THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 247, Ko. 17, Issue of September 10, pp. 5511-5519, 1972

Printed in U.S.A.

Studies on Calciferol Metabolism

IV. SUBCELLULAR LOCALIZATION OF 1,25-DIHYDROXY-VITAMIN D, IN INTESTINAL MUCOSA AND CORRELATION WITH INCREASED CALCIUM TRANSPORT*

(Received for publication, March 9, 1972)

HUAN C. TSAI, RICHARD G. WONG, AND ANTHONY W. NORMANI

From the Department of Biochemistry, University qf California, Riverside, California 92502

SUMMARY

It has been previously established that vitamin DB (cho- lecalciferol) must undergo an obligatory two step metabolism to first 25hydroxycholecalciferol and then 1,25-dihydroxy- cholecalciferol prior to the initiation in the intestine of in- creased calcium transport. These steps are mediated suc- cessively by the liver and the kidney. The present report compares the time course of appearance of vitamin D me- tabolites in various subcellular fractions of the intestinal mucosa with the onset of increased intestinal calcium trans- port after physiological doses of radioactive 25-hydroxy- cholecalciferol or 1 ,25-dihydroxycholecalciferol. With both steroids, the intestinal nucleus and its chromatin fraction, but not that of the liver or kidney, preferentially accumulated 65 to 75% of the radioactivity present in the tissue. This radioactivity was found by chromatographic analysis to be exclusively 1 ,25-dihydroxycholecalciferol. Maximal nu- clear accumulation (4.5 to 6 pmoles per chick intestinal chromatin) occurred after 10 to 12 hours or only 4 hours with doses of 0.32 nmole of 25-hydroxycholecalciferol or 1 ,25-dihydroxycholecalciferol, respectively. Under these conditions increased intestinal calcium transport is maximal 28 or 9 hours after 25-hydroxycholecalciferol or 1 ,25-dihy- droxycholecalciferol, respectively. Additionally it was found that the binding capacity of the nuclear chromatin for the 1,25-dihydroxycholecalciferol became saturated after a dose of 1.3 nmoles of 25-hydroxycholecalciferol or a dose of only 0.025 nmole of 1 ,25-dihydroxycholecalciferol. This corre- lates closely with the doses of these steroids required to elicit a maximum intestinal calcium transport response. These results collectively support the concept that the nu- clear localization of 1 ,25-dihydroxycholecalciferol in the in- testinal mucosa is an integral step in the development of the physiological response to vitamin D by this tissue.

The administration of radioactive cholecalciferol (vitamin I&) to rachitic chicks or rats results in the production of two

* This work was supported in part by Public Health Service Grants AM-09012 and AM-14,750. The preceding paper in this series is Reference 1. A portion of this work was presented at the 1972 FASEB meetings held in Atlantic City, New Jersey.

$ To whom reprint requests should be addressed. Recipient of a Public Health Service Career Research Development Award l-K4-AM-13,654.

major metabolites and a number of more minor metabolites (2, 3). The predominant metabolite present in the blood is 25- hydroxycholecalciferol (4). It has been shown to be produced by the liver (4). Myrtle et al. (3) found that the major metabo- lite present in the target intestinal mucosa was chemically differ- ent from both 25-hydroxycholecalciferol and the parent chole- calciferol. This substance, originally designated Metabolite 4B, was shown to be produced from 25-hydroxycholecalciferol by the kidney (5, 6) and to localize stereospecifically in the in- testinal nucleus and its chromatin fraction (2), where it was as- sociated noncovalently with an acidic, nonhistone receptor pro- tein (7). Myrtle and Norman (8) reported that metabolite 4B was four to five times as effective as cholecalciferol and over two times as effective as 25-hydroxycholecalciferol in stimulating intestinal calcium transport 24 hours after administration. Re- cently Norman et al. (9) and a number of other laboratories (10, 11) have reported the chemical structure of this metabolite to be 1,25-dihydroxycholecalcifero1.l

An important problem now is to determine which of the chole- calciferol metabolites or its further derivatives is the primary initiator of the physiological response in the intestine attributed to vitamin D. Specific information as to how steroids2 may mediate their characteristic physiological response has tradi- tionally been to study the time course of their appearance in tissues, their subcellular localization and possible further me- tabolism. Such studies have been reported for estrogen (16), aldosterone (17), testosterone (18), ecdysone (19), etc. In this communication we wish to report the comparative time course of appearance of radioactive compounds and their subcellular localization and relationship to stimulation of calcium transport after physiological doses3 of radioactive 25-hydroxycholecalcif-

1 Norman et al. (1) have reported that the metabolite 4B of Haussler et al. (2) is chromatographically identical with the peak P of Lawson et al. (12), peak V of Mawer (13), and peak 5 of Pon- chon and DeLuca (14). All except the Mawer metabolite have subsequently been chemically characterized to be 1,25-dihydroxy- cholecalciferol (9-11).

2 According to the International Union of Pure and Applied Chemistry Commission on the Nomenclature of Biological Chem- istry (15) cholecalciferol (vitamin Dp) is defined as a steroid. The chemical name is 9,10-secocholesta-5,7,10(19)-trien-3-D-01.

3 One international unit of cholecalciferol (vitamin D3) is equiv- alent to 0.025 pg or 0.065 nmole (20). The minimum daily require- ment for cholecalciferol in the chick is 0.65 to 1.30 nmoles (10 to 20 i.u.) (21). No formal definition of units has been defined for 25.hydroxycholecalciferol or 1,25-dihydroxycholecalciferol. For this report 1.0 unit of each of these compounds was arbitrarily defined as being 0.065 nmole.

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erol or 1,25-dihydroxycholecalciferol to rachitic chicks. These results support the concept that 1,25-dihydroxycholecalciferol plays a prominent if not exclusive role in the development in the intestine of the physiological response to vitamin D.

MATERIALS AND METHODS

dnimals-One-day-old White Leghorn cockerels (generously donated by 1-I and N of California, Inc.) were raised on a vitamin D-deficient diet described elsewhere (21, 22). Chicks were uti- lized in their fourth week after they had become severely rachitic.

ChemicaZs-[4-14C]Vitami~l-D~ (Philips-Duphar, Amsterdam), specific activity 20.7 mCi per mmole or 46,150 dpm per nmole and 25.hydroxy[26, 27-3H]cholecalcifero1 (Amersham-Searle, Chi- cago) specific activity 328 mCi per mmole or 730,000 dpm per nmole were employed in the metabolism studies. 1,25-Dihy- droxy[26, 27-3H]cholecalcifero1 was synthesized enzymatically in our laboratory with a specific activity of 20.5 mCi per mmole or 45,660 dpm per nmole (6). [4-14C]Cholesterol (New England Nuclear Corporation, Boston), specific activity 57.5 mCi per mmole or 127,600 dpm per nmole was used as a “control” steroid in several experiments.

Preparation of 1 ,25-Dihydroxy[S?6 ,b7-3H]cholecalciferol-l ,25- Dihydroxy[26, 27-3H]cholecalcifero1 (2960 dpm per 65 pmoles) was routinely prepared by incubation of 25.hydroxy[26, 27-3H]- cholecalciferol (20.5 mCi per mmole) with homogenates or mito- chondria prepared from kidneys obtained from 3-week-old ra- chitic chickens according to previously published techniques (6). Each individual incubation contained 2700 pmoles of 25-hydroxy- [26, 27-3H]cholecalcifero1 as substrate and other incubation com- ponents as described previously (4). After 400 to 500 incuba- tions the media were pooled and extracted with chloroform- methanol (1:2, v/v). Next the organic solvent was removed in ouc~o, and the lipid residue dissolved in 5 to 15 ml of 100% di- ethylether and applied to a column (1 x 80 cm) of activated silicic acid.’ The column was eluted successively with 400 ml of 100% ether, 200 ml of diethylether-dichloroethane (1: 1, v/v), and lastly 200 ml of 100% methanol. Fractions of 10 ml were collected. After removal of lo-p1 aliquots and location of the fractions containing the 1,25-dihydroxycholecalciferol, these tubes were pooled, the solvent evaporated under nitrogen, and the steroid dissolved in 2 to 3 ml of hexane-choloroform (35:65, v/v). This was applied to a column (1 x 80 cm) containing Sephadex LH-20 which was equilibrated with the same solvent. One hundred 3.0-ml fractions were collected. After removal of lo+1 aliquots, the tubes containing the 1, 25-dihydroxy[3H]- cholecalciferol were pooled, the solvent evaporated under nitro- gen, and the steroid dissolved in a small volume of methanol and stored in the dark at -5”. Each batch of 1 ,25-dihydroxy[3H]- cholecalciferol was assayed for biological activity according to previously described methods (8) in groups of five to eight ra- chitic chicks in terms of stimulating the intestinal absorption of doses of 45Ca2f in vivo.

Preparation of Xubcellular Practions-All chicks were injected intracardially with the appropriate steroid dissolved in 0.2 ml of 1,2-propanediol. After the prescribed time interval, the chicks were killed and the tissue of interest excised. All subse- quent operations were carried out at 1 to 4”. Liver and kidney were rinsed and washed several times with 0.25 M sucrose in 0.05 M Tris-HCl, pH 7.5, 0.025 M KCl, and 9.005 M MgCle (0.25 M

sucrose-Buffer A). The small intestine was rinsed with 0.25 M

sucrose-Buffer A and then the mucosa was scraped free from the serosa with a microscope slide. After preparation of a 10% ho-

mogenate in 0.25 M sucrose-Buffer 9, the crude nuclei were iso- lated by centrifugation at 800 x g for 10 min in a Beckman model J-21 centrifuge. Mitochondria were isolated from the resulting supernatant by centrifugation at 8,000 x g for 20 min. The supernatant fluid remaining after sedimentation of mitochondria was then centrifuged in a Beckman model L preparative ultra- centrifuge at 105,000 x g for 1 hour to yield a microsomal pellet and a final supernatant fraction. Purified chromatin4 was pre- pared from the crude nuclei essentially by the procedure of Haussler et al. (2, 22). The crude receptor fraction was prepared from purified chromatin as described by Haussler and Norman

(7) Radioactivity Measurement-The radioactive steroids of all

tissue and subcellular fractions were extracted according to a slight modification of the Bligh and Dyer technique (23) as modified by Haussler et al. (2). The chloroform layer was evap- orated to dryness with air in a liquid scint’illation vial and 10 ml of a counting solution consisting of 100 mg of POPOP (1,4-bis- [2-(5.phenyloxazolyl)]benzene) and 3 g of PPO (2,5-diphenyl- oxazole) per liter of toluene was added. The sample was counted to 2% error in a Beckman CPM-200 liquid scintillation counter. The number of disintegrations per min of tritium and 14C present was determined by use of subsequently added internal standards and a computer program designed to process double label count- ing. The internal standards used were [3H]toluene (New Eng- land Nuclear Corporation) and [14C]benzene (Amersham-Searle) with respective specific activities of 34,548 and 9800 dpm per 10 /.Ll.

A model 1042 Nuclear-Chicago planchet counter was used to determine 45Ca2+ radioactivity.

Chromatography Procedure-Radioactive metabolites were separated on column (1 x 80 cm) of Sephadex LH-20 equili- brated with 35% hexane in 65% chloroform, v/v (24). The same solvent system was used for elution, and 3-ml fractions were collected.

Measurement of Calcium Transport-The rate of transport of calcium in vitro through segments of chick ileum was deter- mined using the techniques described by hdams and Norman (25). This method employs an apparatus with two chambers which are separated from one another by a lucite diaphragm in which is mounted a segment of ileal tissue. Both chambers are filled with Krebs-bicarbonate buffer + 0.1 mM CaClz and the solution then continuously gassed with 95% 0,-5% COZ. Then 45Ca2f (100 PCi) is added to the solution bathing the mucosal surface of the ileal segment. After 30 min, five 0.10.ml samples were removed every 10 min and placed on planchets. The cal- cium flux, J,,, (from the mucosal-to-serosal side of the tissue) was determined by a linear regression analysis of the radioactivity appearing in the serosal side of the tissue.

RESULTS

Fig. 1 shows the chromatographic purity of the 1,25-dihy- droxy[3H]cholecalcifero1 used in these studies. All batches of 1,25-dihydro&H]cholecalciferol were always chromatographed on at least two columns before use. In addition, each batch was always tested for its biological activity in terms of its ability

4 The term chromatin is employed in this paper to define the subnuclear fraction obtained from the intestinal mucosa after go- ing through the steps for its preparation which are outlined in Reference 2. This fraction has been examined by the electron microscope (22) and found to be relatively free of contaminating membranes. The ratio of RNA to DNA was rolltinely found to be 0.15.

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TABLE I

Distribution of radioactivity between the chloroform and methanol- Hz0 layers after a dose of 25-hydrnxy[3H]cholecalciferol or

1 ,25-dihyclroxy[3H]cholecalciferol

Groups of three to five chicks received intracardially 5 units (0.325 nmole) of 2%hydroxy[3H]cholecalciferol or 1,25-dihy-

droxy[3H]cholecalciferol. They were killed at the time in- dicated. The total lipid of the tissues was extracted and the radioactivity content of both layers determined. Each number

is the average of duplicate determinations.

Percentage of radioactivity isolated

Intestine _-

Liver Hours 1

.-

Total dpm/ chick

:HCla (

-I-

HgO- MeOH

Total dpm/ chick

2790 75 25 6550 2840 83 17 4760 4070 80 20 3090

760 74 26 900 1220 71 29 550

250 64 36 190

CHClz HO MeOH

1 68 32 4 78 22

20 88 12 1 83 17

4 84 18 20 76 24

25.Hydroxy-

cholecalciferol

1,25-Dihydroxy- cholecalciferol

FRACTK3~ ,:,,,, m

FIG. 1. Chromatographic analysis of 1,25-dihydroxy[3H]chole- calciferol. 1,25-Dihydroxy[sH]cholecalciferol was prepared in vitro from 25-hydroxy [26,27-3Hlcholecalciferol by incdbation of the latter with either chick kidney homogenates or mitochondria as described under “Materials and Methods.” The lipid extract of the incubation media was first chromatographed on a column of silicic acid. As indicated by the arrows in the top chromato- gram, the material migrating between Fractions 18 and 30 on the silicic acid column was rechromatographed on a Sephadex LH-20 column (bottom panel). The material migrating in Fractions 60 to SO, the region of migration of authentic 1,25-dihydroxychole- calciferol, was pooled and tested for biological activity in terms of stimulating intestinal calcium transport in viva. It is a char- acteristic of 1,25-dihydroxycholecalciferol, as contrasted with 2.5-hydroxycholecalciferol or cholecalciferol, that it will mediate a maximum stimulation in 8 to 10 hours, whereas 20 to 30 hours are required for the latter steroids.

HOURS

to stimulate intestinal calcium transport in &JO before use (6, 26). This combined chromatographic purification coupled with biological testing was deemed mandatory for every preparation of 1 ,25-dihydroxy[3H]cholecalciferol since it is not yet une- quivocally established that this is the only steroid produced from 25-hydroxycholecalciferol by the kidney.

In all of the following experiments the radioactive steroids of all tissues and subcellular fractions were extracted according to a slight modification (2) of the procedure of Bligh and Dyer (23). The distribution of radioactivity between the chloroform and the methanol-H20 layers was examined. The results are re- ported in Table I. In the intestine, approximately 80% of the radioactivity was found in the chloroform layer, whereas in the liver only 65 to 75% of the radioactivity is found in the chloro- form layer. No attempts were made to further analyze the nature of the radioactivity in the water-methanol layer.

A study was made to correlate the time course of appearance of increased intestinal calcium transport with the appearance of radioactive cholecalciferol metabolites in the intestinal mucosa after separate intracardial doses of 25-hydroxy[3H]cholecalciferol or 1, 25-dihydroxy[3H]cholecalciferol. Also the time course of appearance of radioactivity in the liver was assessed for com- parative purposes. Fig. 2 depicts the results obtained after

administration of 5 units of 25-hydroxy[3H]cholecalciferol. Fig. 3 depicts the results obtained after administration of 5 units of

TIME (hours)

FIG. 2. Time course of localization of radioactivity in mucosa and liver after a dose of 25-hydroxy[3H]cholecalciferol (A) and its relationship to stimulation of intestinal calcium trans- port (B). A, all chicks for radioactivity determinations received a dose of 5 units (0.325 nmole) intracardially and were killed at the time indicated. Each number represents an average of two to four separate determinations. B, the ileal calcium flux, J,,, was measured in vitro at the indicated time interval following ad- ministration of 50 units (3.25 nmoles) of 25.hydroxycholecalciferol to rachitic chicks. Each point represents the average of three to four separate determinations.

in the liver, after a dose of 25-hydroxy[311]cholecalciferol, rises rapidly so that within 1 hour of dosing 0.084 unit or 1.7% of the dose was present. It then falls gradually with time up to 12

1, 25-dihydroxy[3H]cholecalciferol. The radioactivity localized hours and remains relatively unchanged between 12 to 48 hours.

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4 8 12

HOURS

TIME (hours)

FIG. 3. Time course of localization of radioactivity in mucosa and liver after a dose of l,25-dihydroxy[3H]cholecalciferol and its relationship to stimulation of intestinal Ca++ transport. A, all chicks for radioactivity determinations received a dose of 5 units (0.325 nmole) of 1,25-dihydroxy[3H]cholecalciferol intracardially and were killed at the time interval indicated. Each point is an average of two to four determinations. B, the ileal caIcium flux, J,,, was measured in vitro at the indicated time interval following a dose of 2 units (0.130 nmole) of 1,25-dihydroxy[3H]cholecalcif- erol. Each point represents the average of four separate determinations.

In contrast, the radioactivity in the target intestine increases gradually with time and reaches a maximum between 8 and 16 hours so that 0.06 unit or 1.2% of the dose is present. Hereafter, it stays relatively constant up to 48 hours. In the case of 1,25- dihydroxycholecalciferol (Fig. 3), the radioactivity isolated in the target intestine reaches a maximum within only 4 hours. At this time, 0.26 unit or 5.1 y0 of the dose is present. Then there is a relatively rapid fall of radioactivity so that by 64 hours only a minute amount remains in the intestine. The radioactivity in the liver follows a similar pattern but the maximum is reached by 30 min after dosing when only 0.19 unit or 3.8% of the dose is bound.

The time course of stimulation of calcium transport after a dose of 25-hydroxycholecalciferol (Fig. 2B) or 1,25-dihydroxy- cholecalciferol (Fig. 3B) was also studied. For 25-hydroxychole- calciferol, a maximum transport response was not obtained until 28 hours after dosing, which is some 10 to 12 hours after maximal localization of radioactivity in the intestine. In contrast, for 1,25-dihydroxycholecalciferol a maximum calcium transport response was obtained within only 8 hours, which was only 4 hours after maximum accumulation of radioactivity by the in- testine. As will be shown later, the major form of radioactivity associated with the mucosa after both doses of 25-hydroxycho- lecalciferol and 1,25-dihydroxycholecalciferol is 1,25-dihydroxy- cholecalciferol. Thus, the maximum stimulation of calcium

TIME IN HOURS

FIG. 4. Time course of appearance of 25-hydroxycholecalcif- erol and its metabolites in rachitic chick intestinal mucosa after a dose of 5 units (0.325 nmole) of 25.hydroxy[3H]cholecalciferol. The total lipids were extracted from each sample and chromatographed on a Sephadex LH-20 column as described in the text. A chloro- form-hexane 65:35 elution solvent system was used and 3-ml frac- tions were collected. O---O, 1,25-dihydroxycholecalciferol migrating in Fractions 81 to 100; O--U, 25.hydroxycholecalcif- erol migrating in Fractions 21 to 30; A--A, an unidentified sub- stance, possibly an ester of 25hydroxycholecalciferol, migrating in Fractions 9 to 15.

transport seems to closely follow the event of incorporation of 1,25-dihydroxycholecalciferol into the intestine.

Fig. 4 shows the time course of appearance of 25-hydroxychole- calciferol and its metabolites in the intestinal mucosa after a dose of 25-hydroxy[3H]cholecalciferol. An unidentified substance, possibly an ester of 25-hydroxycholecalciferol, appears first and then decreases sharply. Also the bulk of the 2Bhydroxychole-

calciferol is gone by 20 hours. The 1,25-dihydroxycholecalcif- erol increases gradually with time and reaches a maximum at 16 hours. It is of interest to note that this maximum level was maintained for at least 48 hours.

When the same type of time course study was carried out after a dose of 1 ,25-dihydroxy[3H]cholecalciferol, only one compound, 1,25-dihydroxycholecalciferol, was found in the intestine between 4 to 48 hours after dosing.5 More detailed results obtained after analysis of the subcellular localization are reported below. This strongly suggests that 1,25-dihydroxycholecalciferol was not further metabolized in intestine and that it is the functional form of cholecalciferol in the intestine.

The subcellular localization of radioactivity in the intestinal mucosa, liver, or kidney was studied after a dose of 25.hydroxy- [3H]cholecalciferol or 1 ,25-dihydroq[3H]cholecalciferol. The results are presented in Fig. 5. With both steroids, the crude nuclear fraction of the intestinal mucosa accumulated 75% of the radioactivity present. Neither the liver nor the kidney crude nucleus fractions contained such a large proportion of the total radioactivity. This agrees with the earlier findings obtained by Haussler and Norman (27) when they followed the subcellular distribution of radioactivity resulting after a dose of [3H]chole- calciferol. It is interesting that the kidney mitochondrial fraction

5 H. G. Nowicki and A. W. Norman, unpublished observa- tions.

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a Nuclei W Microsomes

q Mitochondria Elsupernate

80 1,25-diOH-CC

HIJCOSCI

Liver Kidney

25-OH-CC

Liver Kidney

FIG. 5. The subcellular distribut.ion of radioactivity in rachitic chick intestinal mucosa, liver, and kidney after doses of 25.hy- droxy[3H]cholecalciferol or 1 ,25-dihydroxy[3H]cholecalciferol. A dose of 5 units (0.325 nmole) of 25-hydroxy[3H]cholecalciferol or 1,25-dihydroxy[3H]cholecalciferol was administered intracardially 8 hours or 4 hours, respectively, before killing. The subfractiona- tion of tissue, extraction of lipids, and determination of radio- activity were carried out as described in the text. Each number is the average of two to four separate determination of samples from four to eight chicks.

does not demonstrate a preferential accumulation of radioactivity, since it is believed to be the site of conversion of 25-hydroxy- cholecalciferol to 1,25-dihydroxycholecalciferol.6 When the

subcellular distribution of radioactivity was followed with time (Table II), the relative distribution of radioactivity in the sub- cellular fractions of the liver stays constant. On the other hand, in the target intestine, the radioactivity found in the nucleus increases with time so that by 4 to 5 hours it has accumulated 75% of the total radioactivity isolated.

Previous work from this laboratory (2) indicated that the administration of a physiological dose of radioactive cholecalcif- erol to vitamin D-deficient chicks resulted in the stereospecific localization of a biologically active vitamin D metabolite (desig- nated Metabolite 4B) with the intestinal nuclear chromatin frac- tion. Further we have shown (7) that a crude protein receptor fraction with only a finite number of binding sites could be solubilized from the chromatin. Accordingly it was of interest in the present studies to ascertain if a similar chromatin localiza- tion of a cholecalciferol metabolite would result after administra- tion of 25-hydroxy[3H]cholecalciferol or 1 ,25-dihydroxy[3H]- cholecalciferol. The specificity of the localization was tested by the simultaneous administration of [4-14C]cholesterol. A re- ceptor molecule for cholecalciferol or its metabolites should have little affinity for cholesterol. In addition, the [14C]cholesterol which is found in the intestinal mucosa after injection is probably associated with the membrane-rich components of cells via a lipid-lipid interaction. The [r4C]cholesterol present in a given fraction is therefore a means of estimating the amount of steroid which is nonspecifically associated with that fraction. Thus, the

6 A. W. Norman, R. J. Midgett, and J. W. Coburn, unpublished observations.

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TABLE II

Time course of subcellular distribution of radioactivity in rachitic chick mucosa and liver after doses of 25-hydroxy[~H]cholecalciferol

or 1,25-dihydroxy[3H]cholecalciferol

All chicks received 5 units (0.325 nmole) of the appropriate steroid intracardially in 0.2 ml of 1,2-propanediol at the time period indicated before killing. Subcellular fractions were pre-

pared as described in the text. Each number is an average of two

to four determinations on samples from four to eight chicks. The range never exceeded ~t5’% of the indicated mean. The recovery

of the administered dose after a dose of 1,25-dihydroxycholecal- ciferol ranged from 5.07, (4 hours) to 0.6C0 (48 hours) in intestine and 4% (f hour) to 0.87, (48 hours) in liver. In the case of 25- hydroxycholecalciferol, the recovery of the administered dose

ranged from 0.6y0 (4 hours) to 1.27, (48 hours) in intestine and 1.770 (2 hours) to 1% (48 hours) in the liver.

1,25-Dihydroxy- [3H]cholecalcif-

erol

25-Hydroxy[%- cholecalciferol

Time .fter dose

hrs

t 1 4 8

16 48 65

2 4 8

12

20 48

Percentage of radioactivity isolated

Nuclei

31 48 17 70 49 8 75 44 6

64 40 13

64 35 12 36 57 12 31 35 17 57 46 10 63 49 9 71 46 8

73 33 7 75 56 7 62 44 12

7 17 15 6 21 12 7 26 16

12 17 12 31 14 21 24 14 28

11 18 22

9 16 19 7 15 14

7 15 13 7 9 12

11 15 14

18 17 15 13

19 15 22

27 28 20

27 23 22

ratio of 3H-bound steroid to r4C-bound steroid will increase when the nuclear fraction is further purified and an infinite ratio will be obtained when a pure receptor molecule specific for cholecalciferol or its metabolites is isolated. Table III summerizes the results obtained. Due to nonspecific binding of steroids, the ratio of %steroid to 14C-steroid in the homogenate or crude nuclear fraction is 0.91 or 8.53 after doses of 25-hydroxy[3H]cholecalcif- erol or 1 ,25-dihydroxy[3H]cholecalciferol, respectively. When the chromatin fraction was prepared, the ratio increased signif- icantly. The chromatin was then purified by washing with 1%

Triton X-100 three times in order to remove nonspecifically bound steroids. The resulting ratio of 3H:14C is 5 to 18 times above the value for the crude nuclear fraction. This suggests that only the %steroid is tightly bound to the chromatin and that it cannot be removed simply by washing with Triton X-100. As shown in Table II, there is also possibly a cholecalciferol-re- lated receptor in the supernatant fraction. In both experiments the 3H :r4C ratio of the supernatant fraction was higher than that of the starting homogenate sample. Similiar experiments have been done on liver. The ratio of 3H :r*C remains unchanged be- tween the homogenate and chromatin fraction. This suggests that the 3H-steroid bound to the liver is a nonspecific binding.

If it is assumed that receptor sites for cholecalciferol metabo- lites reside in the intestinal mucosal chromatin, then preliminary treatment with [14C]cholecalciferol should prevent the binding

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of 25-hydroxycholecalciferol and its metabolites or 1,25-dihy- droxycholecalciferol and its metabolites to the chromatin fraction. Table IV shows the results of an experiment where rachitic chicks were previously treated with [14C]cholecalciferol

TABLE III Relative amounts of 25-hydroxy[3H]cholecalciferol and its metabolites

or 1 ,25-dihydroxy[3H]cholecalciferol and its metabolites to [4-‘4C]- cholesterol and its metabolites in subcellular fractions obtained

from rachitic chick intestinal mucosa

Vitamin D-deficient chicks received 5 units (0.325 nmole) of 25- hydroxy[3H]cholecalciferol or 1,25-dihydroxy[3H]cholecalciferol and an equivalent amount of [4-WJcholesterol in 0.2 ml of 1,2- propanediol via intracardial injection 16 hours or 8 hours, respec- tively, prior to death. Cellular subfractions were prepared as

outlined in the text. All purified subnuclear fractions were pre-

pared from the crude nuclear fraction, not necessarily from the

fraction listed previously in the table. Each number represents

an average of four to eight separate determinations. The ratio of absolute amount of 25-hydroxy[3H]cholecalciferol and its metabo- lites or 1,25-dihydroxy[JH]cholecalciferol and its metabolites to [4-Wlcholesterol and its metabolites in the fraction is computed

from the ratio of tritium to 14C and the specific activities of the parent molecules. Dilution of the [4J‘C]cholesterol by endoge- nous cholesterol is nealected.

Cell fraction Ratio of 25.OH[~H]CP Ratio of 1,25-

and metabolites to diOHPH]CC and [I-“Clcholesterol and metabolites to

metabolites [CWlcholesterol and metabolites

I I

Homogenate............. 0.91 8.53 Crude nuclear fraction.. 1.43 9.58

Mitochondrial fraction. 0.48 5.36 Microsomal fraction. 0.49 5.66 Supernatant fraction.. 4.50 11.7 Chromatin fraction. 2.00 12.5 Purified chromatinb. 17.7 36.4

a 25-OH[3H]CC, 25-hydroxy[3H]cholecalciferol; 1,25-diOH[aH]- CC, 1,25-dihydroxy[3H]cholecalciferol.

b Each chromatin fraction was washed three times with 1.0% Triton X-100 in 0.01 M Tris, pH 7.5, before the radioactivity con-

tent was determined.

TABLE IV E$ect oj preliminary i,.eatment with [4J4C]cholecalciferol on the

binding of radioaciil?ty of intestinal mucosal chromatin after doses of 25-hytlroxy[3H]cholecalciferol or

1,25-dihytl~~oxy[3H]cholecalciferol Rachitic chicks received 50 i.u. (3.25 nmoles) of [4-14C]cholecal-

ciferol intracardially 8 hours or 16 hours prior to administration

of 5 units of 25-hydroxy[3H]cholecalciferol or 1,25-dihydroxy[3H]- cholecalciferol, respectively. The chicks were then killed 16 hours or 4 hours after the dose of 25-hydroxy[3H]cholecalciferol or l,25-dihydroxy[3H]cholecalciferol, respectively, and the chro-

matin fraction was prepared, extracted, and assayed for radio- activity by the usual method. Each number presented is the

average of four to six determinations.

Homogenate.................. 0.21 0.74

Purified chromatin.. . . . . . . . . 0.34 1.00

a 25-OH[3H]CC, 25-hydroxy[3H]cholecalciferol; 1,25-diOH- [“H]CC, 1,25-dihydroxy[3H]cholecalciferol; [‘%]CC, [ltC]chole- calciferol.

either 8 or 16 hours before dosing with 25-hydroxy[3H]choleca1cif- erol or 1, 25-dihydroxy[3H]cholecalciferol. When the 3H :14C ratios for the previously cholecalciferol-treated chicks are com- pared with the values obtained for [14C]cholesterol-treated chicks (Table II), it is apparent that the dose of cholecalciferol but not cholesterol is capable of filling the specific receptor sites in the chromatin fraction which exist for the 25-hydroxycholecalcif- erol or 1,25-dihydroxycholecalciferol and their metabolites.

As a result of the foregoing studies, it was important to deter- mine the nature of the 3H-steroid(s) associated with the intestinal mucosal chromatin. Fig. 6 presents the results of an experiment where the lipid extract from intestinal mucosal chromatin ob- tained 4 hours after dosing with 1, 25-dihydroxy[3H]cholecalciferol was chromatographed on a Sephadex LH column. There is only one peak of tritium. Similar results have been obtained with chromatin lipid extracts obtained after dosing with 25-hydroxy- [3H]cholecalciferol. Thus the cholecalciferol-related receptor site in the intestinal chromatin fraction is specific for 1,25-dihy- droxycholecalciferol.

Haussler and Norman (7) have previously reported on the ex- istence of an acidic protein associated with the intestinal chro- matin fraction which had a high affinity for binding non- covalently Metabolite 4B or 1,25-dihydroxycholecalciferol. The binding capacity of this receptor molecule, which was purified some 167-fold, became saturated after administration of a phys- iological dose of 50 i.u. (3.25 nmoles) of cholecalciferol. In the present studies, when a dose of 5 units (0.325 nmole) of 1,25- dihydroxy[3H]cholecalciferol was given to four rachitic chicks, and this receptor fraction prepared, 0.08 unit (5.2 pmoles) of steroid was found per receptor fraction from the intestinal chro- matin of one chick. This is in good agreement with the previous studies (7) where after administration of 20 i.u. (1.30 nmoles) of [3H]cholecalciferol, 0.055 i.u. (3.6 pmoles) of steroid metabolite was found bound to the intestinal chromatin receptor from one chick.

In Figs. 7 and 8 are shown the results of experiments where the saturability of the intestinal chromatm receptor was tested after

P

1,25-diOWC O”

I\ 0 0

p a3-

0

I I ,',I,

0

40 80 120

FRACTION NUMBER (3ml)

FIG. 6. Chromatographic analysis of intestinal chromatin- bound radioactivity after a dose of 1,25-dihydroxy[3H]cholecalcif- erol. Doses of 3 units (0.325 nmole) of 1,25-dihydroxy[3H]chole- calciferol were given intracardially to a group of rachitic chicks 4 hours before death. The chromatin fraction was then prepared from the intestinal mucosa, and the radioactivity was extracted wit.h organic solvents and chromatographed on a column of Seph- adex LH-20 as described under “Materials and Methods.” Under

these conditions only 1,25-dihydroxycholecalciferol migrates in Fractions 70 to 85.

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increasing doses of either 25-hydroxy[3H]cholecalciferol or 1,25- dihydroxy[3H]cholecalciferol, respectively. The steroids were given intracardially to groups of four to six rachitic chicks and Triton X-100 washed chromatin prepared.

It, was found that a dose of 25 to 30 units (1950 pmoles) of 25- hydroxycholecalciferol will saturate the chromatin (Fig. 7) but that only a 0.4 unit (25 pmoles) dose of 1,25-dihydroxycholecal- ciferol was required to saturate the binding sites in chromatin (Fig. 8A). Also shown in Figs. 7 and 8 are the dose-response curves of intestinal calaium transport, measured in. vitro as the

DOSE(UNIT)

FIG. 7. Saturation of intestinal mucosal chromatin after in- creasing doses of 25-hydroxy[3H]cholecalciferol and its relation- ship to increased intestinal calcium transport. A, the calcium

flux, Jm, was determined as described under “Materials and Methods.” Each point is the mean f S.E. of determinations on ileal tissue from four chicks. B, the indicated doses of 25-hy- droxy[3H]cholecalciferol were administered intracardially 16 hours before killing. After preparation of intestinal mucosal chromatin, it was washed (via homogenization) three times in 1.0% Triton X-100. Each point is the average of t.wo to four separate determinations.

DOSE (UNIT)

FIG. 8. Saturation of intestinal mucosal chromatin after creasing doses of 1,25-dihydroxy[3H]cholecalciferol and its rela- tionship to stimulation of intestinal Ca++ transport. A, the cal- cium flux measurement, J,,,, was done as described previously under “Materials and Methods.” Each number is the mean f S.E. of determinations on ileal tissue from four to five chicks. B, the indicated doses of 1,25-dihydroxy [3H]cholecalciferol were administered intracardially 9 hours before killing. The Triton X-100 washed chromatin was prepared as described under “Ma- terials and Methods.” Each point is the average of two to four separate determinations.

5517

dose of 25-hydroxy[3H]cholecalciferol or 1) 25-dihydroxy[3H]cho- lecalciferol is increased. There is a clear and obvious correlation between saturation of the intestinal chromatin fraction with 1,25-

dihydroxycholecalciferol and the appearance of a maximal cal- cium transport response. With 1, 25-dihydroxy[3H]cholecalcif- erol the receptor is saturated after a close of only 0.4 unit (25 pmoles), a dose which also gives a maximum calcium transport response. These results and the time course of appearance of 1,25-dihydroxycholecalciferol in the chromatin fraction strongly suggest that there is a cause and effect relat,ionship between the appearance of this steroid in the intestinal chromatin fraction and development of the biological response.

It is known that cholecalciferol (vitamin Ds) plays as essential role in mediating the intestinal absorption of calcium (26, 28, 29). It has been postulated that cholecalciferol may generate this characteristic physiological response via it:: ability to activate or stimulate the biochemical expression of genetic information to induce the synthesis of enzymes or the alteration of membrane structure necessary for calcium absorption. This hypothesis was primarily based on the findings that actinomycin D com- pletely blocked the intestinal response to cholecalciferol (30). Furthermore, cholecalciferol was found to stimulate the incorpo- ration of [%]uridine or [3H]orotic acid into intestinalmucosa RNA (31, 32). More recently additional evidence was obtained which was consistent with the vitamin D induction hypothesis. After a physiological dose of [3H]cholecalciferol, 6.5 to 75% of the radio- activity found in the intestinal mucosa was in the form of a polar metabolite (designated Metabolite 4B in this laboratoryl) which was stereospecifically associated with its chromatin fraction and an acidic, nonhistone receptor protein (7). Myrtle and Norman (8) as well as Haussler et al. (33) subsequently reported that Metabolite 4B was some four times more biologically active than its parent cholecalciferol and two times more active than the intermediate 25-hydroxycholecalciferol in terms of stimulating intestinal calcium transport. Additionally, this chromatin- localized metabolite was found to be capable of producing a maximal physiological response in only 8 to 9 hours, whereas cholecalciferol and 25-hydroxycholecalciferol both required 25 to 35 hours.

Our results described in this communication, which were obtained after dosing with radioactive 25-hpdroxycholecalciferol or 1,25-dihydroxycholecalciferol, further emphasize the possible importance of the cholecalciferol metabolite being in close asso- ciation with the genome of its target organ. With both of these steroids, as reported in Figs. 4 and 6, chromatographic analysis of the radioactivity present in the intestinal mucosa after doses of radioactive 25-hydroxycholecalciferol or 1,25-dihydroxychole-

calciferol, indicated that at all time period:: essentially only one compound was present, 1,25-dihydroqcholecalciferol. Further- more it was exclusively associated with the nucleus and its chro- matin fraction. Also as reported in Table I, there were only minimal amounts of radioactivity in the methanol-water layer of the organic solvent extracted mucosa. These results strongly suggest that the intestinal mucosa does not further metabolize 1,25-dihydroxycholecalciferol and that’ this is the final form of vitamin D present in the target intestinal mucosa. This is consistent with the recent findings of Frolick and DeLuca (34) and Nowicki and Norman.5 No other metabolites were found by them in the intestinal mucosa 12 hours after dosing with 1,25- dihydroxycholecalciferol. Thus if 1,25-dihydroxycholecalcif- erol is the final form of vitamin D found in the target intestine,

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5518

and since it has biological activity much greater than the parent vitamin, and can develop a physiological response much more rapidly than the vitamin, then it seems virtually certain that this is the biologically active form of vitamin D in the mucosa.

An important and as yet unanswered question then is by what biochemical mechanism(s) does 1,25-dihydroxycholecalciferol mediate a physiological response. One approach to this problem is to correlate the subcellular localization of this steroid with the onset of the physiological response. The results shown col- lectively in Figs. 2 to 5, suggest that the association of 1,25-dihy- droxycholecalciferol with the intestinal nucleus and its chromatin fraction precede the development of the physiological response. This has been found to be the case for all three steroids, chole- calciferol (2, 3), 25.hydroxycholecalciferol (Fig. 2) and 1,25- dihydroxycholecalciferol (Fig. 3). The decreasing lag of 30, 28, and 9 hours for development of the maximal physiological re- sponse after administration of cholecalciferol, 25-hydroxychole- calciferol, or 1,25-dihydroxycholecalciferol, respectively, is in accord with their sequential order of metabolism by the liver and kidney and localization in the intestinal mucosa.

Another approach to understanding the mechanism of action of 1,25-dihydroxycholecalciferol is to correlate the magnitude of the dose required for saturation of the chromatin binding sites with the magnitude of the dose required for development of the maximum biological response. These results are summarized in Table V and Figs. 7 and 8. Again there is excellent agreement between the results obtained from dosing separately with the three steroids. In each instance the dose required to produce the maximum stimulation of the intestinal transport of calcium also produced a saturation of the chromatin binding sites with 1,25- dihydroxycholecalciferol. Also, as reported in Tables III and IV, the chromatin fraction of the mucosa has the highest speci- ficity of binding sites for 1,25-dihydroxycholecalciferol. HOW-

ever, preliminary treatment of the rachitic chicks with [‘“Cl- cholecalciferol several hours prior to dosing with 25-hydroxy- [3H]cholecalciferol or 1 ,25-dihydroxy[3H]cholecalciferol (Table IV) prevented the subsequent localization of any tritium-labeled steroids. Taken together these results are strong circumstantial evidence that the chromatin localization of 1,25-dihydroxychole- calciferol is an important if not essential prerequisite for the development of the characteristic response in the intestine to vitamin D.

The production of the steroid, 1,25-dihydroxycholecalciferol by the kidney and its selective accumulation by the target organ support the concept that this compound should be regarded as a hormone. This suggestion is emphasized by the results shown in Figs. 2 and 3. When a single dose of 5 units (0.325 nmole) of the hormone, 1 ,25-dihydroxy[3H]cholecalciferol, was given, intracardially, maximal amounts of this steroid were pres- ent in the intestinal nucleus by 4 to 5 hours; but this decayed away rapidly so that by 24 to 30 hours less than 10% of the maximum level was present. In sharp contrast are the results obtained after administration of the pro-hormone, 25.hydroxy- [3H]cholecalciferol. Here the maximal accumulation of the hormone, 1,25-dihydroxycholecalciferol, did not cccur until 8 to 12 hours after the dose. More importantly, maximal levels of the hormone in the intestinal chromatin were maintained for up to 36 hours; by 48 hours after the dose of the pro-hormone there was still 50 to 60% of the hormone associated with the intestinal chromatin. Thus the continued availability of the pro-hormone and the likely occurrence of a homeostatic mechanism regulating the metabolism of 25-hydroxycholecalciferol permit the produc- tion in a highly modulated manner of just enough 1,25-dihy- droxycholecalciferol to be taken up by its receptor sites.

Thus it appears that the mechanism of action of calciferol (vitamin D) may be analogous to that of other steroid hormones such as estrogen (16)) hydrocortisone (35)) testosterone (18)) and aldosterone (17). All of these steroids are believed by virtue of their association with the nucleus of their respective target tissues to activate or stimulate the biochemical expression of genetic in- formation. It was also recently reported by Steggles et al. (36, 37) that there is hormone receptor complex in the cytoplasm of the cell and that the genome is programmed to receive the com- plex as it is transferred into the nucleus. Our present data are consistent with this same possibility. In Tables II and III, the data indicate that there is an increase of radioactivity in nucleus and a decrease of radioactivity in the supernatant with time, while the amount of radioactivity present in the other subcellular fractions remains constant over the same time interval. Also as shown in Table IV, there is a high ratio of 3H-steroid to 14C-ste- roid in the supernatant fraction as well as the chromatin fraction, which is indicative of a preferential binding of vitamin D-related steroids. The studies in vitro of the transfer of such steroids as testosterone (36) and progesterone (37) between their cytoplas-

TABLE V Summary of results obtained after closing with cholecalciferol, 25-hydroxycholecalciferol, or 1,25-&hydroxycholecalciferol

Compound rtdministered

Parameter measured

Dose in picomoles necessary to produce a maximal Ca2+ transport response. ._ . . . . . . . . . .

Dose in picomoles necessary to saturate chromatin binding sites..................................................

Lag time in hours for maximal Ca2+ transport response. Lag time in hours before chromatin binding sites are satu-

rated. . ..____...................................... Evidence for specificity of chromatin binding for 1,25-di-

hydroxycholecalciferol.. Maximum amount in picomoles of 1,25-dihydroxychole-

calciferol bound to the chromatin fraction from one

chick intestinal mucosa. -

Cholecalciferol

19500

1950 (41)

36-48 (8)

15-20 (40)

Yes (3, 7)

7.8 (40)

--

-

25.Hydroxycholecalciferol

1400 (Fig. 7A)b

1400 (Fig. 7A) 20-30 (8) (Fig. 2)

12-16 (Fig. 2)

Yes (Tables III and IV)

6.5 (Fig. 7B)

1,25-Dihydroxycholecalciferol

26 (Fig. 8B)

26 (Fig. 8B) 8-10 (8) (Fig. 3)

4 (Fig. 3)

Yes (Tables III and IV)

5.2 (Fig. 8B)

a R. G. Wong and A. W. Norman, Unpublished observations. b The informat,ion in parentheses is either the reference citation to other published papers from this laboratory or the table or

figure number of this paper which reports the data.

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5519

mic receptors and their subsequent stereospecific binding to target tissue chromatin provides a useful model for us to study further the action of cholecalciferol at the molecular level. Al- though the results reported by Gray and DeLuca (38) and Ta- naka et al. (39) were interpreted as possibly questioning the validity of the genome induction model for the mechanism of cholecalciferol, the earlier findings from this laboratory (2, 3, 7), the recent reported by Lawson et al. (41) of the association of 1,25-dihydroxycholecalciferol with bone cell nuclei, and the present data all support the validity of the genome induction hypothesis as the most acceptable explanation for the mode of action of vitamin D. Certainly the prospect now exists that with the availability of 1, 25-dihydroxy[3H]cholecalciferol that new experiments can be devised to critically test this model.

Acknowledgments-We wish to acknowledge the excellent technical assistance of Miss Patricia Roberts and Miss June Bishop.

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Huan C. Tsai, Richard G. Wong and Anthony W. NormanCORRELATION WITH INCREASED CALCIUM TRANSPORT

1,25-DIHYDROXY-VITAMIN D3 IN INTESTINAL MUCOSA AND Studies on Calciferol Metabolism: IV. SUBCELLULAR LOCALIZATION OF

1972, 247:5511-5519.J. Biol. Chem. 

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