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A New Role for the Parathyroid Glands in Calcium Homeostasis
Vol. 97, June
THE JOURNAL OF UROLOGY
Copyright © 1967 by The Williams & Wilkins Co.
Printed in U.S.A.
A KEW ROLE FOR THE PARATHYROID GLA::"JDS IK CALCIUM HOMEOSTASIS RUBEN F. GITTES,* SAMUEL A. WELLS
AND
GEORGE L. IRVIN, III
From the Urology Service, Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts and the Surgery Branch, National Cancer Institute, Bethesda, Maryland
An understanding of the normal physiology of calcium homeostasis is a prerequisite to the interpretation and control of the derangements of calcium metabolism commonly encountered in urology~hypercalciuria, nephrocalcinosis and renal lithiasis. In the past 4 years, the accepted concept of the hormonal mechanisms involved in calcium homeostasis has been extensively revised. The established theory accounted for the maintenance of normal calcium values by the activity of just one hormone, the classical calciumraising hormone from the parathyroid gland, or parathormone. Its secretion was postulated to increase in response to the lowering of the circulating level of ionized calcium below the narrow range of normal and, conversely, to turn itself off when that level rose above normal. The present concept invokes the presence of two antagonistic hormonal mechanisms, one to raise and another to actively lower the calcium level whenever that transgresses the limits of the normal physiological range. However, the published reports which have unveiled the existence of a calcium-lowering mechanism in calcium homeostasis have presented conflicting evidence and have arrived at opposite conclusions as to the relative roles of the thyroid and parathyroid glands in the normal response to hypercalcemia. This essay is in two parts. The first reviews the apparently conflicting reports of previous investigators and presents our new hypothesis which reconciles the conflicting observations and incorporates their evidence in a theory of parathyroid and thyroid interaction in calcium Accepted for publication September 27, 1966. Read at annual meeting of American Urological Association, Inc., Chicago, Illinois, May 30-June 2, 1966. Editor's note. Doctor Gittes' paper was awarded first prize for laboratory research in the essay contest sponsored annually by the American Urological Association, Inc. * Current address: Department of Urology, Massachusetts General Hospital, Boston, Massachusetts 02114.
homeostasis. The second part outlines the results of new experiments which have been designed to test our hypothesis and which appear to provide some further support to it. REVIEW OF PREVIOUS EXPERIMENTAL AND
POSTULATlON
OF
EXISTENCE
EVIDENCE OF
PARA-
THYROID THYROCALCITONIN-RELEASING FACTOR
The concept of a calcium-lowering hormone was introduced by Copp and associates. 1 After extensive experience with physiological perfusion techniques in studies of calcium homeostasis, they noted important observations. 1) Some extracts of para thyroid tissue had an initial hypocalcemic effect when injected in large doses intravenously into normal dogs, a drop of less than 1 mg. per 100 ml. in the plasma content of calcium which lasted 30 to 40 minutes and was rapidly superseded by the hypercalcemia expected from the classical parathormone. They were apparently able to separate some such extracts into a fraction that would only lower and another which would only raise the plasma calcium of injected dogs. 2) The perfusion of the thyroid and parathyroid glands on one side of the neck with hypercalcemic blood in vivo in dogs, ingeniously arranged so that any calcium added to the animals by the perfusate was subtracted with an equivalent drip of intravenous ethylenediaminetetracetic acid (EDTA), actually resulted in a drop in the animal's circulating calcium levels. It is to be noted that the glands on the other side of the neck were left intact. The perfusion of the thyroid gland after removal of its parathyroids did not result in a systemic calcium drop. Copp ascribed the calcium-lowering activity to a second principle in the parathyroid which he named calcitonin. Then in 1963, Hirsch and associates made a discovery which seemed to demand a role for the 1 Copp, D. H., Cameron, E. C., Cheney, B. A., Davidson, A. G. and Henze, K. G.: Evidence for calcitonin-a new hormone from the parathyroid that lowers blood calcium. Endocrinology, 70: 638, 1962.
1082
CALCIUM HOMEOSTASIS
thyroid in the calcium-lowering response.2 They noted that rats which were parathyroidectomized by cauterizing the parathyroids embedded on the surface of their thyroid had a much more rapid and greater drop in their calcium level than rats in which the parathyroids had been removed. Probing for the source of this difference, they found that the thyroid gland contained a substance which had a potent calcium-lowering activity and which apparently was released from the thyroid tissue injured by the cautery. They named this substance thyrocalcitonin. Subsequent investigations have shown thyrocalcitonin to be a very active polypeptide which is present in the thyroid tissue of every mammalian species tested, including man, and which lowers both the calcium and the phosphorus levels after injection into rats, even parathyroidectomized or nephrectomized rats. 3- 5 Other investigators soon carried out experiments in search of physiological evidence for the participation of thyrocalcitonin in a calciumlowering response. Talmage and associates found an impaired handling of injected calcium in normocalcemic rats which had been thyroidectomized but not parathyroidectomized, a procedure made possible by previous autotransplantation of the parathyroids. 6 They did find that hypocalcemic parathyroidectomized rats disposed of a large calcium injection more rapidly than hypocalcemic thyroparathyroidectomized rats, suggesting that the residual thyroid played a part in the observed response to the exogenous hypercalcemia. In 1964, two apparently conflicting reports appeared. In repeated careful experiments, Foster and associates could not obtain any calcium-lowering response from the hypercalcemic perfusion of the operatively isolated parathyroid gland in goats-a procedure made possible by 2 Hirsch, P. F., Gauthier, G. F. and Munson, P. L.: Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy in rats. Endocrinology, 73: 244, 1963. 3 Hirsch, P. F., Voelkel, E. F., Savery, A. and Munson, P. L.: Partial purification of thyrocalcitonin. Fed. Proc., 23: 204, 1964. 4 Aliapoulios, M.A. and Munson, P. L.: Thyrocalcitonin. Surg. Forum, 16: 55, 1965. 5 Tenenhouse, A., Arnaud, C. and Rasmussen, H.: The isolation and characterization of thyrocalcitonin. Proc. Nat. Acad. Sci., 53: 818, 1965. 6 Talmage, R. V., Neuenschwander, J. and Kraintz, L.: Evidence for the existence of thyrocalcitonin in the rat. Endocrinology, 76: 103. 1965.
1083
the presence in goats of superior parathyroid glands with a blood supply separate from that perfusing the thyroid and inferior parathyroid glands. 7 They did obtain a prompt and marked response when they similarly perfused a thyroid lobe along with its inferior parathyroid. Yet Copp and Henze, using sheep for the same anatomical advantage, consistently noted a definite and prompt lowering of the systemic plasma calcium levels in response to the in situ perfusion of the superior parathyroid gland alone. 8 They obtained no response from perfusion of thyroid tissue alone after removal of its parathyroid. We noted in their protocols that whenever Copp's group perfused the superior parathyroid alone, they had left the contralateral thyroid lobe and parathyroid glands intact, while Foster and associates had removed all of the animal's thyroid tissue. Thus, both investigators would be correct in their observations if we postulated that the parathyroid did respond to hypercalcemia by secreting a thyrocalcitonin-releasing factor (TCRF), without intrinsic calcium-lowering activity. This factor would have been detected only in Copp's perfusions by reaching the contralateral thyroid tissue and there releas-ing the potent calcium-lowering polypeptide, thyrocalcitonin. This hypothesis has been presented in a preliminary report. 9 When our hypothesis was first formulated, all of the evidence was consistent with the further postulate that the thyroid itself was incapable of releasing thyrocalcitonin in the absence of TCRF. Thus Copp and associates had repeatedly failed to note a response from the hypercalcemic perfusion of thyroid alone either in the dog1 or in the sheep. 8 Although Talmage and associates had indeed demonstrated that hypocalcemic thyroparathyroidectomized rats did not dispose of a calcium infusion as rapidly as parathyroidectomized rats, they did not report controlling the metabolic effect of thyroidectomy and it seemed possible that incipient myxedema might be the cause of that discrepancy. 6 7 Foster, G. V., Baghdiantz, A., Kumar, M.A., Slack, E., Soliman, H. A. and MacIntyre, I.: Thyroid origin of calcitonin. Nature, 202: 1303,
1964.
8 Copp, D. H. and Henze, K. G.: Parathyroid origin of calcitonin-evidence from perfusion of sheep glands. Endocrinology, 75: 49, 1964. 9 Gittes, R. F. and Irvin, G. L.: Thyroid and parathyroid roles in hypercalcemia: Evidence for a thyrocalcitonin-releasing factor. Science, 148:
737, 1965.
1084
GITTES, WELLS AND IRVIN
More recently, evidence has accumulated that thyroid tissue alone can make some response to an ambient hypercalcemia. Our own experiments in rats to be presented herein, have demonstrated such activity in the absence of the postulated TCRF. Care reported the hypercalcemic perfusion of thyroid tissue alone in pigs, noting a drop in the systemic circulation, but did not report the perfusion of both thyroid and parathyroid tissue for comparison. 1° Foster and associates did perfuse the thyroid gland alone in 7 dogs immediately after parathyroidectomy and found that there was a calcium-lowering response which was delayed and significantly diminished compared to the response seen in 7 dogs in which both the thyroid and its parathyroids were perfused. 11 Similarly, Copp has reported to us that using his hypercalcemic perfusion system he now can obtain some response from thyroid tissue alone but, again, it is greatly delayed and much diminished response compared to that found with the perfusion of both tissues.12 However, in these perfusions the additional trauma to the thyroid from the parathyroidectomy, a factor that was not controlled, may have played a part, rather than just the absence of TCRF.u, 12 Our present theory of the relative roles of parathyroid and thyroid tissue in calcium homeostasis postulates that in hypercalcemia the parathyroid may produce a humoral thyrocalcitonin releasing factor which acts upon the thyroid to increase the secretion of the demonstrated stores of the rapidly acting calcium-lowering hormone thyrocalcitonin. At the same time, in hypercalcemia, the parathyroid reduces the production of classical calcium-mobilizing parathormone, but this hormone's prolonged effect restricts the contribution of this mechanism to the observed stability of the normal circulating level of calcium. In hypocalcemia, secretion of classical parathormone is increased while the TCRF-thyrocalcitonin system is probably suppressed to insignificant levels. 1 ° Care, A. D.: Secretion of thyrocalcitonin. Nature, 205: 1289, 1965. 11 Foster, G. V., Kumar, A., Baghdiantz, H., Soliman, H., Slack, E., DeBats, ~. _andlVIac~nty!e, I.: Preliminary studies on the ongm of calc1tonm. In: Proceedings of the Second European Symposium on Calcified Tissues, Liege,. 19~5, p. 401. 12 Copp, D. H.: Personal commumcat10n.
EXPERIMENTAL
EVIDENCE
FOR
PARATHYROID
THYROCALCITONIN-RELEASING FACTOR
Although the apparently conflicting observations of other investigators are reconciled by our hypothesis of the presence of a parathyroid hormone which acts as a thyrocalcitonin-releasing factor and in that sense such work is evidence for its existence, we have carried out some experiments whose design promised additional and more direct evidence for the postulated factor. In their presentation here much of the detail as to the materials and methods used will be abbreviated. However, the results will be detailed and discussed in relation to the working hypothesis presented. Experiment 1. Effect of cross-transfusion experiments between parathyroid-loaded and thyroidloaded strictly inbred guinea pigs. If the parathyroid secretes TCRF in response to hypercalcemia, then the blood of an animal bearing a large number of parathyroid transplants and made hypercalcemic should contain a high concentration of the factor. Such blood transfused into another animal bearing a large number of thyroid transplants should produce a marked drop in the recipient's circulating calcium level by releasing thyrocalcitonin from the accumulated thyroid tissue. On the other hand, such blood transfused into a thyroidectomized animal should not affect the calcium level if TCRF has no intrinsic calcium-lowering activity. The design of this experiment, based on this reasoning, is illustrated (fig. 1). As donors, strictly inbred strain 13 guinea pigs were transplanted intramuscularly with 40 parathyroid glands collected from strain 13 guinea pigs sacrificed in other experiments. Such small endocrine grafts are uniformly successful and functional, 13 and cannot be differentiated from in situ glands even under the electron microscope.14 In 3 to 4 weeks following transplantation, such guinea pigs were thyroidectomized and 1 day later injected intravenously with 2.0 mg. calcium per 100 gm. body weight. One hour later their inferior vena cava, draining blood fron'l the thigh muscles containing the 13 Russell P. S. and Gittes, R. F.: Parathyroid transplants 'in rats. A comparison of their survival time with that of skin grafts. J. Exper. Med.,
109: 571, 1959. 14 Wetzel, B. K. and Gittes, R. F.: Unpublished data.
1085
Cl,LCIUM HOMEOSTASIS
DONOR 40 PTH's
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Carotid Blood Samples
FIG. 1. Diagrammatic outline of experimental design in experiment 1. Eflluent venons blood from 40
isologons para thyroid transplants in thyroidectomi½ed guinea pig was collected 1 hour after calcium injection and infused into normocalcemic recipient bearing 40 transplanted thyroid lobes, as shown on right. As control procedure, same or similar blood was iufnsecl into normocalcemic th)'roidectomi½ed guinea pig as shown on left.
transplants, was cannulated and 12 to 24 ml. blood drawn for transfusion to the prepared recipients. Two main types of recipients were used. Thymid-loaded recipients were prepared by transplanting 40 isologous thyroid lobes, each sliced into 6 pieces to insure adequate vascularization, into the thigh muscles of a strain 13 recipient. An inlying pol?ethylene catheter was placed 3 to 4 weeks later into one carotid to permit frequent sampling of the blood. After establishing the presence of a stable baseline of calciurn levels for at least 2 hours, they were infused intravenously with 9 to 12 ml. of the donor's blood, usually within 1 hour after it was drawn, and samples of their carotid blood was taken every 5 to 10 minutes for 1 hour, then again at 90 to 120 minutes after the start of the infusion, As the principal type of control transfusion, also illustrated in figure 1, totally thyroiclectomized guinea 11igs, kept normocalcemic residual parathyroid tissue in the neck or by a parathyroid transplanted in their thigh, received a similar infusion of blood from a parathyroid-loaded donor and were bled from their carotid at the same intervals. Other control donor-recipient combinations used included hypercalcemic normals to intact
normals and normocalcemic normals to loaded guinea pigs. Also tested wa~ the effect of the infusion into thyroid-loaded reeipients o[ 12 ml. normal saline with calt:ium addecl to a concentration of 12 to 13 mg. per 100 ml., the usual level in the hypercalcemic donor bloocl. Also tested, in order to contrnl the response noted in the parathyroid-loaded to thyroidloaded combination, was the effect on thyroidloaded guinea pigs of nembutal multiple bleedings and rough manipulation oI their transplant-bearing leg muscles. The serial camtid blood samples, about 1 m-1. each, and a sample of the transfused blood in each case, were assayed for total calcium content using the semi-automated micro-method of Copp.15 The effects of the parathyroid-loaded to thyroid-loaded and of the control transfusions are summarized in figure 2. The transfusion of slightly hypercakemic blood from a parathyroidloaded donor pmclucecl a prompt drop in the calcium level of the thyroid-loaded but no effect in the thyroiclectomized recipients. The maximum effect was noted at 40 to 50 mill·· 15 Copp, D. I-I.: Simple and precise rnicrornethod for EDTA titration of calcium. ,J, Lab. Cli1L Med., 61: 1029, 1963.
1086
GITTES, WELLS AND IRVIN SUMMARY OF HYPERCALCEMIC TRANSFUSIONS
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MINUTES AFTER BLOOD INFUSION
FIG. 2. Changes in plasma calcium level of transfused recipients in experiment 1, as outlined in figure 1. Values are plotted as changes from pre-infusion baseline levels. Responses of 2 groups are significantly different (p < .001).
utes, with variable recovery thereafter presumably due to a parathormone response. The responses were statistically different at a confidence level of p < .001. The various control procedures previously outlined failed to provoke the consistent calciumlowering response seen with the ideal parathyroidloaded to thyroid-loaded combination. In one of 4 calcium-saline infusions an unexplained marked drop in the recipient's calcium level occurred, a drop which seemed to start during the baseline bleedings before the infusion was begun. Histological examination of the parathyroid and thyroid transplants demonstrated their intact morphology 7 months after transplantation. There was no evidence of an immunological reaction, as expected in this isologous combination (fig. 3). The thyroid tissue removed from the parathyroid-loaded guinea pigs was examined carefully for evidence of hypertrophy or hyperplasia of whatever cell type produced thyrocalcitonin. Prominent islands of parafollicular or light cells were seen (fig. 4) which may well be the source of the calcium-lowering hormone, 16 • 17 but 16 Pearse, A. G.: The cytochemistry of the thyroid C cells and their relationship to calcitonin. Proc. Roy. Soc. Biol., 164: 478, 1966. 17 Wetzel, B. K. and Gittes, R. F.: Changes in the light (parafollicular) cells of the rat thyroid gland following parathyroidectomy. In: Proceedings of Endocrine Society, 48th Meeting, Chicago, 1966.
no quantitation of their size or number was done. It should be noted that a second distinct cell type has not been identified in the parathyroid of these animals. Experiment 2. E.fiect of the injection of a crude extract of guinea pig parathyroid glands on the calcium level of thyroid-loaded guinea pigs. About 300 parathyroid glands were harvested from 150 freshly killed strain 13 guinea pigs used in other experiments and stored frozen at -120C. They were homogenized at OC into 6 ml. of 0.15 M phosphate buffer with a pH of 7.2. The hmnogenate was centrifuged at 2000 revolutions per minute for 2 hours at 5C and the supernatant was used as the extract for injection. Two thyroid-loaded guinea pigs, transplanted with 40 thyroid lobes as described in experiment 1, and 1 guinea pig thyroidectomized 8 hours earlier were each infused intravenously with 2 ml. of the extract after a prolonged baseline of stable calcium values obtained hourly for at least 4 hours was obtained on each one. The plasma calcium level was monitored as before by taking several samples from an inlying polyethylene carotid catheter. The animals remained awake in the confines of a small cage throughout the procedure. The serial calcium values on the 3 infused animals are shown in figure 5. It is evident that the thyroid-loaded recipients responded to the small dose of isologous crude extract with a profound and prolonged drop in their circulating calcium level, from which they recovered by the next day. The thyroidectomized recipient did not have a calcium-lowering response. Because of the limitation on the available amount of extract, statistical analysis of the data was not indicated. Experiment 3. E.fiect of thyroidectomy alone on the response to hypercalcemia in rats. An experiment was carried out to determine whether thyroidectomy alone, without the development of classical hypothyroidism, would impair the response to hypercalcemia. Female rats, 10 to 12 weeks old, all previously parathyroidectomized at age 6 weeks and maintained on a normal diet, underwent either a total surgical thyroidectomy or a sham thyroidectomy and 12 to 18 hours later were tested with a dose of calcium. For this test they were bled by cardiac puncture, immediately injected intraperitoneally with a solution of calcium chloride, 2.5 mg. calcium per milliliter of normal saline, for a total dose of 5.0 mg. of calcium per 100 gm. of body
CALCIUM HOMEOSTASIS
1087
FIG. 3. A, histological appearance of strain 13 guinea pig parathyroid gland 2 months after intramuscular transplantation. Gland's structure and cellular morphology are indistinguishable that of in situ gland. Large artery and small vein are demonstrated connecting transplant to host mus cle. H & E, 54 X. B, isologous thyroid transplant 2 months after intramuscular transplantation in strain 13 guinea pig. Subdivision of each thyroid lobe into 6 slices for transplantation prevented development of central ischemic necrosis in transplants. H & E, 40X.
weight. Exactly 1 hour later each rat was lightly anesthetized and bled in the same manner. The response to an intraperitoneal injection of calcium in rats either thyroidecto1nized or shamoperated several weeks after parathyroidectomy is illustrated in the table. The data show that in the absence of any source of circulating TCRF, removal of the thyroid permitted a hypercalcemia
which was statistically significant whether corn· pared in terms of the calcium level attained at 60 minutes (p < .001) or in terms of the increase in each rat's level above the pre-injection hypo. calcemic baseline values (p < .0025). Since the thyroidecto1ny was carried out hours these rats could be presumed to be euthyroid in the classical sense.
1088
GITTES, WELLS AND IRVIN
Fm. 4. Cluster of parafollicular or light cells from thyroid of guinea pig transplanted with 40 parathyroids. This second cell type in thyroid may be source of thyrocalcitonin. Such clusters were also found in normal guinea pigs. H & E, 500X. INFUSION OF ISOLOGOUS GUINEA PIG PARATHYROID HOMOGENATE
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Fm. 5. Response of plasma calcium level in each of 3 guinea pigs (2 thyroid-loaded and 1 thyroidectomized) to infusion of 2 ml. crude neutral extract prepared from 100 guinea pig parathyroid glands as described in experiment 2. DISCUSSION
The broad interpretation of our experimental evidence has been anticipated in part 1 of this essay. Some of the specific findings outlined deserve further comment.
The transfusion experiments between parathyroid-loaded and thyroid-loaded guinea pigs would seem to be a direct demonstration of the presence in the circulation of the donors of a substance which had no intrinsic calcium-lowering
108D
CALCIUM HOMEOSTASIS
Comparison of hypercalcemia induced by intraperiloneal injection of calcium vs. thyroparalhyroidectomized rats* Group
Parathyroidectomized Thyroidectomized and parathyroidectomized
18 20
parathyroidectomized
Calcium Content of Plasma (mg./100 ml.)t
No.
Rats
in
Before Injection
60 :Mins After Injection
Increase
± .20 7.89 ± .18
11.51 ± .75
3.98 ± .63
14.08 ± .34 p < .001
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7 54
p
<
± .30 .0025
--------------------------------------------------
* All rats were parathyroidectomized 4 weeks earlier and then either surgically thyroidecl.omized sham-operated 16 hours before injection of 5 mg. calcium per 100 gm. body weight. t All values expressed are mean ± standard error of mean.
acfo·ity in thyroidectomized test animals, but did trigger a hypocalcemic response in the already normocalcemic thyroid-loaded test aninials. The possibility that the response seen might be simply a response of the recipient's thyroid to the hypercalcemic transfusion rather than to a TCRF had to be considered. However, the 9 to 12 ml. trans-· fused, with a total calc:ium level of 12 to 13 mg. per 100 ml., would only raise the systen1ic calcium level by a maximum of 0.5 mg. per 100 ml. in the recipients if contained in the vascular compartment and by less than 0.2 mg. when equilibrated with the extracellular fluid. Furthermore, 3 infusions of thyroid-loaded guinea pigs with calcium in saline gave no response; and 7 calcium tolerance tests in thyroid-loaded guinea pigs using a high dose of 2 mg. per 100 gm. body weight intravenously, failed to show any h:niocalcemic overshoot following the elimination of the calcium loacl.18 Finally, the parathyroid homogenate in experiment 2 clearly was not a hypercalcemic stimulus to the thyroid-loaded recipients and yet the expected response occurred. The findings of Copp previously outlined,1· 8 demonstrating a progressive calcium-lowering effect in animals without a systemic hypercalcemia, and our own experiments demonstrating the effect of a parathyroid homogenate in normocalcemic guinea pigs, point to the presence of a thyrocalcitonin-releasing rather than a thyrocalcitonin-enhancing factor. A direct measurement of the thyrocalcitonin content of the thyroid 18 Gittes, R. F. and Wells, S. A.: Unpublished data.
OJ'
gland in hypercalcemia with or without the para thyroids present appears to be a feasible experiment and should provide further eYiclence on the interaction of the 2 glands. Since the thyroid gland is the actual origin of the hormone v,ith intrinsic calcium-lmrnring activity, thyrocalcitonin is nmY established. \vl1ilc we have shown that the parathyroid may play a role in hypercalcemia which is mediated the thyroid, our rat experiments also demonstrate that the thyroid alone can engineer some response in the absence of any parathyroid tissue or TCRF in the circulation. This finding is consonanl with the finding of a decreased but real response from the perfusion of thyroid tissue alone. 10 - 1" Also it is apparent that Talmage and associates had drn.1n1 a valid conclusion about the ability of the alone to accelerate the disposal of a calrium infusion even though their rats mar ha,·e demonstrated the additive effects of lack of th)TOcalcitonin and incipient classical hypothyroidism. 6 A more positive chemical identification of the postulated parathyroid TCRF, best approached through the fractionation of active parathyroid. homogenates, and more direct eYidence of its action on the thyroid gland will be required before its role can be accepted. For the present, our hypothesis and experimental evidence have permitted a. unified concept on the mechanism of the calcium-lowering response in calcium homeostasis and hence reconciled the findings of established investigators which appeared to be in direct conflict.
THE JOURNAL OF UROLOGY
Copyright © 1967 by The Williams & Wilkins Co.
Printed in U.S.A.
A KEW ROLE FOR THE PARATHYROID GLA::"JDS IK CALCIUM HOMEOSTASIS RUBEN F. GITTES,* SAMUEL A. WELLS
AND
GEORGE L. IRVIN, III
From the Urology Service, Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts and the Surgery Branch, National Cancer Institute, Bethesda, Maryland
An understanding of the normal physiology of calcium homeostasis is a prerequisite to the interpretation and control of the derangements of calcium metabolism commonly encountered in urology~hypercalciuria, nephrocalcinosis and renal lithiasis. In the past 4 years, the accepted concept of the hormonal mechanisms involved in calcium homeostasis has been extensively revised. The established theory accounted for the maintenance of normal calcium values by the activity of just one hormone, the classical calciumraising hormone from the parathyroid gland, or parathormone. Its secretion was postulated to increase in response to the lowering of the circulating level of ionized calcium below the narrow range of normal and, conversely, to turn itself off when that level rose above normal. The present concept invokes the presence of two antagonistic hormonal mechanisms, one to raise and another to actively lower the calcium level whenever that transgresses the limits of the normal physiological range. However, the published reports which have unveiled the existence of a calcium-lowering mechanism in calcium homeostasis have presented conflicting evidence and have arrived at opposite conclusions as to the relative roles of the thyroid and parathyroid glands in the normal response to hypercalcemia. This essay is in two parts. The first reviews the apparently conflicting reports of previous investigators and presents our new hypothesis which reconciles the conflicting observations and incorporates their evidence in a theory of parathyroid and thyroid interaction in calcium Accepted for publication September 27, 1966. Read at annual meeting of American Urological Association, Inc., Chicago, Illinois, May 30-June 2, 1966. Editor's note. Doctor Gittes' paper was awarded first prize for laboratory research in the essay contest sponsored annually by the American Urological Association, Inc. * Current address: Department of Urology, Massachusetts General Hospital, Boston, Massachusetts 02114.
homeostasis. The second part outlines the results of new experiments which have been designed to test our hypothesis and which appear to provide some further support to it. REVIEW OF PREVIOUS EXPERIMENTAL AND
POSTULATlON
OF
EXISTENCE
EVIDENCE OF
PARA-
THYROID THYROCALCITONIN-RELEASING FACTOR
The concept of a calcium-lowering hormone was introduced by Copp and associates. 1 After extensive experience with physiological perfusion techniques in studies of calcium homeostasis, they noted important observations. 1) Some extracts of para thyroid tissue had an initial hypocalcemic effect when injected in large doses intravenously into normal dogs, a drop of less than 1 mg. per 100 ml. in the plasma content of calcium which lasted 30 to 40 minutes and was rapidly superseded by the hypercalcemia expected from the classical parathormone. They were apparently able to separate some such extracts into a fraction that would only lower and another which would only raise the plasma calcium of injected dogs. 2) The perfusion of the thyroid and parathyroid glands on one side of the neck with hypercalcemic blood in vivo in dogs, ingeniously arranged so that any calcium added to the animals by the perfusate was subtracted with an equivalent drip of intravenous ethylenediaminetetracetic acid (EDTA), actually resulted in a drop in the animal's circulating calcium levels. It is to be noted that the glands on the other side of the neck were left intact. The perfusion of the thyroid gland after removal of its parathyroids did not result in a systemic calcium drop. Copp ascribed the calcium-lowering activity to a second principle in the parathyroid which he named calcitonin. Then in 1963, Hirsch and associates made a discovery which seemed to demand a role for the 1 Copp, D. H., Cameron, E. C., Cheney, B. A., Davidson, A. G. and Henze, K. G.: Evidence for calcitonin-a new hormone from the parathyroid that lowers blood calcium. Endocrinology, 70: 638, 1962.
1082
CALCIUM HOMEOSTASIS
thyroid in the calcium-lowering response.2 They noted that rats which were parathyroidectomized by cauterizing the parathyroids embedded on the surface of their thyroid had a much more rapid and greater drop in their calcium level than rats in which the parathyroids had been removed. Probing for the source of this difference, they found that the thyroid gland contained a substance which had a potent calcium-lowering activity and which apparently was released from the thyroid tissue injured by the cautery. They named this substance thyrocalcitonin. Subsequent investigations have shown thyrocalcitonin to be a very active polypeptide which is present in the thyroid tissue of every mammalian species tested, including man, and which lowers both the calcium and the phosphorus levels after injection into rats, even parathyroidectomized or nephrectomized rats. 3- 5 Other investigators soon carried out experiments in search of physiological evidence for the participation of thyrocalcitonin in a calciumlowering response. Talmage and associates found an impaired handling of injected calcium in normocalcemic rats which had been thyroidectomized but not parathyroidectomized, a procedure made possible by previous autotransplantation of the parathyroids. 6 They did find that hypocalcemic parathyroidectomized rats disposed of a large calcium injection more rapidly than hypocalcemic thyroparathyroidectomized rats, suggesting that the residual thyroid played a part in the observed response to the exogenous hypercalcemia. In 1964, two apparently conflicting reports appeared. In repeated careful experiments, Foster and associates could not obtain any calcium-lowering response from the hypercalcemic perfusion of the operatively isolated parathyroid gland in goats-a procedure made possible by 2 Hirsch, P. F., Gauthier, G. F. and Munson, P. L.: Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy in rats. Endocrinology, 73: 244, 1963. 3 Hirsch, P. F., Voelkel, E. F., Savery, A. and Munson, P. L.: Partial purification of thyrocalcitonin. Fed. Proc., 23: 204, 1964. 4 Aliapoulios, M.A. and Munson, P. L.: Thyrocalcitonin. Surg. Forum, 16: 55, 1965. 5 Tenenhouse, A., Arnaud, C. and Rasmussen, H.: The isolation and characterization of thyrocalcitonin. Proc. Nat. Acad. Sci., 53: 818, 1965. 6 Talmage, R. V., Neuenschwander, J. and Kraintz, L.: Evidence for the existence of thyrocalcitonin in the rat. Endocrinology, 76: 103. 1965.
1083
the presence in goats of superior parathyroid glands with a blood supply separate from that perfusing the thyroid and inferior parathyroid glands. 7 They did obtain a prompt and marked response when they similarly perfused a thyroid lobe along with its inferior parathyroid. Yet Copp and Henze, using sheep for the same anatomical advantage, consistently noted a definite and prompt lowering of the systemic plasma calcium levels in response to the in situ perfusion of the superior parathyroid gland alone. 8 They obtained no response from perfusion of thyroid tissue alone after removal of its parathyroid. We noted in their protocols that whenever Copp's group perfused the superior parathyroid alone, they had left the contralateral thyroid lobe and parathyroid glands intact, while Foster and associates had removed all of the animal's thyroid tissue. Thus, both investigators would be correct in their observations if we postulated that the parathyroid did respond to hypercalcemia by secreting a thyrocalcitonin-releasing factor (TCRF), without intrinsic calcium-lowering activity. This factor would have been detected only in Copp's perfusions by reaching the contralateral thyroid tissue and there releas-ing the potent calcium-lowering polypeptide, thyrocalcitonin. This hypothesis has been presented in a preliminary report. 9 When our hypothesis was first formulated, all of the evidence was consistent with the further postulate that the thyroid itself was incapable of releasing thyrocalcitonin in the absence of TCRF. Thus Copp and associates had repeatedly failed to note a response from the hypercalcemic perfusion of thyroid alone either in the dog1 or in the sheep. 8 Although Talmage and associates had indeed demonstrated that hypocalcemic thyroparathyroidectomized rats did not dispose of a calcium infusion as rapidly as parathyroidectomized rats, they did not report controlling the metabolic effect of thyroidectomy and it seemed possible that incipient myxedema might be the cause of that discrepancy. 6 7 Foster, G. V., Baghdiantz, A., Kumar, M.A., Slack, E., Soliman, H. A. and MacIntyre, I.: Thyroid origin of calcitonin. Nature, 202: 1303,
1964.
8 Copp, D. H. and Henze, K. G.: Parathyroid origin of calcitonin-evidence from perfusion of sheep glands. Endocrinology, 75: 49, 1964. 9 Gittes, R. F. and Irvin, G. L.: Thyroid and parathyroid roles in hypercalcemia: Evidence for a thyrocalcitonin-releasing factor. Science, 148:
737, 1965.
1084
GITTES, WELLS AND IRVIN
More recently, evidence has accumulated that thyroid tissue alone can make some response to an ambient hypercalcemia. Our own experiments in rats to be presented herein, have demonstrated such activity in the absence of the postulated TCRF. Care reported the hypercalcemic perfusion of thyroid tissue alone in pigs, noting a drop in the systemic circulation, but did not report the perfusion of both thyroid and parathyroid tissue for comparison. 1° Foster and associates did perfuse the thyroid gland alone in 7 dogs immediately after parathyroidectomy and found that there was a calcium-lowering response which was delayed and significantly diminished compared to the response seen in 7 dogs in which both the thyroid and its parathyroids were perfused. 11 Similarly, Copp has reported to us that using his hypercalcemic perfusion system he now can obtain some response from thyroid tissue alone but, again, it is greatly delayed and much diminished response compared to that found with the perfusion of both tissues.12 However, in these perfusions the additional trauma to the thyroid from the parathyroidectomy, a factor that was not controlled, may have played a part, rather than just the absence of TCRF.u, 12 Our present theory of the relative roles of parathyroid and thyroid tissue in calcium homeostasis postulates that in hypercalcemia the parathyroid may produce a humoral thyrocalcitonin releasing factor which acts upon the thyroid to increase the secretion of the demonstrated stores of the rapidly acting calcium-lowering hormone thyrocalcitonin. At the same time, in hypercalcemia, the parathyroid reduces the production of classical calcium-mobilizing parathormone, but this hormone's prolonged effect restricts the contribution of this mechanism to the observed stability of the normal circulating level of calcium. In hypocalcemia, secretion of classical parathormone is increased while the TCRF-thyrocalcitonin system is probably suppressed to insignificant levels. 1 ° Care, A. D.: Secretion of thyrocalcitonin. Nature, 205: 1289, 1965. 11 Foster, G. V., Kumar, A., Baghdiantz, H., Soliman, H., Slack, E., DeBats, ~. _andlVIac~nty!e, I.: Preliminary studies on the ongm of calc1tonm. In: Proceedings of the Second European Symposium on Calcified Tissues, Liege,. 19~5, p. 401. 12 Copp, D. H.: Personal commumcat10n.
EXPERIMENTAL
EVIDENCE
FOR
PARATHYROID
THYROCALCITONIN-RELEASING FACTOR
Although the apparently conflicting observations of other investigators are reconciled by our hypothesis of the presence of a parathyroid hormone which acts as a thyrocalcitonin-releasing factor and in that sense such work is evidence for its existence, we have carried out some experiments whose design promised additional and more direct evidence for the postulated factor. In their presentation here much of the detail as to the materials and methods used will be abbreviated. However, the results will be detailed and discussed in relation to the working hypothesis presented. Experiment 1. Effect of cross-transfusion experiments between parathyroid-loaded and thyroidloaded strictly inbred guinea pigs. If the parathyroid secretes TCRF in response to hypercalcemia, then the blood of an animal bearing a large number of parathyroid transplants and made hypercalcemic should contain a high concentration of the factor. Such blood transfused into another animal bearing a large number of thyroid transplants should produce a marked drop in the recipient's circulating calcium level by releasing thyrocalcitonin from the accumulated thyroid tissue. On the other hand, such blood transfused into a thyroidectomized animal should not affect the calcium level if TCRF has no intrinsic calcium-lowering activity. The design of this experiment, based on this reasoning, is illustrated (fig. 1). As donors, strictly inbred strain 13 guinea pigs were transplanted intramuscularly with 40 parathyroid glands collected from strain 13 guinea pigs sacrificed in other experiments. Such small endocrine grafts are uniformly successful and functional, 13 and cannot be differentiated from in situ glands even under the electron microscope.14 In 3 to 4 weeks following transplantation, such guinea pigs were thyroidectomized and 1 day later injected intravenously with 2.0 mg. calcium per 100 gm. body weight. One hour later their inferior vena cava, draining blood fron'l the thigh muscles containing the 13 Russell P. S. and Gittes, R. F.: Parathyroid transplants 'in rats. A comparison of their survival time with that of skin grafts. J. Exper. Med.,
109: 571, 1959. 14 Wetzel, B. K. and Gittes, R. F.: Unpublished data.
1085
Cl,LCIUM HOMEOSTASIS
DONOR 40 PTH's
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Calcium
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2.0mg/100g
TO
JUGULAR
IV
\
RECIPIENT
RECIPIENT
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---~rH
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Carotid Blood Samples
FIG. 1. Diagrammatic outline of experimental design in experiment 1. Eflluent venons blood from 40
isologons para thyroid transplants in thyroidectomi½ed guinea pig was collected 1 hour after calcium injection and infused into normocalcemic recipient bearing 40 transplanted thyroid lobes, as shown on right. As control procedure, same or similar blood was iufnsecl into normocalcemic th)'roidectomi½ed guinea pig as shown on left.
transplants, was cannulated and 12 to 24 ml. blood drawn for transfusion to the prepared recipients. Two main types of recipients were used. Thymid-loaded recipients were prepared by transplanting 40 isologous thyroid lobes, each sliced into 6 pieces to insure adequate vascularization, into the thigh muscles of a strain 13 recipient. An inlying pol?ethylene catheter was placed 3 to 4 weeks later into one carotid to permit frequent sampling of the blood. After establishing the presence of a stable baseline of calciurn levels for at least 2 hours, they were infused intravenously with 9 to 12 ml. of the donor's blood, usually within 1 hour after it was drawn, and samples of their carotid blood was taken every 5 to 10 minutes for 1 hour, then again at 90 to 120 minutes after the start of the infusion, As the principal type of control transfusion, also illustrated in figure 1, totally thyroiclectomized guinea 11igs, kept normocalcemic residual parathyroid tissue in the neck or by a parathyroid transplanted in their thigh, received a similar infusion of blood from a parathyroid-loaded donor and were bled from their carotid at the same intervals. Other control donor-recipient combinations used included hypercalcemic normals to intact
normals and normocalcemic normals to loaded guinea pigs. Also tested wa~ the effect of the infusion into thyroid-loaded reeipients o[ 12 ml. normal saline with calt:ium addecl to a concentration of 12 to 13 mg. per 100 ml., the usual level in the hypercalcemic donor bloocl. Also tested, in order to contrnl the response noted in the parathyroid-loaded to thyroidloaded combination, was the effect on thyroidloaded guinea pigs of nembutal multiple bleedings and rough manipulation oI their transplant-bearing leg muscles. The serial camtid blood samples, about 1 m-1. each, and a sample of the transfused blood in each case, were assayed for total calcium content using the semi-automated micro-method of Copp.15 The effects of the parathyroid-loaded to thyroid-loaded and of the control transfusions are summarized in figure 2. The transfusion of slightly hypercakemic blood from a parathyroidloaded donor pmclucecl a prompt drop in the calcium level of the thyroid-loaded but no effect in the thyroiclectomized recipients. The maximum effect was noted at 40 to 50 mill·· 15 Copp, D. I-I.: Simple and precise rnicrornethod for EDTA titration of calcium. ,J, Lab. Cli1L Med., 61: 1029, 1963.
1086
GITTES, WELLS AND IRVIN SUMMARY OF HYPERCALCEMIC TRANSFUSIONS
+1.00
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thyroid looded (6)
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MINUTES AFTER BLOOD INFUSION
FIG. 2. Changes in plasma calcium level of transfused recipients in experiment 1, as outlined in figure 1. Values are plotted as changes from pre-infusion baseline levels. Responses of 2 groups are significantly different (p < .001).
utes, with variable recovery thereafter presumably due to a parathormone response. The responses were statistically different at a confidence level of p < .001. The various control procedures previously outlined failed to provoke the consistent calciumlowering response seen with the ideal parathyroidloaded to thyroid-loaded combination. In one of 4 calcium-saline infusions an unexplained marked drop in the recipient's calcium level occurred, a drop which seemed to start during the baseline bleedings before the infusion was begun. Histological examination of the parathyroid and thyroid transplants demonstrated their intact morphology 7 months after transplantation. There was no evidence of an immunological reaction, as expected in this isologous combination (fig. 3). The thyroid tissue removed from the parathyroid-loaded guinea pigs was examined carefully for evidence of hypertrophy or hyperplasia of whatever cell type produced thyrocalcitonin. Prominent islands of parafollicular or light cells were seen (fig. 4) which may well be the source of the calcium-lowering hormone, 16 • 17 but 16 Pearse, A. G.: The cytochemistry of the thyroid C cells and their relationship to calcitonin. Proc. Roy. Soc. Biol., 164: 478, 1966. 17 Wetzel, B. K. and Gittes, R. F.: Changes in the light (parafollicular) cells of the rat thyroid gland following parathyroidectomy. In: Proceedings of Endocrine Society, 48th Meeting, Chicago, 1966.
no quantitation of their size or number was done. It should be noted that a second distinct cell type has not been identified in the parathyroid of these animals. Experiment 2. E.fiect of the injection of a crude extract of guinea pig parathyroid glands on the calcium level of thyroid-loaded guinea pigs. About 300 parathyroid glands were harvested from 150 freshly killed strain 13 guinea pigs used in other experiments and stored frozen at -120C. They were homogenized at OC into 6 ml. of 0.15 M phosphate buffer with a pH of 7.2. The hmnogenate was centrifuged at 2000 revolutions per minute for 2 hours at 5C and the supernatant was used as the extract for injection. Two thyroid-loaded guinea pigs, transplanted with 40 thyroid lobes as described in experiment 1, and 1 guinea pig thyroidectomized 8 hours earlier were each infused intravenously with 2 ml. of the extract after a prolonged baseline of stable calcium values obtained hourly for at least 4 hours was obtained on each one. The plasma calcium level was monitored as before by taking several samples from an inlying polyethylene carotid catheter. The animals remained awake in the confines of a small cage throughout the procedure. The serial calcium values on the 3 infused animals are shown in figure 5. It is evident that the thyroid-loaded recipients responded to the small dose of isologous crude extract with a profound and prolonged drop in their circulating calcium level, from which they recovered by the next day. The thyroidectomized recipient did not have a calcium-lowering response. Because of the limitation on the available amount of extract, statistical analysis of the data was not indicated. Experiment 3. E.fiect of thyroidectomy alone on the response to hypercalcemia in rats. An experiment was carried out to determine whether thyroidectomy alone, without the development of classical hypothyroidism, would impair the response to hypercalcemia. Female rats, 10 to 12 weeks old, all previously parathyroidectomized at age 6 weeks and maintained on a normal diet, underwent either a total surgical thyroidectomy or a sham thyroidectomy and 12 to 18 hours later were tested with a dose of calcium. For this test they were bled by cardiac puncture, immediately injected intraperitoneally with a solution of calcium chloride, 2.5 mg. calcium per milliliter of normal saline, for a total dose of 5.0 mg. of calcium per 100 gm. of body
CALCIUM HOMEOSTASIS
1087
FIG. 3. A, histological appearance of strain 13 guinea pig parathyroid gland 2 months after intramuscular transplantation. Gland's structure and cellular morphology are indistinguishable that of in situ gland. Large artery and small vein are demonstrated connecting transplant to host mus cle. H & E, 54 X. B, isologous thyroid transplant 2 months after intramuscular transplantation in strain 13 guinea pig. Subdivision of each thyroid lobe into 6 slices for transplantation prevented development of central ischemic necrosis in transplants. H & E, 40X.
weight. Exactly 1 hour later each rat was lightly anesthetized and bled in the same manner. The response to an intraperitoneal injection of calcium in rats either thyroidecto1nized or shamoperated several weeks after parathyroidectomy is illustrated in the table. The data show that in the absence of any source of circulating TCRF, removal of the thyroid permitted a hypercalcemia
which was statistically significant whether corn· pared in terms of the calcium level attained at 60 minutes (p < .001) or in terms of the increase in each rat's level above the pre-injection hypo. calcemic baseline values (p < .0025). Since the thyroidecto1ny was carried out hours these rats could be presumed to be euthyroid in the classical sense.
1088
GITTES, WELLS AND IRVIN
Fm. 4. Cluster of parafollicular or light cells from thyroid of guinea pig transplanted with 40 parathyroids. This second cell type in thyroid may be source of thyrocalcitonin. Such clusters were also found in normal guinea pigs. H & E, 500X. INFUSION OF ISOLOGOUS GUINEA PIG PARATHYROID HOMOGENATE
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Fm. 5. Response of plasma calcium level in each of 3 guinea pigs (2 thyroid-loaded and 1 thyroidectomized) to infusion of 2 ml. crude neutral extract prepared from 100 guinea pig parathyroid glands as described in experiment 2. DISCUSSION
The broad interpretation of our experimental evidence has been anticipated in part 1 of this essay. Some of the specific findings outlined deserve further comment.
The transfusion experiments between parathyroid-loaded and thyroid-loaded guinea pigs would seem to be a direct demonstration of the presence in the circulation of the donors of a substance which had no intrinsic calcium-lowering
108D
CALCIUM HOMEOSTASIS
Comparison of hypercalcemia induced by intraperiloneal injection of calcium vs. thyroparalhyroidectomized rats* Group
Parathyroidectomized Thyroidectomized and parathyroidectomized
18 20
parathyroidectomized
Calcium Content of Plasma (mg./100 ml.)t
No.
Rats
in
Before Injection
60 :Mins After Injection
Increase
± .20 7.89 ± .18
11.51 ± .75
3.98 ± .63
14.08 ± .34 p < .001
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7 54
p
<
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--------------------------------------------------
* All rats were parathyroidectomized 4 weeks earlier and then either surgically thyroidecl.omized sham-operated 16 hours before injection of 5 mg. calcium per 100 gm. body weight. t All values expressed are mean ± standard error of mean.
acfo·ity in thyroidectomized test animals, but did trigger a hypocalcemic response in the already normocalcemic thyroid-loaded test aninials. The possibility that the response seen might be simply a response of the recipient's thyroid to the hypercalcemic transfusion rather than to a TCRF had to be considered. However, the 9 to 12 ml. trans-· fused, with a total calc:ium level of 12 to 13 mg. per 100 ml., would only raise the systen1ic calcium level by a maximum of 0.5 mg. per 100 ml. in the recipients if contained in the vascular compartment and by less than 0.2 mg. when equilibrated with the extracellular fluid. Furthermore, 3 infusions of thyroid-loaded guinea pigs with calcium in saline gave no response; and 7 calcium tolerance tests in thyroid-loaded guinea pigs using a high dose of 2 mg. per 100 gm. body weight intravenously, failed to show any h:niocalcemic overshoot following the elimination of the calcium loacl.18 Finally, the parathyroid homogenate in experiment 2 clearly was not a hypercalcemic stimulus to the thyroid-loaded recipients and yet the expected response occurred. The findings of Copp previously outlined,1· 8 demonstrating a progressive calcium-lowering effect in animals without a systemic hypercalcemia, and our own experiments demonstrating the effect of a parathyroid homogenate in normocalcemic guinea pigs, point to the presence of a thyrocalcitonin-releasing rather than a thyrocalcitonin-enhancing factor. A direct measurement of the thyrocalcitonin content of the thyroid 18 Gittes, R. F. and Wells, S. A.: Unpublished data.
OJ'
gland in hypercalcemia with or without the para thyroids present appears to be a feasible experiment and should provide further eYiclence on the interaction of the 2 glands. Since the thyroid gland is the actual origin of the hormone v,ith intrinsic calcium-lmrnring activity, thyrocalcitonin is nmY established. \vl1ilc we have shown that the parathyroid may play a role in hypercalcemia which is mediated the thyroid, our rat experiments also demonstrate that the thyroid alone can engineer some response in the absence of any parathyroid tissue or TCRF in the circulation. This finding is consonanl with the finding of a decreased but real response from the perfusion of thyroid tissue alone. 10 - 1" Also it is apparent that Talmage and associates had drn.1n1 a valid conclusion about the ability of the alone to accelerate the disposal of a calrium infusion even though their rats mar ha,·e demonstrated the additive effects of lack of th)TOcalcitonin and incipient classical hypothyroidism. 6 A more positive chemical identification of the postulated parathyroid TCRF, best approached through the fractionation of active parathyroid. homogenates, and more direct eYidence of its action on the thyroid gland will be required before its role can be accepted. For the present, our hypothesis and experimental evidence have permitted a. unified concept on the mechanism of the calcium-lowering response in calcium homeostasis and hence reconciled the findings of established investigators which appeared to be in direct conflict.