Vol. 169, No. 3, 1990 June 29. 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 91 O-91 5

BIOCHEMICAL

UP-REGULATION

OF THE INTESTINAL

1,25-DIHYDROXWITAMIN

RECEPTOR DURING HYPERVITAMINOSIS BETWEEN VITAMIN M. J.

Beckman,t

*National

Animal

Disease

USDA, Department

Received

May 3,

J 2 T. A. Reinhardt,* D. C. Beitz t Agriculture

of Agriculture,

State

D3l

*

Center,

t Department Iowa

D: A COMPARISON

D2 AND VITAMIN

R. L. Horst, and

D

Ames,

of Animal

University,

Research

Service,

IA 50010

Science, Ames,

IA 50011

1990

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 + 24, vitamin D2-treated; 525 +_ 41, The 1,25-dihydroxyvitamin D vitamin D3-treated; and 249 k 19, control). receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D. 01990 Academic Press, Inc. Typical vitamin

of other

steroid

hormones,

D, 1,25-dihydroxyvitamin

receptor

protein

biologic

responses

(VDR)

in

the

physiologically

D (l,25-(OH)2D), target

by modulating

Several

investigators

have

elicits

homologous

up-regulation

provided

cells.

active

binds

The VDR/hormone

transcription evidence

that

complex

suggests (3)

of

to an intracellular

of specific

of VDR in vitro

form

as well

genes

initiates (1,2).

l,25-(OH)2D as in vivo

1 Journal Paper No. J-13970 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2504. Mention of a trade name, proprietary product, or vendor does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may be suitable. The U.S. Government's right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. 2To whom correspondence should be addressed. Abbreviations used: 1,25-(OH)2D, 1,25-dihydroxyvitamin D; 25-OH-D, 25-hydroxyvitamin D; 24,25-(OH)2D, 24,25-dihydroxyvitamin D; VDR, l,25-(0H)2D-receptor. ooo6-291x/90 Copyright All rights

$1.50

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

910

Vol.

169, No. 3, 1990

BIOCHEMICAL

Homologous

(4,5,6). systems

for

both

glucocorticoid regulation

(9,10), (12),

Under

responsiveness in

cell

undergo

other

cantly

(15)

that

l,25-(OH)2D3

in all

In chronic

resulted

Hughes

Therefore,

from

control) rats

was the

infused

plasma

are

increased

respon-

recent were

work that

greater

with

a dose

concentrations

of

signifi-

showed

was significantly were

imply

in an increase

They also

D, 1,25-(OH)2D is

(18)

have

in young

of

of

that

plasma

and,

toxicity

bone

in vitamin

if death

D toxicity, of 25-OH-D. 25-OH-D

calcium

other

and,

and ultimately

concentrations,

possibly,

relatively

occurs

concentrations

at stimulating

transport,

is

dysfunction

suggested

at pharmacological

concentration that

renal

to high

1,25-(OH)2D

calcium

so elevated

in severe

responding

when present

intestinal

in

logic

to adults.

in comparable

result

et al. are

to VDR and mimic

numbers

resulting

result

this

would target-cell

VDR concentrations

young

calcium

will

tissues

and

rats.

plasma

unattended,

(11)

Indeed,

receptor

likely

with

relative

the

down-

as androgen

an event

intestinal

rats

receptor the

VDR concentrations

when

would

hypervitaminosis but

(16,17).

showed

when

1,25-(OH)2D3

unaffected,

impaired

Therefore,

of VDR (change in adults

such

to 1,25-(OH)2D.

Consistent

that

in young

up-regulation

target

is

other

up-regulation.

tissue

(13,14).

in

For example,

ligand-dependent

increased

VDR concentrations

greater than

receptors,

ligand-dependent

to l,25-(OH)2D. et al.

left

receptors

to 1,25-(OH)2D

or lacking

rats

(7,8).

of target

decreased siveness

demonstrated

hormones

conditions,

sensitivity

target

has been

whereas undergo

physiological

enhanced

also

RESEARCH COMMUNICATIONS

and peptide

and estrogen

prolactin

Horst

regulation

steroid

AND BIOPHYSICAL

can bind

resorption,

functions

attributed

to

1,25-(OH)2D. The purpose vitaminosis intestinal

of this

D, like VDR.

vitamin

D3 in

excess

of both

Also,

the

paper,

therefore,

1,25-(OH)2D, rat

forms

because (19),

this

of vitamin

was to determine

can cause vitamin

homologous

D2 is

study

also

putatively compared

if

hyper-

up-regulation less the

of

toxic

effects

than of dietary

D on VDR regulation.

MATERIALS

AND METHODS

Animals and diet: Male albino Holtzman rats (4 weeks old) were purchased Rats were randomized in groups of 6 and from Holtzman Company (Madison, WI). fed a normal rat diet (Teklad 4%) for 2 weeks before initiation of the experiment. At 6 weeks of age (200-250 g body weight), two groups were treated orally with 25,000 IU/rat daily of vitamin D2 or vitamin D3 in 100 ~1 of cottonseed oil for 6 days. Materials: All vitamin D3 compounds were standardized by using a molar extinction coefficient (Ese,) of (18,200 M-lcrn-l) in a Beckman Model DU-70 recording spectrophotometer (Beckman Instruments, The Es64 used for vitamin D2 compounds was Fullerton, CA). (19,600 M-'cm-'). High-pressure liquid chromatography (HPLC) was performed with a Waters AlGPC 204 liquid chromatograph fitted with

911

a modal

Vol.

169,

No.

BIOCHEMICAL

3, 1990

AND

810-B Waters intelligent sampling vitamin D3 standards were detected

BIOPHYSICAL

RESEARCH

processor (WISP 810-B). at 254 nm.

COMMUNICATIONS

Vitamin

D2 and

Intestinal VDR assay: Twenty-four hours after the last dosage, rats from each group were anesthetized with C02/02 (50:50), weighed, and killed by decapitation. Blood was collected in heparinized tubes, cooled to 4"C, and centrifuged to separate plasma. To eliminate extraneous proteins and contaminants, intestinal mucosa was washed in 3 ml of TED buffer (50 mM Tris/HCl, 5 mM dithioltreitol, 1.5 mM EDTA, and 200 pg/ml soybean trypsin inhibitor, pH 7.4), and centrifuged at 5000 x g for 10 minutes. Intestinal mucosa was prepared as a 20% homogenate in TED buffer containing 500 mM potassium chloride, and VDR was quantitated as previously described (15). Ouantitation of plasma vitamin D metabolites: Plasma vitamin D metabolites were extracted by using the procedure described by Reinhardt and Horst (20) and were prepared for HPLC as described previously (21). Briefly, vitamin D2 metabolites were separated from vitamin D3 metabolites by using straight-phase Zorbax Sil columns developed in hexane/isopropanol (98:2, v/v) for 25-hydroxyvitamin D (25-OH-D) analysis and methylene chloride/isopropanol v/v) for 1,25-(OH)2D analysis. (97/3, General Perkin

techniques: Elmer Model

Plasma calcium concentration was determined 5000-AA spectrophotometer at wavelength 422.7

by using nm.

a

RESULTS The effects weight,

plasma

Table

I.

calcium,

Animals

perienced were,

of hypervitaminosis treated

g less

when compared analysis

(rig/ml)

in rats

Table 1. in rats

Changes treated

D3-treated

dosed

in for

vitamin

either

relative between

was decreased

the vitamin

plasma either Plasma Ow/dl)

D2

calcium, 25,000

(466

D3 ex-

treated

(p < .Ol)

(p < .Ol)

or control

groups.

greater

concentrations

+ 36)

or

vitamin

25-OH-D (x/ml)

1,25-(0H)zD h/ml)

20 2

f

230 17

11.8a f 0.6

466a f 36

f

123 12

*

201a 18

12.0a k 0.9

506a f 67

f

150b*c 8

between the mean A S.E.

912

D2-

the

Plasma D3

f

of

50

and plasma vitamin D metabolites IU/d of vitamin D2 or vitamin D3

Ca

significance

Body

and was about

9.5 f 0.7

ap < 0.01. Statistical control group. b, < 0.05. 'Statistical significance The data represent the

There

groups.

251 5

Ds-treated

in

to controls.

the

D2-treated

vitamin

are presented

D2 and vitamin

hypercalcemia

significantly

body weight, six days with wt

D metabolites

differences rats

D3 on body

f D-r-treated

the

with

Body w

Control

to

with

revealed

vitamin

excess

(p < .Ol)

of vitamin

25-OH-D

with

no significant

weight

Group

and plasma

significant

however,

D2 and hypervitaminosis

treated and

D3-treated

112 f 11

groups

relative groups

(n=6)

Vol.

BIOCHEMICAL

169, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Day 6 of Treatment

(506

Fie. 1 Intestinal VDR in rats treated vitamin D2 or vitamin D3 relative to (a) Vitamin-D3-treated significantly (p < .05).

6 days with 25,000 IU/d of either the response in age-matched controls different from vitamin-D2-treated

f

2).

67)

25-OH-D

relative

The

unchanged, these

only

increases

in

that

animals

1 demonstrates

Treatment