1 Periparturient Diseases of Cattle Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010; and 2 Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Protein expression of plasma membrane Ca2+-ATPases (PMCAs) and the putative Golgi secretory pathway Ca2+-ATPase (SPCA) was examined in rat mammary tissue. As lactation started, PMCA protein expression increased dramatically, and this increased expression paralleled milk production. Mammary PMCA was primarily PMCA2b but was ~4,000 daltons larger than expected. RT-PCR showed that the primary mammary PMCA2b transcript was alternatively spliced, at splice site A, to include an additional 135 bp, resulting in the insertion of 45 amino acids. This splice form is designated 2bw. PMCA2bw is secreted into milk, associated with the milk fat globule membrane. Therefore, PMCA2bw is located on the apical membrane of the secretory cell. Smaller amounts of PMCA1b and 4b protein were found in mammary tissue. PMCA4b was the major PMCA expressed in developing tissue, and its level declined as lactation started. PMCA1b expression increased moderately during lactation. SPCA protein expression increased 1 wk before parturition and increased further as lactation proceeded. The abundance and cell location of PMCA2b suggest that it is important for macro-Ca2+ homeostasis in lactating tissue. The pattern of expression and abundance of SPCA suggest that it is a candidate for the Golgi Ca2+-ATPase.
breast; mammary gland; calcium pumps; calcium-transporting ATPase
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INTRODUCTION |
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THE MAMMARY GLAND transports large amounts of Ca2+ from the blood to milk via mammary secretory cells. These transcellular Ca2+ fluxes, associated with lactation, must be rigorously regulated to prevent Ca2+ cytotoxicity (29, 41, 44). This is a formidable task, because the mammary gland in mice and cows stores 12-30 µmol Ca2+/g tissue compared with <1 µmol/g in nonmammary tissues (4, 39, 41). The large intracellular Ca2+ pool in the mammary gland is thought to be concentrated in the Golgi apparatus (10), where millimolar Ca2+ is essential for normal protein synthesis, processing, and secretion (8, 15). Therefore, a mechanism must be in place to move Ca2+ into the Golgi.
Despite the importance of mammary cell Ca2+ homeostasis, little is known about the proteins involved in Ca2+ transport and their regulation (44). Biochemical evidence exists for a P-type Ca2+-ATPase in the Golgi of mammary tissue with characteristics slightly different from either the plasma membrane Ca2+-ATPase (PMCA) or the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) (4, 5, 15, 40, 50). This mammary Golgi secretory pathway Ca2+-ATPase (SPCA) could be a homologue to the yeast Golgi Ca2+-ATPase PMR1 (16, 45), such as the putative rat SPCA (26). It has also been suggested that there is no unique mammalian Golgi Ca2+-ATPase but, rather, that SERCAs in the endoplasmic reticulum (ER) and PMCAs in transit to the plasma membrane maintain the Ca2+ concentrations found in the Golgi (49). Previous work has shown that the putative rat SPCA mRNA is expressed in the mammary gland and is associated with milk production (44). Furthermore, the pattern of SPCA expression is consistent with the biochemical evidence for a Golgi SPCA in lactating tissue.
In addition to being pumped into the Golgi, Ca2+ must be effectively removed from the mammary cell and pumped into the milk. PMCAs represent a ubiquitous, high-affinity system for removal of Ca2+ from the cell. PMCAs, along with SERCAs, are responsible for the long-term setting and maintenance of intracellular Ca2+ levels (1, 11-13, 23, 28). Mammalian PMCAs are encoded by a multigene family consisting of four members termed PMCAs 1-4 (7, 11, 23). Cloning has revealed some 20-30 isoforms of the pump, generated by the alternative splicing of the primary gene transcripts at two sites identified as A and C (2, 20, 27, 28, 34, 38, 48). Alternative splicing of site C (COOH-terminal tail) has been shown to alter the regulatory properties of PMCA isoforms, particularly with respect to phosphorylation and calmodulin stimulation (17, 18, 23). Alternate splicing at site A has been shown to occur in PMCA2 (2, 27). No specific function has yet been found for this splice site (28), and only the z splice form of the protein has been demonstrated (20). The distribution of PMCA mRNA in rat and human tissues reveals that gene products 1 and 4 are ubiquitous, whereas gene products 2 and 3 are tissue specific and are found enriched in nervous tissue (22). Early protein studies have confirmed this pattern of expression for the four isoforms. It has also been shown that the splice variants of each isoform are expressed in a tissue- and cell type-specific manner (20, 23, 46-48).
Previous work has shown that PMCA isoforms 1b, 2b, and 4b, but not 3, are expressed in rat mammary tissue (44). PMCA2b mRNA was the most abundant PMCA mRNA expressed, and its expression was high in the lactating mammary gland of the rat (44). These data suggested that PMCA2b mRNA expression in the mammary gland is highly regulated by a factor(s) controlling milk production. This is the first evidence of significant expression of the PMCA2 isoform in a tissue other than brain. Thus it is suggested that PMCA2b might play a key role by regulating intracellular Ca2+ concentrations where large transcellular Ca2+ fluxes are required for milk production.
In this paper we present evidence for high expression of SPCA and PMCA2b protein in rat lactating mammary tissue. Interestingly, we have evidence that alternate splicing at site A in the mammary gland is different than that observed in the brain, where the shorter z splice form of PMCA2b is predominant (2, 20). We will also demonstrate that the PMCA2bw transcript and the correspondingly larger 2bw protein are the primary PMCA forms observed in lactating mammary tissue, whereas PMCA4b is the primary PMCA protein in developing mammary tissue.
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MATERIALS AND METHODS |
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Animals.
The National Animal Disease Center's Animal Care and Use Committee
approved all animal procedures. Confirmed-pregnant Sprague-Dawley rats
were purchased from Harlan Sprague Dawley (Madison, WI). Rats were
housed, individually, in hanging basket cages with sawdust bedding.
Starting 11 days after the breeding date, rats were killed at 10, 7, and 1 day prepartum and 1, 7, 14, 18, and 21 days postpartum. Data are
presented as days, with parturition as the reference point: negative
days indicate days before parturition, and positive days indicate days
of lactation (i.e., after parturition). Rats were anesthetized with a
50:50 mix of C02-O2 and then decapitated. Rats
were confirmed pregnant by inspection of the uterus. Mammary tissue and
other tissues were removed, flash frozen in liquid N2, and
stored at 70°C until RNA or membranes were prepared.
Tissue and milk fat globule membrane preparation.
Tissue microsomes were prepared as previously described
(14). Briefly, tissue was homogenized in 10 vol of
buffer A, containing 10 mM Tris · HCl, 2 mM
MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 4 µg/ml aprotinin, and 4 µg/ml leupeptin at pH 7.5. An aliquot of the
homogenate was saved for DNA analysis (9). The homogenate
was mixed with an equal volume of buffer B (buffer A containing 0.3 M KCl) and centrifuged at 4,000 g for
10 min. The supernatant was collected, adjusted to 0.7 M KCl by the
addition of solid KCl, and centrifuged at 100,000 g for
1 h. The supernatant was discarded, and the pellets were
resuspended in buffer C (buffer A containing 0.15 M KCl). Membrane preparations were stored at 70°C until assayed.
Gel electrophoresis and Western blotting. The methods were basically as described previously (20). Briefly, microsomes were incubated for 15 min at room temperature in a modified Laemmli buffer containing 150 mg/ml urea and 65 mM dithiothreitol. Samples were then electrophoresed for 1.5 h at 125 V in a 6% Tris-glycine gel (Novex, San Diego, CA). Proteins were transferred to nitrocellulose membranes for 1 h at 25 V in 0.7 M glycine-0.025 M Tris at pH 7.4. Blots were developed with Supersignal (Pierce, Rockford, IL) by using the protocol provided by the manufacturer. Anti-PMCA antibodies 5F10, NR1, NR2, and NR4, described previously (20), were used at 1:20,000, 1:1,000, 1:1,000, and 1:5,000 dilutions, respectively. Antibody 220 (final dilution 1:5,000) is a PMCA2-specific antibody similar to the previously described NR2 (20). Antibody 220 was prepared by using the same peptide sequence (TNSDFYSKNQRNESSC) used to make NR2. The peptide sequence used to make the SPCA antibody (final dilution 1:5,000) was VARFQKIPNVENETMIC (26). Rabbits were immunized with peptide, which was synthesized by Multiple Peptide Systems (San Diego, CA) and conjugated to keyhole limpet hemocyanin. The general procedures were described previously (35). Antibodies 220 (anti-PMCA2) and anti-SPCA were characterized as follows. Antibody 220 gave the same results as the previously characterized NR2, and preincubation of antibody 220 with the PMCA2 peptide TNSDFYSKNQRNESSC eliminated specific banding on Western blots. Preincubation of antibody 220 with an irrelevant peptide yielded normal PMCA2 blots. Preincubation of SPCA antibody with the SPCA peptide VARFQKIPNVENETMIC eliminated specific banding on Western blots. Preincubation of the SPCA antibody with an irrelevant peptide yielded normal SPCA blots with bands of the predicted size.
RNA preparation and Northern blot analysis. Total RNA and DNA were isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction using Trizol reagent (GIBCO-BRL, Gaithersburg, MD). Poly(A)+ RNA was prepared using the Poly A Tract mRNA Isolation System IV (Promega, Madison, WI). Poly(A)+ RNA (2-3 µg) was fractionated in formaldehyde-agarose gels, and blotting and hybridization were performed as previously described (31). PMCA2 and SPCA probes were prepared by RT-PCR as described previously. After hybridization, the blots were imaged and quantitated on an InstantImager (Packard Instruments, Downers Grove, IL).
RT-PCR and sequencing. The primers used for RT-PCR of splice site A in PMCA2b were 5'-TGAAAGCTCGCTCACAGGG-3' and 5'-TCAGAGGCTGCATTTCCATAG-3', as described previously (2). PCR was carried out in a GeneAmp 9600 PCR System (Perkin Elmer, Foster City, CA). The RT-PCR reaction master mix (99 µl) consisted of 10 µl of 10× reaction buffer (Boehringer Mannheim), 77.75 µl of diethyl pyrocarbonate-treated water, 8 µl of dNTPs of 10 mM stock (Amresco, Solon, OH), 1 µl at 100 pmol/µl of each of the specific primers (IDT, Coralville, IA), 0.25 µl (50 units) MMLV (GIBCO-BRL), 0.5 µl (20 units) RNAsin (Promega), and 0.5 µl (2.5 units) Taq polymerase (GIBCO-BRL). To this master mix we added 1 µl of template (1 µg RNA). The RT reaction was incubated for 40 min at 56°C, followed by 4 min at 95°C. PCR was 25 cycles at 94°C for 10 s, 60°C for 30 s, and 72°C for 30 s. The run was completed with a 20-min extension step at 72°C. PCR products of the expected band size were sampled and reamplified using the same primers. The reamplified products were purified and submitted to the Iowa State University Sequencing Facility for direct PCR sequencing.
Statistical analysis. Differences in pump expression, as affected by day, were analyzed by least-squares ANOVA. When day effects were significant, specific comparisons were made using Tukey's test. All analysis was performed using StatView (SAS Institute, Cary, NC).
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RESULTS |
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PMCA expression in rat mammary tissue as affected by pregnancy and
lactation.
The patterns of Ca2+-ATPase protein expression in mammary
tissue of rats 11 days pregnant to 21 days lactating were
examined by Western blot. Figure
1A shows a typical blot
representing the expression of total PMCA. Using an antibody (5F10)
that recognizes all PMCA isoforms, we found small amounts of PMCA
protein in the mammary gland before parturition. It was not until
lactation started that total PMCA protein increased significantly
(P < 0.1). PMCA protein expression increased 15 times
by day 18 of lactation (P < 0.001) compared
with that at day 11 of pregnancy (Fig. 1). This increase in
total PMCA paralleled milk production. Using isoform-specific antibodies (Fig. 2), we found that PMCA1b
protein was present at low levels on day 10. PMCA1b protein doubled
in late pregnancy and almost doubled again by peak lactation on
day 18 (P < 0.01) (Fig. 2A). As
shown in Fig. 1, the developing mammary gland of pregnant rats
expressed relatively low amounts of PMCA compared with the levels
expressed in the lactating mammary gland. Figure 2B also
shows that this relatively small amount of PMCA in developing mammary
tissue is primarily PMCA4b. PMCA4b expression increased 2.5 times
(P < 0.05) from day 11 of pregnancy to
day 1 of lactation. As lactation increased, PMCA4b
expression in the mammary gland declined significantly
(P < 0.001). Since PMCA1b (Fig. 2A)
increased only moderately during lactation, while PMCA4b was rapidly
declining in lactation (Fig. 2C), the large increase in PMCA
cannot be attributed to isoforms 1 and 4. Probing the blots with
anti-PMCA2 antibodies showed that the large increase in PMCA protein
expression is due primarily to PMCA2b (Fig. 2B). PMCA2b
protein expression increased significantly on day 1 of
lactation (P < 0.001) and increased almost 100 times
by day 18 of lactation (P < 0.001) compared
with that at day 11 of pregnancy.
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Evidence for alternate splicing of PMCA2b, at splice site A, in
mammary tissue.
Western blotting showed that mammary PMCA2b was ~4,000 daltons larger
than expected compared with brain PMCA2b (Fig.
3A) or recombinant PMCA2bz
(data not shown). RT-PCR showed that the primary mammary PMCA2b
transcript was alternatively spliced, at splice site A, to include an
additional 135 bp resulting in the insertion of 45 amino acids (Fig.
3B). This splice form is designated 2bw and accounts for the
4,000-dalton increase observed for mammary PMCA2b. The identity of the
PCR products in Fig. 3 was confirmed by size and sequencing. RT-PCR
showed that the w splice form was the predominant PMCA2 transcript in
the mammary gland, kidney, uterus, and liver (Fig. 3B).
However, only the mammary gland expressed PMCA2bw protein in
abundance, with small amounts of PMCA2bw seen in the liver (Fig.
3A).
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Putative (Golgi) SPCA expression in rat mammary tissue as affected
by pregnancy and lactation.
Using an antibody specific for the putative rat SPCA, we found that
SPCA protein expression was evident in the mammary gland at day
11 (day 10) of pregnancy (Fig.
5). SPCA protein expression increased
three times (P < 0.01) by day 14 (day
7) of pregnancy. SPCA protein increased further at the start of
lactation and remained elevated (P < 0.001) at six to
seven times the level seen at day 11 of pregnancy through
day 18 of lactation (Fig. 5, A and B). Western blotting showed that a form of mammary SPCA was ~2,000 daltons larger than expected when compared with brain or testes SPCA
(Fig. 5C). RT-PCR, with primers covering most of the open reading frame (bases 205-2,858) of rat SPCA (GenBank accession no.
M93017), failed to find evidence for an alternatively spliced form of
SPCA. This suggests that alternate splicing occurs outside the region
examined or that the increased size of mammary SPCA is due to
posttranslational modifications. Preincubation of the SPCA antibody
with the SPCA peptide used to prepare the antibody eliminated specific
banding on the Western blots (data not shown). The data suggest a
mammary-specific SPCA splice form because the mammary form was not
found in any other tissues tested. The splice must occur at the extreme
amino or carboxyl terminus.
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Secretion of Ca2+-ATPases into milk
on the MFGMs.
The fat in milk is a lipid droplet surrounded by a membrane. The source
of this membrane is the apical membrane of the milk secretory cell.
Therefore, examination of MFGMs yields information about the proteins
located on the apical membrane of the mammary secretory cell. Using
antibody 5F10, which recognizes all PMCA isoforms, we showed that
significant amounts of PMCAs were associated and secreted into milk
with the MFGM (Fig. 7, top
left). PMCAs in MFGMs were enriched eight times (Fig. 7,
bottom) more than PMCA measured in mammary tissue membranes
(see Fig. 7 legend for protein loading differences between blots and
membrane sources). Anti-PMCA2 antibody showed that PMCA2 was 21 times
more concentrated in MFGMs than in mammary tissue membranes. Small
amounts of PMCA1b and 4b were associated with the MFGM (data not
shown), but PMCA2 accounted for the majority of PMCA in MFGMs. In
contrast to the enrichment of PMCAs in MFGMs, SPCA (Fig. 7, top
right) was found to be primarily associated with mammary membranes
with very little SPCA found associated with MFGMs.
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Comparison of Ca2+-ATPases expression
in mammary tissue to other tissues.
Figure 8 shows the relative expression of
PMCAs, PMCA2, PMCA4, and SPCA in mammary tissue compared with that in
the brain, kidney, uterus, liver, and intestine (note different amounts
on membranes loaded by tissue type). Except for the brain, the
lactating mammary gland expresses more Ca2+-ATPases than
most tissues.
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DISCUSSION |
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The lactating mammary gland is the site of significant Ca2+ storage and transport, but despite the importance of mammary cell Ca2+ homeostasis, little is known about the proteins that regulate it or their expression (29, 41). Previously, we identified the Ca2+-ATPases (PMCA1b, 2b, 4b, and SPCA) present in mammary tissue and examined their mRNA expression during development and lactation (44). We concluded that (on the basis of Ca2+ pump mRNA expression) the comparatively low expression of PMCA1b and 4b limited the potential role of these two Ca2+- ATPases in macro-Ca2+ transport in the mammary gland. The high level of expression of PMCA2b and the putative SPCA mRNAs, as well as the timing of their expression relative to Ca2+ transport and storage, suggested that these Ca2+ transporters are key regulators of macro-Ca2+ transport in the mammary gland. These studies did not address whether the mRNA expression data was reflected in protein expression for the Ca2+-ATPases. The present study shows that our previous mRNA data are generally correlated with pump protein expression and provide new information about PMCA2 splice forms and their cellular location in mammary tissue. The exception is PMCA4b. PMCA4 mRNA was increased in lactating tissue, but PMCA4 protein declined significantly during lactation.
Using 5F10, an antibody that binds all PMCAs equally, we found that PMCA protein expression increased significantly and paralleled milk production (Fig. 1). This increased PMCA expression in mammary tissue was primarily PMCA2b (Fig. 2), because PMCA1b increased only slightly and PMCA4b declined significantly as lactation increased. The observed downregulation of PMCA4b, while PMCA2b was developmentally upregulated, in lactating mammary tissue resembles the regulation of these PMCA2 and 4 isoforms in cultured cerebellar granule cells as they develop (24, 25). The downregulation of PMCA4 and upregulation of PMCA2 in granule cells is associated with increased influxes of Ca2+, which are constantly occurring in lactating mammary tissue. Peak lactation in mammary tissue resulted in higher total PMCA despite the marked downregulation of PMCA4. Furthermore, the major portion of total mammary pump protein was PMCA2, similar to findings in cultured granule cells at peak development.
The significant expression of PMCA2 transcripts and protein in lactating mammary tissue (Figs. 2 and 3) is contrary to the popular belief that PMCA2 is only present in significant amounts in brain (23). Figure 4 shows that PMCA2 mRNA in the mammary gland is four times that observed in brain, whereas PMCA2 protein levels are one-third that seen in brain. The higher mRNA but lower PMCA2 protein seen in mammary tissue vs. brain suggests a much shorter half-life for mammary PMCA2 than the reported 12-day half-life for brain PMCAs (19). A significantly shorter half-life for PMCA2 protein in the mammary gland is supported by the finding that PMCA2 is concentrated and secreted into the milk on the MFGM (Fig. 7). The source of MFGM is the apical membrane of the mammary secretory cell. This apical membrane surrounds milk fat as it is secreted into milk, and it is estimated that the apical membrane is completely lost and replaced three times a day as a part of the process of milk fat secretion (21, 32, 33). Consequently, the high expression of PMCA2 mRNA in the mammary gland is explained by the need to rapidly replace PMCA2 lost to milk secretion. The concentration of PMCA2 in the MFMG suggests that PMCA2 is primarily located on the apical membrane. The mammary secretory cell transports Ca2+ from the basolateral to the apical side of the cell. The apical location of most of the mammary PMCA contrasts with that in the kidney and intestine, where PMCAs are localized in the basolateral membrane and are also involved in macrotranscellular Ca2+ fluxes (6, 51). This raises the question as to how PMCAs might be selectively targeted to apical or basolateral membranes in polarized epithelial cells.
Isoform-specific antibodies showed that mammary PMCA was primarily PMCA2b but that it was ~4,000 daltons larger than expected. RT-PCR showed that the primary mammary PMCA2b transcript was alternatively spliced, at splice site A, to include an additional 135 bp resulting in the insertion of 45 amino acids. This splice form is designated 2bw (2) and accounts for the 4,000-dalton increase in size of mammary PMCA2b. Only the mammary gland expressed PMCA2bw protein in abundance, with small amounts of PMCA2bw found in the liver (Fig. 3). The significance of this splice form remains to be determined because no functional differences resulting from alternate splicing at site A have been detected with the methods used (28). Although its role is yet unknown, the w splice form could play a role in targeting PMCA2 to the apical membrane of the mammary secretory cell.
The putative Golgi Ca2+-ATPase, SPCA, is the only protein that increased significantly before parturition (Fig. 5). This finding is similar to results in our previous study in which the mRNA for SPCA was the only transcript to increase before parturition (44). This is significant because this is the time when the animal becomes hypocalcemic due to rapid influx of Ca2+ to mammary stores. Clear biochemical evidence exists for a Golgi Ca2+-ATPase important to mammary function in general and casein micelles synthesis in particular (4, 5, 8, 15, 39, 40, 50). In yeast, the PMR1 gene product is a Ca2+-ATPase located in the Golgi complex of yeast (3, 16, 42, 45). Rat, human, and bovine homologues of the yeast PMR1 have been detected by cloning, but their subcellular location and specific function remain to be determined (26, 30). Mutations in the human PMR1 homologue have been shown to be the cause of Hailey-Hailey disease, in which cultured keratinocytes from these patients have impaired regulation of cytoplasmic Ca2+ (30). Together, the data for yeast and humans suggest that the mammalian PMR1 homologue could be a mammalian Golgi Ca2+-ATPase. The opposing argument put forth by Taylor et al. (49) is that the Golgi complex does not contain a unique "resident" Ca2+-ATPase. They state that all Ca2+ uptake into Golgi can be attributed to PMCAs in transit to the plasma membrane and to SERCAs that are not restricted to the ER. The presence of a unique resident Ca2+-ATPase in the Golgi remains to be determined, but data presented here and elsewhere suggest that SPCA is a candidate (26, 30, 44, 45).
Whether SPCA is determined to be a Golgi Ca2+-ATPase or not, transport and retention of calcium by the Golgi is important to mammary cell function (8, 15) and to cell function in general (36, 37, 43). As noted above, the high expression of PMCA2 mRNA in the mammary gland is explained by the need to rapidly replace PMCA2 protein lost to milk secretion. This need to rapidly replace PMCA2 lost to the rapid turnover of the apical membrane of the mammary secretory cell also should result in significant PMCA2 in transit in the Golgi. It is reasonable, on the basis of the findings of Taylor et al. (49), that the large amount of PMCA2 in transit to the apical membrane may make a significant contribution to Golgi Ca2+ in the mammary gland. The contribution of PMCA2s to Golgi Ca2+ would appear to be restricted to after the start of lactation, because there is little PMCA2 present before significant milk production (Fig. 2). The mammary gland needs to accumulate Ca2+ before the initiation of lactation. The significant expression of SPCA before the start of parturition and lactation is therefore significant, because this putative Ca2+-ATPase is the only Ca2+-ATPase to increase significantly before parturition.
In summary, this study demonstrates that the initiation of lactation and increasing milk production are associated with significant PMCA expression in the mammary gland. The primary PMCA expressed is the unique isoform PMCA2bw. PMCA2bw is apparently synthesized at a high rate to maintain its concentration in the apical membrane of the secretory cell, where it is rapidly lost to milk secretion. The abundance, cell location, high affinity for Ca2+, and high constitutive activity of PMCA2bw suggest that PMCA2b is important for macro-Ca2+ homeostasis in lactating tissue. The putative Golgi Ca2+-ATPase, SPCA, is the only Ca2+-ATPase whose expression increases significantly before parturition at a time of significant Ca2+ accumulation by the mammary gland. The pattern of expression and the abundance of SPCA suggest that it is a candidate for the Golgi Ca2+-ATPase shown to be important in maintaining the Golgi Ca2+ concentration required for casein synthesis. Several questions remain to be addressed, specifically with regard to the nature and function of SPCA and whether the large amount of PMCA2bw being made contributes to Golgi Ca2+ as it transits to the plasma membrane.
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ACKNOWLEDGEMENTS |
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We thank Becky Baccam and Derrel Hoy for excellent technical assistance and Annette Bates for expert preparation of the manuscript.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. A. Reinhardt, Periparturient Diseases of Cattle Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA 50010 (E-mail: treinhar{at}nadc.ars.usda.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 April 2000; accepted in final form 26 June 2000.
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