Plasma membrane calcium pump expression in human placenta and small intestine

Vol. 183, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 16, 1992 Pages 499-505

PLASMA MEMBRANE CALCIUM PUMP EXPRESSION IN HUMAN PLACENTA AND SMALL INTESTINE

Alison Howard, Stephen Legon*, and Julian RF. Walters1

Gastroenterology Unit, Department of Medicine, and *Department of Chemical Pathology, Royal Postgraduate Medical School, London W12 ONN, U.K.

Received January 21, 1992

To identify the forms of the plasma membrane calcium pump present in tissues that transport calcium, cDNA from human placenta and proximal small intestine was amplified by the polymerase chain reaction using a pair of mixed primers based on all the known human and rat plasma membrane calcium pump sequences. Clones were identified from the two human forms HPMCAI and HPMCA4, but no new sequences were found in either tissue. RNA blots probed with HPMCAl showed two bands in both tissues; probing with HPMCA4 gave a single, larger species. In placenta, HPMCA4 was the more abundant form and similar expression was found in full-term and second-trimester placentas. In contrast, in the small intestine, HPMCAl was more abundant, suggesting that calcium absorption is not associated with any one specific isoform in calcium transporting cells. D 1992 Academic Press, 1°C.

Transcellular transport of Cazt takes place across the polarized epithelial

cells of placenta, intestine and kidney. In placenta, Ca2+ IS transported from the maternal

circulation by the fetal trophoblasts [l], in the proximal small intestine, dietary Ca2+ is

absorbed by enterocytes, and in the kidney, reabsorption of filtered Ca2+ occurs in the

tubules [2,3]. In all, the energy-dependent final step is the extrusion of Ca2+ from the cell

across the basolateral membrane, and in membrane vesicles prepared from all these

tissues, ATP-dependent transport of Ca2+ has been demonstrated [4-6). It has not been

established whether, in these cells with vectorial Ca2+ transport, the plasma membrane

Ca2+-pump differs from that found in other cells such as erythrocytes or neurones, where

it maintains the low cytoplasmic Ca2+ concentration necessary for cell viability and used in

hormonal signalling [7].

1 To whom correspondence should be addressed.

ABBREVIATIONS

PCR, polymerase chain reaction; bp, base pairs; kb, kilobases.

0006-291 X/92 $1.50

499 Copyright 0 1992 by Academic Press, Inc.

All rights of reproduction in any .forrn twervrd.


It is now apparent that there are several isoforms of the plasma membrane

Ca’+-pump [7]. In rats, cDNA sequences from three separate genes have been found

(RPMCAl, RPMCA2, and RPMCAB) [8,9] and tissue-specific expression has been

described [9]. In humans, one of the published sequences, HPMCAl, is homologous with

RPMCAI [lo], whereas another, HPMCA4, is distinct [I I]. Further complexity is produced

by alternative splicing of the transcripts near the 3’ end [8,9.12] which modifies suggested

cyclic-nucleotide and calmodulin regulatory sequences. Sequences homologous to

RPMCAl have also been found in rabbit and pig smooth muscle [13,14].

In human tissues, little is known about the tissue-specific expression of the

forms of the Ca*+-pump, and studies have not addressed whether any particular form is

found in Ca*+-transporting cells. We have therefore looked in human placenta and

proximal small intestine for transcripts expressed from the different genes using a strategy

based upon the polymerase chain reaction with mixed oligonucleotide primers designed to

amplify DNA of known rat and human plasma membrane Ca’+-pumps.

EXPERIMENTAL

CDNA Preparation

Human placental tissue was obtained from normal full-term deliveries and mid-trimester therapeutic abortions. Proximal small intestinal mucosa was from patients undergoing pancreatic surgery and normal colonic mucosa from a patient having resec- tion for cancer. RNA was obtained by minor modifications of the method of acid guan- idinium thiocyanate-phenol-chloroform extraction [15]. Poly (A)+ RNA was prepared by affinity chromatography on an oligo-dT cellulose column [16] and cDNA templates for the PCR were prepared using AMV reverse transcriptase and random hexanucleotide primers.

Polymerase Chain Reaction

The mixed sequence oligonucleotides described in Fig. 1 were synthesised on a Biosearch Cyclone, deblocked, ethanol precipitated three times and used without further purification. The amplification reaction used Taq DNA polymerase (Amersham) and the buffer and nucleotide concentrations recommended by the supplier. Primers were present at 27 pg/ml (5’ primer mix) and 11 pg/ml (3’ primer mix) reflecting the different degrees of complexity of the two mixtures. The PCR conditions were as follows: denaturation, 30 set at 95°C; annealing, 3 min at 5O”C, and extension, 30 set at 70°C. Products were extracted with chloroformiisoamyl alcohol, isopropanol precipitated and restricted with EcoRl before analysis on agarose gels.

identification of PCR products

A band of the appropriate size was identified by ethidium staining, cloned into the Eco Rl site of Ml 3 mpl9 and transfected into E coli XL1 B using standard procedures [17]. Filter lifts were taken and all the white plaques were eluted into 2001.11 of buffer [17]. 1 ~1 of each was spotted onto a XL16 lawn and filter lifts were taken of the resultant ‘macro-plaques’. 10 ng of the original gel band was labelled [18], using the PCR primers (100 pg/ml), and was then used to probe the filter lifts. Hybridisation was at 60°C in 125 mM Na phosphate buffer, pH 7.2, 500 mM NaCI, 2.5 mM EDTA, 25 FM aurin tricarboxylic acid, 5% w/v SDS, 20 pg/ml carrier RNA and 0.5 % w/v dried milk powder.

500


Filters were washed at 60°C for 60 min in the wash buffer described in [I91 followed by a stringent wash at 70°C for 60 min in 0.2 x SSC [17], 0.2% SDS. Major species were identified by strong hybridisation and 8 such clones were sequenced using the ‘dideoxy’ method [20] following the ‘extended Klenow’ procedure recommended by GibcoiBRL. The inserts from sequenced clones were themselves used as probes to the ‘macro-plaque’ lifts, a further 8 non-hybridising clones were picked for sequencing, and by repeated use of this strategy, all the clones were identified.

Northern blotting

Aliquots of 2.5 - 3 pg of poly (A)+ RNA were fractionated on formaldehyde/ agarose gels and blotted [21]. Probe sequences were labelled using the 3’ primer mix to generate an antisense probe [18]. Hybridisation and washing conditions were as described for plaque lifts but omitting the NaCl from the hybridisation mix and with all washes at 60°C. Quantification was by densitometry performed on autoradiograms of blots which were hybridised, washed and exposed in parallel. Corrections were made for variable loading of the lanes with mRNA by reprobing with an oligo-dT probe.

RESULTS AND DISCUSSION

&+-pump sequence amplification

Figure 1 shows the primer sequences used in the PCR. All the possible

coding sequences were synthesised for two conserved hexapeptides found in all five

published rat and human plasma membrane Ca*’ -pump sequences upstream from the

putative calmodulin binding site near the C-terminus [lo]. There was 256-fold degener-

acy of the 5’ primer and 96-fold degeneracy of the 3’ primer. In addition to the 17-base

Pump Initial residue Membrane 9

Rat

RPMCAl 1004 RPMCA2 982 RPMCA3 987

Human

HPMCAl 1004 HPMCA4 992

Conserved amino acid sequences

Nucleotide sequences

Proposed domains

Membrane 10 Calmodulin

binding site

FGGKPF

1

5' (X)-TTYGGNGGNAARCCNTT 3' +

EIDHAE

I

GARATHGAYCAYGCNGA 3' CTYTADCTRGTRCGNCT-(X) 5'

m. Partial sequences of rat and human plasma membrane Ca”-pumps showing the conserved regions used to construct the mixed oligonucleotide primers. Sequences for FiPMCAl and RPMCA2 are from ref. (81; RPMCA3 from [9]; HPMCAl from [lo]; and HPMCA4 from Ill]. The number of the initial amino acid residue is shown as are two of the putative transmembrane domains and the proposed calmodulin binding-site. In the oligonucleotide sequences, (X) represents the primer extension containing an EcoRl restriction site: N = A.C,G or T; R = A or G: Y = C or T; H = AC or T; D = A, G or T.

501


TABLE I IDENTITY OF CLONES AMPUFIED BY PCR

USING Ca2+-PUMP PRIMERS

Sequence Total number of clones

Placenta Small intestine

HPMCAl 27 51 HPMCM 101 10

Other known sequencesa Unidentified

(15 different species) Not possible to sequence

62 11 9 14

2 3

a 2% ribosomal RNA, mitochondrial tRNA, and Ml 3 sequences

coding sequence, the oligonucleotides had g-base 5’ extensions which included EcoRl

restriction sites. They were predicted to amplify a sequence of 209 bp from HPMCA4 and

215 bp from the other sequences.

Amplification of cDNA from both full-term placenta and proximal small

intestine gave two major bands of approximately 210 bp and 120 bp on agarose gels (data

not shown). Table I shows the analysis of clones identified from the 210 bp band. The

predicted sequences of both HPMCAl [lo] and HPMCA4 [l I] were found. In placental

cDNA, HPMCA4 sequences predominated, whereas in proximal small intestine HPMCAl

sequences were more common.

As well as these pump sequences, DNA identified as part of 28s ribosomal

RNA was amplified with the 5’ primer at each end. This insert was smaller than the two

pumps and Southern blots indicated that this was found predominantly in the 120 bp PCR

product though streaking on the gel resulted in contamination of the larger band.

Sequences of mitochondrial tRNA and Ml3 were also amplified. These have limited,

chance homology with the 3’ end of one or other primer sequence which ranges from 13

bases out of 15 to just 7 out of 8.

In the other unidentified sequences, homology with known Ca2+ pumps was

limited to the primer regions. No sequences were found which corresponded to the two

others found in rats (RPMCA2 and RPMCA3) [8,9]. RPMCA2 has been shown to be

predominantly in brain and heart, and RPMCA3 appears to be in brain, skeletal muscle

and testes [9], but it is unknown if either of these is present in rat placenta in addition to

PMCAI [22]. This experiment of course does not exclude the possibility that there may

exist other plasma membrane Ca*+ -pump sequences which do not share these

502


HPMCAl

123

HPhlCA4

123 HPMCAl HPMCA4

123C 1 2 3 c

1

0 3

m. Blots of poly (A)+ placental RNA (2.5 pg per lane) hybridised with probes for HPMCAl or HPMCA4. The position of 28s rRNA is indicated. Different exposures of the two autoradiograms are shown. Lane 1, 14 wk; lane 2. 17 wk; lane 3. full-term placenta.

Eigu~Q. Blots of poly (A)+ intestinal RNA (3 /.fg per lane) hybridised with probes for HPMCAl or HPMCM. Lanes 1,2 and 3 are samples of proximal small intestine from three different subjects; C is large intestine.

homologous primer regions or are so similar to the species detected that they cross-

hybridise even under the high stringency conditions used in the screening procedure.

Calcium pump transcripts in placenta

The specific pump sequences were used to probe blots of poly (A)+ RNA

from full-term and mid-trimester placentas. Despite the increase in Cazt transport

occurring near full-term [l], a similar pattern was found at both stages of development

(Fig. 2). The HPMCAl probe detected two bands; the smaller being more intense. This

pattern is similar to the findings in rat tissue where RPMCAI hybridizes with bands of

approximately 5.5 - 6 and 7.6 - 7.8 kb [9]. The presence of two sizes of transcripts of

HPMCAl in human tissues was not reported previously [ll], and as has been suggested

in the rat [9], may represent alternative polyadenylation sites.

The band detected with the probe for HPMCA4 was larger in size than both

those of HPMCAi and was of greater intensity on densitometry (approximately twice that

of the smaller band). A size difference has been noted previously [I l], and as the

translated regions of HPMCAl and HPMCA4 are similar, is a result of differences in

nontranslated regions. The intensities of the bands indicate that in the placenta, the

transcript for HPMCA4 is more abundant than those of HPMCAl, confirming the less

reliable indication from the number of clones obtained by PCR.

503


Calcium pump transcripts in intestine

On intestinal blots, bands of similar size to those in placenta were detected

(Fig. 3). The density of the bands seen with the HPMCAl probe in all samples of small

intestine was much greater than that found with the HPMCA4 probe, although there was

considerable variation between different preparations. After correction for small loading

differences, the densitometry values for the smaller band of HPMCAl varied up to 4-fold

between subjects, though there was approximately 3-fold greater density of this band than

that found in blots probed with HPMCA4. In contrast, in a preparation of colonic mRNA,

HPMCA4 was the predominant transcript detected.

It is of interest to note that the HPMCA4 sequence was first identified in a

small intestinal cDNA library [l I] although our present results would suggest that it is in

fact the minor form in that tissue. In rat small intestine, RPMCAI was much more

abundant than RPMCA2 or RPMCA3 [9], and vitamin D responsiveness has been reported

recently using a probe that detects the RPMCAl isoform [23].

There is no evidence from these experiments to support the hypothesis that

any particular form of the plasma membrane Ca*+ -pump is found in cells involved with the

absorption of Ca*+ as both HPMCAI and HPMCA4 were found but the predominant form

differed in placenta and small intestine. The possibility remains that there are more subtle

differences in tissue-specific expression of pump transcripts than would have been

detected here. Slight differences in the size of the phosphorylated pump proteins have

been detected in some tissues [24] indicating that there may be tissue-specific, or

functional, changes such as those produced by alternative splicing of the 3’ region of the

transcript [12]. This remains to be explored in Ca*+ -absorbing tissues such as the

placenta, kidney and intestine.

ACKNOWLEDGMENTS

We are grateful to Dr. Clare Selden for gifts of placental tissue, to Dr. Nick Davidson for advice on extracting human small intestinal RNA, and to Miss Gwen Ferrier for synthesis of the oligonucleotides. This work is supported by a MRC Project grant to JRFW and by the Trustees of Hammersmith and Queen Charlotte’s Special Health Authority.

REFERENCES

1. 2.

Pitkin, R.M. (1985) Am. J. Obstet. Gynecol. 151, 99-l 09. Bronner, F. (1990) Calcium transport and intracellular calcium homeostasis. (Pansu, D. and Bronner, F. Eds.), pp. 199-223, Springer-Verlag, Berlin

3. Van OS, C.H. (1987) Biochim. Biophys. Acta 906, 195-222. 4. Fisher, G.J., Kelley, L.K. and Smith, C.H. (1987) Am. J. fhysiol. 252, C38-C46. 5. Nellans, H.N. and Popovitch, J.E. (1981) J. Biol. Chem. 256, 9932-9936. 6. Gmaj, P., Murer, H. and Kinne, R. (1979) Biochem. J. 178, 549-557. 7. Carafoli, E. (1991) Physiof. Rev. 71, 129-153.

504


8. 9. 10.

11.

12.

13.

14. 15. 16. 17.

18. 19. 20.

21. 22.

23.

24.

Shull, G.E. and Greeb, J. (1988) J. Biol. Chem. 263, 8646-8657. Greeb, J. and Shull, G.E. (1989) J. Viol. Chem. 264, 18569-18576. Verma, A.K., Filoteo, A.G., Stanford, DR., Wieben, E.D., Penniston, J.T., Strehler. E.E., Fischer, R., Heim, Ft., Vogel, G., Matthews, S., Strehler-Page. M.A., James, P.. Vorherr. T., Krebs, J. and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-l 4159. Strehler, E.E., James, P., Fischer, R., Heim, Ft., Vorherr, T., Filoteo, A.G., Penni- ston, J.T. and Carafoli, E. (1990) J. Biol. Chem. 265, 2835-2842. Strehler, E.E., Strehler-Page, M.A.. Vogel, G. and Carafoli, E. (1989) Proc. Nat/. Acad. Sci. U.S.A. 86, 6908-6912. De Jaegere, S.D., Wuytack, F.. Eggermont, J.A., Verboomen, H. and Casteels, R. (1990) Biochem. J. 271, 655-660. Khan, I. and Grover, A.K. (1991) Biochem. J. 277, 345-349. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-l 59. Aviv, H. and Leder, P. (1972) froc. Nat/. Acad. Sci. U.S.A. 69, 1408-1412. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning: a laborafory manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 132, 6-13. Reed, K.C. and Mann, D.A. (1985) Nucleic Acids Res. 13. 7207-7221. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Nat/. Acad. Sci. U.S.A. 74, 5463-5467. Thomas, P.S. (1980) Proc. Nat/. Acad. Sci. U.S.A. 77, 5201-5205. Glazier, J.D., Thornburg, K.L., Sharpe, P.T.. Boyd, R.D.H. and Sibley. C.P. (1991) Placenta 12, 390-391 Zelinski. J.M., Sykes. D.E. and Weiser, M.M. (1991) Biochem. Biophys. Res. Commun. 179, 749-755. Kessler, F., Bennardini, F., Baths, O., Serratosa, J.. James, P., Caride, A.J., Gazzotti, P., Penniston, J.T. and Carafoli, E. (1990) J. Viol. Chem. 265, 16012- 16019.

505

Plasma membrane calcium pump expression in human placenta and small intestine

Documents

Transcript of Plasma membrane calcium pump expression in human placenta and small intestine