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Role of thyrocalcitonin in the regulation of the blood calcium level
RIOCHEMICAL
MEDICINE
Role
1,
of
261-279 (1967)
Thyrocalcitonin of the
F. BRONNER, Department
Blood
P. J. SAMMON,? of Physiology Scl~ool
of Medicine,
and
in the Calcium
Regulation
Level1
R. E. STACEY,” Biophysics, Louisville.
Received November
UniL~ersity
Kentucky
AKD
B. G. PHA%H’
of Lotrist~ill~~
$I202
14, 1967
Calcium homeostasis in the young growing rat has been considered as effected by a regulator-type system, with a feedback loop involving a proportional control in thyroparathyroidectomized animals and a combination of proportional and integral controls in the normal intact animal (1). At the time the model was formulated, parathyroid hormone alone WZIS considered responsible for the integral control, as the mode of action of thyrocalcitonin (2,3), a hypocalcemic polypeptidc (4-7) was unknown, although the existence of a hypocalcemic factor of parathyroid origin had been postulated (8). Current evidence indicates that thyrocalcitonin acts in a direction opposite to that of parathyroid hormone, i.e., it inhibits bone resorption (9-12). This paper is concerned with an attempt to take into account the existence and mode of action of thyrocalcitonin. In the t,ypc of system postulated (Fig. I) the blood plasma is the controlled system, the blood calcium level, [Ca,], is the controlled signal, and the level around which regulation takes place is the reference value [U] . The net amount of calcium that enters the system is the disturbing signal Si, eyual to net calcium absorption from the gut. Two controlling systems have been considered, the skeleton and the kidney. Of these, the skeleton is by far the principal regulator, as the intensity of the renal signal (i.e., urinary calcium excretion, zj,,) in normal (13, 14) and in TPTX (15, 16) rats is so faint in relation to the disturbing signal as to be negligible. The net controlling signal of the skeleton is the calcium balance A, made up of two constituent signals acting in opposite directions, calcium deposition in bone zj,,+, and calcium removal from bone vn-. ‘Supported by USPHS grant AM-07983, by Training grant 1 TI DE 160 and by :I grant from the Kentucky Chapter, The Arthritis Foundation. ’ Predoctoral Trainee (USPHS Training grant 1 Tl DE 160). ,IPostdoctoral Trninec (USPHS Training grant 1 Tl DE 160). ’ Present address: Nutrition Division, Food and Drug Research Laboratory, Tunney’s Pasture, Ottama 3, Canada. 261
262
BRONNER
ET
AL.
In a system with proportional control, an error exists in the controlled system; this error was defined as the difference between the actual [Ca,] and the reference [U] values of the controlled signal, i.e., t.he plasma calcium concentration. At steady state, the error is directly proportional to the intensity of the steady state disturbing signal Si, defined as net, calcium absorption from the intestine. In animals with functional parathyroids, the feedback loop due to the skeleton was thought to involve a combination of proportional plus to the action of parathyroid hormone, causes the error in the controlled to the action of parathyroid hormone, causes the error in the controlled system to be virtually nil over a wide range of net absorption. In brief, in the model parathyroid hormone was considered responsible for the maintenance of the high level of the reference value and for keeping the error near zero.
Controlled System BLOOD 7,
1,
_
Controlling System
A
BONE
[ul 11 FIQ. 1. Feedback loop of regulation of the blood calcium level by bone metabolism in young rats. Si = The net disturbing signal of the system. It equals v,, - undo (See Fig. 2). A = The net controlling signal. It equals vO+- vO- (See Fig. 2). CCa.1 = Blood plasma calcium concentration, the controlled signal. [UI = The reference value of the plasma calcium around which regulation occurs in normal animals. The kidney, a second controlling system, has been ignored because it contributes less than 5% of the total controlling signal in young rats. For further explanation, see text. Adapted from Aubert and Bronner (1).
Theoretically, a hormone that acts on bone in a direction opposite to that of parathyroid hormone may: (a) affect the level of the reference value; (b) be a component, of the integral control. For example, the mathematical expression chosen to describe the rate of appearance of parathyroid hormone in blood might in fact be the resultant of the appearance rates of two hormones; (c) play a subsidiary role, refining the action of parathyroid hormone. For example, such a hormone might reduce the delay with which the controlled signal returns to its st,eady state value following an acute disturbance.
THYROCALCITONIN
AND
BLOOD
CA
REGULATIOX
3G3
We are now presenting evidence concerning the action of endogenous thyrocalcitonin on the blood calcium level of rat,s. It, will be shown that of the three hypotheses enumerated above, the third is the most likely. MBTERIALS
AND
METHODS
Male Sprague-Dawley rats (purchased from Sprague-Dawley, Wisconsin) were utilized in all experiments. The following were common lo the three types of studies described below: Diets. A calcium-deficient semisynthetic diet (purchased from Nutritional Biochemicals, Cleveland, Ohio, or General Biochemicals, Chagrin Falls, Ohio) containing 24% vitamin-free test casein, 67% sucrose, 5% corn oil, 3% salt mixture5 and 1% vitamin supplement9 was modified by t,he addition of calcium carbonate (analytic reagent), monobasic calcium phosphate (analytic reagent) and/or monobasic and dibasic potassium phosphates and, when intended for thyroidectomized animals, by supplementation with L-thyroxin. Table 1 lists the analytically determined Ca and P content of the diets used in these experiments. The intake data used to calculat,e balances were based on these analyses. Surgical Procedures. Parathyroid glands were identified under the dissecting microscope, removed with the aid of jewelers’ forceps No. 5, placed in iced saline solution, and transplanted to a pocket located either in the gluteus maximus or along the interior of the thigh just under the femoral vessels. When thyroidectomy was intended, the thyroids were extirpated directly after parathyroid transplantation and care was t’aken to minimize blood loss and to avoid injury to the recurrent laryngeal nerve. After surgery, the effectiveness of the transplantation was judged by periodic blood analysis. Only animals whose blood calcium remained above 0.08 mg/ml plasma were considered to have functioning parathyroid glands. Histological sections obtained on some of these animals after the experiments confirmed the presence of parathyroid tissue. Analytical Techniques. Most of the analytical techniques have been reported previously (13). Plasma was analyzed directly; urine was wct’ Composition of salt mixture in gm/kg of diet: magnesium chloride, 14.250; potassium citrate, 6.210; potassium chloride, 5.910; sodium chloride, 2.460; potassium sulfate, 0.630; ferric citrate, 0.480; potassium iodide, 0.012 : sodium floridc, 0.012; manganous sulfate, 0.0906; and potassium alum, 0.0924. ‘Composition of vitamin supplement in mg/kg of dirt : para-aminobenzoic acid, 110.23; ascorbic acid, 1017.52; biotin, 0.44; calcium pantothenate, 66.14; choline dihydrogen citrate, 3715.20; folic acid, 1.98; i-inositol, 110.23; menadione, 49.60; nicotinic acid, 99.21; pyridoxine hydrochloride, 22.05 ; riboflavin, 22.05; thiamine hydrochloride, 22.05; vitamin A (500,000 USP U/gm) dry stabilized, 39.68; vitamin B1? (0.1% trituration w/mannitol), 29.76; vitamin D, (Dry, 500,000 USP U/gm). 4.41; and vitamin E acetate, 25% (250 IU of vitamin E/gm), 485.02.
BRONNER
ET AL.
THYROCALCITONIN
AND
m d
I
I I
I % 6 2 6 I I
BLOOD
CA
REGULATION
265
266
BRONNER
ET
AL.
ashed and feed and feces were dry-ashed. Calcium was determined by atomic absorption spectrophotometry, phosphate by the method of Chen et al. (17). Calcium-45 content of plasma samples was determined on a low-background planchet counter in the presence of suitable standards. Calcium-4.5 content of urine- and fecal-ash solutions was determined with a liquid-scintillation well counter using Bray’s solution (18)-each sample being run in duplicate, together with its own internal standard. To study the physiological role of endogenous thyrocalcitonin, three types of studies were undertaken: (n) [Ca,] RS a Function of Si The steady-state effect of thyrocalcitonin on the reference value and error of t-he blood calcium level, [Ca,], was studied by comparing the effect of an increase in the net amount of calcium absorbed from the intestine on the blood calcium level in three groups of 6-7-week-old rats: normal (N) , thyroidectomized (TX), and thyroparathyroidectomized (TPTX). Animals intended for study in the intact state were upon receipt divided into two or three lots, placed on a semisynthetic diet in metabolic cages and, beginning 10 days later, a balance experiment (13) was begun. Animals intended for surgery were placed on Diet II (Table 1) until after recovery from the operation, i.e., when their body weight again equaled that before surgery. They were then divided into lots, placed on the intended semisynthetic diets supplemented with 5.0 mg L-thyroxin/kg diet and, 10 days later, the balance experiment, was begun. The blood calcium levels, considered the controlled signal, were measured on the 3 days of the balance and then averaged for each animal. The average net calcium absorption for these 3 days, considered the disturbing signal, Si = Us - v,,~~ (Fig. 2)) was also determined for each animal. For each of the three groups of animals regression equations were then derived between the individual values of Si and [Ca,]. The reference value of [Gas] is defined as that value of [Ca,] when Si = 0; the steady state error is defined as the difference between a given [Ca,] for any Xi > 0 and the referehce value. (b) Calcium Parameters Thyrocalcitonin j
in Animals
with and without
Endogenous
The effect of t,he removal of endogenous thyrocalcitonin on the steady state intensity of the parameters of calcium metabolism (Fig. 2) was studied in two experiments, one comparing normal animals (hi) with thyroidectomized animals bearing parathyroid autografts (TX), the other comparing thyroid-intact controls bearing parathyroid autografts (C) with thyroidectomized animals bearing such autografts (TX). Since the administration of exogenous thyrocalcitonin to intact rats is thought to
THYROCALCITONIN
inhibit
calcium removal
AND
BLOOD
CA
REGULATION
267
from bone, uO-, and to increase calcium balance,
A, (12)) it seemed logical to study the effect of the removal of endogenous thyrocalcitonin, because it might lead to a decrease in A and therefore to
an increase in vO- in rats whose vO- was very low and whose A was very high. This was accomplished by studying animals on Diet III (13, 14).
i I
“F
“”
FIG. 2. One model of calcium metabolism in the rat. E, = compartment containing the rapidly exchangeable calcium, including blood plasma calcium (units: mg Ca). IX’*= compartment containing the slowly exchangeable calcium, most of which is presumed to be in bone (units: mg Cal. The symbol 2r denotes rates, expressed in mg Ca/day, and refers to the following processes: va zexchange between E1 and E’*; V. = absorption from intestine; v. = urinary excretion; VM,, = fecal endogenous excretion; vu+ = deposition in bone; vO- = resorption from bone; vc = intake ; VF = fecal excretion.’ Pool = E1 + El*; A = balance = DC- (v. + VR) = vO+- II?-. Pool size is constant during experiment. Adapted from Bronner and Aubert (13).
To this end, 6-week-old rats were upon receipt divided into two weightmatched groups and either one or both groups were operated upon by the procedures detailed above. From receipt through recovery, i.e., when body weight again equaled that before surgery, the animals were fed Purina Chow (approximately equal in Ca content to Diet III) .7 Thereafter they were placed on a semisynthetic diet (III), supplemented in the case of the thyroideatomized animals with 0.5 mg L-thyroxin/kg feed.8 After 10 days on Diet III, a combined Kinetic and balance experiment was begun. As previously (13)) this consisted of an intravenous injection of 45Ca, repeated blood sampling and a Ca balance measured between carmine markers administered at 6 and 72 hours after isotope injection. AH kinetic ‘Better results, as judged by rate of survival and of recovery, were obtained b> keeping animals on Purina Chow rather than on Diet II during the period of operat,ion and recovery. ‘Our rats typically ate 12 gm feed/day. On the assumption that 50% of the ingested L-thyroxin is absorbed, an intake of 0.5 mg/kg feed corresponds to a dail? thyroxin level of 3 pg per rat. This was judged to be more physiologiiaal and proved to be adequate for maintaining weight gain.
268
BRONNER
ET
AL.
and balance data reported were obtained during that 3-day period. Data were analyzed according to a two-compartment model (14) (Fig. 2). Curve fitting and numerical analysis were done with an IBM computer (1620, later 1160). (c) Loading
Experiments
In acute experiments designed to study the effect of an intraperitoneal calcium load on the blood calcium level, use was made of the thyroidectomized and control animals with parathyroid autografts which had been studied in the kinetic experiments above. When those experiments were completed, the animals were placed on Diet II. Twenty-four hours before each test they were switched to the unmodified calcium-deficient semisynthetic diet (<0.02% Ca) . The test, done repeatedly over a period of weeks, consisted of an intraperitoneal injection of 4.1 mg Ca (as calcium gluconate)/lOO gm body weight. Blood samples were collected just before and 30, 60, 90, 120, 180, and 240 minutes after the injection, as under our condit.ions the blood calcium level 15 minutes after the injection was always lower than at 30 minutes. In a second set of loading experiments, intact (N) and thyroidectomized animals bearing parathyroid autografts (TX) were subjected to an intraperitoneal injection of 8.2 mg Ca (as calcium gluconate)/lOO gm body weight. The animals weighed 130-140 gm when received and were placed on Purina Chow. Select.ed animals were operated upon and after being allowed to recover for 6 days they, t’ogethcr with the controls, were transferred to Diet II, supplemented with 0.5 mg L-thyroxin/kg diet in the case of the TX animals (Table 1). Approximat,ely 3 weeks later they were subjected to three loading experiments, done at an interval of 2 days. Following these experiments, the TX animals were tested for a hypocalcemic response to crude exogenous rat thyrocalcitonin in the form of an extract prepared according to Hirsch et al. (3). The animals were bled from t,he tail and then received by subcutaneous injection 0.5 ml of a graded dose of the extract, equivalent either to 0.33 or 0.66 of a rat gland. Sixty mi&tes later the animals were bled again. The response to the exogenous thyrocalcitonin injection was evaluated in terms of the drop of [Ca,] from the initial value. At the completion of these experiments, the animals were killed and their tibias and fibulas removed for calcium and phosphorus analysis. RESULTS
[Ca,] as a Function of S; Figure 3 shows the relationship between net absorption, the average disturbing signal S; = (@,, - v,,,,,,) , and t,hc blood calcium level, [Ca,]. the
THYROCALCITONIN
AND
BLOOD
CA
269
REGULATIOX
average controlled signal, in normal, thyroidectomized (TS), and thyroparathyroidectomized (TPTX) animals. In the normal animals, the blood calcium level remained nearly invariant as the intensity of the disturbing signal increased from 0 to 100 mg Ca/day. Even though the slope of the relationship differed very significantly from 0, the steady state error, i.e., the difference between [Ca,] when Ai was zero and when it was large, was less than 3%. The 95% confidence limits of the slope of the relationship for the normal animals were 0.0013 and 0.0055; the 95% confidence limits of the intercept, (i.e., when Si = 0) were 10.63 and 10.88. In the TPTX animals, on the other hand, the blood calcium level rose markedly as the intensity of the disturbing signal increased, the steady state error increasing proportionately with t.he disturbing signal. Both intercepts and slopes of the regression equations describing [Ca,] as a function of Si differed significantly in the TPTX animals from those in the intact rats. It is, therefore, apparent that double ablation removed a control which, when present, almost completely suppressed fhe occurrence of a steady state error in the blood calcium level. 12
- 1 -.
\
a
J,,
E ‘;:-
A
-_--S--A-----.---- l
NORMAL A
A
.
A
- I ._________
---__
B
----------TX n
-1
n
a
t 5.;/
NORMAL [&I
“, 0 m
TPTX RATS (28) [cas] = 5.52 + 0.035
RATS (IOIl = ID.76 + 0 0034
s, s,
4-l 0
10
20
30 s,
40 50 (v, -Vndc,.
60 m g /day)
70
a0
90
3 100
FIG. 3. Relationship between net absorption, the avrragc net disturbing signal and the average controlled signal, blood calcium concentration (SC = U’J - t’“,,“), I:Ca,l, in normal, thyroparathyroidectomized (TPTS), and thyroidectomizcd (TX) :mimals. The TPTS and TX animals received L-thyroxin in their diets. The lenstsquare lines for the normal and TPTX animals are shown with 95% confidence limits. Thp number of rats is indicated in parentheses.
270
BRONNER
ET
AL.
Results presented in Fig. 3 show that in thyroidectomized animals with parathyroid autografts and supplied with L-thyroxin, the [Ca,] did not increase as Si increased. The dashed line was calculated by the method of least squares; its slope did not differ significantly from zero. The 95% confidence limits of the slope were -0.0079 to -0.020 and of the intercepts 9.38 and 10.62. There was no overlapping with the intercepts of the normal animals, but the absolute difference between the values of the normal and TX animals was small; moreover, the values of the TX animals were lower. Hence, neither the reference value nor the steady state error of the blood calcium level of animals without endogenous thyrocalcitonin differed to any important degree from those of the intact. animals. Calcium Parameters Thyrocalcitonin
in Animals with and without Endogenous
Table 2 lists the terms of the disappearance equations and Tables 3 and 4 the numerical values of the parameters derived (Fig. 2) for animals with and without an endogenous source of thyrocalcitonin. Table 2 shows that the disappearance equations describing the time course of the injected 45Ca in the plasma of the N and TX animals, on the one hand, and of the C and TX animals on the other, did not differ significantly. -4s shown in Table 3, there was no significant difference in the intensity of most parameters in intact and ablated animals. Not’e in particular the similarity of the blood calcium level, [CaS], of the balance, L!., and of urinary excretion, v,. In addition, the rat,es of deposition of calcium in bone, vO+,and of its removal from bone, vO-, were similar. Table 4 shows the results of a similar experiment, but one where both groups of animals carried autogenous parathyroid grafts. Here the tendency was great,er than in the preceding experiment for many parameters of the TX animals to be lower in intensity than those of the controls. As a result, more of the differences assumed st,atistical significance, although none was very large. Comparison of the data obtained on the two control groups (Tables 3 and 4) with the mean and individual lot parameters of 29 ot’her rats on Diet III studied over the past 2 years showed that the values of the t,wo control groups fell at the exkemes of the normal range and that the differences between the two control groups could have resulted from differences among lots of animals (obtained from the same supplier and raised under similar conditions). While it is not permissible to pool the results of the two experiments, it is apparent that removal of the endogenous supply of thyrocalcitonin had either no or smalI effects on the intensity of the steady state parameters of calcium met.abolism.
THYROCALCITONIN
AND
BLOOD
TABLE TERMS
N
Normal TX* Control” TX*
8 8 11 9
FOLLOWING
IV IN.W~TION
OF ““C.%
= AeBal + Becat
A
SE
a
SE
B
SE
b
4.52 4.83 3.62 4.44
0.35 0.33 0.15 0.64
7.74 7.17 8.71 9.05
0.13 0.34 0.34 0.96
0.59 0.67 0.38 0.43
0.05 0.04 0.02 0.03
0.66 0.71 0.67 0.73
a R. = ‘j?, dose/mg Ca and t = days. b TX = tjhyroidectomized and lrthyroxin c With sut,ogenous parathyroid grafts.
EFFECT
271
REGULATION
2
OF DISAPPEARANCE EQUATIONS EQUATION”: R.
Type
CA
OF THYROIDECTOMY
Mean Body weight, gm Plasma Ca, [CaJ, mg/lOO ml Compartment E1, mg Ca Compartment Elp, mg Ca Pool, E, + E’z, mg Ca Rate of exchange between El and E’z, v., mg Ca/day Intake, vi, mg Ca/day Urinary Ca, v,, mg/day Fecal Ca, VP, mg/day Balance, A, mg Ca/day AbFrption, v,, mg Ca/day y0 Absorption, a Fecal endogenous Ca, v.dO, w/day Ca into bone, vO+,mg/day Ca out of bone, v.-, mg/day
(8)” SE
Mean
20.2 88.0 108.2 70.1
239 10.99 18.9 73.1 92.0
189.9
5.4
1.4
0.1
178.2 1.7
133.9
4.0 4.3 4.3 1.6 0.5
11.13
+54.6 65.9 34.5 9.8 58.0 3.4
3.4
3.8
PARAMETERS
TX (8)
2.1 0.07 1.3 6.4 7.4 4.8
247
0.03 0.02 0.03 0.03
in feed.
TABLE 3 (TX) ON CALCIUM
Normal
SE
55.4b
119.8 $56.7 66.2 37.2 7.9
53.9 -2.8
SE
P
5.0 0.17
1.5 5.2 6.3 4.4
0.05
> p > 0.02
0.02
> p > 0.01
8.9
0.4 7.4 5.5 4.9 2.2 0.6 3.3 7.6
Note.-TX animals had parathyroid autografts and received 0.5 mg kthyroxin/kg feed. For further explanation of parameters, see Fig. 2 and text,. R Number of animals shown in parentheses. 6 Significantly lower than corresponding value of normal.
Loading
Experiments
The data displayed in Figs. 4 and 5 indicate that when hypercalcemia was induced by subjecting animals to intraperitoneal calcium loads, their [Ca,] returned to the steady state value more quickly if they had a source of endogenous thyrocalcitonin. Thus, when the calcium load was
272
Body weight, gm Plasma Ca, [Ca,], mg/lOO ml Compartment Er, mg Ca Compartment E’2, mg Ca Pool, El + E’j, mg Ca Rate of exchange between El and E’z, ve, mg Ca/day Intake, vi, mg Ca/day Urinary Ca, vu, mg/day Fecal ca, vF, mg/day Balance, A, mg Ca/day Absorption, v,, mg Calday To Absorption, 01 Fecal endogenous Ca, l)n&,, w/day Ca int)o bone, ZL,+, mg/day Ca out of bone, IL-, mg/day
BRONNER
ET
Cout~rols
(11 I’(
Mean
SE
AL.
P
239 10.49 25.4 128.6 154.0 96.8
1.9 0.10 1.0 6.5 7.0 1.9
“26b 10.95 22.6 108.2 130.8 78.3”
2.7 0.09 2.1 8.4 8.9 2.5
213.7 2.1 159.8 +51.8 69.0 32.3 15.0
4.6 0.2 5.7 5.3 5 4 2.4 0.8
190.4b 3.9* 144.3 +42.2 59.6 31.3 13.5
9.5 0.5 9.1 7.4 7.3 3.5 0.6
0.05
> p > 0.02
3.1 7.8
0.05
> p > 0.02
84.7 32.9
1.4 5.6
a Number of animals shown in parentheses. All grafts. TX animals received 0.5 mg L-thyroxin/kg parameters, see Fig. 2 and text. * Significantly different, from control value.
77.1 34.9
animals had autogenous parathyroid feed. For further explanation
of
4.1 mg/lOO gm body weight, the [Ca,] of the normal animals had returned to its preinjection (or steady state) level in approximately 21/ hours! while that of the TX animals took approximately 3jh hours to return to this level. When the calcium load was doubled (Fig. 5) the delay in both groups was markedly increased and appears approximately to have doubled. Moreover, the difference in peak values was also increased, from 3.3 (164.3 I!Z 0.8 - 161.0 t 0.7, p < 0.01) to 13.1 (225.7 Z!Z 4.2 - 212.6 rt 3.6, p _< 0.02), the TX animals in both instances reaching somewhat higher relative peaks. This positive effect can also be taken as evidence that all TX animals responded to the absence of thyrocalcitonin. Moreover, this response appeared relatively independent of age and weight, as the animals whose responses are displayed in Fig. 4 were heavier and older than those whose responses are shown in Fig. 5. The data shown in Table 5 give added evidence of the ability of the TX animals to respond to exogenous rat thyrocalcitonin. The findings set, out in Table 6 indicate that the tibias and fibulas of bot,h groups of animals had the same phosphorus content, but that the
THYROCALCITONIN
AND
BLOOD
CA
bones of the normal animals contained slightly, but calcium than those of the TX animals and had higher small differences in calcium content are consistent with values of the pool, of vuc, and of vu+, found in either TABLE RESPONSE
Fraction of gland=
OF THYROIDECTOMIZED
N
13
5
3
4
RATS
273
REGULATIOK
significantly mow Ca/P ratios. Thcee the slightly higher the normal or the
5 TO EXOGENOUS
THYROCALCITOSIN
Mean percent drop in (SE) 1CaJ
Mean weight, gm (SE) 275(5.41 282(10.3)
5.0 14.0
(1.7) (1.7)
Note.-The animals had previously been used in loading experiments (Fig. 5). They were thyroidectomized and bore parathyroid autografts. a Fraction of gland refers to a fraction of rat gland, extracted according t’o Hirsch el al. (3). The animals were bled from the tail before and 60 minutes after a subcut,aneous injection of thyrocalcitonin in 0.5 ml.
I , 0 ILOAD
30
60
120 Time (ml 1
180
240
PIG. 4. The blood calcium level, l’Ca.1, of thyroidectomized (TX) and control rats as a function of time, following an intraperitoneal injection of 4.1 mg Ca (as Ca gluconate) per 100 gm body weight. Both groups of animals carried parathyroid autografts and had been maintained on Diet II (Table 1) until 24 hours before each loading experiment, when they were placed on a very low Ca (<0.02%) diet. Means and standard errors for each group at each time were calculated from the percent rise of individual animals on successive experiments.
[Ca,], mg/lOO ml Type
N
Base
(SE)
Peak
(SE)
TX Control
10 17
10.54 10.26
(0.20) (0.07)
17.32 16.52
(0 08) (0.07)
274
BRONNER
ET
TABLE
AL.
6
COMPARISON OF CA AND P CONTENT OF TIBIAS ASD FIBULAH OF NORMAL AND THYRO~DECTOMIZED (TX) RATS
Calcium
Weight,
Phosphorus
gm (SE)
Total mg (SE)
M&m (SE)
12
0.536 (0.013)
11
0.527
78.8 (1.5) 74.48
147.6 (2.0) 141.3”
Group
12:
Normal TX
(0.007)
(1.2)
(2.4)
Ca/P (SE)
Total mg Mg/gm (SE) (SE) 34.7 (0.5) 33.9 (0.4)
64.4 (0.8) 65.1 (1.1)
2.27 (0.015) 2.19* (0.013)
Note.-The animals had previously been used in loading experiment’s (Fig. rj). The TX animals bore parathyroid autografts. a Significantly lower than normal (0.05 > p > 0.02). b Significantly lower than normal (p < 0.01).
I I 0
I
30
60
LOfnO
120 itme (Inn)
180
240
5. The blood calcium level, CCa.1, of thyroidectomized rats with parsthyroid autografts (TX) and of intact animals (N) as a function of time, following an intraperitoneal injection of 8.2 mg Ca (as Ca gluconate) per 199 gm body weight. Both groups were maintained on Diet II (Table 1) throughout the experiment. Means aad standard errors for each group at each time were calculated from the percent rise of individual animals in three successive experiments. FIG.
[Ca.], mg/lOO ml
Type
N
Base
(SW
Peali
(SE)
TX Normal
27 25
10.62 10.89
(0.12) (0.09)
23.88 23.12
(0.32) (0.33)
TEYRCCALCITONIN
AND
BLOOD
CA
REGULATION
275
control animals, as compared with the TX animals (Tables 3 and 4). Whether these small differences can be attributed to differences in parathyroid function that may have resulted from differences in the efficiency of the autograft or whether they were the direct result of the absence of thyrocalcitonin cannot be determined without measurement of the steady state parathyroid hormone level. DISCUSSION
On the basis of the above results it is possible to classify the action of endogenous thyrocalcitonin with respect to the three modes of action outlined in the introduction as follows. (a) If thyrocalcitonin acted on the reference value of the blood calcium, the reference value should have changed when the endogenous supply of the polypeptide was removed. It can be seen from the data reported here that the [Ca,] values of normal and TX animals were similar when net absorption, Si, was near zero. Thus, in the two groups of animals used in the loading experiments (Figs. 4 and 5) and in the group whose calcium parameters are shown in Table 3, there were no significant differences in [Ca,] between animals wit,h and wit.hout an endogenous source of thyrocalcitonin. In the case of the animals whose data are displayed in Fig. 3 and of those whose calcium parameters are shown in Table 4, the differences were statistically significant. However, the differences were small and their direction was not the same and therefore, it appears that thyrocalcitonin does not act on the reference value of the blood calcium. (b) If thyrocalcitonin were a component of the integral control, removal should affect the steady state error of the blood calcium, as well as ot,her steady state parameters. Figure 3 indicates that the response of thyroidectomized animals to an increase in Si was similar to that of normal rats. This confirms and extends the observation by Talmage et al. (19) on the similarity of the blood calcium values of animals with a,nd without endogenous thyrocaIcitonin on a normal calcium intake (see also 20). If the controlled signal [Ca,] is unchanged by the removal of thyrocalcitonin, the net controlling signal, A, should also be unchanged. This was indeed the case (Tables 3 and 4). Failure to affect h does not preclude alterations in the constituent signals, v,+ and z)~-. However, these also did not change markedly. That z)~+is normal in thyroidectomieed animals supplied with L-thyroxin has also been reported by Payne and Sansom (21), who found that in two thyroidectomixed goats on 1 mg thyroxin,/day the values for A (the rate of accretion, equivalent to v,+)
276
BRONNER
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AL.
and E (the size of the exchangeable pool, equivalent to E, + Etz) were normal or slightly higher t,han normal. It might be argued that the values of uuo-arp lower in these animals than consistent with normal growth and bone remodeling. The problem inherent in the analysis has recently been discussed at length (14). However, even if the values for u,- were erroneously low, the failure to observe differences in A and in ZJ,+,two independent set’sof measurements, makes it unlikely that removal of endogenous thyrocalcitonin affected v,- =
(A -
vo+) .
In the analysis of calcium homeostasis (1) the rate of calcium a,bsorption, v,, has been considered an acute disturbing signal, even though it was recognized that this parameter may be subject to long-term regulation (22). It, is, therefore, worth noting that calcium absorption appeared unaffected by the removal of the source of thyrocalcitonin (v, and cy, Tables 3 and 4). Whereas the administration of thyrocalcitonin does not necessarily result in the opposite of what happens when the source of endogenous thyrocalcitonin is removed, it is interesting to note that Wase et al. (23) report that, thyrocalcitonin was without effect on calcium absorption; on the other hand, Milhaud and Moukhtar (24) observed an increase in absorption following administration of a long-acting preparation. In the normal animal, the kidney, a second controlling system, has been ignored (1) becauseurinary calcium excretion is so small in relation to t,he balance, the major controlling signal. This situation was not changed by the removal of t’he source of thyrocalcitonin, as the ratio of v,/A was only 0.03 (Table 3) and 0.09 (Table 4) in the TAX animals, compared with 0.03 and 0.04 in the normal and control animals, respectively. As none of the st.eady state parameters of calcium metabolism was changed appreciably by removal of the source of thyrocalcitonin (Tables 3 and 4), it may be concluded that thyrocalcitonin does not constitute a major portion of the integral control. Removal of the parathyroids, on the other hand, resulted in a large drop of the reference value, from 10.74 to 5.52 mg Ca/lOO ml (Fig. 3), and in a rise of the controlled signal [Ca,], when the disturbing signal Xi rose. This indicates that the parathyroids are responsible for the integral control. Moreover, the fact that a rise in Si produced a linear rise in [Ca,] (see also 25) can be considered support for the existence of a proportional control of [Ca,] in TPTX animals. While removal of the source of thyrocalcitonin is without obvious effect on steady-state parameters in animals with functioning parat’hyroids, it has been reported (19) that the [Ca,] is lower in parathyroidectJomized than in TPTX animals. We have maintained these two groups of ablated
THYROCALCITONIN
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RFxXJLATIOi’G
animals for many weeks and have not observed differences in either their fasting or their peak blood calcium levels after ingestion of high-calcium diets (Table 7). This suggests that the same differences in the regulation of [Ca,] that have been observed to exist between TX and TPTX animals (Fig. 3) may also exist between TX and PTX animals. BLOOD
CALCIUM
TABLE 7 LEVELS [CA,] OF THYROPARATHYROIDECTOMIZED AND PARATHYROIDECTOMIZED (PTX) RATS [Ca,ly
mg Ca/lOO
ml plasma
Fastming Diet III
Animals
(TPTX)
Peak
(N)
Mean
SE
Mean
SE
TPTX (6) PTX (4)
5.88 5.68
0.24 0.40
6.75 6.67
0.23 0.27
Time of peak (hours) 4 4
Note.-The animals were trained to eat their daily ration in 2 hours and blood samples were taken just before (“fasting”) and at varying periods after feeding. The values reported are the fasting and the peak values. TPTX animals received L-thyrosin in their diets.
(c) Thus, if thyrocalcitonin plays a role in calcium homeostasis, we are left with the third alternative, that it plays only a subsidiary role, such as reducing the delay with which the controlled signal returns to its reference value following an acute disturbance. This is brought out by the loading experiments (Figs. 4 and 5) which indicate that t,he overshoot of the blood calcium level induced by an intraperitoneal injection of calcium gluconate subsided more slowly in animals deprived of their endogenous supply of thyrocalcitonin than in normal controls (see also 19, 26). It is therefore apparent that the presence of thyrocalcitonin improvcn the effectiveness of the control system in terms of the rate of return. The physiological significance of a shorter delay in the return to the steady state level of plasma calcium requires further study. SUMMARY
The role of thyrocalcitonin in calcium homeostasis of rats has been studied in three types of experiments. (a) Measurement of the effect on the blood calcium level, [Ca,], of varying net calcium absorption in normal (N), thyroparathyroidectomized (TPTX) and thyroidectomized animals with autogenous parathyroid transplants (TX). These studies showed that the values of [Ca,] were similar in N and TX animals at all levels of net absorption over a
278
BRONNEB ET AL.
nearly lOO-fold range. Moreover, the error, i.e., the difference between [Ca,] when net absorption was zero and when it was verjr high, was close to zero. In TPTX animals, on the other hand, the values for [Ca,] were markedly lower than in the other two groups at all levels of net absorption and the error of [Ca,] increased linearly and proportionately with increased absorption. (b) Determination of the intensity of the steady-state parameters of calcium metabolism (pool size, rates of Ca absorption, deposition in and removal from bone, urinary output) in N, in controls with parathyroid autografts (C), and in TX failed to reveal any important differences between either N and TX or C and TX. (c) Measurement of the return of [Ca,] to its base value following an acute calcium surcharge by intraperitoneal injection in N, C, and in TX animals indicated a greater delay in the return in the TX than in the C or N animals. It is concluded that thyrocalcitonin is not involved in the regulation of the steady state value of the blood calcium or of the steady-state parameters of calcium metabolism, including turnover and amount of bone calcium. Rather, thyrocalcitonin appears to refine the action of parathyroid hormone in terms of its effects on bone, the primary regulator of blood calcium. ACKNOWLEDGMENTS We thank Dr. J.-P. Aubert for valuable discussions and critique of the manllscript. Lawrence Boram prepared histological sections. Dr. R. C. Kelsay and Mrs. Kay Shotts helped with the statistical analyses. REFERENCES 1. AUBERT, 6.-P., AND BRONNER, F., Biophys. J. 5, 349 (1965). 2. FOSTER, G. V., BAGHDIANTZ, A., KUMAR, M. A., SLACK, E., SOLIMAN, H. A., .~ND MACINTYRE, I., Nature 202, 1303 (1964). 3. HIRSCH, P. F., VOELREL, E. F., AND MUNSON, P. L., Science 146, 412 (1964). 4. TENENHOUSE, A., ARNAUD, C., AND RASMUSSEN, H., Proc. Natl. Acad. Sci. (USA) 53, 818 (1965). 5. MUNSON, P. L., POTTS, J. T., JR., REISFELD, R. A., COOPER, C. W., AND VOELKEL, E. F., Science 15% 425 (1966). 6. O’RIORDAN, J. L. H., TASEJIAN, A. H., JH., MUNSON, P. L.. CONDLIFFF,,P. G., AND AURBACH, G. D., Science l!%, 885 (1966). 7. PUTTER, J., KACZKA, E. A., HARMAN, A. E., RICKES, E. L., KEMPF, A. J., CHAIET, L., ROTHROCK, J. W., WASE, A. W., AND WOLF, F. ‘J.. J. .4m. Chem. Sot. 89, 5301 (1967). 8. COPP, D. H., CAMERON, E. C., CHANCY, 13. A., IX~VIDSON, A. C. F., AND HENZE, K. ‘4., Endocrinology 70, 638 (1962). 9. ALIAPOULIOS, &I. A., GOLDHABER, P., .4xD MUNSON, P. L., &knre 151, 330 (1966). 10. FRIEDMAN, J., .~ND RAISZ. L. G., Science 150, 1465 (1965).
THYROCALCITONIN
11.
MILIIAUD,
(l-‘ari.s) 12.
13. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25. 26.
PERAULT,
A.-M.,
AND
BLOOD
MOUKIITAR,
CA
M.
279
REGULATIOS
S., Compl.
Rend.
Acad.
Sci.
813 (1965).
G., MOUHHTAR, M. S., CHERIAN, G., .4ND PERAliLT, A.-hf., corn)?/. Rrnrl. Sci. (Paris) 262,511 (1966). BRONNER, F., AND AUBERT, J.-P., Am. J. Physiol. 209,887 (1065). BRONNER, F., Trans. N. Y. Acad. Sci. (Ser. II) 29,502 (1967). AUBERT, J.-P., CHERIAN, A. G., MOUKHTAR, M. S., AND MILH.~UD, (;., Hio~h~t~?. Pharmacol. 13, 31 (1964). SAMMON, P. J., BOR~M, L., MOODY, C., AND BRONNER, F., Federation Proc. 25, 495 (1966). CHEN, P. S., JR., TORIBARA, T. Y., AND WAHNER, H., Anal. Chem. 28, 1756 (1956). BRAY, G. A., Anat. Biochem. 1,279 (1960). TALMAGE, R. V., NEUENSCHWANDER, J., AND KFLAINTZ, L., Endocrinology 76, 103 (1965). BRONNER, F., SAMMON, P. J., NICHOLS, C., ST.4CEy, R. E.. .ISD SHAH, 13. G.. Proc. Third Pamthyroid Conference, Mont Gabriel, Canada (1967). (in press). PAYNE, J. M., AND SANSOM, B. F., J. Physiol. (London) 184, 433 (1966). BRONNER, F., in “Mineral Metabolism-An Advanced Treatise” (C. L. Comar and Felix Bronner, eds.), pp. 314414. Vol. 2A. Academic Press, Kcw York (1964). WASE, A. W., PETERSON, A., RICKES, E., AND SOLO~SKI, J., Endocrinology 79, 687 ( 1966). MILHAUD, G., AND MouKHTAR, M. S., Proc. Sot. Exptl. Biol. Med., 123, 207 (1966). COPP, D. H., in “Bone Biodynamics” (H. M. Frost, ed.), p. 441. Lit.tlr, Brown, Boston, Massachusetts, 1964. CARE, A. D., ~XINCAN, T., AND WEBSTER, D., .l. Endocrinol. 34, xi ( 1966). MILHAUD,
Acad.
14.
G.,
261,
AND
MEDICINE
Role
1,
of
261-279 (1967)
Thyrocalcitonin of the
F. BRONNER, Department
Blood
P. J. SAMMON,? of Physiology Scl~ool
of Medicine,
and
in the Calcium
Regulation
Level1
R. E. STACEY,” Biophysics, Louisville.
Received November
UniL~ersity
Kentucky
AKD
B. G. PHA%H’
of Lotrist~ill~~
$I202
14, 1967
Calcium homeostasis in the young growing rat has been considered as effected by a regulator-type system, with a feedback loop involving a proportional control in thyroparathyroidectomized animals and a combination of proportional and integral controls in the normal intact animal (1). At the time the model was formulated, parathyroid hormone alone WZIS considered responsible for the integral control, as the mode of action of thyrocalcitonin (2,3), a hypocalcemic polypeptidc (4-7) was unknown, although the existence of a hypocalcemic factor of parathyroid origin had been postulated (8). Current evidence indicates that thyrocalcitonin acts in a direction opposite to that of parathyroid hormone, i.e., it inhibits bone resorption (9-12). This paper is concerned with an attempt to take into account the existence and mode of action of thyrocalcitonin. In the t,ypc of system postulated (Fig. I) the blood plasma is the controlled system, the blood calcium level, [Ca,], is the controlled signal, and the level around which regulation takes place is the reference value [U] . The net amount of calcium that enters the system is the disturbing signal Si, eyual to net calcium absorption from the gut. Two controlling systems have been considered, the skeleton and the kidney. Of these, the skeleton is by far the principal regulator, as the intensity of the renal signal (i.e., urinary calcium excretion, zj,,) in normal (13, 14) and in TPTX (15, 16) rats is so faint in relation to the disturbing signal as to be negligible. The net controlling signal of the skeleton is the calcium balance A, made up of two constituent signals acting in opposite directions, calcium deposition in bone zj,,+, and calcium removal from bone vn-. ‘Supported by USPHS grant AM-07983, by Training grant 1 TI DE 160 and by :I grant from the Kentucky Chapter, The Arthritis Foundation. ’ Predoctoral Trainee (USPHS Training grant 1 Tl DE 160). ,IPostdoctoral Trninec (USPHS Training grant 1 Tl DE 160). ’ Present address: Nutrition Division, Food and Drug Research Laboratory, Tunney’s Pasture, Ottama 3, Canada. 261
262
BRONNER
ET
AL.
In a system with proportional control, an error exists in the controlled system; this error was defined as the difference between the actual [Ca,] and the reference [U] values of the controlled signal, i.e., t.he plasma calcium concentration. At steady state, the error is directly proportional to the intensity of the steady state disturbing signal Si, defined as net, calcium absorption from the intestine. In animals with functional parathyroids, the feedback loop due to the skeleton was thought to involve a combination of proportional plus to the action of parathyroid hormone, causes the error in the controlled to the action of parathyroid hormone, causes the error in the controlled system to be virtually nil over a wide range of net absorption. In brief, in the model parathyroid hormone was considered responsible for the maintenance of the high level of the reference value and for keeping the error near zero.
Controlled System BLOOD 7,
1,
_
Controlling System
A
BONE
[ul 11 FIQ. 1. Feedback loop of regulation of the blood calcium level by bone metabolism in young rats. Si = The net disturbing signal of the system. It equals v,, - undo (See Fig. 2). A = The net controlling signal. It equals vO+- vO- (See Fig. 2). CCa.1 = Blood plasma calcium concentration, the controlled signal. [UI = The reference value of the plasma calcium around which regulation occurs in normal animals. The kidney, a second controlling system, has been ignored because it contributes less than 5% of the total controlling signal in young rats. For further explanation, see text. Adapted from Aubert and Bronner (1).
Theoretically, a hormone that acts on bone in a direction opposite to that of parathyroid hormone may: (a) affect the level of the reference value; (b) be a component, of the integral control. For example, the mathematical expression chosen to describe the rate of appearance of parathyroid hormone in blood might in fact be the resultant of the appearance rates of two hormones; (c) play a subsidiary role, refining the action of parathyroid hormone. For example, such a hormone might reduce the delay with which the controlled signal returns to its st,eady state value following an acute disturbance.
THYROCALCITONIN
AND
BLOOD
CA
REGULATIOX
3G3
We are now presenting evidence concerning the action of endogenous thyrocalcitonin on the blood calcium level of rat,s. It, will be shown that of the three hypotheses enumerated above, the third is the most likely. MBTERIALS
AND
METHODS
Male Sprague-Dawley rats (purchased from Sprague-Dawley, Wisconsin) were utilized in all experiments. The following were common lo the three types of studies described below: Diets. A calcium-deficient semisynthetic diet (purchased from Nutritional Biochemicals, Cleveland, Ohio, or General Biochemicals, Chagrin Falls, Ohio) containing 24% vitamin-free test casein, 67% sucrose, 5% corn oil, 3% salt mixture5 and 1% vitamin supplement9 was modified by t,he addition of calcium carbonate (analytic reagent), monobasic calcium phosphate (analytic reagent) and/or monobasic and dibasic potassium phosphates and, when intended for thyroidectomized animals, by supplementation with L-thyroxin. Table 1 lists the analytically determined Ca and P content of the diets used in these experiments. The intake data used to calculat,e balances were based on these analyses. Surgical Procedures. Parathyroid glands were identified under the dissecting microscope, removed with the aid of jewelers’ forceps No. 5, placed in iced saline solution, and transplanted to a pocket located either in the gluteus maximus or along the interior of the thigh just under the femoral vessels. When thyroidectomy was intended, the thyroids were extirpated directly after parathyroid transplantation and care was t’aken to minimize blood loss and to avoid injury to the recurrent laryngeal nerve. After surgery, the effectiveness of the transplantation was judged by periodic blood analysis. Only animals whose blood calcium remained above 0.08 mg/ml plasma were considered to have functioning parathyroid glands. Histological sections obtained on some of these animals after the experiments confirmed the presence of parathyroid tissue. Analytical Techniques. Most of the analytical techniques have been reported previously (13). Plasma was analyzed directly; urine was wct’ Composition of salt mixture in gm/kg of diet: magnesium chloride, 14.250; potassium citrate, 6.210; potassium chloride, 5.910; sodium chloride, 2.460; potassium sulfate, 0.630; ferric citrate, 0.480; potassium iodide, 0.012 : sodium floridc, 0.012; manganous sulfate, 0.0906; and potassium alum, 0.0924. ‘Composition of vitamin supplement in mg/kg of dirt : para-aminobenzoic acid, 110.23; ascorbic acid, 1017.52; biotin, 0.44; calcium pantothenate, 66.14; choline dihydrogen citrate, 3715.20; folic acid, 1.98; i-inositol, 110.23; menadione, 49.60; nicotinic acid, 99.21; pyridoxine hydrochloride, 22.05 ; riboflavin, 22.05; thiamine hydrochloride, 22.05; vitamin A (500,000 USP U/gm) dry stabilized, 39.68; vitamin B1? (0.1% trituration w/mannitol), 29.76; vitamin D, (Dry, 500,000 USP U/gm). 4.41; and vitamin E acetate, 25% (250 IU of vitamin E/gm), 485.02.
BRONNER
ET AL.
THYROCALCITONIN
AND
m d
I
I I
I % 6 2 6 I I
BLOOD
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265
266
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ET
AL.
ashed and feed and feces were dry-ashed. Calcium was determined by atomic absorption spectrophotometry, phosphate by the method of Chen et al. (17). Calcium-45 content of plasma samples was determined on a low-background planchet counter in the presence of suitable standards. Calcium-4.5 content of urine- and fecal-ash solutions was determined with a liquid-scintillation well counter using Bray’s solution (18)-each sample being run in duplicate, together with its own internal standard. To study the physiological role of endogenous thyrocalcitonin, three types of studies were undertaken: (n) [Ca,] RS a Function of Si The steady-state effect of thyrocalcitonin on the reference value and error of t-he blood calcium level, [Ca,], was studied by comparing the effect of an increase in the net amount of calcium absorbed from the intestine on the blood calcium level in three groups of 6-7-week-old rats: normal (N) , thyroidectomized (TX), and thyroparathyroidectomized (TPTX). Animals intended for study in the intact state were upon receipt divided into two or three lots, placed on a semisynthetic diet in metabolic cages and, beginning 10 days later, a balance experiment (13) was begun. Animals intended for surgery were placed on Diet II (Table 1) until after recovery from the operation, i.e., when their body weight again equaled that before surgery. They were then divided into lots, placed on the intended semisynthetic diets supplemented with 5.0 mg L-thyroxin/kg diet and, 10 days later, the balance experiment, was begun. The blood calcium levels, considered the controlled signal, were measured on the 3 days of the balance and then averaged for each animal. The average net calcium absorption for these 3 days, considered the disturbing signal, Si = Us - v,,~~ (Fig. 2)) was also determined for each animal. For each of the three groups of animals regression equations were then derived between the individual values of Si and [Ca,]. The reference value of [Gas] is defined as that value of [Ca,] when Si = 0; the steady state error is defined as the difference between a given [Ca,] for any Xi > 0 and the referehce value. (b) Calcium Parameters Thyrocalcitonin j
in Animals
with and without
Endogenous
The effect of t,he removal of endogenous thyrocalcitonin on the steady state intensity of the parameters of calcium metabolism (Fig. 2) was studied in two experiments, one comparing normal animals (hi) with thyroidectomized animals bearing parathyroid autografts (TX), the other comparing thyroid-intact controls bearing parathyroid autografts (C) with thyroidectomized animals bearing such autografts (TX). Since the administration of exogenous thyrocalcitonin to intact rats is thought to
THYROCALCITONIN
inhibit
calcium removal
AND
BLOOD
CA
REGULATION
267
from bone, uO-, and to increase calcium balance,
A, (12)) it seemed logical to study the effect of the removal of endogenous thyrocalcitonin, because it might lead to a decrease in A and therefore to
an increase in vO- in rats whose vO- was very low and whose A was very high. This was accomplished by studying animals on Diet III (13, 14).
i I
“F
“”
FIG. 2. One model of calcium metabolism in the rat. E, = compartment containing the rapidly exchangeable calcium, including blood plasma calcium (units: mg Ca). IX’*= compartment containing the slowly exchangeable calcium, most of which is presumed to be in bone (units: mg Cal. The symbol 2r denotes rates, expressed in mg Ca/day, and refers to the following processes: va zexchange between E1 and E’*; V. = absorption from intestine; v. = urinary excretion; VM,, = fecal endogenous excretion; vu+ = deposition in bone; vO- = resorption from bone; vc = intake ; VF = fecal excretion.’ Pool = E1 + El*; A = balance = DC- (v. + VR) = vO+- II?-. Pool size is constant during experiment. Adapted from Bronner and Aubert (13).
To this end, 6-week-old rats were upon receipt divided into two weightmatched groups and either one or both groups were operated upon by the procedures detailed above. From receipt through recovery, i.e., when body weight again equaled that before surgery, the animals were fed Purina Chow (approximately equal in Ca content to Diet III) .7 Thereafter they were placed on a semisynthetic diet (III), supplemented in the case of the thyroideatomized animals with 0.5 mg L-thyroxin/kg feed.8 After 10 days on Diet III, a combined Kinetic and balance experiment was begun. As previously (13)) this consisted of an intravenous injection of 45Ca, repeated blood sampling and a Ca balance measured between carmine markers administered at 6 and 72 hours after isotope injection. AH kinetic ‘Better results, as judged by rate of survival and of recovery, were obtained b> keeping animals on Purina Chow rather than on Diet II during the period of operat,ion and recovery. ‘Our rats typically ate 12 gm feed/day. On the assumption that 50% of the ingested L-thyroxin is absorbed, an intake of 0.5 mg/kg feed corresponds to a dail? thyroxin level of 3 pg per rat. This was judged to be more physiologiiaal and proved to be adequate for maintaining weight gain.
268
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ET
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and balance data reported were obtained during that 3-day period. Data were analyzed according to a two-compartment model (14) (Fig. 2). Curve fitting and numerical analysis were done with an IBM computer (1620, later 1160). (c) Loading
Experiments
In acute experiments designed to study the effect of an intraperitoneal calcium load on the blood calcium level, use was made of the thyroidectomized and control animals with parathyroid autografts which had been studied in the kinetic experiments above. When those experiments were completed, the animals were placed on Diet II. Twenty-four hours before each test they were switched to the unmodified calcium-deficient semisynthetic diet (<0.02% Ca) . The test, done repeatedly over a period of weeks, consisted of an intraperitoneal injection of 4.1 mg Ca (as calcium gluconate)/lOO gm body weight. Blood samples were collected just before and 30, 60, 90, 120, 180, and 240 minutes after the injection, as under our condit.ions the blood calcium level 15 minutes after the injection was always lower than at 30 minutes. In a second set of loading experiments, intact (N) and thyroidectomized animals bearing parathyroid autografts (TX) were subjected to an intraperitoneal injection of 8.2 mg Ca (as calcium gluconate)/lOO gm body weight. The animals weighed 130-140 gm when received and were placed on Purina Chow. Select.ed animals were operated upon and after being allowed to recover for 6 days they, t’ogethcr with the controls, were transferred to Diet II, supplemented with 0.5 mg L-thyroxin/kg diet in the case of the TX animals (Table 1). Approximat,ely 3 weeks later they were subjected to three loading experiments, done at an interval of 2 days. Following these experiments, the TX animals were tested for a hypocalcemic response to crude exogenous rat thyrocalcitonin in the form of an extract prepared according to Hirsch et al. (3). The animals were bled from t,he tail and then received by subcutaneous injection 0.5 ml of a graded dose of the extract, equivalent either to 0.33 or 0.66 of a rat gland. Sixty mi&tes later the animals were bled again. The response to the exogenous thyrocalcitonin injection was evaluated in terms of the drop of [Ca,] from the initial value. At the completion of these experiments, the animals were killed and their tibias and fibulas removed for calcium and phosphorus analysis. RESULTS
[Ca,] as a Function of S; Figure 3 shows the relationship between net absorption, the average disturbing signal S; = (@,, - v,,,,,,) , and t,hc blood calcium level, [Ca,]. the
THYROCALCITONIN
AND
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269
REGULATIOX
average controlled signal, in normal, thyroidectomized (TS), and thyroparathyroidectomized (TPTX) animals. In the normal animals, the blood calcium level remained nearly invariant as the intensity of the disturbing signal increased from 0 to 100 mg Ca/day. Even though the slope of the relationship differed very significantly from 0, the steady state error, i.e., the difference between [Ca,] when Ai was zero and when it was large, was less than 3%. The 95% confidence limits of the slope of the relationship for the normal animals were 0.0013 and 0.0055; the 95% confidence limits of the intercept, (i.e., when Si = 0) were 10.63 and 10.88. In the TPTX animals, on the other hand, the blood calcium level rose markedly as the intensity of the disturbing signal increased, the steady state error increasing proportionately with t.he disturbing signal. Both intercepts and slopes of the regression equations describing [Ca,] as a function of Si differed significantly in the TPTX animals from those in the intact rats. It is, therefore, apparent that double ablation removed a control which, when present, almost completely suppressed fhe occurrence of a steady state error in the blood calcium level. 12
- 1 -.
\
a
J,,
E ‘;:-
A
-_--S--A-----.---- l
NORMAL A
A
.
A
- I ._________
---__
B
----------TX n
-1
n
a
t 5.;/
NORMAL [&I
“, 0 m
TPTX RATS (28) [cas] = 5.52 + 0.035
RATS (IOIl = ID.76 + 0 0034
s, s,
4-l 0
10
20
30 s,
40 50 (v, -Vndc,.
60 m g /day)
70
a0
90
3 100
FIG. 3. Relationship between net absorption, the avrragc net disturbing signal and the average controlled signal, blood calcium concentration (SC = U’J - t’“,,“), I:Ca,l, in normal, thyroparathyroidectomized (TPTS), and thyroidectomizcd (TX) :mimals. The TPTS and TX animals received L-thyroxin in their diets. The lenstsquare lines for the normal and TPTX animals are shown with 95% confidence limits. Thp number of rats is indicated in parentheses.
270
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ET
AL.
Results presented in Fig. 3 show that in thyroidectomized animals with parathyroid autografts and supplied with L-thyroxin, the [Ca,] did not increase as Si increased. The dashed line was calculated by the method of least squares; its slope did not differ significantly from zero. The 95% confidence limits of the slope were -0.0079 to -0.020 and of the intercepts 9.38 and 10.62. There was no overlapping with the intercepts of the normal animals, but the absolute difference between the values of the normal and TX animals was small; moreover, the values of the TX animals were lower. Hence, neither the reference value nor the steady state error of the blood calcium level of animals without endogenous thyrocalcitonin differed to any important degree from those of the intact. animals. Calcium Parameters Thyrocalcitonin
in Animals with and without Endogenous
Table 2 lists the terms of the disappearance equations and Tables 3 and 4 the numerical values of the parameters derived (Fig. 2) for animals with and without an endogenous source of thyrocalcitonin. Table 2 shows that the disappearance equations describing the time course of the injected 45Ca in the plasma of the N and TX animals, on the one hand, and of the C and TX animals on the other, did not differ significantly. -4s shown in Table 3, there was no significant difference in the intensity of most parameters in intact and ablated animals. Not’e in particular the similarity of the blood calcium level, [CaS], of the balance, L!., and of urinary excretion, v,. In addition, the rat,es of deposition of calcium in bone, vO+,and of its removal from bone, vO-, were similar. Table 4 shows the results of a similar experiment, but one where both groups of animals carried autogenous parathyroid grafts. Here the tendency was great,er than in the preceding experiment for many parameters of the TX animals to be lower in intensity than those of the controls. As a result, more of the differences assumed st,atistical significance, although none was very large. Comparison of the data obtained on the two control groups (Tables 3 and 4) with the mean and individual lot parameters of 29 ot’her rats on Diet III studied over the past 2 years showed that the values of the t,wo control groups fell at the exkemes of the normal range and that the differences between the two control groups could have resulted from differences among lots of animals (obtained from the same supplier and raised under similar conditions). While it is not permissible to pool the results of the two experiments, it is apparent that removal of the endogenous supply of thyrocalcitonin had either no or smalI effects on the intensity of the steady state parameters of calcium met.abolism.
THYROCALCITONIN
AND
BLOOD
TABLE TERMS
N
Normal TX* Control” TX*
8 8 11 9
FOLLOWING
IV IN.W~TION
OF ““C.%
= AeBal + Becat
A
SE
a
SE
B
SE
b
4.52 4.83 3.62 4.44
0.35 0.33 0.15 0.64
7.74 7.17 8.71 9.05
0.13 0.34 0.34 0.96
0.59 0.67 0.38 0.43
0.05 0.04 0.02 0.03
0.66 0.71 0.67 0.73
a R. = ‘j?, dose/mg Ca and t = days. b TX = tjhyroidectomized and lrthyroxin c With sut,ogenous parathyroid grafts.
EFFECT
271
REGULATION
2
OF DISAPPEARANCE EQUATIONS EQUATION”: R.
Type
CA
OF THYROIDECTOMY
Mean Body weight, gm Plasma Ca, [CaJ, mg/lOO ml Compartment E1, mg Ca Compartment Elp, mg Ca Pool, E, + E’z, mg Ca Rate of exchange between El and E’z, v., mg Ca/day Intake, vi, mg Ca/day Urinary Ca, v,, mg/day Fecal Ca, VP, mg/day Balance, A, mg Ca/day AbFrption, v,, mg Ca/day y0 Absorption, a Fecal endogenous Ca, v.dO, w/day Ca into bone, vO+,mg/day Ca out of bone, v.-, mg/day
(8)” SE
Mean
20.2 88.0 108.2 70.1
239 10.99 18.9 73.1 92.0
189.9
5.4
1.4
0.1
178.2 1.7
133.9
4.0 4.3 4.3 1.6 0.5
11.13
+54.6 65.9 34.5 9.8 58.0 3.4
3.4
3.8
PARAMETERS
TX (8)
2.1 0.07 1.3 6.4 7.4 4.8
247
0.03 0.02 0.03 0.03
in feed.
TABLE 3 (TX) ON CALCIUM
Normal
SE
55.4b
119.8 $56.7 66.2 37.2 7.9
53.9 -2.8
SE
P
5.0 0.17
1.5 5.2 6.3 4.4
0.05
> p > 0.02
0.02
> p > 0.01
8.9
0.4 7.4 5.5 4.9 2.2 0.6 3.3 7.6
Note.-TX animals had parathyroid autografts and received 0.5 mg kthyroxin/kg feed. For further explanation of parameters, see Fig. 2 and text,. R Number of animals shown in parentheses. 6 Significantly lower than corresponding value of normal.
Loading
Experiments
The data displayed in Figs. 4 and 5 indicate that when hypercalcemia was induced by subjecting animals to intraperitoneal calcium loads, their [Ca,] returned to the steady state value more quickly if they had a source of endogenous thyrocalcitonin. Thus, when the calcium load was
272
Body weight, gm Plasma Ca, [Ca,], mg/lOO ml Compartment Er, mg Ca Compartment E’2, mg Ca Pool, El + E’j, mg Ca Rate of exchange between El and E’z, ve, mg Ca/day Intake, vi, mg Ca/day Urinary Ca, vu, mg/day Fecal ca, vF, mg/day Balance, A, mg Ca/day Absorption, v,, mg Calday To Absorption, 01 Fecal endogenous Ca, l)n&,, w/day Ca int)o bone, ZL,+, mg/day Ca out of bone, IL-, mg/day
BRONNER
ET
Cout~rols
(11 I’(
Mean
SE
AL.
P
239 10.49 25.4 128.6 154.0 96.8
1.9 0.10 1.0 6.5 7.0 1.9
“26b 10.95 22.6 108.2 130.8 78.3”
2.7 0.09 2.1 8.4 8.9 2.5
213.7 2.1 159.8 +51.8 69.0 32.3 15.0
4.6 0.2 5.7 5.3 5 4 2.4 0.8
190.4b 3.9* 144.3 +42.2 59.6 31.3 13.5
9.5 0.5 9.1 7.4 7.3 3.5 0.6
0.05
> p > 0.02
3.1 7.8
0.05
> p > 0.02
84.7 32.9
1.4 5.6
a Number of animals shown in parentheses. All grafts. TX animals received 0.5 mg L-thyroxin/kg parameters, see Fig. 2 and text. * Significantly different, from control value.
77.1 34.9
animals had autogenous parathyroid feed. For further explanation
of
4.1 mg/lOO gm body weight, the [Ca,] of the normal animals had returned to its preinjection (or steady state) level in approximately 21/ hours! while that of the TX animals took approximately 3jh hours to return to this level. When the calcium load was doubled (Fig. 5) the delay in both groups was markedly increased and appears approximately to have doubled. Moreover, the difference in peak values was also increased, from 3.3 (164.3 I!Z 0.8 - 161.0 t 0.7, p < 0.01) to 13.1 (225.7 Z!Z 4.2 - 212.6 rt 3.6, p _< 0.02), the TX animals in both instances reaching somewhat higher relative peaks. This positive effect can also be taken as evidence that all TX animals responded to the absence of thyrocalcitonin. Moreover, this response appeared relatively independent of age and weight, as the animals whose responses are displayed in Fig. 4 were heavier and older than those whose responses are shown in Fig. 5. The data shown in Table 5 give added evidence of the ability of the TX animals to respond to exogenous rat thyrocalcitonin. The findings set, out in Table 6 indicate that the tibias and fibulas of bot,h groups of animals had the same phosphorus content, but that the
THYROCALCITONIN
AND
BLOOD
CA
bones of the normal animals contained slightly, but calcium than those of the TX animals and had higher small differences in calcium content are consistent with values of the pool, of vuc, and of vu+, found in either TABLE RESPONSE
Fraction of gland=
OF THYROIDECTOMIZED
N
13
5
3
4
RATS
273
REGULATIOK
significantly mow Ca/P ratios. Thcee the slightly higher the normal or the
5 TO EXOGENOUS
THYROCALCITOSIN
Mean percent drop in (SE) 1CaJ
Mean weight, gm (SE) 275(5.41 282(10.3)
5.0 14.0
(1.7) (1.7)
Note.-The animals had previously been used in loading experiments (Fig. 5). They were thyroidectomized and bore parathyroid autografts. a Fraction of gland refers to a fraction of rat gland, extracted according t’o Hirsch el al. (3). The animals were bled from the tail before and 60 minutes after a subcut,aneous injection of thyrocalcitonin in 0.5 ml.
I , 0 ILOAD
30
60
120 Time (ml 1
180
240
PIG. 4. The blood calcium level, l’Ca.1, of thyroidectomized (TX) and control rats as a function of time, following an intraperitoneal injection of 4.1 mg Ca (as Ca gluconate) per 100 gm body weight. Both groups of animals carried parathyroid autografts and had been maintained on Diet II (Table 1) until 24 hours before each loading experiment, when they were placed on a very low Ca (<0.02%) diet. Means and standard errors for each group at each time were calculated from the percent rise of individual animals on successive experiments.
[Ca,], mg/lOO ml Type
N
Base
(SE)
Peak
(SE)
TX Control
10 17
10.54 10.26
(0.20) (0.07)
17.32 16.52
(0 08) (0.07)
274
BRONNER
ET
TABLE
AL.
6
COMPARISON OF CA AND P CONTENT OF TIBIAS ASD FIBULAH OF NORMAL AND THYRO~DECTOMIZED (TX) RATS
Calcium
Weight,
Phosphorus
gm (SE)
Total mg (SE)
M&m (SE)
12
0.536 (0.013)
11
0.527
78.8 (1.5) 74.48
147.6 (2.0) 141.3”
Group
12:
Normal TX
(0.007)
(1.2)
(2.4)
Ca/P (SE)
Total mg Mg/gm (SE) (SE) 34.7 (0.5) 33.9 (0.4)
64.4 (0.8) 65.1 (1.1)
2.27 (0.015) 2.19* (0.013)
Note.-The animals had previously been used in loading experiment’s (Fig. rj). The TX animals bore parathyroid autografts. a Significantly lower than normal (0.05 > p > 0.02). b Significantly lower than normal (p < 0.01).
I I 0
I
30
60
LOfnO
120 itme (Inn)
180
240
5. The blood calcium level, CCa.1, of thyroidectomized rats with parsthyroid autografts (TX) and of intact animals (N) as a function of time, following an intraperitoneal injection of 8.2 mg Ca (as Ca gluconate) per 199 gm body weight. Both groups were maintained on Diet II (Table 1) throughout the experiment. Means aad standard errors for each group at each time were calculated from the percent rise of individual animals in three successive experiments. FIG.
[Ca.], mg/lOO ml
Type
N
Base
(SW
Peali
(SE)
TX Normal
27 25
10.62 10.89
(0.12) (0.09)
23.88 23.12
(0.32) (0.33)
TEYRCCALCITONIN
AND
BLOOD
CA
REGULATION
275
control animals, as compared with the TX animals (Tables 3 and 4). Whether these small differences can be attributed to differences in parathyroid function that may have resulted from differences in the efficiency of the autograft or whether they were the direct result of the absence of thyrocalcitonin cannot be determined without measurement of the steady state parathyroid hormone level. DISCUSSION
On the basis of the above results it is possible to classify the action of endogenous thyrocalcitonin with respect to the three modes of action outlined in the introduction as follows. (a) If thyrocalcitonin acted on the reference value of the blood calcium, the reference value should have changed when the endogenous supply of the polypeptide was removed. It can be seen from the data reported here that the [Ca,] values of normal and TX animals were similar when net absorption, Si, was near zero. Thus, in the two groups of animals used in the loading experiments (Figs. 4 and 5) and in the group whose calcium parameters are shown in Table 3, there were no significant differences in [Ca,] between animals wit,h and wit.hout an endogenous source of thyrocalcitonin. In the case of the animals whose data are displayed in Fig. 3 and of those whose calcium parameters are shown in Table 4, the differences were statistically significant. However, the differences were small and their direction was not the same and therefore, it appears that thyrocalcitonin does not act on the reference value of the blood calcium. (b) If thyrocalcitonin were a component of the integral control, removal should affect the steady state error of the blood calcium, as well as ot,her steady state parameters. Figure 3 indicates that the response of thyroidectomized animals to an increase in Si was similar to that of normal rats. This confirms and extends the observation by Talmage et al. (19) on the similarity of the blood calcium values of animals with a,nd without endogenous thyrocaIcitonin on a normal calcium intake (see also 20). If the controlled signal [Ca,] is unchanged by the removal of thyrocalcitonin, the net controlling signal, A, should also be unchanged. This was indeed the case (Tables 3 and 4). Failure to affect h does not preclude alterations in the constituent signals, v,+ and z)~-. However, these also did not change markedly. That z)~+is normal in thyroidectomieed animals supplied with L-thyroxin has also been reported by Payne and Sansom (21), who found that in two thyroidectomixed goats on 1 mg thyroxin,/day the values for A (the rate of accretion, equivalent to v,+)
276
BRONNER
ET
AL.
and E (the size of the exchangeable pool, equivalent to E, + Etz) were normal or slightly higher t,han normal. It might be argued that the values of uuo-arp lower in these animals than consistent with normal growth and bone remodeling. The problem inherent in the analysis has recently been discussed at length (14). However, even if the values for u,- were erroneously low, the failure to observe differences in A and in ZJ,+,two independent set’sof measurements, makes it unlikely that removal of endogenous thyrocalcitonin affected v,- =
(A -
vo+) .
In the analysis of calcium homeostasis (1) the rate of calcium a,bsorption, v,, has been considered an acute disturbing signal, even though it was recognized that this parameter may be subject to long-term regulation (22). It, is, therefore, worth noting that calcium absorption appeared unaffected by the removal of the source of thyrocalcitonin (v, and cy, Tables 3 and 4). Whereas the administration of thyrocalcitonin does not necessarily result in the opposite of what happens when the source of endogenous thyrocalcitonin is removed, it is interesting to note that Wase et al. (23) report that, thyrocalcitonin was without effect on calcium absorption; on the other hand, Milhaud and Moukhtar (24) observed an increase in absorption following administration of a long-acting preparation. In the normal animal, the kidney, a second controlling system, has been ignored (1) becauseurinary calcium excretion is so small in relation to t,he balance, the major controlling signal. This situation was not changed by the removal of t’he source of thyrocalcitonin, as the ratio of v,/A was only 0.03 (Table 3) and 0.09 (Table 4) in the TAX animals, compared with 0.03 and 0.04 in the normal and control animals, respectively. As none of the st.eady state parameters of calcium metabolism was changed appreciably by removal of the source of thyrocalcitonin (Tables 3 and 4), it may be concluded that thyrocalcitonin does not constitute a major portion of the integral control. Removal of the parathyroids, on the other hand, resulted in a large drop of the reference value, from 10.74 to 5.52 mg Ca/lOO ml (Fig. 3), and in a rise of the controlled signal [Ca,], when the disturbing signal Xi rose. This indicates that the parathyroids are responsible for the integral control. Moreover, the fact that a rise in Si produced a linear rise in [Ca,] (see also 25) can be considered support for the existence of a proportional control of [Ca,] in TPTX animals. While removal of the source of thyrocalcitonin is without obvious effect on steady-state parameters in animals with functioning parat’hyroids, it has been reported (19) that the [Ca,] is lower in parathyroidectJomized than in TPTX animals. We have maintained these two groups of ablated
THYROCALCITONIN
AND
BLOOD
CA
277
RFxXJLATIOi’G
animals for many weeks and have not observed differences in either their fasting or their peak blood calcium levels after ingestion of high-calcium diets (Table 7). This suggests that the same differences in the regulation of [Ca,] that have been observed to exist between TX and TPTX animals (Fig. 3) may also exist between TX and PTX animals. BLOOD
CALCIUM
TABLE 7 LEVELS [CA,] OF THYROPARATHYROIDECTOMIZED AND PARATHYROIDECTOMIZED (PTX) RATS [Ca,ly
mg Ca/lOO
ml plasma
Fastming Diet III
Animals
(TPTX)
Peak
(N)
Mean
SE
Mean
SE
TPTX (6) PTX (4)
5.88 5.68
0.24 0.40
6.75 6.67
0.23 0.27
Time of peak (hours) 4 4
Note.-The animals were trained to eat their daily ration in 2 hours and blood samples were taken just before (“fasting”) and at varying periods after feeding. The values reported are the fasting and the peak values. TPTX animals received L-thyrosin in their diets.
(c) Thus, if thyrocalcitonin plays a role in calcium homeostasis, we are left with the third alternative, that it plays only a subsidiary role, such as reducing the delay with which the controlled signal returns to its reference value following an acute disturbance. This is brought out by the loading experiments (Figs. 4 and 5) which indicate that t,he overshoot of the blood calcium level induced by an intraperitoneal injection of calcium gluconate subsided more slowly in animals deprived of their endogenous supply of thyrocalcitonin than in normal controls (see also 19, 26). It is therefore apparent that the presence of thyrocalcitonin improvcn the effectiveness of the control system in terms of the rate of return. The physiological significance of a shorter delay in the return to the steady state level of plasma calcium requires further study. SUMMARY
The role of thyrocalcitonin in calcium homeostasis of rats has been studied in three types of experiments. (a) Measurement of the effect on the blood calcium level, [Ca,], of varying net calcium absorption in normal (N), thyroparathyroidectomized (TPTX) and thyroidectomized animals with autogenous parathyroid transplants (TX). These studies showed that the values of [Ca,] were similar in N and TX animals at all levels of net absorption over a
278
BRONNEB ET AL.
nearly lOO-fold range. Moreover, the error, i.e., the difference between [Ca,] when net absorption was zero and when it was verjr high, was close to zero. In TPTX animals, on the other hand, the values for [Ca,] were markedly lower than in the other two groups at all levels of net absorption and the error of [Ca,] increased linearly and proportionately with increased absorption. (b) Determination of the intensity of the steady-state parameters of calcium metabolism (pool size, rates of Ca absorption, deposition in and removal from bone, urinary output) in N, in controls with parathyroid autografts (C), and in TX failed to reveal any important differences between either N and TX or C and TX. (c) Measurement of the return of [Ca,] to its base value following an acute calcium surcharge by intraperitoneal injection in N, C, and in TX animals indicated a greater delay in the return in the TX than in the C or N animals. It is concluded that thyrocalcitonin is not involved in the regulation of the steady state value of the blood calcium or of the steady-state parameters of calcium metabolism, including turnover and amount of bone calcium. Rather, thyrocalcitonin appears to refine the action of parathyroid hormone in terms of its effects on bone, the primary regulator of blood calcium. ACKNOWLEDGMENTS We thank Dr. J.-P. Aubert for valuable discussions and critique of the manllscript. Lawrence Boram prepared histological sections. Dr. R. C. Kelsay and Mrs. Kay Shotts helped with the statistical analyses. REFERENCES 1. AUBERT, 6.-P., AND BRONNER, F., Biophys. J. 5, 349 (1965). 2. FOSTER, G. V., BAGHDIANTZ, A., KUMAR, M. A., SLACK, E., SOLIMAN, H. A., .~ND MACINTYRE, I., Nature 202, 1303 (1964). 3. HIRSCH, P. F., VOELREL, E. F., AND MUNSON, P. L., Science 146, 412 (1964). 4. TENENHOUSE, A., ARNAUD, C., AND RASMUSSEN, H., Proc. Natl. Acad. Sci. (USA) 53, 818 (1965). 5. MUNSON, P. L., POTTS, J. T., JR., REISFELD, R. A., COOPER, C. W., AND VOELKEL, E. F., Science 15% 425 (1966). 6. O’RIORDAN, J. L. H., TASEJIAN, A. H., JH., MUNSON, P. L.. CONDLIFFF,,P. G., AND AURBACH, G. D., Science l!%, 885 (1966). 7. PUTTER, J., KACZKA, E. A., HARMAN, A. E., RICKES, E. L., KEMPF, A. J., CHAIET, L., ROTHROCK, J. W., WASE, A. W., AND WOLF, F. ‘J.. J. .4m. Chem. Sot. 89, 5301 (1967). 8. COPP, D. H., CAMERON, E. C., CHANCY, 13. A., IX~VIDSON, A. C. F., AND HENZE, K. ‘4., Endocrinology 70, 638 (1962). 9. ALIAPOULIOS, &I. A., GOLDHABER, P., .4xD MUNSON, P. L., &knre 151, 330 (1966). 10. FRIEDMAN, J., .~ND RAISZ. L. G., Science 150, 1465 (1965).
THYROCALCITONIN
11.
MILIIAUD,
(l-‘ari.s) 12.
13. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25. 26.
PERAULT,
A.-M.,
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MOUKIITAR,
CA
M.
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S., Compl.
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G., MOUHHTAR, M. S., CHERIAN, G., .4ND PERAliLT, A.-hf., corn)?/. Rrnrl. Sci. (Paris) 262,511 (1966). BRONNER, F., AND AUBERT, J.-P., Am. J. Physiol. 209,887 (1065). BRONNER, F., Trans. N. Y. Acad. Sci. (Ser. II) 29,502 (1967). AUBERT, J.-P., CHERIAN, A. G., MOUKHTAR, M. S., AND MILH.~UD, (;., Hio~h~t~?. Pharmacol. 13, 31 (1964). SAMMON, P. J., BOR~M, L., MOODY, C., AND BRONNER, F., Federation Proc. 25, 495 (1966). CHEN, P. S., JR., TORIBARA, T. Y., AND WAHNER, H., Anal. Chem. 28, 1756 (1956). BRAY, G. A., Anat. Biochem. 1,279 (1960). TALMAGE, R. V., NEUENSCHWANDER, J., AND KFLAINTZ, L., Endocrinology 76, 103 (1965). BRONNER, F., SAMMON, P. J., NICHOLS, C., ST.4CEy, R. E.. .ISD SHAH, 13. G.. Proc. Third Pamthyroid Conference, Mont Gabriel, Canada (1967). (in press). PAYNE, J. M., AND SANSOM, B. F., J. Physiol. (London) 184, 433 (1966). BRONNER, F., in “Mineral Metabolism-An Advanced Treatise” (C. L. Comar and Felix Bronner, eds.), pp. 314414. Vol. 2A. Academic Press, Kcw York (1964). WASE, A. W., PETERSON, A., RICKES, E., AND SOLO~SKI, J., Endocrinology 79, 687 ( 1966). MILHAUD, G., AND MouKHTAR, M. S., Proc. Sot. Exptl. Biol. Med., 123, 207 (1966). COPP, D. H., in “Bone Biodynamics” (H. M. Frost, ed.), p. 441. Lit.tlr, Brown, Boston, Massachusetts, 1964. CARE, A. D., ~XINCAN, T., AND WEBSTER, D., .l. Endocrinol. 34, xi ( 1966). MILHAUD,
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