Indi an Journal Ex perimental Bio logy Vol. 41 , January 2003, pp. 4 1-46

Effect of oophorectomy on expression of calcium sensing receptor mRNA in rat duodenal mucosa

M Mehrotra, S K Gupta"', S Ti wari , A Agarwalt , K Kumar, P K Awasthi & M M Godbole

Departments o f Medica l Endocrino logy and t Surgieal Endocrinology

Centre for Endocrine Sc ie nces, Sanjay Gandhi Postg raduate Institute o f Medica l Sc iences, Luek now 226 01 4, Indi a

Receil'ed 4 Deeeli/ber 200 / ; re l,ised 28 GC/ober 2002

Calcium sensing receptor (Ca R) in duodenal mucosa may be in volved in ac ti ve cal c ium absorpt ion. Estrogen de fi ­c iency resuits in decreased intestina l calc ium absorpti on. Effec ts o f bila te ra l oophorectomy (OVX) have been studied on calc ium homeostas is, bone mineral density ( BMD) and CaR mRN A le vels in duodenal mucosa at 4 weeks in adult female Sprague Dawley ra ts and compJ red with those in sham-operated Jnd control group. There was no s ignifi cant change in se­rum correc ted calc ium , inorganic pho"phorous, cale idi o l and int act parJthyro id hormo ne in all the three groups. O VX rats had a significant dec line in serum estrogen (E2) levels and a lka line phosphatase. They J lso had a s ignificant decrease in BMD (DX A) at lumba r spine ill vivo, and prox imal and d ista l ti bia ill vil ro while there was no s ignificant c hange in serum E2 and BMD paramete rs in sham-operated and cont ro l rats. Northern blot anal ys is revea led no signi ficant change in the Ca R mRN A ex pression in duodenal mucosa in a ll three groups. T he resuits sugges ts that Ca R mRN A ex press ion in duodenJ I mucosa is not J ffec ted by phys io logical circul ating concen trations o f es tradi o l in ra ts.

Estrogen defi ciency is a major cause of osteoporosis in postmenopausal women. It causes bone loss by di ­rect or cytokine medi ated increased bone resorption I .

In addition, estrogen defi ciency has al so been impli­cated in negative calcium balance and is associated with decreased intestinal calcium absorption 2

. How­ever, the mechani sms remain unclear. It is poss ible that estrogen defi ciency causes bone resorpti on thereby increased extracellular calcium levels, de­creased parathyroid hormone (PTH ) secreti on and reduced calcitriol synthesis. Thus, decreased intestinal calcium absorption is a consequence of bone resorp­tion . Estrogen deficiency may cause decreased ca l­cium absorption by reduced renal I-a hydroxyl ase activit/ and intestinal resistance to action of cal cit­rioI4

,5. Estrogen also increases vitamin D receptors (YDR) expression and bioresponse in rat duodenal mucosao. However, ev idences suggest th at es trogen itself acts directl y on the intestinal cell s to stimulate calcium absorption rather than th rough calcitriol7

.

Estrogen receptor mRNA and immunoreactivity has been demonstrated in the rat intestinal mucosal cell sx

and it has also been shown that the cells respond to estrogen with enhanced calcium transport9

.

*Correspondent author Tel: +91 -522-668004-8 ex tn . 2395 Fax: +91 -522-668078/17 E-mail: sushil @sgpgi. ae. in

Calcium sensing receptor (CaR) is ex pressed in the organs in volved in calcium homeostas is like parathy­roid gland , renal tubules, bone cell s and intestine lo

.

Role of CaR in the intestine is largely unknown but It has been show n to regulate fun cti ons like gastric ac id and gastrin secreti on" -13

, intes tinal motility, nutrient absorption in the villi and secreti on of chloride and other ions in crypt cell s l4

. There is no proven regul a­tor or its expression reported till date. Thus, it is pos­sible that estrogen may be exerting its role in calcium absorption through CaR also. Further, transcellular active cal cium absorption occurs mainl y in duodenal mucosa 15 and the effect of estrogen defi ciency on CaR express ion may be seen predominantly in duodenal mucosa.

The objecti ve of the present study is to demonstrate the effect of estrogen defi ciency on CaR mRNA lev­els in duodenal mucosa of rats.

Matedal and Methods Sprague dawley adult female rats (250-290 g and

year of age) were maintained in separate cages at 12: 12 hI' L: D cyc les and had free access to chow di et and tap water. They were divided randomly into fol­low ing 3 groups of 4 animals each (a) Group I control rats: (Control ); (b) Group II oophorectomi zed rats (OYX): oophorectomy was performed through single ventral incision under ketamine (im) and open mask ether anaesthesia; (c) Group III sham operated rats

42 !NOIAN J EXP BIOL. JANUA RY 2003

(Sham): sham operation was done by exteriorising the ovari es bri efl y. The ex perimental protoco l was ap­proved by the In stitute Ethical Committee. The care of ex perimental anima ls conformed to the Guidelines 0 11 the Handling and Training of Laboratory Animals

Blood was coll ected at baseline and 4 weeks, se­rum was separated and stored at -70°C till the time of assay. Whole body ill vi vo area l bone mineral density (aBM D) and bone mineral contents (BMC) were done at base line and at 4 weeks. The animals were sacri­ficed at 4 weeks by decapitation. The duodenum was dissected out, the mucosa was harvested , fro zen in liqu id nitrogen and stored at -70°C fo r RNA ex trac­ti on. The femora and tibia were di ssected out for ill I'i lm BMD measurement. It decided to keep the time period of the ex periment as 4 weeks as prev ious stud­ies have suggested th at signifi cant bone loss at lumbar spine and prox imal tibi a is observed in oophorec-

. I I " . Iln.IX toml zee rats at t liS tllne penoe . Biochelll ical alld fl o rillollol esl illlol iOlls - Seru m

total calc ium, albumin and inorganic phosphorus (iP) (co lori metric method) and alkal i ne phosphatase (A LP) (colorimetric ki net ic method) were es ti mated by commerciall y available kits (S igma Di agnostics, MO, USA). Serum calcidiol (RI A, Diasorin , Minne­sota , USA), intact PTH (iPTH , human equival ent , IRM A, Diagnosti c Systems Lab, Texas, USA) and estrad iol (E2) (RI A, Di agnosti c Systems Lab, Texas, USA) were measured . The sensiti vity of ca lcidiol, iPTH and E2 were 1.5 ng/ml , 6 pg/ml , and Spg/ml re­specti ve ly. The interass,:y and intrassay coe ffi cient of va riati on were: calc idiol (int ra- 10.S %, inter 12%), iPTH (intra- 5%, inter- 6.5 %) and E2 (intra- 7%, inter­S.I %)

Bon e mineral densilY (BMD) alld bOlle milleral cOlllem (BMC) qualllilalion -Areal BMD was meas­ured by DX A (Dual Energy X-ray Absorptiometry, Hologic QDR 4S00A , Waltham, MA, USA) using sma ll animal so ftware. The anaestheti sed rats were placed on the scan tabl e in a supine position and the whole body scan of the rats was obtained. In vivo BMD measurements were analysed in whole body (WB), lumbar spine (LS , LI -L4), whole femur (WF) and whole tibia (WT).

Femora and tibia were di ssected out , attached soft ti ssue was scrapped off using a sca lpel and ad herent fatty ti ssue was removed by placing bones in eth anol and diethyl ether. Indi vidual bone was placed in water and ill vilro BMD measurements were obtained. All the bones were measured on the same day . Sub­regional ana lyses of femoral neck and prox imal, mid

and di stal regions of femur and tibi a were performed. To assess the short-term drift of DEXA measure­ments, 2 rats and 2 femora were measu red fi ve ti mes on same day and coefficient of va ri ati on (% CY) ill vi vo and ill vil ro was detennined. Simi larly , for long term drift , DEXA was ca librated with small animal phantom dai ly. The co-effi cient of va ri at ion of ill- I'iv() and iII- vi I ro measurement was 0.56 and 0.6 I % respec­ti ve ly . The areal BMD results are ex pressed as g/cm2 and the BMC in grams.

Norlh em Blo! procedure-Total RNA was ex­tracted from duodenal mucosa accord ing to single step RN A isolati on meth od l9. Total RNA samples (SOj.lg) were denatured and size fractiona ted in 1% agarose, 2.2 M formaldehyde denatu ring ge l and transferred to nylon membrane (Hybond N, Amer­sham Inc. UK). Gel purifi ed CaR (600 bp rat CaR cDNA cloned in pBluescript (S K±) vector in restri c­ti on site Bgi II/Sac I) and ~ Actin cD A probe frag­ments were labelled by random primed sy nthesis with p:11 labeled deoxy-CTP to a specifi c act ivity of greater than 10'Jcp m/p.g. The protocol for orthern hybridi sa-. . . d . I . d' I() ~()~ I tl on IS In concor ance Wltl prevIOus stu les '- '- .

Briefl y, pre-hybridi sation or blots WJS carri ed out at 42°C for 2-4 hI' in a solution containing Sx SSPE, S% SDS, 5x Denhardt's reagen t, 100 p.g/m l soni cated sa lmon sperm DNA and 50% fo rmarnide. After thi s, the so lution was replaced with fres h so lution contain­ing the rad iolabeled probe ( I 06cpm/ml ) and hybridi sa­tion was carri ed out at 42°C for IS-24 hI' with con­tinu ous shak ing. After hybridi sa ti on, membranes were washed in 2xSSPE, O. I % SDS twi ce fo r 10 min at room temperature and in 0.1 X SSPE, 0. 1 % SDS twice for 10 mi n at roo m temperature. The me mbranes were then wrapped in saran wrap and autoradiographed at -70°C for S-6 days.

Slatislico l anolys is -.Th e data were analysed usin g so ftware "SPSS for windows 9.0" (S PSS, Chicago, IL, USA ). The results of biochemical, hormonal and densitometric estimati ons are expressed in mean ± SD. Data groups were co mpared using Students t test. P va lues <0.05 were considered signi fica nt.

Results

Basel ine There was no signiticant difference in weights, serum

corrected calcium, iP, ALP, calcidiol, iPTH and E2 among the three groups (P>O.05) . III IJilJO BMD and BMC of whole body and at lumbar spine, whole tibia and femur were similar in all the three groups (hoO.OS).

MEHROTRA el al: OOPHORECTOMY & CaR mRN A IN RAT DUODENAL MUCOSA 43

At 4 weeks General and biochelllical- Weights and behav­

iour remained unchanged in all the groups. There was no significant change in serum E2 from baseline to 4 weeks peri od in Control and Sham rats. At 4 weeks, OVX group had undetectabl e serum E2 levels (base­line Vs 4 weeks: P<O.OOS) . There was no signifi cant difference in serum E2 levels at 4 weeks between Control and Sham rats. There was no signi ficant change in serum corrected calcium, iP, ca lcidiol and iPTH in Control , OVX and Sham rats between base­line and at 4 weeks (Tab le 1). Serum ALP showed a signifi cant decrease in the OVX group when com­pared to the Control or Sham operated group (P<O.OOS ).

BOlle lIIineml density qll(fll titatioll III vivo - WB BMD (Tab le 2) and BMC (data not

shown) remained unchanged in all the three groups (P>O.OS). OVX rats had significant dec line in BMD at LS at 4 weeks (-1 4.3 %) as compared to baseline (P<O.OOS ) while there was no signifi cant change in LS BMD in Control and Sham rats (Tablc 2). BMD at WF and WT in OVX group was red uced though in­signifi cantl y whil e there was a trend for progressive increase in Contro l and Sham rats.

III l'itro - At 4 weeks, sub-reg ional anal yses of tibia showed a signifi cant decline in the BMD at proximal (- 14.98%) and distal tibia (-15.25 %) in OYX group (P < 0.005) as compared to Contro l and Sham groups while no significant change was ob­served in mid-tibi a (Tab le 3). There was no signifi-

cant diffe rence in the BMD at any of thc sub-reg ions of femur in any of the three groups (P> 0.05).

Effect of estrogen deficiency 0 11 CaR transcrip­tion- Prev ious studi es on rat intestine in re lat ion to CaR have demonstrated the presence of transc ri pts with molecul ar sizes of 7.5 and 4. 1 kb in the du ode­num. Since the 4. 1 kb transcript encodes the entire functional CaR protein, the significance of larger 7.5 kb transcript remains uncertain. In the present experi­ments, Northern Blot analysis of mRNA transcri pts of CaR in duodenal mucosa indicated that there was no change in the ex pression of the both transcripts after OVX . Three Northern hybridi zati ons were done to confirm the results, which are represented in Fig. La. Densitometric quantificati on of the signals produced by hybridi sati on in each treatment group normali sed according to ~ Actin mRNA ex press ion indicated that there was no change in the CaR mRN A content in es trogen defici ent (OVX) rats as compared to Control or Sham rats (Fig. Lb).

Discussion This present result demonstrate signifi can t dec line

in BMD at lumbar spine and tibia (proximal and dis­tal ) in OVX rats after 4 weeks as comparcd to Sham and Cont ro l rats. There was no significant change in serum corrected calcium, iP, calcidiol and iPTH in all the three groups. Northern blot and densitometric analys is of CaR mRNA contents in duodenal mLlcosa revealed no alteration in OYX rats as well as in Con­trol or Sham rats.

Table I ~ Biochemica l profile in rats in vari ous ex perimental groups

Groups

Weight (g)

Serum corrected calcium (mg/dl)

Serum iP (mg/d l)

Serum alkaline phos­phatase (I U/L)

Serum c. li cidiol (ng/ml)

Serum iPTH (pg/ml) (Human equ iva lent )

Serull1 es tradiol (pg/ml )

P va lue: *< 0.005.* *<0.00 1

[Values are mean ± SD from 4 observati ons in each group [

Contro l Sham o~erated

Baseline 4 weeks base line 4 weeks

239±7.18 227 ± 9.06 253 ± 11.08 252 ± 20.46

7.90 ± 0.70 8. 17 ± 0.20 8.32 ± 0.65 9.62 ± 1.0 I

6.2 1 ± 0.94 4 .38 ± 0.1 1 6.25 ± 1.45 5.58 ± 0.37

87.48 ± 7.54 124.53 ± 0.45 102.23 ± 9.94 130.68 ± 5.39

2.60 ± 0. 18 2.33 ± 0.26 2.30 ± 0.34 1.70 ± 0. 16

<6 < 6 < 6 < 6

40.00 ± 0.98 39.00 ± 1.1 0 40.00 ± 0.98 4 1.00 ± 1.8 1

OVX baseline 4 weeks

248 ± 6.29 235± 8.2 1

7.44 ± 0.38 8.1 3± 0.96

6.07 ± 0.96 4.3 1± 0.68

100. 10± 1.68 77.10± 1.27 *

2.90± 0. 15 1.93 ± 0.09

<6 <6

40.00± 0.98 Undetectable < 8 pg/ml *,:'

44 INDI AN J EXP BIOL, JANUARY 2003

Estrogen defi ciency is an establi shed cause of os­teopeni a in females. It induces a high turnover of bone remodelling where bone resorption exceeds bone formati on. Signi ficant dec line in BMD at LS, proximal and di stal tibia l 6

, whole femur and di stal femur l ?22 and femoral neck 18 has been observed, after 4 weeks of OYX, whereas signifi cant dec line was observed in the present study in BMD at 4 weeks only at LS and proximal and distal tibi a, areas predomi ­nently compri sing of trabecular bone.

In addition, estrogen deficiency results in negati ve calcium balance. It reduces intes tinal ca lcium absorp­tion th rough unknown mechani sm. Intestinal ca lcium absorption takes pl ace vi a 2 main mechanisms: pas­sive diffusion and acti ve absorption. The ac ti ve ab­sorption occurring predominantl y in duodenum, is a complex process and is mediated by cal eitrio l th rough its receptors. Studies have demonstrated that intestinal calcium absorpti on decreases with age both in hu-

?3 d · ?4 0 h . , mans- an In rats-. op orectomy In young rats leads to increased intestinal calcium secretion detec t­able at 3 to 6 weeks posl-OYX which is attenuated at 9 weeks25 but in adult rats OYX did not have any ef­fect on intestinal calcium secreti on26

. Kalu el ([ t .27

have reported 30% decrease in intestin al calcium ab­sorpti on in OYX rats. However, Bolscher et aex have demonstrated no change in intestinal calcium absorp­tion at 8 weeks post-OYX in ad ult rat. There are con­fli cting reports of urinary calcium excretion after OYX, viz., increased excretioll'l, no efTect25

.30 and

inconsistent effect31• J n postmenopausa l women, there

is increased rena l excreti on32 and reduced intestinal absorpti on of ca lcium2

.

Estrogen receptors in the intestinal mucosa? modu­lates the calc ium, however, the mechanisms remain still unclear. E2 deficiency results in increased bone resorpti on, hypercalcemi a, suppressed PTH secreti on and decl ine in pl asma I ,25(OHh D3

33 Seru m calcitriol leve ls increase after E2 treatment in postmenopausal women34

• Calcitriol admini stration normali zes the ca lcium absorpti on in· postmenopausa l women35

.

However studies in OYX rats have shown no altera­ti on in serum PTH and free I ,25(OHh D3 2X . Estrogen modulates intes tin al vitamin D receptors (YDR) and ca lbindi n D9K expression. In rats, OYX induced re­duction in intestinal YDR expression is reversible with es trogen treatment. This is probabl y due to direct modulating acti on of estrogen on the action of en-

Tablc 2 - BM D mcasurcmcnl ill -vi vo in va ri ous cx pcrimcntal groups

[Valucs arc mean ± SO from 4 obscrva ti ons in each groupl

BMD (g/cm2)

Globa l

Lumbcr Spi nc

Wholc Fcmur

Whole Tibia

*P< 0.005

Cont ro l Sham OVX Basc linc 4 wccks Basc li nc 4 wccks Basc linc

0. 14 t6±0.003 O. 1402± 0.003 0. 1393± 0.002 O. IS I4± 0.005 0. 1449± 0.003

0.I S3S± 0.00 I 0. 1639± 0.002 0. 1627± 0.006 o 168 1± 0.007 0. 1702± 0.004

0. 1448± 0.00 I 0. IS I3±0.008 0. 1473± 0.008 0. IS94± 0.00 I O. IS II ± 0.006

0. 1036± 0.009 0. 1 060± 0.00 I O. I 033± 0.006 0. 1329± 0.002 O. 1237± 0.006

Tab le 3 - I II viI ro 13 M 0 mcasu rcmcnt s of d issccted fcmora and ti bia at 4 wceks in va ri ous cxperi mcnta l groups

IValucs arc mcan±SD fro m 4 observations in cac h group]

Cont ro l Sham OVX

Prox imal fcmur 0 .1 8 13±0.005 0 .1 90 1± 0.004 O. I 820± 0.007

Mid fcm ur 0. 16SS± 0.004 0.1 665± 0.007 o. lno± 0.004

Di stal fc mur 0. ln 7± 0.02 0. 1945± 0.009 0. 18 11± 0.004

Femur ncck 0 .1 778± 0.008 0. 1997± 0.0 I 0. 1743± 0.005

Prox imal ti bia 0. 1769± 0.004 0.1739± 0.002 0. 1504± 0.002*

Mi d ti bia 0 .1 746±0.00 1 0. !711 ± 0.004 0.I S28± 0.002

Distal ti bia 0. 1744± 0.002 0. 1702± 0.003 0. 1478± 0.002*

* P <0.005

4 wccks

0. IS03± 0.003

O. t4S8± 0.006*

0. 1399± 0.005

0. 11 45± 0.002

MEHROTRA et a/: OOPHORECTOMY & CaR mRNA IN RAT DUODENAL MUCOSA 45

(0 )

2 3

1.2 l b)

P>O.05 ..-

,----

.08

ci cj

0.6

0.4

0.2

0 S 0 C

Fig. I - (a): Northern blot analysis of total RNA (50Ilg/lane) from rat duodenum probed with 32 P_labeled rat CaR eDNA probe and exposed for 6 days. Lane I-Sham ; Lane 2-0YX ; Lane 3-Control. Results were normali sed to f) -Actin ex pression. (b): Den­sitometric analysis of CaR in duodenal mucosa in various experi ­mental groups ISham(S), OYX(O) and Control (C)l The intensi­ties of the transcripts were quantified and normali sed to the re­spective intensities of the f)-actin mRNA prod ucts obtained in the different experimental groups.

dogenous 1,25(OH h D3 on intestinal yDR6. Pure es­trogen receptor antagonist lCI 182780 prevents ri se in estrogen but not calcitriol induced rise in duodenal calcium absorption in OYX rats2X

.

The CaR is expressed in all intestinal segments on the basal surface of the absorpti ve cells of small i ntes­tinal vi lli , small intestinal and colonic crypt ce ll s and in Aurbach's and Meissner's pl ex uses 1o

. The present study demonstrated no alteration in the expression of CaR in the duodenal mucosa in OYX rats as com­pared to Control and Sham rats. It seems apparent that CaR expression is not effected by physiological concentrations of E2 in rats. Whether pharmacological doses of estrogens have any effect on CaR express ion

in the intest ine needs to be explored further. It is pos­sible that es trogens may influence the post-receptor signalling of CaR through various post-receptor pathways. Recently, calcium transport prote in (CaT I ) in small intestine enterocytes has been shown to be a

I' I I I I · b . )(, 37 I . part 0 transce u ar ca clum a sorpttol1' .. . t IS POS-

sible that estrogen may effect the CaR evoked changes in the intrace llular calcium and signalling . Therefore, further studies, both in vivo and ill vilro , are required to study the rol e of CaR in the in test ina l calc iulll absorption.

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