined. Both normal and adenomatous tissue were stimulated to secrete cyclic AMP and parathyroid hormone in the presence of the p agonist, isoproterenol. Halfmaximal stimulation for adenomas occurred at 1 w. Propranolol, a /3-adrenergic inhibitor, completely prevented the response, with half-maximal inhibition occurring at 0.1 w. Under basal conditions, normal explants secreted significantly more cyclic AMP and parathyroid hormone than adenomas. The results indicate that both normal and adenomatous human parathyroid glands can be maintained in organ culture for up to 4 weeks and that P-adrenergic catecholamines can stimulate the secretion of cyclic AMP and parathyroid hormone.

parathyroid glands was established to evaluate the role of catecholamines in the secretion of parathyroid hormone. These studies show that the P-adrenergic catecholamines do stimulate the secretion of parathyroid hormone from human parathyroid explants. They also suggest that the technique of long-term organ culture of parathyroid tissue may be potentially applicable to studies related to the problem of heterotopic parathyroid gland transplantation.

INTRODUCTION

Although calcium has long been considered to be the principal regulator of parathyroid hormone secretion [ 1,13,16,30-32, 36,371, recent studies have shown that other agents such as magnesium [28], calcitonin [15], la,25-dihydroxyvitamin D [ll], cortisol [2], dopamine [7], and the /3adrenergic system [9, 16, 171may also play a role in the regulation of parathyroid gland function. The P-adrenergic catecholamines are of particular interest because physioMATERIALS AND METHODS logical studies suggest an influence upon the secretion of parathyroid hormone in vivo (-)-Epinephrine, (-)-norepinephrine, [ 14, 381. However, information pertinent to and (-)-phenylephrine were obtained from the adrenergic control of human parathyroid Sigma Chemical Company (St. Louis, MO.); tissue is limited. In the present study, organ minimal essential medium (MEM), Grand culture of normal and adenomatous human Island Biological Company (Grand Island, 1 A preliminary report of this work was presented at N. Y .); cyclic AMP binding protein, Calbiothe Annual Meeting of the American Federation for them (La Jolla, Calif.); cellulose nitrate filClinical Research, May 1977, Washington, D. C. ters (0.8 pm), Millipore Corporation (Bed2 Present address: St. Luke’s Hospital Center, New ford, Mass.); and Gelman A/E glass filters, York, N. Y. 10025. through A. H. Thomas (Philadelphia, Pa.). 3 Present address: The Nichols Institute for EndoCyclic[3H]AMP was purchased from New crinology, San Pedro, Calif. 90731. 0022-4804/81/010086-10$01.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

86

DERENONCOURT r= + 99 (pc

E 140 P

ET AL.: PARATHYROID

,011

1

t

ADENOMAS

(mglexplanl)

FIG. 1. Correlation of adenoma weight with cyclic AMP secretion. Human parathyroid adenomas of different weights were placed into organ culture. Basal cyclic AMP secretion in Week 1 was determined in medium exposed to explants for 24 hr (see Materials and Methods). Symbols represent the average 2 SEM of the number of glands studied at that weight (in parentheses). The glands were pooled into indicated weight groups ?20%. Coefficient of correlation (r) was determined by linear regression analysis.

EXPLANTS AND CATECHOLAMINES

87

tive sample for morphologic study, the gland was weighed and cut into l- to 2-mm pieces each weighing approximately l-2 mg. These pieces were placed upon triangular steel grids and then into petri dishes. Normal explants contained IO-20 mg of parathyroid tissue whereas adenomatous explants contained 20-60 mg. Over the ranges of sample weights used, basal cyclic AMP secretion was directly related to the amount of tissue (Fig. 1). The calcium concentration was physiological (1.2 mM) throughout these experiments. Medium (700 ~1) bathing the explants was changed every 48 hr. It contained fetal calf serum (lo%), penicillin (100 Units/ml), streptomycin (0.1 mg/ml), and amphotericin B (2.5 &ml). Tissue was maintained in an atmosphere of O2 (95%) and CO, (5%). Explants were exposed to the indicated agents for 24 hr, or as otherwise indicated, once per week and the medium was stored at -20°C until assays for cyclic AMP and parathyroid hormone were performed.

England Nuclear Corporation (Boston, Mass.). The following compounds were generous gifts from the sources indicated: Measurement of Cyclic AMP (-)- and (+)-propranolol (Ayerst PharmaceuCyclic AMP was determined by a ticals, New York, N. Y .); (-)-isoproterenol competitive binding assay [ 171. This assay (Sterling-Winthrop, Rensselaer, N. Y.). detected cyclic AMP with a sensitivity of 1 pmole . Preparation of Parathyroid Glands for Organ Culture Measurement of Phosphodiesterase Normal and adenomatous human paraActivity thyroid glands were obtained from patients Chromatographically pure cyclic[3H]in the course of surgery for primary hyperparathyroidism. Another source of normal AMP (0.2 PM) was added to culture fluid parathyroid tissue was a cadaveric donor. removed from normal or adenomatous Two hundred and seven adenomatous ex- explants and incubated at 37°C for 24 hr. plants from 23 adenomas, and 26 explants Experiments were performed with and withfrom 6 normal glands were cultured. Ap- out theophylline (10 n&f) added to the culproximately half the normal tissue was ob- ture fluid. Aliquots (100 ~1) were then astained during surgery and the other half from sayed for remaining intact cyclic[3H]AMP the cadaver+ donor. The data from normal by sequential Dowex and alumina chromaglands obtained at surgery and those from tography [29]. The enzymatic breakdown the fresh cadaver were essentially in- of cyclic AMP was determined by comparing the recovery of nonincubated, intact distinguishable. Under sterile conditions, cyclic[3H]AMP (30-40%) to the recovery of the parathyroid tissue was immediately placed in cold minimal essential medium cyclic AMP after exposure to culture fluid (MEM). After removal of a representa- (g-25%).

88 Measurement

JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 1, JANUARY

of Parathyroid

Hormone

Parathyroid hormone was measured in an assay previously described [3, 5, 211. The antibody is multivalent with recognition sites for both amino and carboxy-terminal fragments of parathyroid hormone [21]. Samples were run in duplicate; the interassay variation was lo- 15%. Parathyroid hormone secretion, as detected in this radioimmunoassay, was inversely related to changes in the calcium concentration [3, 5, 211. To corroborate these data, selected samples from medium bathing normal tissue were measured independently in another, well-characterized radioimmunoassay for parathyroid hormone [20]. This assay employs an antibody that also has recognition sites for both carboxy- and amino-terminal portions of parathyroid hormone.4 Absolute values for 11 coded samples as determined independently showed remarkably close agreement to within 20% of each other. In one series of experiments, parathyroid hormone was determined with a carboxyterminal-specific antiserum, anti-PI’H 1. The binding of 1251-labeled parathyroid hormone to anti-PTH 1 is inhibited 85% by the carboxy-terminal parathyroid hormone fragment, 53-W5 Protein was determined by the method of Lowry et al. [23]. RESULTS Normal Parathyroid

Explants

Normal human parathyroid glands secreted cyclic AMP under basal conditions at a constant rate for up to 4 weeks (Fig. 2). Over this period, the explants were morphologically intact although some cellular fragmentation and central necrosis were evident by the end of the fourth week (Fig. 3). Isoproterenol significantly stimulated the secretion of cyclic AMP which mediates its physiological effects (Fig. 2). The isoproterenol-stimulated increase in cyclic 4 Gerald A. Williams, personal communication. 5 S. Krutzik, unpublished observations.

I1.T TT

WEEK

1981 NORMAL GLANDS

I

WEEK 2

WEEK 3

WEEK 4

FIG. 2. Cyclic AMP secretion by normal parathyroid

explants. Cyclic AMP secretion was measured under basal conditions (B: open bars); after exposure to isoproterenol alone, 500 m (I: diagonal bars); or with theophylline, 10 mM (I+T: stippled bars); or with isoproterenol, theophylline, and propranolol, 500 N (I+T+P; black bars). Experiments were performed at Weeks 1 through 4 of culture. The data represent the mean + SEM. Number in parentheses indicates how many explants were studied. Significance from baseline values (P < .Ol), established by Student’s f test, is indicated by asterisk. Cyclic AMP (pmole/mg/24 hr) was determined according to Materials and Methods.

AMP was augmented by theophylline, a phosphodiesterase inhibitor. Propranolol, the P-adrenergic inhibitor, completely prevented the increase in cyclic AMP secretion stimulated by isoproterenol alone or with theophylline. Data obtained in normal glands obtained from a cadaveric donor were similar to those in normal glands obtained during surgery. Adenomatous

Parathyroid

Explants

Human parathyroid adenomas also secreted cyclic AMP under basal conditions at a constant rate for up to 4 weeks (Fig. 4). Similar to the normal glands, the adenomas retained morphological integrity for most of this period (Fig. 5). The degree of cellular breakdown was qualitatively similar between normal and adenomatous explants. During the first 2 weeks, isoproterenol significantly stimulated cyclic AMP secretion, but theophylline was necessary to

FIG. 3. Morphological appearance of normal human parathyroid glands after 1 or 4 weeks in organ culture. Age of explants (weeks) is indicated. x400

90

JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 1, JANUARY 1981

demonstrate a significant isoproterenol effect during Weeks 3 and 4 (Fig. 4). The increase in cyclic AMP secretion due to isoproterenol alone or with theophylline was completely prevented by propranolol. Propranolol appeared to inhibit basal secretion of cyclic AMP during the first week of culture. The effect of the 24-hr period of culture on the ultimate detection of cyclic AMP in culture fluid was explored by comparing the rate of cyclic AMP production at 1 FIG. 4. Cyclic AMP secretion in adenomatous paraand 24 hr. The rate of basal and iso- thyroid explants. Details of the protocol and explanaproterenol-stimulated cyclic AMP produc- tion of symbols are noted in the legend to Fig. 2. tion was identical when short and long normal and adenomatous explants, the posincubation periods were compared. sibility that adenomas might secrete into the medium much more phosphodiesterase Sensitivity of Adenomas to Stimulation by activity than normal explants was conZsoproterenol and Inhibition by sidered. However, fluid bathing the Propranolol adenomatous explants did not contain sigAdenomas exposed to increasing concennificantly more phosphodiesterase activity trations of isoproterenol released progresthan fluid bathing the normal glands. It was sively greater amounts of cyclic AMP (Fig. also observed that theophylline inhibited 6A). Half-maximal stimulation occurred at over 75% of the phosphodiesterase activity approximatley 1 @f. Increasing concentrain each case. tions of propranolol led to progressive inWith respect to the apparent greater hibition of isoproterenol-induced cyclic amount of parathyroid hormone secreted AMP secretion (Fig. 6B). When the iso- per milligram of normal tissue than proterenol concentration was 50 a, half- adenomatous tissue, it seemed possible maximal inhibition of cyclic AMP occurred that the adenomas could have been assowhen the concentration of propranolol was ciated with more extensive metabolism of 0.1 PM. parathyroid hormone and, therefore, be associated with less immunoreactive paraSecretion of Parathyroid Hormone by thyroid hormone. However, parathyroid Normal and Adenomatous Glands hormone assayed using two different antiParathyroid hormone was secreted under sera, one with specificity for the carboxybasal conditions by both normal and terminal fragment (anti-PTH 1) and the adenomatous explants (Fig. 7). In Week other with dual specificity (GP 101) gave 4, basal secretion persisted but was 75% similar results. There was not more carboxyless than in Week 1 (data not shown). For terminal-specific parathyroid hormone in both normal and adenomatous explants, adenomatous explant fluid using an assay with sole specificity for the fragment. isoproterenol stimulated and propranolol inhibited parathyroid hormone secretion. DISCUSSION It can be seen further that for both paraRecent investigations concerning the thyroid hormone and cyclic AMP, normal tissue appeared to secrete approximately 10 regulation of parathyroid hormone secretimes more per milligram than adenomatous tion have drawn attention to the importance tissue (Fig. 8). To account for the differ- of P-adrenergic catecholamines [9, 14, 18, ences in cyclic AMP measured between 371. Catecholamines augment the secretion

DERENONCOURT

ET AL.:

PARATHYROID

EXPLANTS

AND CATECHOLAMINES

91

92

JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 1, JANIJARY

FIG. 6. Sensitivity of parathyroid adenomas to stimulation by isoproterenol and inhibition by propranolol. (A) Adenomatous explants were exposed to increasing concentrations of isoproterenol and cyclic AMP in the medium was determined (see Materials and Methods). (B) Adenomatous explants were exposed to isoproterenol, 50 m, and increasing concentrations of propranolol.

of parathyroid hormone via increases in cyclic AMP. The results reported here demonstrate in human parathyroid explants that the P-adrenergic catecholamine, isoproterenol, stimulates the secretion of cyclic AMP and parathyroid hormone from normal and adenomatous tissue. The degree of isoproterenol-induced cyclic AMP stimulation of normal glands far exceeded the increment in parathyroid hormone secretion caused by the p agonist. It is unlikely that the differences in the magnitude of these responses can be accounted for by the admixture of fat cells in normal parathyroid glands-which could potentially contribute cyclic AMP but not parathyroid hormone-because studies in dispersed bovine parathyroid cells, devoid of adipocytes, are consistent with the results of this investigation [7]. Furthermore, in our studies, there was a similar, greater increase in cyclic AMP than in parathyroid hormone secretion in adenomatous explants which do not contain adipocytes. This observation is general to many biological systems in which the capacity of the cell to respond to an agonist with an increase in cyclic AMP far exceeds its capacity to generate a cyclic AMP-mediated response [27]. Direct studies with human adenomas [ 121and bovine explants [l] have confirmed that cyclic AMP directly mediates the secre-

-I

1981

RMAL

ADENOMA

FIG. 7. Stimulation ofparathyroid hormone in normal and adenomatous explants by isoproterenol. Oneweek-old explants were exposed to isoproterenol alone, 500 PM (diagonal bars), or to isoproterenol and propranolol, 500 @4 (black bars). Parathyroid hormone was determined according to Materials and Methods. Basal, nonstimulated secretion (open bars). Parentheses indicate number of explants studied.

tion of parathyroid hormone. It is likely, therefore, that in human parathyroid glands, /3-adrenergic catecholamines directly stimulate parathyroid hormone through a cyclic AMP-mediated pathway. I

I

8-

- 400

m %iiC

1

_

PTH

FIG. 8. Comparison of basal cyclic AMP and parathyroid hormone secretion by normal and adenomatous explants. Basal cyclic AMP and parathyroid hormone were measured in normal (open bars) and adenomatous (black bars) explants and expressed per milligram of

DERENONCOURT

ET AL.: PARATHYROID

EXPLANTS AND CATECHOLAMINES

93

Under basal conditions, human para- adenomas may not be as autonomous as thyroid ademonas appeared to secrete less was previously thought and that neither parathyroid hormone per milligram of tis- normal nor adenomatous tissue can be sue than normal glands. The physiological completely suppressed [4, 6, 8, 10, 25, 36, significance of this observation should be 381. These results combined with those interpreted in light of the methodology used relative to the possible lower secretory rate in these experiments. For example, these of parathyroid hormone and cyclic AMP for results could be due to differences in the adenomas are consistent with the suggestion metabolism of secreted parathyroid hor- that the primary index of adenomatous mone among normal and adenomatous ex- activity is related more to the number of plants [19, 331. However, a number of functioning cells rather than to any intrinsic related observations suggest that these difference in the control of parathyroid findings do have physiological relevance. hormone secretion by calcium [4]. Finally, the long-term culture of human First, similar results obtained using antisera with limited specificity for the carboxyparathyroid tissue may be useful in the determinal fragment of parathyroid hormone velopment of techniques for successful suggests that peptide breakdown, to the ex- heterotopic parathyroid gland transplantatent resolvable by mono- and multivalent tion. Other endocrine tissues have been antisera, is not significantly different be- shown to have markedly decreased imtween normal and adenomatous explants. munogenicity after 3 weeks of organ culture Second, results of short-term incubation of [22, 34, 351. The successful long-term parathyroid tissue are consistent with culture of human parathyroid glands, as these data [4]. In those experiments, it was demonstrated in this study, suggests that shown that secreted parathyroid hormone studies along these lines are now feasible. comigrates on gel filtration analysis with immunoreactivity of native parathyroid ACKNOWLEDGMENTS hormone [4]. Third, the greater elaboration of parathyroid hormone in normal explant We are grateful to Dr. Gerald Williams for permedium was mirrored by a similarly in- forming corroborative parathyroid hormone radiocreased secretory rate of cyclic AMP in immunoassay on selected samples and to Ms. Sandra normal explants. It is unlikely that dif- Rivera for technical assistance. This work was supported by Grants HL 12738, HL 20859, AM 19652, ferences in the metabolism of cyclic AMP and Training Grant TI-AM 07271 from the National account for these differences because phos- Institutes of Health. John P. Bilezikian is the recipient phodiesterase activity released into the of Research Career Development Award 5KO4 HL medium was similar in obth normal and 00383 (National Institutes of Health). adenomatous explants. Finally, clinical studies relating the weight of human REFERENCES adenomas to the concentration of circulating parathyroid hormone have shown that 1. Abe, M., and Sherwood, L. M. Regulation of parathyroid hormone secretion by adenyl cyclase. the lo- to 20-fold increase in adenoma Biochem. Biophys. Res. Commun. 48: 396, weight over normal glandular weight is 1972. usually accompanied by a much smaller 2. AU, W. Y. W. Cortisol stimulation of parathyroid increase in circulating parathyroid hormone hormone secretion by rat parathyroid glands in [24-261. Thus, the secretory rate of paraorgan culture. Science 193: 1015, 1976. 3. Bilezikian, J. P., Canfield, R. E., Jacobs, T. P. thyroid hormone from adenomatous tissue, Polay, J. S., D’ Adamo, A. P., Eisman, J. A., and when expressed per unit of tissue weight, DeLuca, H. F. The Response of la,25Dihydroxyappears to be less than that of normal tissue. vitamin D3 to Hypocalcemia in Human Subjects. Recent observatioins comparing adenoN. Engl. J. Med. 299: 437, 1978. mas and normal glands indicate that 4. Bimbaumer, M. E., Schneider, A. B., Palmer, D.,

94

JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 1, JANUARY

Hanley, D. A., and Sherwood, L. M. Secretion of parathyroid hormone by abnormal human parathyroid glands in vitro. .I. Clin. Endocrinol. Metab. 45: 105, 1977. 5. Broadus, A. E., Horst, R. L., Lang, R., Littledike, T., and Rasmussen, H. Circulating 1,25-dihydroxyvitamin D in primary hyperparathyroidism. N. Engl. J. Med. 302: 421, 1980. 6. Brown, E. M., Brennan, M. F., Hurwitz, S., Windeck, R., Marx, S. J., Spiegel, A. M., Koehler, J. O., Gardner, D. G., and Aurbach, G. D. Dispersed cells prepared from human parathyroid glands: Distinct calcium sensitivity of adenomas vs primary hyperplasia. J. C&n. Endocrinol. Metab. 46: 267, 1978. 7. Brown, E. M., Carroll, R. J., and Aurbach, G. D. Dopaminergic stimulation of cyclic AMP accumulation and parathyroid hormone release from dispersed bovine parathyroid cells. Proc. Nat/. Acad. Sci. USA 74: 4210, 1977. 8. Brown, E. M., Hurwitz, S., and Aurbach, G. D. Preparation of viable isolated bovine parathyroid cells. Endocrinology 99: 1582, 1976. 9. Brown, E. M., Hurwitz, S., and Aurbach, G. D. Beta-adrenergic stimulation of cyclic-AMP content and parathyroid hormone release from isolated bovine parathyroid cells. Endocrinology 100: 1696, 1977. 10. Blum,.J. W., Fischer, J. A., Hunziker, W. H., Binswanger, U., Picolti, G. B., DaPrada, M., and Guillebeau, A. Parathyroid hormone responses to catecholamines and to changes of extracellular calcium in cows. J. C/in. Invest. 61: 1113, 1978. 11. Chertow, B. S., Baylink, D. J., Wergedal, J. E., Su, M. H. H., and Norman, A. W. Decrease in serum immunoreactive parathyroid hormone in rats and in parathyroid hormone secretion in vitro by 1,25-dihydroxycholecalciferol, J. Clin. Invest. 56: 668, 1975. 12. Dietel, M., Dom, G., Montz, R., and Altenahr, E. The effect of calcium and dibutryl-CAMP on the secretion of parathyroid hormone by human parathyroid adenomas in organ cultures. Acta Endocrinol. 85: 541, 1977. 13. Feinblatt, J. D., Lih-Ruey, T., and Kenny, A. Avian parathyroid glands in organ culture: Secretion of parathyroid hormone and calcitonin. Endocrinology 96: 282, 1975. 14. Fischer, J. A., Blum, J. W., and Binswanger, U. Acute parathyroid hormone response to epinephtine in vivo. J. Clin. Invest. 52: 2434, 1973. 15. Fischer, J. A., Oldham, S. B., Sizemore, G. W., and Amaud, C. D. Calcitonin stimulation of parathyroid hormone in vitro. Horm. Metab. Res. 3: 223, 1971. 16. Fugita, T. Kazutoshi, O., Orimo, H., and Yoshikawa, M. Release of immunoreactive parathyroid hormone from rat parathyroid glands in organ culture. Endocrinology 95: 30, 1974. 17. Gilman, A. G. A protein binding assay for

1981

adenosine 3’,5’ cyclic monophosphate. Proc. Nat/. Acad. Sci. USA 61: 305, 1970.

18. Hanley, D. A., Takatsuki, K., Schneider, A. B., Sherwood, L. M. Comparison of low calcium and beta-agonist stimulation of parathyroid hormone secretion. Clin. Res. 25: 522A, 1977. 19. Hanley, D. A., Takatsuki, K., and Sherwood, L. M. Evidence for release of fragments of parathyroid hormone during “perifusion” of bovine parathyroid glands in vitro. In Program, 59th Annual Meeting, Endocrine Chicago, III., 1977. P. 255A.

Society,

20. Hargis, G. K., Bowser, F. N., Henderson, W. J., and Williams, G. A. Radioimmunoassay of rat parathyroid hormone in serum and tissue extracts. Endocrinology 94: 1644, 1974. 21. Krutzik, S., and Potts, J. T., Jr. Parathyroid hormone. In A. L. Nichols and G. C. Nelson (Eds.), Radioimmunoassay Manual, 4th ed., Los Angeles: Hillside Graphics, 1977. P. 105. 22. Lafferty, K. J., Cooley, M. A., Woolnough, J., and Walker, K. Z. Thyroid allograft immunogenicity is reduced after a period in organ culture. Science 188: 259, 1975. 23. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the folin phenol reagent.J. Bio/.Chem. 193: 265, 1951. 24. Mallette, L. E., Bilezikian, J. P., Heath, D. A., and Aurbach, G. D. Primary hyperparathyroidism: Clinical and biochemical features. Medicine 53: 127, 1974. 25. Mayer, G. P. Habener, J. F., and Potts, J. T., Jr. Parathyroid hormone secretion in vivo. J. C/in. Invest. 57: 678, 1976.

26. Pumell, D. C., Smith, L. H., Scholz, D. A., Elvebach, L. R., and Amoud, C. D. Primary hyperparathyroidism: A prospective clinical study. Amer. J. Med. 50: 670, 1971. 27. Robison, G. A., Butcher, R. W., and Sutherland, E. W. Cyclic AMP. Annu. Rev. Biochem. 37: 149, 1968. 28. Rude, R. K., Oldham, S. B., and Singer, F. R. Functional hypoparathyroidism and parathyroid hormone end-organ resistance in human magnesium deficiency. C/in. Endocrinol. 5: 209, 1976. 29. Salomon, Y., Londos, C., and Rodbell, M. A highly sensitive adenylate cyclase assay. Anal. Biochem. 58: 541, 1974. 30. Sherwood, L. M., Lundberg, W. B., Targovnik, J. H., Rodman, J. S., and Seyfer, A. Synthesis and secretion of parathyroid hormone in vitro. Amer. J. Med. 50: 658, 1971. 31. Sherwood, L. M., Mayer, G. P., Ramberg, C. F., Jr., Komfeld, D. S., Aurbach, G. D., and Potts, J. T., Jr. Regulation of parathyroid hormone secretion: Proportional control by calcium, lack of effect of phosphate. Endocrinology 83: 1043, 1968. 32. Sherwood, L. M., Potts, J. T., Jr., Care, A. D., Mayer, G. P., and Aurbach, G. D. Evaluation by radioimmunoassay of factors controlling the secre-

DERENONCOURT

33.

34.

35.

36.

ET AL.: PARATHYROID

tion of parathyroid hormone. Nature (Lond.) 209: 52, 1966. Sherwood, L. M., Rodman, J. S., and Lundberg, W. B. Evidence for a precursor to circulating parathyroid hormone. Proc. Natl. Acad. Sci. USA 67: 1631, 1970. Solhnger, J. W., Buckholder, P. M., Rasmus, W. R., and Bach, F. H. Prolonged survival of xenografts after organ culture. Surgery 81: 74, 1977. Talmadge, D. W., Dart, G., Radovich, J., and Lafferty, K. J. Activation of transplant immunity: Effect of donor leukocytes in thyroid allograft rejection. Science 191: 385, 1976. Targovnik, J. H., Rodman, J. S., and Sherwood,

EXPLANTS AND CATECHOLAMINES

95

L. M. Regulation of parathyroid hormone secretion in vitro. End&rinology 88: 1477, 1971. 37. Williams, G. A., Hargis, G. K., Bowser, E. N., Henderson, W. J., and Martinez, N. J. Evidence for a role of adenosine 3’,5’-monophosphate in 92: parathyroid hormone release. Endocrinology 687, 1973. 38. Williams, G. A., Hargis, G. K., Vora, N. M., Bowser, E. N., Kukreja, S. C., Jackson, B., Kawahara, W., and Henderson, W. J. Parathyroid hormone secretion in primary hyperparathyroidism: Retained control by calcium with impaired control by beta-adrenergic system. In Program 59th Annual Meeting, Endocrine Society, Chicago, Ill., 1977. P. 233A.