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Biochimica et Biophysica Acta, 497 (1977) 805--811 © Elsevier/North-Holland Biomedical Press

BBA Repor t

BBA 21447

1,25-DIHYDROXYVITAMIN D STIMULATION OF SPECIFIC MEMBRANE PROTEINS IN CHICK INTESTINE

P.W. WILSON and D.E.M. LAWSON

Dunn Nutrition Unit, University of Cambridge and Medical Research Council, Milton Road, Cambridge (U.K.)

(Received January 12th, 1977)

Summary

Vitamin D-deficient chicks were injected intracardially with physiological doses of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and the formation of intestinal brush-border proteins was followed in vitro. Within 4 h of receiving the hormone the incorporation of radioactive leucine into at least two proteins in the brush-borders was increased. The apparent molecular weights of these proteins were 45 000 and 84 000. The change in the synthesis of these pro- teins was followed with time and compared with the concomitant changes in intestinal calcium transport. The relationship of these changes is such that there is a strong possibility that the proteins are involved in calcium absorption.

A number of biochemical changes occur in the intestine of vitamin D- deficient chicks before the stimulation of calcium absorption which follows the administration of either vitamin D or 1,25-(OH)2D3. One of the effects of 1,25-(OH)2D3 is to initiate calcium-binding protein synthesis by inducing the expression of the calcium-binding protein gene [1 ]. Although there is signifi- cant evidence in favour of a role for this protein in calcium transport [2] we have recently shown that other vitamin D-dependent factors must also be in- volved in this process since changes in calcium-binding protein levels and the rate of calcium absorption are not in phase [ 3]. Thus in the chicken, intestinal calcium transport showed a significant stimulation 2 h after a dose of 1,25- (OH)2D3 (125 ng) whereas the synthesis of calcium-binding protein was just detectable after 3--4 h and did no t reach its maximum synthetic rate until 13 h after the dose. This rate of synthesis gave rise to the highest concentra- tion of calcium-binding protein in the cytosols 24--48 h after administration of the hormone, by which time calcium transport had returned to its base level.

A b b r e v i a t i o n : 1,25-(OH) 2 Ds, 1,25-dihydroxycholecalciferol.

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Fig. 1. So luble 14 C. a n d 3H-labelled b r u s h - b o r d e r f r a c t i o n p r o t e i n s f r o m i n c u b a t e d je juna l slices o f v i t a m i n D - d e f i c i e n t c h i c k s b e f o r e a n d 3 h a f t e r 1 , 2 5 - ( O H ) 2 D 3 ( 1 2 5 ng ) r e spe c t i ve ly w e r e m i x e d a n d e l e c t r o p h o r e s e d on a 11% a c r y l a m i d e gel as desc r ibed in the t ex t . a. P a t t e r n o f s t a ined p r o t e i n bands . b. A b s o r b a n c e scan o f t h e s t a i ned gel. c. 3H/14C r a t i o in each 1 m m slice o f the gel. d. D i s t r i b u t i o n o f to ta l 3H d p m t h r o u g h o u t the gel.

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We therefore investigated the possibilities of a more rapid formation of other proteins in the intestinal mucosal cells of the chick.

Flasks containing 0.5 g of everted chick jejunal slices in 10 ml of Krebs Improved Ringer 1 media [4], were incubated for 2 h with shaking under an atmosphere of 95% 02, 5% CO2 at 37°C. Slices from vitamin D-deficient chicks or 1,25-(OH)2D3-dosed chicks were incubated with [14C]leucine (25/~Ci) or [3H]leucine (125 pCi), respectively. At the end of the incubation the slices (1 g) were washed with saline and used for tissue fractionation. "Purif ied" brush-border fractions were prepared by the method of Forstner et al. [5] using 50 ml of 5 mM EDTA buffer, pH 7.4, per g of intestinal slices and sus- pending the recovered brush-borders in 2 ml of EDTA buffer. Cytosols were prepared from slices homogenised in a buffer containing 0.137 M Tris chloride, 0.12 M NaC1 and 4.74 mM KC1, pH 7.4, by centrifuging at 7000 × g for 10 rain and collecting the supernatant.

A sample (0.1 ml) of the 14C-labelled brush-border fraction from defi- cient chicks was combined with an equal volume of the 3H-labelled brush- border preparation from dosed chicks. The mixed brush-border pellet ob- tained by centrifugation was dissolved in 0.2 ml of a solution containing 12 mg Tris, 15 mg dithiothreitol, 10 mg SDS and 0.1 ml glycerol per ml (dissoci- ating solution) for 2 h at 37°C. The now soluble brush-border proteins (50 pl) plus 5 pl 0.05% bromophenol blue indicator were run on a discontinuous SDS gel electrophoresis system [6]. The upper gel was prepared from a solu- tion of 0.2% N,N'-methylene-bis-acrylamide (bis) and 3.0% acrylamide and the lower gel from 0.1% bis and 11.0% acrylamide or 0.25% bis and 5.0% acrylamide. They were run at room temperature for approximately 2 h in a Shandon disc electrophoresis apparatus at 3 mA/gel. The gels were stained with 0.25% Coomassie Blue in 50% methanol/10% acetic acid for 1 h and destained overnight in 7% acetic acid/5% methanol. They were scanned in a Joyce Loebl Scan 400, frozen with solid CO2 and 1-mm slices prepared using a Macrotome GTS, Yeda Research and Development Co. Ltd. Each slice was placed in a glass scintillation vial with 0.5 ml 90% NCS, heated at 60°C for 2 h and after cooling a further 0.5 ml NCS and 10 ml scintillation solution (0.01% POPOP, 0.4% PPO in toluene) were added and cooled before counting in a Packard Tricarb Scintillation Spectrometer model 3375. Quenching correction was calculated by means of automatic external standardisation and correlation curves for combined 14C and 3H.

As shown in Fig. 1 the gel system used in these experiments resolved the solubilised membrane proteins into at least 33 bands. The distribution of 3H in these proteins is also shown. Expression of the radioactivity distributed throughout the gel as a 3H/laC ratio indicates by an increased ratio any change in protein synthesis due to 1,25-(OH)2D3. The 3H/14C ratio of each gel slice is shown in Figs. 2a--g for the fractionated brush-border proteins from birds treated for up to 10 h with the hormone. In these experiments spurious varia- tions in ratio could be greatly decreased when care was taken to measure radio- activity with maximum accuracy (i.e. with the accumulation of 10 000 counts at least). Changes in ratio were only regarded as significant when they occurred in the same direction in adjacent slices thereby producing a peak. Ratio changes were ignored in regions of little radioactivity as sometimes occurs at either end

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of the gel. The small variation in the ratio which can be obtained (Fig. 2a) illustrates the sensitivity of the technique. Although after 1 h exposure to the hormone more than one protein peak with an increased 3H/14C ratio appeared to be present, at subsequent time-intervals only in the regions of peak A and then of peak B did the changes in ratio increase with time after t reatment with the hormone, thereby indicating the significance of these peaks.

The increase in the synthesis of the protein(s) in peak A could be seen after t reatment with 1,25-(OH)2D3 for 1 h and reached a peak at 3 h. Changes in the synthesis of protein(s) in peak B occurred later being first observed 3 h after t reatment with 1,25-(OH)2D3 and reaching a peak at 8 h. Using the formula 3H dpm -- (~4C dpm × average 3H/14C) it is possible to calculate the amount of 3H in these areas of the gel giving rise to the two peaks A and B. The changes in the amounts of 3H present in these proteins at various time intervals after 1,25-(OH)2D~ treatment are shown in Fig. 3 together with the changes in calcium absorption as measured by the everted intestinal sac technique [ 3].

The molecular weight of these proteins was assessed by comparison with a series of proteins of known molecular weight. Protein(s) in peaks A and B have apparent molecular weights of 45 000 and 84 000, respectively. The effect of 1,25-(OH)2D3 seems to involve the stimulation of de novo synthesis of these two membrane proteins since an intraperitoneal injection of actino- mycin D (100 pg/100 g body wt. for 1 h before the hormone) significantly decreased this stimulation.

Two reports have appeared recently relating to calcium transport and other activities of intestinal brush-borders. In one report calcium-binding protein was detected immunologically in a brush-border fraction and in another [8] a particle with calcium-binding activity was described. The relationship of the proteins in peak A and B with these other observations is not clear but we have established that peak A does not cross-react with calcium-binding protein anti-sera and the other aspects are under investigation.

Changes in the synthesis of cytoplasmi c proteins in response to these steroids are to be published separately but in essence we have found that calcium-binding protein is the only cytoplasmic protein whose synthesis is affected by 1,25-(OH)~D3.

The incorporation of [3H]leucine into brush-border fraction proteins could be due to the synthesis of a new protein in response to a physiological dose of 1,25-(OH)2D3, or the alteration of the rate of synthesis of an existing protein or rapid changes in the pool size of precursors or certain protein sub- units. This effect of 1,25-(OH)2D3 appears to require mRNA synthesis as actinomycin D lowers the rate of leucine incorporation into these particular proteins more than the other intestinal brush-border fraction proteins. This suggests that the hormone induces the synthesis of these proteins in a similar manner to the induction of calcium-binding protein synthesis.

Whether these proteins are directly involved with the transport of calci- um across the membrane will need further research but their rapid appearance in response to 1,25-(OH)2D3, their position in the mucosal cell membrane and the relationship of their rate of synthesis with the rate of calcium absorption

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Fig. 2. I n f l u e n c e of 1 , 2 5 - ( O H ) 2 D 3 on syn thes i s o f b r u s h - b o r d e r f r ac t i on p r o t e i n s ana ly sed by the doub le label l ing t e c h n i q u e . J e juna l slices f r o m v i t a m i n D-def i c i en t ch i cks t r e a t e d w i t h 1 , 2 5 - ( O H ) 2 D 3 or the d i luen t o f th i s s te ro id for v a r y i n g t imes , were i n c u b a t e d w i t h [ 3H] leuc ine or [ i 4 C ] l e u c i n e , r e spec t ive ly . B r u sh -bo rde r f r a c t i o n s were p r e p a r e d , m i x e d , d isso lved , e l ec t rop l io resed , a n d the 3H/~4C ra t io ca lcu la ted for each slice, a, 1 li; b, 2 h; c, 3 h; d, 4 h; e, 6 h; f, 8 h; g, 10 h. Gels a - -c and d ~ were o b t a i n e d us ing 5% or 11% a c r y l a m i d e , r e spec t ive ly .

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Fig. 3. C h a n g e s in t he r a t e o f s y n t h e s i s o f b r u s h - b o r d e r f r a c t i o n p r o t e i n s in p e a k s A a n d B also in c a l c i u m t r a n s p o r t in r e s p o n s e to 1 , 2 5 - ( O H ) 2 D 3. A p r o t e i n b a n d A; e , p r o t e i n b a n d B; i , 4 SCa t r a n s p o r t b y in v i t ro e v e r t c d i n t e s t i na l sac. Bars i n d i c a t e t i m e of i n c u b a t i o n .

indicates a role for these proteins in the biochemical processes by which 1,25-(OH)~D3 stimulates calcium absorption.

References

1 S p e n c e r , R. , C h a r m a n , M., E m t a g e , J .S . a n d L a w s o n , D.E.M. ( 1 9 7 6 ) Eur . J . B i o c h e m . 71 , 3 9 9 - - 4 0 9 2 W a s s e r m a n , R . H . , T a y l o r , A .N. a n d F u l l m e r , C.S. ( 1 9 7 4 ) in The M e t a b o l i s m a n d F u n c t i o n o f

V i t a m i n D, B i o e h e m . Soc . Spec . Pub l . ( F r a s e r D . R . , ed . ) , Vol . 3, p p . 5 5 - - 7 4 3 S p e n c e r , R. , C h a r m a n , M., Wilson, P. a n d L a w s o n , E. ( 1 9 7 6 ) Na tuxe 2 6 3 , 1 6 1 - - 1 6 3 4 D a w s o n , R.M.C. ( 1 9 6 9 ) in D a t a fo r B i o c h e m i c a l R e s e a r c h ( D a w s o n , R .M.C. , E l l io t t , D.C. , E l l io t t ,

W.H. a n d J o n e s , K.M. , eds . ) , 2r id e d n . , pp . 4 7 6 - - 5 0 8 , C l a r e n d o n Press , O x f o r d 5 F o r s t n e r , G.G. , Sabes in , S.M. a n d I s se lbache r , K.J . ( 1 9 6 8 ) B i o c h e m . J. 1 0 6 , 3 8 1 - - 3 9 0 6 Nevil le, D.M. ( 1 9 7 1 ) J . Biol . C h e m . 2 4 6 , 6 3 2 8 - - 6 3 3 4 7 Fehe r , J . J . a n d W a s s e r m a n , R . H . ( 1 9 7 6 ) Fed . P roc . 35 , A b s t r . 7 4 5 , p. 3 3 9 8 K o w a r s k i , S. a n d S c h a c t e r , D. ( 1 9 7 5 ) A m . J. Phys io l . 2 2 9 , 1 1 9 8 - - 1 2 0 4