Proc. Nat. Acad. Sci. USAVol. 72, No. 12, pp. 4871-4875, December 1975Biochemistry

Cytoplasmic and nuclear binding components forla,25-dihydroxyvitamin D3 in chick parathyroid glands

(vitamin D/receptors/parathyroid hormone)

PETER F. BRUMBAUGH, MARK R. HUGHES, AND MARK R. HAUSSLERDepartment of Biochemistry, College of Medicine, University of Arizona, Tucson, Ariz. 85724

Communicated by John H. Northrop, September 29, 1975

ABSTRACT Specific binding of la,25-dihydroxyvitaminD3 [la,25(OH)2D3J to macromolecular components in the cy-toplasm and nucleus is demonstrated in parathyroid glandsof vitamin-D-deficient chicks. The interaction of la,25-(OH)WD3 with the cytoplasmic binding component is of highaffinity (R4 = 3.2 X 10-10 M) and high specificity [la,25-(OH)2D3 > 25-hydroxyvitamin D3 > la-hydroxyvitamin D3 >vitamin D3 in competing with radioactive la,25(0H)2D31.Both cytoplasmic and nuclear hormone-macromolecularcomplexes sediment at 3.1 S in 0.3 M KCI-sucrose gradients,and agarose gel filtration of the components indicates an ap-parent molecular weight of 58,000. The 3.1S binding mole-cules are not observed in adrenal gland, testes, liver, or kid-ney, but similar receptors for la,25-(0H)2D3 have been foundpreviously in intestine.

Macromolecular species with a high affinity and prefer-ence for 25-hydroxyvitamin D3 [25(0H)D3] are also identi-fied in parathyroid cytosol and differ from the parathyroidla,25(OH)WD3-binding component in that: (1) they sedimentat 6 S in 0.3 M KCI-sucrose gradients, (2) they are observed inall tissues examined, (3) they have a higher affinity for 25-(OH)D3 than la,25-(0H)2D3, and (4) they are not found in thenucleus of the parathyroid glands, in vitro. The discovery ofunique la,25.OH)2D3-binding components in the parathy-roid glands is consistent with the sterol hormone's action atthis endocrine site and possible involvement in the regula-tion of parathyroid hormone synthesis and secretion.

Vitamin D3 action to mobilize calcium and phosphate at in-testine and bone is thought to be mediated by the metabolitela,25-dihydroxyvitamin D3 [la,25-(OH)2D3] (1-4). Its pro-duction from 25-hydroxyvitamin D3 [25-(OH)D3] by therenal la-hydroxylase appears to be regulated by calcium (5),phosphate (6, 7), parathyroid hormone (PTH) (7, 8), and thevitamin D status of the animal (9). Evidence suggests thathypocalcemia stimulates PTH secretion which in turn en-hances the production of la,25-(OH)2D3 at the kidney (7,8). Thus PTH, rather than calcium, may be the dominantmodulator of the renal la-hydroxylase (10) and the findingof abnormal circulating 1a,25-(OH)2D3 in humans withparathyroid disease is consistent with this concept (11, 12).DeLuca has proposed that PTH and phosphate deficiencymay be functioning through a common intracellular mecha-nism to enhance the la-hydroxylase by lowering the phos-phate level in the renal cell (10). Moreover, MacIntyre andassociates (13) have suggested that la,25-(OH)2D3 mightcontrol its own biosynthesis directly at the kidney by a nega-tive feedback mechanism involving the de novo synthesis ofthe la-hydroxylase enzyme. Clearly, the fashion in whichla,25-(OH)2D3, PTH, and phosphate deficiency interact tocontrol the formation of 1a,25-(OH)2D3 at its kidney endo-crine site must be completely understood before the exact

role of 1a,25-(OH)2D3 in the homeostatic regulation of cal-cium and phosphate can be elucidated.Henry and Norman (14) have recently reported that

la,25-(OH)2[3H]Da is localized in chick parathyroid glandsfollowing administration of the sterol, in vivo. In the presentexperiments, specific binding components for 1a,25-(OH)2D3 have been isolated from chick parathyroid glandsand characterized in vitro. Previous reports (15, 16) havedemonstrated that 1la,25-(OH)2D3 interacts with the intes-tine in a manner similar to the binding of steroid hormonesto their respective target organs. 1a,25-(OH)2D3 enters theintestinal cell and binds to a 3.7S cytoplasmic receptor pro-tein (17, 18). The hormone receptor complex then migratesinto the nucleus in a temperature-dependent process, whereit associates with the chromatin (15, 17-19). We report herethat similar intracellular receptor proteins for la,25-(OH)2D3 exist in chick parathyroid glands.

MATERIALS AND METHODS-Materials. Animals used in experiments were White Leg-

horn cockerels (kindly donated by Demler Farms, Anaheim,Calif.) that were raised for 6 weeks on a vitamin-D-deficientdiet (20). 25-Hydroxy[26(27)-methyl-3H]vitamin D3 (6.5 Ci/mmol) was obtained from Amersham-Searle.

Preparation of la,25-Dihydroxy[3Hjvitamin D3, InVitro. la,25-Dihydroxy[26(g7)-methyl-3H]vitamin D3 wasprepared as previously described (19). Radiochemical purityof generated la,25-dihydroxy[3H]vitamin D3 was 98%. 25-Hydroxy[26(27)-methyl-3H]vitamin D3 substrate for the re-action was purified by Celite liquid-liquid partition chro-matography (21). The radiochemical purity of the 25-hy-droxy[3H]vitamin D3 was 95%, and its specific activity wasdetermined by ultraviolet absorbance spectrophotometry at265 nm.

Exposure of Chick Tissue Subfractions to RadioactiveSterols, In Vitro. Homogenates [300 mg wet weight (20parathyroid glands)/3 ml] were made in 0.25 M sucrose,0.05 M Tris-HCI, pH 7.4, 0.025 M KCI, and 5 mM MgC12(0.25 M sucrose-buffer A) with a Potter-Elvehjem homoge-nizer equipped with a Teflon pestle at 00 by six passes, with2 min cooling periods between passes. Homogenates werecentrifuged at 1200 X g for 10 min. Nuclear pellets were re-moved, and the resulting supernatant was centrifuged at100,000 X g for 1 hr at 0° to yield a final supernatant frac-tion (cytosol). The cytosol (0.2-1.0 ml) was incubated withsterol (in 20,l ethanol) for 1 hr at 00 and then analyzed forsterol binding components.

Purified nuclear extracts (chromatin) were prepared fromnuclear pellets by a modification (7) of the method of Haus-sler et al. (1) and were resuspended in 0.01 M Tris-HCl, pH7.5, and centrifuged for 20. min at 48,000 X g. The pelletfrom 300 mg of tissue was reconstituted with cytosol (2.5 ml)

Abbreviations: 25-(OH)D3, 25-hydroxyvitamin D3; la-(OH)D3,la-hydroxyvitamin D3; la,25-(OH)2D3, la,25-dihydroxyvitaminD3; PTH, parathyroid hormone.

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and incubated for 1 hr at 25° with sterol (in 40 Al of etha-nol). Chromatin was harvested and extracted with 0.3 MKC1, 0.01 M Tris-HCl, pH 7.5, 1.5 mM EDTA, 12 mM 1-

thioglycerol (0.3 M KCl-Buffer B). Extracts were centri-fuged at 48,000 X g for 20 min, and the resulting superna-tants were analyzed for sterol-binding activity.

Sucrose Gradient Centrifugation. Linear gradients (5.0ml) of 5-20% sucrose in 0.3 M KCI-Buffer B were preparedwith a Buchler gradient mixer, Auto-Densi Flow, and Poly-staltic pump. Aliquots (0.3 ml) of cytosol or nuclear extractswere layered on gradients and centrifuged at 234,000 X g(average force) for 24 hr at 00 with the use of a BeckmanL3-50 ultracentrifuge and an SW 50.1 rotor. The fractions (6drops each) were counted in 5 ml of liquid scintillation mix-ture A (3% Liquifluor in toluene-Triton X-114, 3:1) in aBeckman LS-233 scintillation counter (35% efficiency). Sedi-mentation coefficients were estimated by comparison withprotein markers (chymotrypsinogen, 2.5 S; ovalbumin, 3.67S; and bovine serum albumin, 4.4 S).Agarose Gel Filtration. All chromatographic procedures

were carried out at 1-3'. Agarose beads (Bio-Gel A-0.5m,100 to 200 mesh from Bio-Rad) were equilibrated with 0.3M KCI-Buffer B and poured into a column (1.6 X 60 cm).Samples (1.0 ml) of nuclear extracts or cytosol incubationswere applied to the column and 1-ml fractions were collect-ed and counted (30% efficiency). The optical density offractions was measured with a Gilford 240 spectrophotome-ter. Column flow rates were maintained at 13-14 ml/hrwith a Polystaltic pump.

Filter Assay for Specific Macromolecular Binding. Sep-aration of bound from free sterol was achieved by the filterassay method of Santi et al. (22). Aliquots of cytosol (0.2 ml)containing la,25(OH)2[3H]D3 and samples containing thesame concentration of 1a,25-(OH)2[3H]Ds plus a 100-foldexcess of unlabeled hormone were incubated at 00 for 2 hr.Cytosol (150 Al) was then applied to DEAE-cellulose filters(Whatman DE 81) and washed with three 1-ml portions of1% Triton X-100 in 0.01 M Tris-HCI, pH 7.5, with the use ofa Millipore sampling manifold. The amount of la,25-(OH)2[3H]D3 specifically bound by the cytosol was deter-mined as previously described (17). Efficiency of the filtra-tion procedure, as determined by measurement of bindingat low hormone concentrations, was 75%.

RESULTS

Initially, parathyroid gland was studied to determine if itcontained a soluble binding component for the Ila,25-(OH)2D3 hormone. Parathyroid cytosol from vitamin-D-de-ficient chicks was incubated at 0° with ia,25-(OH)2[3H]Daand binding of the sterol to macromolecules was analyzedby sucrose gradient centrifugation. As depicted in Fig. 1A,the radioactive hormone interacted with a macromolecularspecies sedimenting at 3.1 S. Parallel cytosol incubationscontaining excess unlabeled 1a,25-(OH)2D3 showed no ra-

dioactive hormone binding in the sedimentation position ofthe 3.1S-binding component (Fig. 1A), demonstrating thatthe interaction of hormone with this cytosol molecule is satu-rable and of high affinity. Sucrose gradients of the la,25-(OH)2D3-binding component in parathyroid cytosol repro-ducibly exhibited a faster-sedimenting shoulder on the pri-

mary peak. When a 2-fold excess of nonradioactive 25-(OH)D3 was included in the incubation (Fig. 1A), the shoul-der was abolished without any detectable effect on la,25-(OH)2[3H]Dj binding to the 3.1S macromolecule. Thus, this

macromolecule selectively binds the hormone and muchhigher concentrations of 25-(OH)D3 are required to effec-

20 TOP 10

FRACTION NUMBER

FIG. 1. Sucrose gradient centrifugation of la,25-(OH)2D3 and25-(OH)D3-binding components in parathyroid glands and nontar-get organs. Incubations were carried out as follows: (A) Parathy-roid cytosol (0.3 ml) at 00 for 1 hr with 3 nM la,25-(OH)2[3H]D3(6.5 Ci/mmol) alone (0), or with 0.3 gM unlabeled la,25-(OH)2D3(0), or with 6 nM nonradioactive 25-(OH)D3 (A). (B) Reconsti-tuted cytosol-chromatin from parathyroid (@), adrenal (A), ortestes (A) was incubated with 6 nM la,25-(OH)2[3H]D3 for 1 hr at250. The chromatin was then extracted with 0.3 M KCl-Buffer B.Parallel incubation with parathyroid cytosol-chromatin, 6 nMlz,25-(OH)2[3H1D3, and 0.6 'M unlabeled la,25-(OH)2D3 was per-formed (0). (C) Parathyroid cytosol with 6 nM 25-(OH)[3H]D3 (6.5Ci/mmol) alone (0), or with 60 nM nonradioactive 25-(OH)D3 (O).or with 60 nM unlabeled la,25-(OH)2D3 (A). (D) Testes cytosolwith 6 nM 25-(OH)[3H]D3 (0); testes cytosol with 6 nM la,25-(OH)2[3H]D3 alone (A) or with 0.6 MM unlabeled la,25-(OH)2D3(A) or with 60 nM nonradioactive 25-(OH)D3 (0). Arrows indicatesedimentation positions of protein standards: 1, chymotrypsino-gen; 2, ovalbumin; 3, bovine serum albumin.

tively compete with 1a,25-(OH)2D3 (see Table 1). Since theshoulder disappeared in the presence of 25-(OH)D3 and cor-responded to the sedimentation position (6S) of the high-af-

Table 1. Influence of unlabeled sterols on the binding oflabeled hormone to cytoplasmic parathyroid

la,25-(OH)2D3-binding component

Unlabeled sterol(concentration, nM) % Binding

None 1001a,25-(OH)2D3 (400) 01,25-(OH)2D3 (4) 5325-(OH)D3 (80) 55la-(OH)D3 (2,000) 95Vitamin D3 (10,000) 98

Aliquots of cytosol were incubated with 4 nM la,25-(OH)2-[3H]D3 and unlabeled sterol for 1 hr at 00. Binding of radioactivesterol to the 3.1S component was determined via sucrose gradientcentrifugation.

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Proc. Nat. Acad. Sci. USA 72 (1975) 4873

II0-J0(I)0

z0

U-LLN

I)I

-J

z

H)0~

-j

C)) z

I

v4

FRACTION NUMBERFIG. 2. Agarose gel filtration of cytoplasmic and nuclear la,25-(OH)2D3-binding components of chick parathyroid glands. Reconstituted

cytosol-chromatin (1 ml) was incubated with la,25-(OH)2[3H]D3 (4 nM) for 1 hr at 250. The chromatin was then extracted with 0.3 M KC1-Buffer B (0-*). One ml of parathyroid cytosol (prepared in 0.3 M KCl-Buffer B) was incubated with la,25-(OH)2[3HJD3 (6 nM) for 1 hr at00 (0-0). (-), relative absorbance at 280 nm of cytosol eluate. Molecular weights of the components were estimated from a linear plot ofMr112 versus the distribution coefficient, KD1 3, with the use of myoglobin, chymotrypsinogen, pepsin, ovalbumin, and bovine serum albuminas standards. Vo and Vt are the void and total volumes, respectively.

finity binding protein for 25-(OH)D3 discovered by Haddadand Birge in the rat (23), it was concluded that this shoulderrepresented association of the hormone with this 6S compo-nent.To further investigate the interrelationship of the 3.1S

binding component and the 6S species which binds both1a,25-(OH)2D3 and 25-(OH)D3, parathyroid cytosol was in-cubated with radioactive 25-(OH)D3. Sucrose gradient anal-ysis (Fig. IC) indicates that 25-(OH)[3H]D3 binds exclusive-ly to a protein sedimenting at 6 S. Parallel incubations con-taining a 10-fold excess of either nonradioactive 25-(OH)D3or la,25-(OH)2D3 show that this protein has a higher affini-ty for 25-(OH)D3 than for the hormone; binding to the 6Speak was reduced by 15% in the presence of 1a,25-(OH)2D3and by about 90% in the presence of 25-(OH)D3 (Fig. IC).The 25-(OH)D3-binding component in parathyroid cytosolis therefore similar to proteins found in a variety of rattissues by Haddad and Birge (23) and to proteins reported inchick intestine, liver, and kidney by Brumbaugh and Haus-sler (24). In all cases, the binding component has a greateraffinity for 25-(OH)D3 than 1a,25-(OH)2D3 and sedimentsat 6 S in sucrose gradients.

Analysis of la,25-(OH)2[3H]Da interactions with other tis-sue cytosols showed that the 3. IS hormone-binding compo-nent exists only in parathyroid gland and is not present intestes (Fig. ID), adrenal gland, liver, or kidney (data notshown). Detailed investigation of testes cytosol (Fig. ID) re-vealed that the la,25-(OH)2D3 hormone associates predomi-nantly with the 25-(OH)Ds-binding protein (sedimenting at6 S). However, the preference of this protein for 25-(OH)D3is demonstrated by the greater potency of unlabeled 25-(OH)D3 over la,25-(OH)2D3 in abolishing this 6S peak (Fig.iD). A small amount of 1a,25-(OH)2[3H]D3 binds to mole-cules in testes cytosol sedimenting at 3-4 S, but this bindingis low affinity and nonspecific because it is not reduced byexcess unlabeled Ia,25-(OH)2D3. Therefore, with the excep-tion of the target intestine, where a selective 3.7S hormonereceptor is found (18), cytosol components specific for thela,25-(OH)2D3 hormone over its 25-(OH)D3 precursor aredetected only in parathyroid glands.The association of la,25-(OH)2D3 with nuclear-binding

components from parathyroid gland was also observed invitro. la,25-(OH)2[3H]D3 was incubated with reconstituted

cytosol-chromatin at 250 for 1 hr and the chromatin wasthen harvested and extracted with 0.3 M KCI-Buffer B. Su-crose gradient analysis of this nuclear extract resulted in a3.1S peak of bound 1a,25-(OH)2[3H]Da (Fig. 1B). Parallelincubations containing a 100-fold excess of unlabeled hor-mone showed a striking reduction in the radioactive sterolbound to this macromolecule. Also, the association of la,25-(OH)2[3H]D3 with a nuclear component was not observed inthe adrenals or testes (Fig. 1B). Therefore, the parathyroidnuclear chromatin contains a tissue-specific, high-affinitybinding component for 1a,25-(OH)2D3 which is indistin-guishable from the cytosol macromolecular species by su-crose gradient centrifugation.The cytoplasmic and nuclear la,25-(OH)2D3-binding

components were also isolated and compared by agarose gelfiltration chromatography in 0.3 M KCI-Buffer B (Fig. 2).The major peak represents [3H]sterol binding to a macro-molecule of apparent molecular weight 58,000, which is re-solved from the major protein peak (eluting in the void vol-ume). No significant or reproducible difference could bedemonstrated between the nuclear and cytoplasmic compo-nents when columns were run under identical, standardizedconditions (Fig. 2) and both peaks of macromolecule-[3H]hormone complexes were abolished when incubationscontaining excess unlabeled la,25-(OH)2D3 were chromato-graphed (data not shown). Thus, the specific nuclear and cy-toplasmic 1a,25-(OH)2D3-binding components could not bedistinguished from each other by the ultracentrifugal andchromatographic techniques employed. These data are con-sistent with the concept that the nuclear component origi-nates in the cytosol and suggest that 1a,25-(OH)2D3 may befunctioning in the parathyroid gland at the level of the cellnucleus.

Next the binding affinity and specificity of the cyto-plasmic-binding components for 1a,25-(OH)2D3 in parathy-roid glands were studied. Incubation of parathyroid cytosolwith increasing concentrations of 1a,25-(OH)2[3H]D3 anddetermination of specific binding by the filter method ofSanti et al. (22) showed the saturation of a limited numberof binding sites (Fig. 3A). Saturation occurs at low concen-trations of hormone (3 X 10-9 M). At this range of hormoneconcentration most of the binding detected by the filterassay is specific for la,25-(OH)2D3, since only a small

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FIG. 3. Determination of dissociation constant for la,25-(OH)2D3-parathyroid cytosol macromolecule interaction. (A) Specific binding.of la,25-(OH)2[3H]D3 by parathyroid cytosol. Aliquots of cytosol (0.2 ml, 2.0 mg of protein per ml) were incubated with increasing amountsof la,25-(OH)2[3H]D3 in the presence (nonspecific) or absence (total) of a 100-fold excess of unlabeled la,25-(OH)2D3 for 2 hr at 00. (B)Scatchard analysis of specific binding in A. Three determinations indicate that the 1a,25-(OH)2[3H]D3 macromolecule interaction has a Kd= 3.2 4 0.2 X 10-10 M at 00, where the uncertainty is expressed as standard deviation.

amount of labeled hormone is bound in the presence of a

100-fold excess of unlabeled hormone (nonspecific binding).Scatchard analysis of the specific binding (total minus non-

specific) is linear (Fig. 3B), suggesting a single class of bind-ing sites. The dissociation constant for the hormone-macro-molecule complex at 00 is 3.2 X 10-10 M.

Analogs of la,25-(OH)-D3 which lack a hydroxyl group atthe la- and/or 25-position were tested for their ability tocompete with the labeled hormone for binding to the 3.1Scytoplasmic component. As is shown in Table 1, the la,25-(OH)2D3-binding macromolecule is highly specific. The ap-

proximate relative affinity of these sterols for the 3.1S mac-

romolecule is 1:1/20:<1/500:<1/2500 for la,25-(OH)2D3:25-(OH)Ds:la-(OH)D3:vitamin D3. These values differfrom the 1:1/500:1/800:<1/20,000 relative affinities forthese sterols' association with the 3.7S receptor of intestine(25). The present results indicate that the parathyroid glandreceptor for la,25-(OH)2D3 differs slightly from the well-characterized hormone receptor in the intestine in terms ofsedimentation coefficient, affinity, and specificity. Yet it isclear that the parathyroid gland should be included with theintestine as a location of specific, high-affinity binding com-

ponents for the la,25-(OH)2D3 hormone.

DISCUSSIONThe existence of a specific la,25-(OH)2D3-binding macro-

molecule has been demonstrated in chick parathyroidglands. Although the function of this binding component isnot known, it has been definitively distinguished from the25-(OH)D3-binding protein which has been found in the ratand chick (23, 24). Table 2 summarizes data on the proper-ties of chick vitamin D metabolite-binding proteins in termsof their sedimentation coefficients and subcellular location.A 6S 25-(OH)Ds-binding component has been identified inthe cytosol of all organs examined in the chick, but this pro-tein is not found in the nucleus. At present, a role for thismacromolecule has not been found, but the fact that it doesnot transport 25-(OH)D3 into the nucleus suggests that it isnot a classic steroid hormone receptor (27, 28). The tissue25-(OH)Ds-binding protein can also be differentiated fromthe serum protein which binds either 25-(OH)D3 or la,25-(OH)2DW and sediments at 4.0 S in sucrose gradients (Table2). We have also observed that although the ubiquitous 6S

protein has a higher affinity for 25-(OH)D3 than the hor-mone, it will also bind la,25-(OH)2[3H]Ds when incubatedwith the sterol at -10-8 M in vitro (Fig. IA and D, Table 2).However, in the target intestine and in the parathyroidgland, unique macromolecules sedimenting at 3.7 S and 3.1S, respectively, are observed to selectively bind la,25-(OH)2D3 and transfer the hormone into the cell nucleus.These binding components are analogous to the classic ste-roid hormone receptors and, based upon the pattern of bind-ing proteins established in Table 2, the parathyroid gland

Table 2. Sedimentation coefficients of high-affinitybinding proteins for 25-(OH)D3 and 1a,25-(OH)2D3

in the chick*

Sedimentationcoefficient,S (±SD)

Sterol Site Cytosol Nuclear Refs.

25-(OH)D3 Intestine 6 t 24Parathyroid 6 tTestes 6 tLiver 6 t 24Kidney 6 t 24Serum 4.0 - 24

± 0.1

lc,25-(OH)2D3 Intestine 3.7 3.7 17, 18± 0.1 ±0.1

Parathyroid 3.1 3.1± 0.2¶ + 0.2¶

Testes 6 § t tLiver 6 § t 26Kidney 6 § t 26Serum 4.0 - 24

± 0.1¶* Data from 0.3 M KCl-sucrose gradients.t Not observed.t Present study.§ Represents binding of la,25-(OH)2[3H]D3 to 25-(OH)D3 bindingprotein.Average of four sucrose gradient experiments; significantly dif-ferent from 3.7S intestinal receptor (P < 0.005).

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LOW CALCIUM

PARATHYROID CELL

PTH NUCLEUS

R ANDSE(-C Xl2

0] (§)-1,25ALCIUM RETENTION \

PTH 1 HOSPHATE EXCRETION

RENAL lIc-OHase (CALCII25-OH-D3 --- I,25-(OH( 3 APHOSF)

I MDib LI

JM/

PHATE -

IZATION

HS- -

LOW PHOSPHATEFIG. 4. Integration of possible 1a,25-(OH)2D3 action at para-

thyroid gland with the homeostatic regulation of serum calciumand phosphate by the concerted functioning of PTH and la,25-(OH)2D3.

should perhaps be classified along with the intestine andbone as a target site for 1a,25-(OH)2D3.

Little information was available until recently on the pos-sible function of la,25-(OH)2D3 at the parathyroid gland.Oldham et al. (29) have detected and characterized a cal-cium-binding protein in parathyroid gland which is similarto the intestinal calcium-binding protein induced by la,25-(OH)2D3 (30, 31). It is possible that the sensing of serum cal-cium by the parathyroids is dependent upon 1a,25-(OH)2D3and the synthesis of a calcium-binding protein in this gland.Also, Chertow et al. (32) have reported that la,25-(OH)2D3suppresses PTH secretion in the rat, in vvo, and in isolatedslices of bovine parathyroid gland. These observationssuggest that 1a,25-(OH)2D3, like other steroid hormones(33), feedback inhibits its trophic counterpart, PTH. Amodel depicting the way in which this feedback loop mayintegrate into the complex scheme for the regulation of cal-cium and phosphate is illustrated in Fig. 4. The two primarysignals in this endocrine system are postulated to be low cir-culating calcium and low phosphate (7, 10). Low calciumenhances the formation of 1a,25-(OH)2D3 via a stimulationof parathyroid hormone secretion (7, 8). The calcium mobi-lized from bone by the synergistic action of PTH and1a,25-(OH)2D3 and that absorbed from intestine under theinfluence of enhanced sterol levels probably closes the hor-mone loop by abolishing further PTH secretion (34). Directfeedback of 1a,25-(OH)2D3 at the parathyroid gland mayalso be involved in the regulation of PTH secretion duringthe correction of low serum calcium.The significance of the operation of 1a,25-(OH)2D3 at the

parathyroid gland may be more profound in terms of phos-phate homeostasis. During phosphate depletion the kidney isstimulated to form more 1a,25-(OH)2D3 (Fig. 4; refs. 6 and7). To maintain the phosphate mobilized by the sterol, PTHsecretion must be curtailed (because PTH causes net phos-phate excretion). Such curtailment can ultimately occur

after hypercalcemia is established by the action of la,25-(OH)2D3, but a more plausible mechanism would be the di-rect inhibition of PTH secretion by 1a,25-(OH)2D3 (Fig. 4).The control of calcium and phosphate is apparently accom-

plished by a complex and delicate interrelationship betweenPTH and 1a,25-(OH)2D3 and a comprehension of this exact

relationship should further our understanding of both thenormal physiology of these ions and of diseases of mineralmetabolism such as primary hyperparathyroidism, idiopath-ic hypercalciuria, and vitamin D-resistant rickets.

We gratefully acknowledge the expert technical assistance of Ms.Kristine Bursac and Ms. Patricia G. Jones. The authors wish tothank the National Institutes of Health, Grant AM-15781-04 andTraining Grant GM-01982, for support of this research.

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Biochemistry: Brumbaugh et al.

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