Journal of Clinical InvestigationVol. 45, No. 12, 1966

Further Studies on the Interrelationship betweenParathyroid Hormone and Vitamin D *

CLAUDEARNAUD,HOWARDRASMUSSEN,tANDCONSTANTINEANASTt(From the Department of Biochemistry, University of Pennsylvania School of Medicine,

Philadelphia, Pa., and the Department of Pediatrics, University of Missouri Schoolof Medicine, Columbia, Mo.)

Calcium homeostasis is achieved by the inter-play of a number of humoral agents (1). Ofcentral importance are the activities of parathyroidhormone (PTH) and of vitamin D. This wasfully appreciated 35 years ago (2), but in spiteof considerable effort, the nature of the relation-ship remains controversial. Recent opinion hasvaried from that of a complete dependence ofhormone action upon the presence of vitamin D(3) to that of a complete independence of action(4, 5). In our initial studies of this problem, weconcluded that in the absence of vitamin D aphysiologic concentration of parathyroid hormoneexerted its usual effect upon the renal excretionof phosphate but was unable to mobilize calciumfrom bone (6).

Since these studies were published, two groupsof investigators have challenged our conclusions(7, 4). Harrison and Harrison reaffirmed theirprevious observations (7) and concluded, as theyhad before, that all of the actions of parathyroidhormone depended upon vitamin D. At the oppo-site extreme, Ney and associates concluded that allof the effects of parathyroid hormone are exertedin the D-deficient organism. These differencesare difficult to explain. However, both studiesdiffered from ours in several respects. In thestudy of Harrison and Harrison (7), no attemptwas made to define or control the level of endoge-nous parathyroid hormone, although we found this

* Submitted for publication April 8, 1966; acceptedSeptember 7, 1966.

Presented in part at the 47th Annual Meeting of theEndocrine Society, June 1965.

Supported by grants from the National Institutes ofHealth (AM-09650, AM-09494-01, and AM-01351-08).

tAddress requests for reprints to Dr. Howard Ras-mussen, Dept. of Biochemistry, University of Pennsyl-vania School of Medicine, Philadelphia, Pa.

t Dept. of Pediatrics, University of Missouri, Columbia,Mo.

to be of prime importance (6). In the work doneby Ney and co-workers a different species wasemployed, and the degree of D deficiency wasassessed by different criteria (4). Nevertheless,both studies raised points that seemed worthy offurther examination.

The development of a technique for study ofthe response of the conscious rat to the long-termperfusion of hormones (8) has allowed us to ex-amine several aspects of this problem more di-rectly. The experimental design permitted theexclusion of altered endogenous secretion of para-thyroid hormone or thyrocalcitonin as variables.The results reaffirm our previous conclusion andexplain, in large part, the previous disparity ofopinion regarding the nature of the parathyroidhormone vitamin D interrelationship.

Methods

Vitamin D deficiency was produced in young growingHoltzman rats as previously described (9). Control ani-mals received the same purified diet but were given 75 Uof vitamin D3 in cottonseed oil by oral intubation dailyfor the week before study. Urinary calcium and phos-phate were measured simultaneously with a TechniconAutoanalyzer (6). Plasma calcium and phosphate weremeasured by the same means except for a manifold modi-fication that allowed for a microdetermination on 50 to100 Id of plasma. Magnesium in the urine was measuredby absorption flame spectrophotometry with a Perkin-Elmer instrument (10). Urinary sodium and potassiumwere measured by emission spectrophotometry with aZeiss instrument. Urinary inulin was measured by anautomated procedure with a Technicon Autoanalyzer (11).

The technique of long-term perfusion of the rat wasdeveloped as a modification of that described by Cotlove(12). With rats under ether anesthesia, a no. 240polyethylene catheter 1 was placed in the urinary bladder,a no. 50 polyethylene catheter 1 was placed in the ex-ternal jugular vein, and the parathyroid or both thy-roid and parathyroid glands were removed by surgicalmeans. In some instances parathyroidectomy was per-

' Clay-Adams Co., New York, N. Y.

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C. ARNAUD, H. RASMUSSEN,AND C. ANAST

TABLE I

Electrolyte balance during period of control infusion

No. ofexperi-ments Na K Mg Ca

/Amoles/hr pmoles/hr jumoles/hr itmoles/hrPTX*

+D 6 +38 -4 4t -11 ±3 +3± 0.5 +10 ± 2-D 5 +26 ± 4 -22 ± 4 +1 ± 0.25 +14 ± 3

High Ca-D 4 + 8 ±1

Sham+D 4 +28 ± 5 -16 ± 4 -2 ±t 0.5 + 8 ± 2-D 4 -15 ±3 - 9 ±2 +4±0.5 +13 ±3

* PTX = parathyroidectomized rats; high Ca = rats fed a highcalcium vitamin D-deficient diet; sham = sham-operated control rats.

t Mean ± standard deviation.

formed with an electric cautery. After surgery, the ani-mal was placed in a cage similar to that designed byCotlove (12) with the jugular vein catheter exitingthrough the top and the bladder catheter and hind legsthrough the rectangular opening in the bottom. Thejugular vein catheter was connected to a peristaltic pumpthat delivered 3.0 to 4.0 ml per hour. The animals wereperfused at this rate with a solution containing 4%o dex-trose, 5 mMcalcium chloride, 5 mMmagnesium chloride,20 mMsodium chloride, 2.5 mMpotassium chloride, and20 mg per L inulin. Urine was collected at half-hour or2-hour intervals. The animals were perfused for 16 hoursafter surgery before measurements of urinary volume anddeterminations of electrolyte content were begun. Collec-tions were then made during a control period of at least10 hours before parathyroid hormone (5 ,ug per hour) wasadded to the perfusate. Hormone infusion was usuallycontinued for 20 hours, and then a period without hor-mone was recorded. Weobtained blood samples by car-diac puncture before surgery in order to measure plasmacalcium and phosphate. This was done to be certain ofthe degree of D deficiency or D repletion. Rats wereconsidered D deficient only if they had stopped gainingweight after 3 weeks of vitamin D-free diet and if theconcentration of calcium in the plasma (plasma calcium)was below 6.5 mg per 100 ml. The mean plasma calciumvalue of the D-fed controls was 10.8 ± 0.4 mg per 100ml before surgery. One group of animals was raised ona high calcium vitamin D-free diet, and their mean pre-operative plasma calcium was 9.6 + 0.3 mg per 100 ml.These animals did not stop gaining weight but were con-sidered vitamin D deficient because littermates raised ona normal calcium D-deficient diet in adjacent individualcages exhibited classic disturbances in growth and plasmacalcium levels. The number of animals studied in eachgroup is indicated in the Figure legends; standard statis-tical methods were used to obtain values for standarderror of the mean and significance of the differencesbetween means (13).

The purified parathyroid hormone used was preparedby the method of Hawker, Glass, and Rasmussen (14).

Results

Initially, the reproducibility of the perfusiontechnique was established. Two problems of im-mediate concern were the stability of the base-linerates of electrolyte excretion and the balance offluid and electrolytes.

Wefound that 10 to 15 hours of perfusion withthe control solution was necessary before the ratesof excretion of the various electrolytes becamestable. Therefore, in all experiments to be re-ported, the animals were perfused for at least 15hours before urine collections were begun. Col-lections over the ensuing 10 hours constituted thecontrol period for each experiment.

After the postsurgical equilibration period, therates of urine flow were from 0.05 to 0.1 mlper hour less than the measured rates of perfusateinfusion in all the animals. However, there ap-peared to be no significant fluid retention. Theanimals weighed 3 to 4 g less after a 50-hourexperiment than before surgery. Postmortem ex-amination revealed no evidence of abnormal fluid

VITAMIN D DEFICIENTPTH 5%pg/HR I

_ ^A ... ,,,..::% >

...494

1 1 I I I I

20 30 0 10 20 30TIME-HOURS

FIG. 1. RATES OF EXCRETION OF INULIN, PHOSPHATE,CALCIUM, MAGNESIUM, POTASSIUM, AND SODIUM BEFOREAND DURING THE INFUSION OF PARATHYROID HORMONE(PTH). Five ,ug per hour PTHwas infused into a D-fed(left) and a D-deficient (right) parathyroidectomizedrat maintained on a constant infusion of glucose andelectrolytes.

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retention, and the rates of infusion were less thanthe maximal rates the rats could tolerate. Ratesof urine flow up to 5.0 to 6.0 ml per hour couldbe produced by increasing the rate of infusion.Because of these facts, it was concluded that theslight discrepancies between rates of infusion andrates of urine flow were accounted for by insen-sible water loss and possibly by slight evaporationfrom the urine samples when they were in thefraction collector. It should be noted, however,that there was usually sodium retention and alwayspotassium loss during the control period (TableI), possibly as a result of adrenal hyperactivitysecondary to the stress of surgery and experi-mentation. In all instances in which sodiumretention occurred, it was followed by sodiumdiuresis during hormone infusion (Figures 1 and2). Magnesium excretion balanced intake, butthere was always calcium retention that was moremarked in the D-deficient than in the D-fed ani-mals (Table I).

Our initial studies were carried out in para-thyroidectomized animals. Two typical experi-ments are shown in Figure 1. In the rat fedvitamin D, the infusion of parathyroid hormoneat a rate of 5 Fug per hour led to a prompt increasein the rate of excretion of phosphate, potassium,and sodium, and an initial fall in magnesium andcalcium excretion (Figure 1). The changes inrates of magnesium and potassium excretion werenot sustained but returned to baseline or slightlygreater values after 4 to 6 hours of perfusion.In contrast, the initial phosphaturia was followedby a sustained phosphate excretion throughoutthe course of the perfusion. Calcium excretion,after decreasing initially, returned to the controlrate and then rose, after 15 to 22 hours of constanthormone perfusion, to a rate considerably higherthan that observed initially. Changes in the rateof sodium excretion were more variable but ingeneral paralleled those of phosphate.

In contrast, the infusion of hormone at the samerate into a D-deficient parathyroidectomized ratled to the initial changes in electrolyte excretionthat were qualitatively similar to those seen in theD-fed animal, i.e., an increased rate of sodium,potassium, and phosphate excretion, and a fallin excretion of calcium and magnesium. However,a different pattern of response emerged during thelater hours of perfusion (Figure 1). The rate of

TIME, HOURS

FIG. 2. PATTERNSOF URINARY ELECTROLYTEEXCRETIONBEFORE, DURING, ANDAFTER THE INFUSION OF PARATHYROID

HORMONE, 5 ,uG PER HOUR, IN PARATHYROIDECTOMIZED(PTrX) AND SHAM-OPERATEDD-DEFICIENT AND D-FED RATS.All animals were maintained on a normal calcium andphosphate diet, except the third group of D-deficientanimals, which were maintained on a high calcium normalphosphate diet. The shaded areas represent the valuesduring hormone infusion. Number of animals in eachgroup: +D, 6; -D, 5; -D high calcium diet, 4; +Dsham, 4; -D sham, 4.

phosphate excretion fell, after the initial increase,to rates only slightly greater than the control rates;the initial decrease in rate of calcium excretion wasfollowed by a gradual return to a rate nearly equalto, but never in excess of, the control. Also,sodium diuresis was not sustained as in the D-fedanimal.

The rates of inulin excretion were also mea-sured in these animals. There was no significantchange in the excretion of constantly infusedinulin during hormone infusion in either animal(Figure 1). Rates of inulin excretion were mea-sured in six additional animals, and in all instancesthe infusion of parathyroid hormone at a rate of5 ug per hour had no effect upon this rate.

The same protocol was followed in six otherD-fed and five other D-deficient animals. Theresults are recorded as the means of the values

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TABLE II

Comparison of responses to parathyroid hormone infusion in parathyroidectomizedvitamin D-fed and D-deficient rats

Urinary electrolyte excretion in 5 hours

Animal* HP04- Ca++ Mg K Na

jumoles jsmoles pmoles Amoles Ismoles+D 24.0 -± 4.2 36.0 it 4.4 69.0 ±- 5.2 108 ±t 10 150 ±- 13-D Ct 7.5 it 3.0$ 27.0 it 3.4 75.0 -- 5.8 150 it 11$ 180 -± 17- D(Ca) 6.1 i 2.6T 34.0 It 4.7

+D 258.0 i 20 7.5 it 2.3 48.0 it 5.8 180 it 12 330 it 12-D 5 120.0 ±i 9$ 3.6 ±t 0.9$ 33.0 4t 4.9§ 165 ±t 11 390 it 14t- D(Ca) 204.0 -- 12§ 8.0 -- 2.5

+D 188.0 it 15 28.0 -- 4.3 81.6 it 6.3 236 -- 15 414 It 16-D 10 65.0 ±t 8$ 17.0 ±- 5.2$ 73.0 ±t 6.2 124 ±t 12$ 580 ±t 23T- D(Ca) 225.0 -- 17 12.0 -- 3.2$

+D 151.0 ± 17 90.3 ±t 8.8 89.4 ±t 7.0 179 ±t 8 571 ±- 29- D 15 44.0 ±t 9t 22.0 ±t 4.0t 73.0 -- 5.8 124 ±t 12$ 521 ±t 20§- D(Ca) 143.0 ±t 16 23.0 -- 5.1$

+D 138.0 -- 16 107.7 -- 9.0 83.2 ±- 5.8 155 ±- 13 487 -- 26-D 20 44.0 i11t 22.0 ±4i 4.3t 69.0 ± 7.1§ 132 ± 14§ 430 ± 12§-D(Ca) 99.0 ± 17§ 23.0 ± 6.6$

+D 30.0 ± 4.2 127.6 ± 11.0 78.2 ± 6.0 55 ± 7 77 ±t 11-D PH 5.0 2.2$ 42.6 ± 7.1t 91.3 ± 8.3 40 ± 8 208 ± 15$-D(Ca) 4.8 ± 2.4$ 51.7 ± 9.3t

* +D = 6 vitamin D-fed controls; -D = 5 vitamin D-deficient rats on normal calcium intake; -D(Ca) = 4vitamin D-deficient rats on high calcium intake.

t C = excretion during 5-hour control period; 5, 10, 15, 20 = excretion during first 5, 10, 15, 20 hours of hormoneinfusion; and PH = excretion during S hours after hormone infusion.

$ p < 0.001 when values (mean ± standard error) between +D and - D animals are compared.§ p < 0.025 when values (mean ± standard error) between +D and -D animals are compared.

for 5-hour periods before, during, and after hor-mone perfusion in Figure 2 (columns 1 and 2).Also shown are the responses of four sham-operated animals (Figure 2, columns 4 and 5),and the changes in urinary calcium and phosphateexcretion on a high calcium, normal phosphate' diet(Figure 2, column 3). Table II summarizes theelectrolyte data and gives the results of the sta-tistical evaluation of the differences between themean values for the various 5-hour periods be-tween D-fed and D-deficient animals. Table IIIshows the mean plasma calcium and phosphatevalues of animals in each group before surgery,during the period of control perfusion, and after12 hours of hormone infusion. All three measure-ments could not be made in all animals in eachgroup. Because of the small number of valuesin some groups, the mean and range in each groupare recorded rather than the standard error.

The plasma values and the changes in electrolyteexcretion were highly reproducible from one ani-mal to the next within a given group but varied

considerably from group to group. Early in thecourse of this project, a single bloodletting fre-quently led to a significant decrease in the urinaryoutput during the next 0.5-hour period, but theurinary output returned to the previous levelthereafter. After further experience, it was pos-sible to obtain three blood samples from a singleanimal within a 24-hour period without greatlyaltering the rate of urine flow. The number ofmeasurements within each group was too few toobtain precise statements of significance, but asseen in Table III, the results were clear. Therewas no significant rise in plasma calcium in theD-deficient animals of any group after hormoneinfusion, although in each instance there was atendency for the value to rise slightly. On theother hand, there was a rise in plasma calcium inthe D-fed animals (Table III). The plasma phos-phate decreased in all animals except in the intactD-deficient ones (Table III).

The slight rise in plasma calcium seen in allD-deficient animals was associated with definite

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TABLE III

Plasma calcium and phosphate values in rats before parathyroidectomy or thyroparathyroidectomy,during the control infusion, and after 12 hours of hormone infusion

Plasma calcium Plasma P04

Status Vitamin D No. rats Pre-op.* Control inf.t PTHJ Pre-op.* Control infQt PTHt

mg/100 ml mg/100 mlPTX + 4 11.1 i 0.4 8.4 ± 0.4 13.1 4L 0.6 11.3 i 0.3 10.2 ± 0.5 7.4 ± 0.4PTX - 4 6.0 ± 0.2 7.0 ± 0.4 8.1 i 0.3 11.4 i 0.3 10.8 ± 0.3 8.4 i 0.3

PTX§ - 4 9.6 ± 0.3 8.3 ± 0.3 8.9 i 0.4 9.3 i 0.4 8.9 i 0.3 6.2 ± 0.5High Ca

Intact + 3 11.4 A 0.3 10.2 i 0.3 12.2 i 0.3 11.3 ± 0.4 10.7 i 0.4 8.6 i 0.1Intact - 2 6.1 4± 0.3 7.1 i 0.2 7.2 ± 0.2 11.8 ± 0.3 10.8 ± 0.4 10.4 ± 0.3

TXPTXII + 4 10.8 i 0.3 8.8 ± 0.3 18.2 i 1.4 11.2 ± 0.4 11.0 ± 0.2 7.2 i 0.4TXPTXf - 4 6.1 i 0.2 9.2 ± 0.4 9.8 i 0.4 10.8 A 0.4 10.6 i 0.3 7.3 ± 0.5

* Values before surgery.t Values after surgery and at least 10 to 12 hours of perfusion with control solution.t Values after 12 hours of parathyroid hormone (PTH) infusion.§ Maintained on a high calcium vitamin D-deficient diet.

Thyroparathyroidectomized.¶ These D-deficient animals were perfused with a solution containing 25 M CaCl2 rather than the usual 5 mM

concentration;

renal retention of calcium during hormone infu-sion and a slight calcium diuresis after the hor-mone infusion stopped (Figure 2, columns 2 and3). This pattern was also seen in the intactD-fed animals, but in the D-fed parathyroidec-tomized animals the initial retention of calciumwas followed by a marked diuresis.

It is noteworthy that the magnitude of the initial(first 5 hours) phosphaturia seen in the D-defi-cient animals (Figure 2, column 2) was approxi-mately 50%o (p < 0.001) and the potassium di-uresis 20%o of that seen in the D-fed animals(Figure 2, column 1). Conversely, the initialmagnesium retention was twice as great in theD-deficient animals as in the D-fed (p < 0.025)(Figure 2, columns 1 and 2). Also, the initialmagnesium retention in the D-fed animals wasfollowed by a diuresis of magnesium during thelater hours of hormone infusion, whereas this didnot occur in the D-deficient animals although thelatter had a magnesium diuresis after the cessationof hormone infusion.

If D-deficient animals were maintained on ahigh calcium diet during the entire period beforestudy, their initial phosphaturia during the first10 hours of hormone perfusion was considerablygreater (p < 0.01) than that seen in D-deficientanimals raised on a normal calcium diet (Figure2, columns 2 and 3), and was approximately 80%o

of that seen in the D-fed animals (Figure 2, col-umns 1 and 3). The results in Figure 2, column3 represent the mean values of four animals.

Animals subjected to a sham operation ratherthan parathyroidectomy responded differently thandid the parathyroidectomized animals. Those fedvitamin D ('Figure 2, column 4) had qualitativelysimilar initial changes (compared to D-fed para-thyroidectomized animals) in rates of electrolyteexcretion. However, the magnitude of the phos-phaturia was significantly less (p < 0.01) thanthat seen in parathyroidectomized animals, andthe marked secondary rise in rate of calciumexcretion, normally seen in the parathyroidec-tomized rats, was not seen in the sham-operatedone. However, after the cessation of hormoneinfusion in these latter animals, a striking calciumdiuresis was always observed. In contrast, D-de-ficient sham-operated animals had no significantchange in potassium or phosphate excretion duringhormone infusion although the rates of excretionof sodium, calcium, and magnesium all fell sig-nificantly (p < 0.01) and rose again only uponcessation of hormone infusion.

The lack of both a rise in plasma calcium(Table III) and a secondary calcium diuresis(Figures 1 and 2, columns 2 and 3) in the D-defi-cient parathyroidectomized animals was inter-preted to mean that the parathyroid hormone was

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TABLE IV

Phosphate balances in D-deficient ratsgiven parathyroid hormone*

Total Excessdecrease urinary

Rats No. Diet ECF HPO4t HPO4-

J.molesPTX 4 Normal Ca 24 150tPTX 3 High Ca 27 330tTXPTX 4 Normal Ca 27 128t

* Calculated from the data in Figure 2, columns 2 and 3;Figure 3, column 2; and Tables II and III.

t ECF = extracellular fluid.t Significantly greater (p < 0.001) than the total de-

crease in ECF phosphate.

unable to mobilize calcium from bone. If this wasthe case, it could be argued that it would also beunable to mobilize phosphate from bone. Thequestion then arose as to the source of the urinaryphosphate seen in the D-deficient animals (Figure2, columns 2 and 3). From the data in Figure 2(columns 2 and 3) and Table III, it was possibleto calculate the excess phosphate appearing in theurine of the D-deficient animals during the first10 hours of hormone infusion. This was done bysubtracting the rates during the control periodfrom those seen in the first two 5-hour periodsof hormone infusion. The difference was takenas a measure of phosphate loss and recorded inTable IV as excess urinary HPO4=. The total lossof phosphate from plasma and extracellular fluidswas calculated from the differences in the plasmaphosphate concentrations seen during the controlinfusion and after 12 hours of hormone infusion(Table I), assuming the plasma and the extra-cellular fluid volume to be 20 % of the bodyweight. These values are recorded in Table IVas total decrease in extracellular fluid (ECF)HP04=. In both instances, the phosphate appear-ing in the urine was considerably in excess of thatwhich disappeared from the extracellular fluids(p < 0.001). This was true even if the totalECF volume was as great as 30% of the bodyweight.

Because of the recent evidence that thyrocalci-tonin suppresses the action of parathyroid hor-mone upon bone resorption (15), it was consideredimportant to determine whether thyrocalcitoninwas involved in the altered responses of the D-de-ficient animals to parathyroid hormone infusion.For this purpose, four D-deficient and four D-fedanimals were thyroparathyroidectomized and per-

fused. The changes in plasma calcium and phos-phate and in the rates of urinary excretion ofcalcium and phosphate are shown in Figure 3 andTable III. In all the D-fed animals, the initialphosphaturia was equal to that seen in the para-thyroidectomized animals (compare Figure 3,column 1, with Figure 2, column 1). However,there was no initial calcium retention as seen inthe parathyroidectomized animals. Calcium ex-cretion rose slightly during the first 6 hours andthen rapidly over the next 6. This was associatedwith a striking rise in plasma calcium [from 8.8to 18.2 as compared to 8.4 to 13.1 in the para-

TIME, HOURSFIG. 3. CONCENTRATIONSOF CALCIUM AND PHOSPHATE

IN PLASMA AND RATES OF EXCRETION OF CALCIUM ANDPHOSPHATEIN THE URINE BEFOREAND DURING THE INFU-SION OF PTH INTO THYROPARATHYROIDECTOMIZEDD-FED(LEFTr) AND D-DEFICIENT (RIGHT) RATS. There werefour animals in each group. The shaded areas along thelower curves represent the range of values, and the heavyline represents the mean. The first pair of plasma valuesin each case represents the values obtained before theanimals were operated on. The latter two pairs repre-sent values taken during the control period and duringthe period of hormone infusion, respectively. Note thatthe D-deficient animals were infused with a more con-centrated solution of calcium chloride so that the concen-tration of calcium in their plasma during the period ofcontrol infusion was nearly identical to that seen in theD-fed controls.

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thyroidectomized animal (Table III) ]. The ratesof excretion of both calcium and phosphate fellduring the last 4 hours of perfusion, but after16 to 20 hours of hormone infusion these animalsdeveloped oliguria and died. The decline in cal-cium and phosphate excretion coincided with theonset of oliguria. The apparent cause of deathwas gross nephrocalcinosis. Because of this, per-fusion could not be continued as long in theseanimals.

In contrast to the D-fed animals, the responsesof thyroparathyroidectomized D-deficient animalswere not very different from those seen in para-thyroidectomized D-deficient animals (compareFigure 3, column 2, with Figure 2, column 2). Inan attempt to make the experimental situation be-tween D-fed and D-deficient animals as comparableas possible, we infused the thyroparathyroidecto-mized D-deficient animals with a solution con-taining 25 rather than 5 mMCaCl2. This wassufficient to raise their plasma calcium concentra-tions to values slightly greater than those of theD-fed animals (Table III and Figure 3). Thisled to a much higher rate of calcium excretion andto the complete disappearance of phosphate fromthe urine during the control period (Figure 3).In spite of the high rate of urinary calcium excre-tion, the animals were in significant positive cal-cium balance, approximately 60 umoles per hour.Parathyroid hormone infusion led to a prompt andstriking fall in urinary calcium, which remainedbelow control values throughout the period of hor-mone infusion. Phosphaturia was also observedand was approximately 40%o of that seen initiallyin the D-fed controls (Figure 3). The total ex-cess phosphate in the urine was significantlygreater (p < 0.001) than that which disappearedfrom the ECF (Table IV). The plasma calciumrose only slightly, 9.2 to 9.8 mg per 100 ml, ascompared to the striking rise, 8.8 to 18.2, seen inthe D-fed animals. In spite of the high initial ratesof calcium infusion and excretion, these animals didnot develop nephrocalcinosis.

Discussion

The present results extend our previous obser-vations (6) and those of Harrison and Harrison(7). In D-deficient rats with intact parathyroidglands, the injection (6, 7) or infusion of para-thyroid hormone (Figure 2, column 5, and Table

II) led to no change in plasma calcium or phos-phate and no change in the urinary excretion ofphosphate. However (Figure 2, column 5), asignificant (p < 0.025) decrease in magnesium andsodium and a slight decrease in calcium excretionwere observed. Removal of the parathyroid orboth thyroid and parathyroid glands of these ani-mals altered this pattern of response. Plasma phos-phate fell after hormone infusion (Table III), andphosphaturia as well as magnesium and calciumretention occurred (Figure 2, columns 2 and 3;and Figure 3, column 2). Plasma calcium roseslightly, but in no instance was this significant(p < 0.2). This slight rise in plasma calcium wasthought to be a consequence of renal retention ofcalcium. Furthermore, the responses of the thyro-parathyroidectomized D-deficient animals (Figure3, column 2) indicate that the lack of calcium mo-bilization during hormone infusion was not dueto the presence of excess thyrocalcitonin. This isin sharp contrast to the results of hormone perfu-sion in D-fed animals. The presence of the thy-roid gland altered the pattern of response of thisgroup (compare Figure 2, column 1, and Figure 3,column 1). In the absence of endogenous thyro-calcitonin, parathyroid hormone led to a morerapid mobilization of calcium from bone. Duringthe same period, the infusion of hormone intoD-deficient animals maintained on a much higherrate of calcium infusion led to no significant hy-percalcemia, but to a striking decrease in the rateof urinary calcium excretion (Figure 3).

The major differences in the responses of theparathyroidectomized D-deficient and D-fed ratsto the long-term infusion of parathyroid hormoneare the less marked initial phosphate and potassiumdiuresis, the lack of delayed hypercalciuria, andthe poorly sustained phosphaturia in the D-defi-cient group (Figures 1 and 2). The latter twofindings probably reflect the failure of the hormoneto induce the mobilization of calcium and phos-phate from bone in the absence of vitamin D. Ifthis conclusion is accepted, then the data shown inTable IV indicate that parathyroid hormone mo-bilizes phosphate from a source other than thebone and extracellular fluids, presumably the in-tracellular fluids. This in turn implies that para-thyroid hormone alters intracellular electrolyteconcentrations as well as those in extracellularfluids, a point that has been discussed previously

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in relation to the effects of this hormone on iso-lated mitochondria (1).

The less marked initial phosphaturia seen inthe D-deficient animals (Figure 2, column 2 vs.column 1; and Figure 3) is difficult to explain.There are several possibilities. It is conceivablethat the rats employed in these studies were notadequately depleted of vitamin D and that thisvitamin is necessary for the action of parathyroidhormone on the renal tubule as well as on bone.However, if one considers the total response ofthe tubule to hormone infusion, then it is apparentthat the quantitative changes in excretion of so-dium and calcium were similar in D-fed and D-de-ficient parathyroidectomized animals; that thechanges in magnesium excretion were greater(p < 0.025) in the D-deficient animals (Figure 2,columns 1 and 2); and that even in D-deficientsham-operated animals (Figure 2, column 5) achange in magnesium and calcium excretion wasobserved. Thus, by some criteria, the kidney ofthe D-deficient animal responds to parathyroidhormone as well as or better than that of the D-fedanimal, but by others it responds less well.

One possibility that may account for the differ-ences in phosphate excretion in the differentgroups of animals is that they had varying de-grees of secondary hyperparathyroidism beforethese experiments were undertaken; it is possiblethat this altered the concentrations of intracellularelectrolytes and consequently renal responses.There is increasing evidence (6, 16, 17) that sig-nificant hyperparathyroidism exists in vitaminD-deficient rats maintained on a normal calciumintake. This appears to be a consequence of thehypocalcemia that results from the vitamin defi-ciency (6, 17) and can be prevented in large partby putting these animals on high calcium intakes(6, 17). In the present context, perhaps the mostimportant consequence of chronic secondary hy-perparathyroidism is possible depletion of intra-cellular phosphate stores. It is important to pointout that, although our animals were studied aftertheir parathyroid glands had been removed, theyreceived no phosphate after this surgery and hencewould not have repleted tissue stores of this sub-stance. The most obvious difference betweenD-deficient animals on a normal and on a highcalcium intake (Figure 2, columns 2 and 3; andTable III) is that the former have low levels of

plasma calcium and the latter have high levels.Thus, the latter would not have secondary hyper-parathyroidism [see also (17)], and the responseof these animals measured in terms of phosphateexcretion (Figure 2, column 3; and Table II)and plasma phosphate (Table III) was similar tothat seen in D-fed controls (Figure 2, column 1).On the other hand, the D-deficient animals withhypocalcemia undoubtedly had secondary hyper-parathyroidism with resultant changes in phos-phate metabolism (6), and their response mea-sured as phosphate excretion (Figure 2, column 2)was significantly less than that of the D-fed con-trols. This difference would account for the factthat parathyroid hormone mobilized less totalphosphate in the D-deficient animals on a normalcalcium intake than it did in the D-deficient animalson a high calcium intake (Table IV). The ac-ceptance of this proposal must await the demon-stration that parathyroid hormone alters intra-cellular phosphate concentrations. These studiesare in progress.

The results with the group of D-deficient ani-mals fed a high calcium diet (Figure 2, column 3;and Table III) were of importance for anotherreason. These animals had the same plasma cal-cium values as the D-fed animals (Table III) dur-ing the period of control infusion (before hormoneinfusion) and the same degree of calcium retentionduring this period (Table I). Nevertheless, theinfusion of hormone into these D-deficient animalsdid not lead to significant rise in plasma calcium(Table III) or delayed hypercalciuria as seen inthe D-fed animals (Figure 2, columns 3 and 1).Also the fall in plasma phosphate concentrationwas as great in this group of animals as that seenin the D-fed ones (2.8 vs. 2.7, Table III). Thus,in terms of initial changes in phosphate metabo-lism, the two groups were nearly identical; inspite of this, parathyroid hormone did not mobilizecalcium from bone. These data constitute thestrongest argument in support of our previousthesis (6) that parathyroid hormone is unable tomobilize calcium from bone in D-deficient ratsbut is still able to exert its usual effects on renalfunction.

This thesis is also supported by the recent stud-ies of the effect of inhibitors of RNAand proteinsynthesis upon the response of rats to parathyroidhormone and vitamin D (8, 18, 19). The infu-

1962

PARATHYROIDHORMONEAND VITAMIN D

sion of parathyroid hormone into an actinomycin-treated rat leads to the same pattern of urinaryelectrolyte changes (8) as seen in the D-deficientrat (Figures 2 and 3), that is, the early renalresponse is normal, but the secondary changes dueto mobilization of calcium and phosphate from boneare absent. Similarly, Zull, Czarnowska-Misztal,and DeLuca (18) and Norman (19) have shownthat prior treatment with actinomycin D blocks thenormal response of vitamin D-deficient rats to theadministration of vitamin D.

Thus, evidence gathered by several differentmeans leads to the conclusion that vitamin D isnecessary for the expression of some, but not all,effects of parathyroid hormone. The problem thatdevelops is that of accounting for the divergentviews of others (4, 5, 7, 20). There is no basicdisagreement between our data and those of Har-rison and Harrison (7). The important point isthat, in a D-deficient rat, exogenous parathyroidhormone has little detectable influence on phos-phate metabolism because of high levels of en-dogenous hormone, which are already exerting anearly maximal effect. Only by removing theparathyroid glands is it possible to demonstrate aneed for, or a response to, exogenous hormone(Figure 2). Other studies in rodents (5, 20),which have led to results different from ours andthose of Harrison and Harrison, can be questionedon their lack of sufficient evidence that the ani-mals involved were completely D deficient. Themost serious challenge to our thesis comes from thework of Ney and associates (4). These authorsconcluded, on the basis of the response of D-defi-cient puppies to parathyroidectomy and to para-thyroid extract, that parathyroid hormone exertsits usual effects on bone even in D deficiency.One possible explanation is that the nature of thehormone-vitamin relationship described in thepresent report is unique to the rat. A more likelyexplanation is that the dogs studied by Ney andco-workers did not have as severe a degree ofD deficiency as that developd by the rats. One ofthe dogs with the most marked hypocalcemia hadno change in plasma calcium after parathyroidec-tomy, and several others had minimal changesboth after operation and after the subsequent in-jection of parathyroid extract. In fact, there wasdirect correlation between the initial level ofplasma calcium and the fall observed after parathy-

roidectomy (omitting animal T782, which was mostunusual), and all animals were initially hypocal-cemic. These results could be accounted for ifthe animals studied had varying degrees of D de-ficiency, and they are similar to our previous ob-servations in the rat (6).

Unfortunately, there is as yet no simple methodby which the degree of D deficiency can be assessedin experimental animals; hence, it is not an easymatter to resolve all controversy. Nevertheless,in the species in which the degree of deficiencycan be best controlled, the rat, it seems clear thatvitamin D is necessary for physiologic concen-trations of parathyroid hormone to mobilize cal-cium and phosphate from bone. On the otherhand, there appears to be no vitamin D require-ment for the expression of parathyroid hormoneaction at the level of the renal tubule.

Summary

The response of the D-deficient parathyroidec-tomized rat to the infusion of parathyroid hormoneis different from that of a D-fed control. Bothexhibited qualitatively similar initial changes inplasma phosphate and in the urinary excretionof calcium, phosphate, magnesium, sodium, andpotassium, but in the D-deficient animals sus-tained phosphaturia and secondary hypercalciuriaand hypercalcemia did not develop as they did inthe D-fed animal.

Initial phosphate excretion was less and initialmagnesium retention greater in D-deficient animalsraised on a normal calcium intake. However,D-deficient animals raised on a high calcium in-take had nearly the same initial values of plasmacalcium and changes in plasma and urinary phos-phate after hormone infusion as D-fed animalsraised on a normal calcium intake. In spite ofthis, the D-deficient animals had no rise in eitherplasma or urinary calcium.

Removal of the thyroid gland did not greatlyalter the response of the D-deficient rat to para-thyroid hormone, but a more immediate andmarked calcium diuresis and hypercalcemia wereobserved in the D-fed animal.

We concluded that vitamin D is necessary forphysiologic concentrations of parathyroid hormoneto mobilize calcium and phosphate from bone butnot for the hormone to act on the renal tubule.

1963

C. ARNAUD, H. RASMUSSEN,AND C. ANAST

AcknowledgmentsWeare indebted to Dr. Maurice Pechet for his advice

in regard to the perfusion technique. Mr. CharlesHawker, Miss Berit Johannson, and Miss Elizabeth Mid-dleton all rendered valuable assistance.

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1964