Endocrinology Vol. 139, No. 5 2615-2621
Copyright © 1998 by The Endocrine Society
Functional Receptors for Atrial Natriuretic Peptide in the Rat Mammary Gland during Lactation1
Katarina Cvek,
Yvonne Ridderstråle and
Rüdiger Gerstberger
Department of Animal Physiology (K.C., Y.R.), Swedish University of
Agricultural Sciences, S-75007 Uppsala, Sweden; and
Max-Planck-Institute for Physiological and Clinical Research (R.G.),
W. G. Kerckhoff Institute, D-61231 Bad Nauheim, Germany
Address all correspondence and requests for reprints to: Dr. Katarina Cvek, Department of Animal Physiology, Swedish University of Agricultural Sciences, Box 7045, S-750 07 Uppsala, Sweden. E-mail:
katarina.cvek{at}djfys.slu.se
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Abstract
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The present study was undertaken: 1) to localize and characterize
atrial natriuretic peptide (ANP) receptors in the rat mammary gland;
and 2) to elucidate ANP-induced cellular formation of cyclic GMP (cGMP)
and alterations in alveolar morphology during both early and late
lactation. Receptor autoradiography, employing rat-specific
[125I]ANP as radioligand, demonstrated binding sites in
the secretory tissue and larger blood vessels of the mammary gland.
Binding of [125I]rANP to membrane fractions was
completely displaced by unlabeled ANP and brain natriuretic peptide.
C-type natriuretic peptide and cANP(4–23) revealed limited
competition with radiolabeled ANP only during early lactation,
indicating a more heterogenous receptor population at that time.
Systemically administered ANP induced cGMP formation in the alveolar
epithelium, as shown with immunohistochemistry, and increased mammary
tissue cGMP concentrations in vivo throughout the
lactation period. Image analysis revealed enlargement of alveolar (but
not epithelial) cell area after ANP stimulation in late lactation,
suggesting altered alveolar filling or myoepithelial cell relaxation.
These results indicate that ANP induces biological effects in the rat
mammary gland through specific ANP-A receptor interaction with
subsequent intracellular cGMP formation. ANP may therefore play a
regulatory role in the control of mammary gland blood supply and
secretory function.
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Introduction
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MILK IS produced in highly vascularized
tubulo-alveolar glands, and the rate of blood flow is closely related
to milk production (1). Milk fluid losses during lactation in the rat
may amount to 20% of the maternal body weight per day (2). The
extracellular fluid compartment is enlarged in the lactating rat; and
cardiac output, mammary blood flow, and milk production increase
continually during the 3-week lactation period (3, 4). Hence, lactation
represents a physiological process that places great demands on the
cardiovascular and fluid regulatory systems. This made the vasoactive,
natriuretic hormone atrial natriuretic peptide (ANP) of special
interest among the many endocrine and paracrine factors involved in
regulating milk formation and secretion.
Detected in (and first isolated from) rat atria, ANP represents a
circulating factor released from atrial myocytes into the plasma under
conditions of local stretch, mainly caused by expansion of the
extracellular fluid compartment (5). Through interaction with specific
receptors coupled to intracellular guanylate cyclase (GC), ANP causes
marked vasodilation in various vascular beds, modifies epithelial ion
transport, and induces renal diuresis and natriuresis (6, 7). The
GC-coupled receptors can be divided into A and B subtypes, depending on
their ligand selectivity (8). In addition, ANP might interact with a
receptor protein that lacks the signal-transducing components and
therefore clears the circulation of ANP, however, with hitherto poorly
specified cellular actions (9, 10). This clearance receptor can be
identified by means of the specific ANP fragment
cANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) (9).
The observation that ANP dilates the internal mammary arteries in
women (11) indicates that ANP may be of importance for the regulation
of the blood supply to the mammary gland and, hence, for milk
secretion. The description of specific [125I]ANP binding
to receptor proteins in the rat mammary gland (12) and the ANP-induced
production of cyclic GMP (cGMP) in cultured rat mammary epithelial
cells (13) were other factors initiating the present study.
Furthermore, the alveoli and ducts of the mammary glands are surrounded
by highly contractile myoepithelial cells, which (at least for the
Bowman’s glands in the olfactory tract) have been described to respond
to C-type natriuretic peptide (CNP) as a member of the ANP peptide
family (14). The aims of the present study, therefore, were: 1) to
localize and characterize the ANP receptors in the rat mammary gland;
and 2) to investigate whether systemically administered ANP might
induce cGMP production in the mammary gland in vivo and
alter mammary gland morphology, thus indicating regulatory actions of
ANP on the secretory or myoepithelial cells. Because milk production
continually increases during lactation in the rat, it was decided to
perform experiments both in early and late lactation periods.
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Materials and Methods
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Animals
Adult female Sprague-Dawley rats were purchased from Alab
(Solna, Sweden), at least 3 days before the experiment, or obtained
from a breeding colony originating from SAVO (Kissleg, Germany). The
animals were kept single under a 12-h light, 12-h dark cycle at 21
± 1 C environmental temperature, and they were fed a diet of standard
rat chow with 1255 kJ/100 g digestible energy, 14.5% crude protein,
and 4.5% crude fat (LAB FOR R70; Lactamin, Stockholm, Sweden); and
they had access to drinking water ad libitum. The rats were
mated, and day 1 of lactation was denoted by the birth of the pups. The
experiments were carried out on mother rats in early (days 5–6) or
late (days 18–20) lactation. The BW of the lactating rats immediately
before the experiments was 377 ± 18 g (n = 10); litter
sizes were 10 ± 1 and 8 ± 1 pups during early (n = 5)
and late lactation (n = 5), respectively. The rats were kept in
their cages, together with their pups, and had free access to food and
water, all the time, until they were anesthetized.
Experimental procedure
Lactating rats were anesthetized by ip injection of
thiobutabarbitone (Inactin; Byk Gulden, Konstanz, Germany) or the
related thiobarbiturate thiopentone (Trapanal, Byk Gulden) (100 mg/kg
in 0.4 ml). The injection site was smeared with a local anesthetic
ointment (Xylocain, Astra Läkemedel, Södertälje,
Sweden) before systemic anesthesia. Immediately after the injection,
the rats were put back in their own cages and were not removed from
there until properly anesthetized. A polyethylene catheter (Intramedic
PE-10, Becton Dickinson, Sparks, Parsippany, NJ) was inserted into the
jugular vein for constant infusion of sterile, pyrogen-free isotonic
saline at 0.1 ml/min (Fresenius, Bad Homburg, Germany). Pieces of
glandular tissue were removed from two or three mammary glands from the
inguinal region (1.2–1.5 g total) after the major artery supplying the
glands was tied off to minimize blood loss. The tissue samples were
prepared immediately for receptor autoradiography, cGMP analysis, and
histological examination. Subsequently, the iv infusion of saline was
stopped, and rat ANP (rANP; Sigma, Munich, Germany) was administered as
bolus application (2 µg/animal) over 30 sec. Within 3 min after the
rANP injection, pieces of mammary gland tissue were dissected from the
contralateral side, as compared with the tissue sampling during
experimental control conditions, and prepared for cGMP analysis and
histological examination. The rats were euthanized after the last
tissue samples had been removed.
[125I]rANP receptor binding to mammary
membranes
Rat-specific ANP (Bachem, Bubendorf, Switzerland) was
radiolabeled employing the chloramine-T method (15), and the
monoradioiodinated ligand was purified using reverse-phase HPLC, as
described before (16, 17). The specific activity of the radioiodinated
rANP proved to be 1,200–1,400 Ci/mmol, as determined by
Bürgisser (18), employing freshly isolated rat glomeruli
(16).
To pharmacologically characterize the rANP-specific binding sites in
rat mammary gland tissue during early and late lactation periods,
competitive displacement experiments were carried out employing an
enriched plasma membrane fraction of the respective tissue samples and
receptor subtype-specific analogs of rANP. Mammary tissue, collected
just before the iv infusion of rANP, was minced and homogenized in
ice-cold, 30 mM Tris-HCl buffer, pH 7.4, containing 100
mM NaCl, 20 mM sucrose, 5 mM
MgCl2, 1 mM EGTA, 0.1 mM
phenyl-methylsulfonylfluoride, and 0.004% bacitracin (all from Sigma),
using a Polytron (Ultra-Turrax; IKA, Germany) and a Teflon-fitted
Elvehjem glass homogenizer. The homogenized tissue was centrifuged at
low speed (480 x g, 8–10 min, 2 C), and the
supernatant was subjected to a high-speed centrifugation (45,000
x g, 25 min, 2 C). The soft top layer of the resulting
pellet, containing membranes of various sources, was resuspended in
ice-cold Tris-HCl buffer and recentrifuged under identical conditions.
Again the soft top layer of the pellet was removed and dispersed in
ice-cold buffer, yielding a protein concentration of 500–600 µg/ml
(BioRad, Munich, Germany).
Competitive binding studies were performed with 30 µg of membrane
protein and 50 pM [125I]rANP as radioligand,
in Tris-HCl buffer containing 0.2% BSA (Sigma), under optimized
conditions (16), at 2 C for 40 min. The unlabeled, receptor
subtype-specific ANP analogs tested (2 x
10-14–10-6 M) were rANP, rat
brain natriuretic peptide (rBNP), rat CNP (rCNP), or the ANP c-fragment
(cANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)), as specific ligand for the clearance
receptor subtype. Peptides were obtained from Saxon Biochemicals
(Hannover, Germany). The incubation was stopped by rapid filtration
through Whatman-GF/C glass microfilters presoaked in 0.1%
polyethylenimin (Sigma), including three subsequent washes in ice-cold
PBS (M-R24; Brandel, Gaithersburg, MD). Filter-bound radioactivity was
determined in a 
-spectrometer (L-51; Berthold, Wildbad, Germany).
The data were analyzed with a modified PC-fitted version of the LIGAND
program (Prism2; GraphPad, San Diego, CA).
[125I]rANP receptor autoradiography
For receptor autoradiography, mammary gland tissue pieces were
immediately frozen in hexane, cooled in powderized dry ice to -50 C,
and stored at -24 C. Tissue sections of 20 µm were cut in a cryostat
(R-2700; Reichert-Jung, Dortmund, Germany) at -24 C and thaw-mounted
on 10% poly-L-lysine-coated slides (Sigma). The sections
were preincubated for 20 min at 2 C in 30 mM Tris-HCl
buffer, as described for membrane binding studies, containing 0.1%
BSA. Incubation was subsequently carried out with 200 µl of 0.3
nM [125I]rANP for 40 min at 2 C, in the
presence (nonspecific binding) or absence (total binding) of
10-6 M unlabeled rANP. After washing in buffer
without BSA for 3 x 2 min and distilled water for another 5 sec,
the sections were dried in a stream of dry air and exposed to an
AgfaScopix XR3 film for 6–7 days.
Analysis of mammary tissue cGMP
Tissue samples, obtained before and 3 min after systemic bolus
application of rANP, were frozen in liquid nitrogen, weighed, and
powderized under liquid nitrogen with a pestle in a mortar. The ground
tissue was then transferred to a teflon-fitted glass homogenizer,
suspended in ice-cold 6% trichloro acetic acid (10:1 vol:wt ratio),
homogenized until it was a milky homogenous fluid, and centrifuged
(2000 x g, 15 min) at 4 C. The supernatant was
recovered and washed four times with five volumes of water-saturated
diethyl ether (Merck, Darmstadt, Germany). The aqueous extract was
dried in a water bath at 45 C under a stream of air. The content of
cGMP was determined using a RIA kit with [125I]cGMP as
tracer (RPA 525; Amersham, Little Chalfont, UK). Before analysis, the
dried extract was dissolved, at a 10:1 vol:original wet wt ratio, in
RIA buffer.
To localize the cells producing cGMP, three additional rats in late
lactation were used for immunohistochemistry, according to Chevalier
et al (19). The rats were anesthetized by an ip injection of
pentobarbital sodium (60 mg/kg), and a catheter was placed in the
aorta. After an intraarterial injection of ANP (80 µg), the rats were
immediately perfused with 4% paraformaldehyde in 0.12 M
phosphate buffer, pH 7.4 for circa 10 min. After the perfusion, pieces
of mammary tissue were obtained and immersed in the fixative for 2
h. Kidney tissue samples were included in the experiment as positive
control tissue. The tissue samples were rinsed in 30% Tris-buffered
sucrose solution, pH 7.4, and immersed in this solution at 6 C
overnight. The tissue was immersed in OCT embedding medium (Tissue-Tek,
Miles, Elkhart, IN) and frozen at -22 C. Tissue sections (7 µm) were
cut in a cryostat and placed on cold poly-L-lysine
coated slides. The sections were air dried and washed in PBS, pH 7.4,
for 15 min (at 6 C), after which they were placed in 0.3% peroxide in
PBS for 30 min. For the immunohistochemical visualization, the
ABC-technique was applied (Vectastain ABC kit, Vector Laboratories,
Burlingame, CA). After being washed in PBS, the slides were blocked for
endogenous avidin and biotin, washed, and treated with rabbit normal
serum, all supplied with the ABC kit. The slides were then incubated
overnight with sheep antibody to cGMP-formaldehyde-thyroglobulin
conjugate, diluted 1:6000. The antibody was kindly supplied by Dr. Jan
de Vente (Department of Pharmacology, Faculty of Medicine, Free
University, Amsterdam, The Netherlands). Control sections were
similarly treated but excluding the specific antibody to cGMP. After
washing with PBS, the slides were incubated with biotinylated antibody
supplied with 10% rat plasma for 45 min, washed again, and treated
with the ABC solution for 60 min. After an additional wash, the
reaction was visualized using diaminobenzidine-PBS (pH 7.6) for 5 min.
After a final wash, some of the sections were also counterstained with
azure blue before being mounted for light microscopical
examination.
Image analysis
Mammary tissue samples from the inguinal region were obtained
from all rats before and after the systemic rANP injection, and
midgland tissue slices (1–2 mm) were cut. The tissue slices were fixed
by immersion in phosphate-buffered 2.5% glutaraldehyde, pH 7.2,
overnight (Sigma). After rinsing in phosphate buffer (0.067
M, 70:30 Na2HPO4:
KH2PO4), pH 7.2, the samples were dehydrated
via graded ethanol concentrations and embedded in water-soluble resin
(Historesin; Leica Instruments, Heidelberg, Germany). Sections, 2-µm
thin, were cut on a microtome (Historange, LKB, Bromma, Sweden), using
a glass knife, and stained with hematoxylin/eosin.
The stained sections were randomly analyzed, using an image analysis
system, with the TEMA program (BioRad Scan Beam, Hadsund, Denmark).
Three areas of equal rectangular size, located at the top, middle, and
lower end of the respective tissue sections, were selected for alveolar
histomorphometric analysis. The total area and the lumen area of the
alveoli were measured, and from these values, the epithelial area was
calculated. The number of alveoli analyzed amounted to 50–60
alveoli/section. To avoid any bias in the analysis of the sections, the
identities of the individual slides were hidden and not disclosed until
all the sections had been analyzed.
Statistics
Using the SigmaStat program (Jandel Scientific Software,
Erkrath, Germany), a paired t test was performed to evaluate
significant differences between treatments. P < 0.05
was considered significant. Values are presented as means with
SEM.
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Results
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Localization and characterization of mammary
[125I]rANP binding sites
Employing monoradioiodinated rANP of high specific
activity as radioligand, binding sites specific for
[125I]rANP could be localized throughout the secretory
tissue of the rat mammary gland (Fig. 1
).
The epithelium of the alveoli and ducts and the larger blood vessels
were markedly labeled, with no obvious difference between tissue
samples examined during stages of early (5–6 days, not shown) and late
(18–20 days) lactation. Connective tissue and fat cells were clearly
devoid of ANP-specific binding sites.
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Figure 1. Receptor autoradiograms showing the distribution
of [125I]rANP specific binding sites in rat mammary
tissue during late lactation. Unfixed cryostat tissue sections
(20-µm) were incubated with 0.3 nM
[125I]rANP. Binding of the radioligand, as indicated by
silver grain deposition, is concentrated at the level of the secretory
epithelium throughout the gland (A and E). In addition, binding proved
to be highly specific because of full displacement of the radioligand
in the presence of 10-6 M unlabeled rANP (B).
The same autoradiogram is shown at higher magnification (C), with the
consecutive tissue section stained for hematoxylin/eosin (D). Larger
blood vessels also exhibit specific binding for radiolabeled rANP, as
can be seen in the middle of the autoradiogram (E) and
the corresponding hematoxylin/eosin-stained section (F).
Bars represents 180 µm in A, B, E, and F; and 90 µm
in C and D.
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Competitive displacement studies, performed with an enriched plasma
membrane fraction to unravel the expression of ANP receptor subtypes in
the rat mammary gland, showed high affinities for rANP and rBNP to
fully displace [125I]rANP from its target (Fig. 2). In both early and late lactation
tissue, the respective EC50 values for rANP were 2.0
± 1.1 nM (n = 5) and 2.6 ± 0.5 nM
(n = 5), whereas rBNP revealing high affinity during early
lactation (EC50 values of 14.4 ± 6.5 nM;
n = 5) became less effective during late lactation
(EC50 values of 68.6 ± 20.4 nM; n =
4). During early lactation, both rCNP and cANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
displaced some 30% of the radioligand, with EC50 values of
0.9 ± 0.5 nM (n = 5) and 0.8 ± 0.2
nM (n = 5), respectively (Fig. 2). Therefore, during
early lactation, the binding data reflect prevalence of the ANP-A
receptor subtype, with both the ANP-B and ANP-C receptor being
expressed to some extent. In the late lactation period, however,
neither rCNP nor the ANP fragment showed consistent displacement of
[125I]rANP, indicative of the sole existence of the ANP-A
receptor subtype.
Systemic ANP induces cGMP production and distention of alveolar
lumen
With functional receptors of the ANP-A and ANP-B subtypes being
coupled to cGMP formation caused by receptor-intrinsic GC activity,
analysis of cellular cGMP content was performed in mammary gland tissue
samples obtained before and after systemic administration of rANP in
anesthetized rats. The results indicate a 4- to 10-fold increase in
cellular cGMP in the tissue samples taken some 3 min after in
vivo stimulation with rANP, as compared with those obtained
shortly before the rANP injection (Fig. 3). The responses were of similar
magnitude in early (0.5 ± 0.08 before, to 3.3 ± 0.6 nmol/g
tissue after iv ANP) and late (0.6 ± 0.05 before, to 3.5 ±
0.3 nmol/g tissue after iv ANP) lactation.
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Figure 3. rANP-induced cGMP synthesis in the rat mammary
gland. cGMP formation was determined in mammary tissue samples obtained
before (Control) and after iv injection of 2 µg rANP (ANP) for female
rats during early (left) and late (right)
lactation periods. The columns represent mean values
with SE of five experiments. **, P <
0.01; ***, P < 0.001 between treatments (paired
t test).
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The immunohistochemical localization of cGMP in mammary tissue sections
revealed positive staining of the alveolar epithelium (Fig. 4A), and occasionally, stained
myoepithelial cells could be distinguished (Fig. 4B). Larger blood
vessels showed staining of the endothelium. Negative controls,
incubated without the primary antibody, indicated no specific staining
(Fig. 4C). The kidney sections included as a positive control showed
distinct staining of the glomeruli and blood vessels, in agreement with
Chevalier et al. (19), proving the specificity of the
method.
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Figure 4. Immunohistochemical localization of cGMP
(brownish stain) in rat mammary gland after rANP
treatment. Paraformaldehyde fixed freeze sections weakly counterstained
with azure blue. Varying amounts of cGMP are present in the alveolar
epithelium (A and B). Stained myoepithelial cells can occasionally be
distinguished (arrow, B). Control sections incubated
without primary antibody show no specific stain; not counterstained
(C). Bars represent 50 µm in A and C, and 10 µm in
B.
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The histological appearance of the mammary tissue was similar before
and after stimulation with rANP in vivo during early
lactation, as confirmed by quantitative image analysis of the tissue
sections. However, in the late lactation period, the alveoli seemed
distended. Image analysis revealed a significant increase in mean
alveolar lumen area (P = 0.03) and a clear, but not
quite significant (P = 0.06), increase in mean total
alveolar area after stimulation with rANP (Fig. 5 and Fig. 6). Distention of the alveoli proved not
to be accompanied by a decrease in the area of the alveolar epithelium
(Fig. 6).
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Figure 5. Photomicrographs of mammary gland tissue before
and after rANP treatment. Thin (2-µm) cross-sections of mammary gland
alveoli and ductules during early (A and B) and late (C and D)
lactation periods, as well as before (A and C) and 3 min after (B and
D) iv bolus application of rANP (2 µg/animal). Only during late
lactation, rANP treatment results in alveolar dilatation (D) throughout
the secretory parenchyma. Glutaraldehyde-fixed sections are stained
with hematoxylin/eosin. The bar represents 180 µm.
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Figure 6. Morphological changes in mammary gland alveoli
caused by rANP treatment during early and late lactation. Employing
image analysis, the areas (µm2 x 1000) of alveoli,
alveolar lumen, and the secretory epithelium were determined for female
rats during early and late lactation periods, as well as before
(Control) and 3 min after iv bolus application of rANP (2 µg/animal)
(ANP). The ANP treatment caused an enlargement of the luminal area and
a tentative, nonsignificant increase of the total area of the alveoli
(top) during late lactation exclusively, whereas the
secretory cell area remained constant. *, P < 0.05
between treatments (paired t test).
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Discussion
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With the principal expression of binding sites for ANP in the rat
mammary gland per se already described by Pelletier (12),
the present study demonstrates, for the first time, the localization of
rANP-specific binding sites associated with the secretory epithelium,
as well as major blood vessels of the rat mammary gland, employing
in vitro receptor autoradiography. The prevalence of the
functional ANP receptor subtypes, during both early and late lactation,
is indicated by markedly enhanced mammary cGMP formation caused by
increased plasma ANP levels in vivo. The high affinity of
both rANP and rBNP (but not rCNP) to displace the radioligand from its
binding sites during late lactation favors the preferential presence of
the ANP-A receptor subtype in the rat mammary gland during maximal milk
production. In the mammary tissue of rats in early lactation, however,
rCNP managed to displace about 30% of the radiolabeled ANP at high
potency, suggestive of partial ANP-B receptor expression (8). The same
sequence of affinities (ANP > BNP >> CNP) has been reported
for the rat uterus, regardless of the stage of the estrous cycle, which
caused Dos Reis and colleagues (20) to suggest that natriuretic
peptides could mediate the changes in uterine water content throughout
the cycle. The fact that the ANP fragment also was able to displace the
radioligand (to a somewhat minor degree but at high affinity) during
early lactation indicates: 1) that a small portion of clearance
receptors might be present in early lactation; and 2) that the receptor
population becomes more homogenous (ANP-A receptors) as lactation
proceeds. In addition, it can be emphasized that most vertebrate organs
predominantly express clearance receptors (9), whereas the mammary
gland and the uterus represent tissues that, in principal, possess
GC-coupled ANP receptors, as shown in the present and earlier studies
for rats (20) and goats (17).
The proof of a functional ANP receptor subtype, present in the rat
mammary blood vessels and secretory tissue, indicates several possible
effects of circulating ANP on the function of the lactating mammary
gland, namely augmented blood supply and modulation of secretory and
myoepithelial cell functions. During lactation, the mammary blood flow
is greatly enhanced, and up to 13% of the cardiac output can be
directed to the mammary glands in the lactating rat (3). The mammary
blood flow has previously been shown to be affected by vasoactive
hormones (21); and since biologically active receptors for ANP were
found in the blood vessels of the rat mammary gland, it is a plausible
postulate that ANP dilates these blood vessels and, thereby,
participates in mammary blood flow regulation.
With regard to the actions of ANP at the level of the epithelium, it
must first be explained that the morphology of the mammary gland varies
greatly, depending on the degree of alveolar filling or hormonal
action. For example, the alveoli are distended, with a flattened
epithelium in an unmilked gland, compared with a newly milked gland,
where the alveoli are collapsed and the secretory cells are
pseudo-columnar in shape (22). In the present study, the pups could
suckle their dam until the last minute before the experiment, which
would minimize filling of alveoli caused by a long separation of pups
and dam. Short-term rANP treatment, in the present study, induced
distention of the alveoli in late lactation exclusively, whereas the
area of the epithelium remained unaffected. This could indicate that
rANP could have relaxed the myoepithelial cells, which are in close
contact with the basal surface of the secretory cells. This is
supported by our immunohistochemical results, showing cGMP to be
present in myoepithelial cells and in secretory cells. It previously
has been reported that vasoactive substances affect myoepithelial
cells, as well as vascular smooth-muscle cells, in the mammary gland
(23). If ANP acts as a relaxing agent on the myoepithelial cells in the
mammary tissue this could be a clue as to why goats showed no changes
in milk sodium, water, or lactose secretion in response to systemic
infusion of 30 ng/min·kg BW ANP (17). Alternatively, the distention
of the alveoli could be a secondary effect of ANP on the cardiovascular
system. It is well known that the tight junctions between epithelial
cells become leaky in late lactation (24, 25), and ANP is well known to
cause a fluid shift from the intravascular compartment to the
interstital space (5). If the tight junctions had started to become
leaky during the late lactation period of our study, the fluid could
have taken the paracellular route into the lumen of the alveoli. This
could also explain why differences in the histological appearance were
not detectable after ANP stimulation in early lactation, a period when
the tight junctions were intact. It seems unlikely that the
morphological index of alveolar distention would represent a filling of
the alveoli because of rapid augmented secretion of milk components and
water. However, the localization of ANP-induced cGMP to secretory cells
indicates an effect of ANP on mammary function. Whether ANP could
affect milk composition in the rat remains to be investigated.
It is concluded that the rat mammary gland expresses biologically
active receptors for ANP throughout the mammary parenchyma, including
its blood vessels, and that increased plasma rANP concentration induced
changes in mammary gland morphology. These results indicate that ANP
could have a role in mammary gland blood supply and secretory
function.
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Acknowledgments
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The excellent technical assistance of Ingrid Wennerberg, Gunilla
Drugge-Boholm, Irene Küchenmeister, and Roswitha Bender is
gratefully acknowledged.
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Footnotes
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1 This study was supported by the Swedish Medical Research Council
(project 3392). The experimental protocol was approved by the Ethical
Committee for Animal Experimentation, in Uppsala, Sweden. 
Received September 30, 1997.
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Top
Abstract
Introduction
), rBNP (
), CNP (•), and cANP(4–23) (
),
revealed prevalence of the ANP-A receptor subtype in mammary gland
tissue during both early and late lactation, with some ANP-C
additionally expressed during early lactation. Data show means ±
SEM of triplicate determinations of a single experiment.
Four to five experiments were performed with each ligand.