P2 purinoceptors regulate calcium-activated chloride and
fluid transport in 31EG4 mammary epithelia
Sasha
Blaug1,*,
Jodi
Rymer1,*,
Stephen
Jalickee2, and
Sheldon S.
Miller1,2
1 Department of Molecular and Cell Biology and
2 School of Optometry, University of California at
Berkeley, Berkeley, California 94720-3200
 |
ABSTRACT |
It has been reported that
secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon
mechanical stimulation. Here we examined the physiological changes
caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia
(31EG4 cells). In control conditions, transepithelial potential (apical
side negative) and resistance were
4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4
· cm2, respectively. The apical
membrane potential was
43.9 ± 1.7 mV, and the ratio of apical
to basolateral membrane resistance (RA/RB) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranes
caused large voltage and resistance changes with an EC50 of
~24 µM (apical) and ~30 µM (basal). Apical ATP/UTP (100 µM)
depolarized apical membrane potential by 17.6 ± 0.8 mV (n = 7) and decreased
RA/RB by a factor of
3. The addition of adenosine to either side (100 µM) had
no effect on any of these parameters. The ATP/UTP responses were
partially inhibited by DIDS and suramin and mediated by a transient
increase in free intracellular Ca2+ concentration (427 ± 206 nM; 15-25 µM ATP, apical; n = 6). This Ca2+ increase was blocked by cyclopiazonic acid, by BAPTA,
or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed
fluid in the resting state, and ATP or UTP increased fluid secretion by
5.6 ± 3 µl · cm
2 · h
1
(n = 10). Pharmacology experiments indicate that 31EG4
epithelia contain P2Y2 purinoceptors on the apical and
basolateral membranes, which upon activation stimulate apical
Ca2+-dependent Cl channels and cause fluid secretion across
the monolayer. This suggests that extracellular nucleotides could play
a fundamental role in mammary gland paracrine signaling and the
regulation of milk composition in vivo.
P2Y purinoceptor; P2U purinoceptor; adenosine trisphosphate; uridine trisphosphate; microelectrodes; mammary physiology; electrophysiology; cystic fibrosis; fluid movement; leaky and tight
epithelia
 |
INTRODUCTION |
EXTRACELLULAR ATP OR
UTP and other purine nucleotides mediate multiple cellular
responses through specific membrane-bound purinoreceptors in diverse
tissues. Purinoceptor stimulation affects antiproliferative and
contractile properties in vascular tissue, endothelial PGI2
release, protein phosphorylation in numerous cell types, bile acid
secretion in the gastrointestinal tract, and an increase in mucociliary
clearance and mucin release in the lung (7, 18, 22, 37, 45,
50). In various epithelia, metabotropic P2 purinoceptors have
been shown to stimulate second messenger-induced activation of Cl (and
K) channels (6, 39, 43, 46, 53, 55).
In mammary epithelia, ATP, UTP, and UDP are released in the
extracellular space after mechanical stimulation (16).
Subsequent ATP-dependent contraction of adjacent myoepithelial cells
can stimulate further nucleotide release (40) and perhaps
lead to increased fluid secretion from mammary cells. Because ATP, UTP, and other P2 agonists are released from cells, their presence in human
cyst fluid and milk secretions is highly likely. Indeed, ATP has been
found in bovine milk (14, 44), and purinoceptors have been
observed in MCF-7 human tumor breast cells (17),
suggesting that paracrine regulation may play a vital role in fluid
homeostasis in the breast. In addition to regulating the composition of
milk, purinoceptors may play a role in the accumulation of abnormal breast fluid common in premenopausal women (5, 36). This cyst fluid consists of a myriad of compounds, including hormones, growth factors, glycoprotein, bile acids, proteins, and glucose.
Previously, we showed that fluid secretion across intact monolayers of
confluent 31EG4 mammary epithelia is in part mediated by apical
membrane Na [epithelial Na channel (ENaC)] and Cl [cystic fibrosis
transmembrane conductance regulator (CFTR)] channels and, possibly,
Ba2+-sensitive K channels (3). We now provide
evidence for P2 purinoceptors at both the apical and basolateral
membranes of 31EG4 cells, using electrophysiological, fluid transport,
and intracellular Ca2+ measurements. The activation of
these receptors by either ATP or UTP significantly increased
intracellular Ca2+ levels
([Ca2+]in), apical membrane conductance, and
steady-state fluid secretion across the epithelium. These data suggest
that in vivo mammary gland function is determined by paracrine,
autocrine, and hormonal activation of metabotropic P2 purinoceptors.
 |
MATERIALS AND METHODS |
Cell culture.
31EG4 cells were cultured in DMEM/F-12 media containing 5% FBS, 5 µg/ml insulin, and 5 µg/ml gentamicin sulfate. Upon reaching confluence, cells were plated on Transwell filters (Costar) at a
density of 105 cells/well. When the cells became confluent
monolayers on the filters, 1 µM dexamethasone in 2% FBS was added to
stop growth and induce differentiation, including formation of tight
junctions (58) and polarization of ion transport
proteins to the apical and basolateral membranes
(49). Transepithelial resistance
(RT) was recorded using an EVOM (Epithelial
Voltammeter; World Precision Instruments, New Haven, CT).
RT-PCR.
Total RNA was extracted from 3lEG4 cells using Qiagen RNeasy per the
manufacturer's instructions. First-strand cDNA synthesis was carried
out with 0.5 pg of total RNA, 20 pM oligo(dT) primers, 0.5 mM dNTP mix,
and 200 units of Moloney murine leukemia virus RT in 20 ml (final
volume) of 50 mM Tris · HCl (pH 8.3), 75 mM KCl,
and 3 mM MgCl2. The RNA and oligo(dT) were annealed by
mixing two items, heating to 70°C for 2 min, and then cooling on ice followed by addition of the remaining reaction components. The mixture
was incubated at 42°C for 1 h for first-strand synthesis and
then heated to 94°C for 4 min to stop the reaction. The mixture was
then diluted 1:5 with sterile distilled water. A control reaction containing no RT was included for each tissue RT reaction to ensure that no genomic DNA was being amplified (data not shown).
PCR was carried out using oligonucleotide primers (sense 5'-GAC ACC ATC
AAT GGC ACC TGG GAT-3'; anti-sense 5'-TGA TGC AGG TGA GGA AGA GGA
TGC-3') designed to cover a 355-bp region of P2Y2. The
primers (0.5 mM final concentration) were added to a 1-ml aliquot of
the first-strand synthesis mixture with the following: 0.2 mM dNTPs
(each), 1.25 units of Pfu turbo polymerase, PCR buffer with
Mg (Boehringer Mannheim), and water to bring to a total volume of 50 µl. The reaction was overlaid with two drops of mineral oil (Sigma),
and the mixture was then incubated in a thermal cycler (Stratagene)
with the following amplification profile: 1 cycle at 94°C for 4 min,
39 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and
1 cycle of 72°C for 10 min. The PCR product was run on a 1.5%
agarose gel and stained with ethidium bromide.
Bathing solutions and materials.
The control Ringer solution for all measurements (intracellular
recording, Ca2+ measurement, and fluid transport) contained
(in mM) 113.5 NaCl, 5 KCl, 26 NaHCO3, 1.8 CaCl2, 0.8 MgSO4, 1.0 NaH2PO4, 5.5 glucose, and 5 taurine, pH 7.4. The following drugs were obtained from Sigma Chemical (St. Louis, MO):
ATP, UTP, ADP, UDP, benzoylbenzoyl-ATP (BzBz-ATP), adenosine
3',5'-diphosphate (A3P5P),
N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate
(MRS2179), ADP
S, 2-methylthio-ATP (2-MeSATP),
,
-methylene ATP
(
,
-MeATP), suramin, and DIDS. H2DIDS and BAPTA-AM
were obtained from Molecular Probes (Eugene, OR), and xestospongin C
was obtained from Calbiochem (La Jolla, CA).
Microelectrode electrophysiology.
Transwells with confluent 31EG4 monolayers and
RT >300
· cm2 were used for
electrophysiology measurements, as previously described (3). Monolayers on filters were mounted on a nylon mesh
support and clamped in a modified Ussing chamber, apical side up. The perfusion of Ringer to each side of the tissue was controlled separately. The apical perfusion rate was ~26 chamber vol/min; the
basal perfusion rate was ~79 chamber vol/min. Ringer solution changes
were delivered to the chamber from a short distance, which delayed
arrival by ~30 s. Calomel electrodes in series with Ringer solution
were used to measure the transepithelial potential (TEP), and the
signals from intracellular microelectrodes were referenced to either
the apical or basal bath to measure the membrane potentials, VA and VB, where TEP = VB
VA
(30, 38). Conventional microelectrodes, with resistances
of 80-250 M
, were made from fiber-filled borosilicate glass
tubing with 0.5 mm inner diameter and 1 mm outer diameter (Sutter
Instrument, Novato, CA) and were back-filled with 150 mM KCl.
The ratio of the apical (RA) to basolateral
(RB) membrane resistance (a value)
and RT were obtained by passing 4 or 8 µA
current pulses (peak to peak) across the tissue and measuring the
resultant changes in TEP, VA, and
VB. Current pulses were bipolar, with a period
of 3 s applied at various time intervals.
RT is the resulting change in TEP divided by the
appropriate current, and a is the absolute value of the
ratio of voltage change in VA divided by the
change in VB (a =
VA/
VB). The
current-induced voltage deflections were digitally subtracted from the
records for clarity.
Calculating membrane and shunt resistances and equivalent
electromotive forces.
The electrical properties of the 31EG4 monolayer can be modeled as a
simple equivalent circuit (3) where the apical and basolateral membranes are electrically coupled by the paracellular shunt pathway and possibly by edge damage around the circumference of
the mounted tissue. The apical and basolateral membranes of the
monolayer are each represented as an equivalent electromotive force
(EMF), EA or EB, in
series with a resistor, RA or
RB, respectively (30, 38). The
paracellular pathway is represented as a shunt resistor,
Rs, which is a parallel combination of the
paracellular resistances between neighboring cells and the resistance
of the mechanical seal around the tissue. In general, a shunt current (IS) will flow around the circuit because
EA
EB.
IS is given by
|
(1)
|
Because the TEP is basolateral side positive,
IS flows through shunt resistance
(RS) and then inward across the apical membrane. This circulating current hyperpolarizes the recorded apical membrane potential (VA) relative to
EA and depolarizes the recorded basolateral (VB) membrane potential relative to
EB. The observed steady-state membrane
potentials, VA and VB,
are related to the respective EMFs by
|
(2)
|
RT and the ratio of apical to basolateral
membrane resistance (a) are expressed in terms of the
membrane and RS as follows
|
(3)
|
The apical and basolateral membrane voltages are electrically
coupled via Rs so that a voltage change at one
membrane will be shunted to the opposite membrane. For example, a
membrane voltage change,
VA, originating at
the apical membrane will produce a smaller change with the same time
course at the basolateral membrane,
VB
(30).
We could calculate RA,
RB, and RS by using
Eqs. 2, 3, and 4 before and after the
addition of a K channel blocker (Ba2+) to the apical bath
(responses in Ba2+ denoted with *). This calculation
assumes that Ba2+ affected only the apical membrane,
whereas RS and RB
remained constant (3, 30)
|
(4)
|
[Ca2+]in.
[Ca2+]in levels were monitored with the
fluorometric ratioing dye fura 2-AM (Molecular Probes) in a modified
Ussing chamber. In this chamber, the tissue is mounted apical side
down. The chamber and recording setup have been described previously
(3, 31, 35, 41). In these experiments, the perfusion of
Ringer solution to each side of the tissue was controlled separately.
The apical perfusion rate was ~20 chamber vol/min and the basal
perfusion rate was ~5 chamber vol/min. Ringer solutions were
delivered over a short distance, delaying the arrival by ~30 s.
Cell monolayers were loaded with dye by bathing them in Ringer solution
containing 8.2-12.5 µM fura 2-AM (dissolved in DMSO + 10%
pluronic acid) for 2 h (8% CO2) at room temperature.
In addition, 1 mM probenicid was included in the apical bath and in all
subsequent apically perfused Ringer solutions to inhibit dye extrusion,
presumably mediated by an organic anion transporter, as in other
epithelia (31). Photic excitation was achieved using a
Xenon light source filtered at
= 340 and 380 nm every 0.5 s; the emission fluorescence was measured at 510 nm with a
photomultiplier tube (Thorn; EMI). The ratio of the fluorescence
intensities (R) at 340/380 nm was determined every second. The
technique and computer software for data acquisition have been
described previously (2). Calibration of
[Ca2+]in was performed at the end of each
experiment by first perfusing both membranes with a
zero-Ca2+ Ringer solution containing 10 mM EGTA, which
chelates any residual free Ca2+, and 10 µM ionomycin, a
Ca2+ ionophore that facilitates the equilibration of
[Ca2+]in and extracellular Ca2+
concentration. After this zero Ca2+ calibration, the tissue
was exposed to a saturating (1.8 mM) concentration of Ca2+.
[Ca2+]in was determined according to the
following equation: [Ca2+]in = K([R
Rmin]/[Rmax
R]), where K = Kd(Fmin/Fmax).
Kd is the dissociation constant for fura 2-AM,
and Fmin and Fmax are the fluorescence
intensities at 380 nm in the presence and absence, respectively, of
saturating Ca2+. With the use of solutions of known
[Ca2+]in and 6 µg/ml digitonin or 20 µM
ionomycin, the value for K was determined to be ~140 nM.
Fluid transport.
The rate of transepithelial fluid flow (JV) was
measured using the capacitance probe technique, as previously described
(25, 29). A monolayer of cells on a filter (0.5 cm2 exposed area) was mounted between two water-impermeable
Kel F half-chambers. JV was determined using a
sensitive oscillator circuit (Acumeasure 1000; Mechanical Technology,
Lantham, NY) connected to two probes, one on either side of the tissue,
that measure the capacitance between the probe tips and the fluid
meniscuses connected to each half-chamber. Net fluid movement across
the epithelium, from the apical to basolateral surface, or vice versa, is recorded by the changes in probe output voltage. This technique has
a resolution of ~1 nl/min. Ports in the bottom of separate half-chambers allow for the perfusion of solutions with different chemical compositions to either side of the tissue. Voltage-sensing and
current-passing agar bridges built in each half-chamber permit continuous monitoring of TEP and RT, the latter
calculated from the TEP voltage deflections in response to
transepithelial current pulses of 4-10 µA.
All data are presented as means ± SD, unless otherwise specified.
Student's unpaired t-test was used to compare groups, and P < 0.05 is considered statistically significant.
 |
RESULTS |
RT-PCR.
RT-PCR was performed on 31EG4 cells grown to confluence in the presence
of dexamethasone to increase differentiation of the cells. The PCR
experiment summarized in Fig. 1 shows
that mRNA for the P2Y2 purinoceptor is present in 31EG4
cells.

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Fig. 1.
PCR product for the P2Y2 purinoceptor (355 bp). The amplified product was run on a 1.5% agarose gel and stained
with ethidium bromide. The 100-bp ladder is on left
(marker).
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ATP- and/or UTP-induced electrical responses.
Figure 2 shows typical intracellular
microelectrode recordings from 31EG4 mammary epithelial cells in the
absence and presence of ATP in the apical (A) or basal
(B) baths. In Fig. 2A, the resting, unstimulated
levels of VA and VB were
37.5 and
33.5 mV, respectively, with a TEP of 4 mV, whereas
RT and
RA/RB were 385
· cm2 and 2.6. The addition of
100 µM ATP to the apical bath transiently increased TEP by 3 mV,
since VA depolarized at a faster rate than VB; concurrently, RT
decreased by 70
· cm2 and
RA/RB decreased from
~2.6 to 0.2 and then slowly returned to baseline. This return was
accelerated by the removal of ATP from the apical bath. Figure
2B shows that a similar set of responses was obtained with
the addition of 100 µM ATP to the basal bath. Basal addition of ATP
caused a rapid increase in TEP of 2.8 mV because
VA depolarized faster than
VB; concomitantly, there was an 80
· cm2 drop in
RT, and
RA/RB decreased from 2.75 to 0.75. The magnitude and direction of the resistance changes provide
strong evidence that ATP (or UTP) increased apical membrane conductance
(42).

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Fig. 2.
A: effects of apical ATP (100 µM) on membrane voltages
and resistances in 31EG4 cells. VA and
VB, apical and basolateral membrane potentials,
respectively; RA/RB,
ratio of apical to basolateral membrane resistance; TEP (=
VB VA),
transepithelial potential; RT, total tissue
resistance (MATERIALS AND METHODS).
B: effects of basal ATP (100 µM) on membrane voltages and
resistances in 31EG4 cells.
|
|
Figure 3, A and B,
shows that apical or basal bath UTP (20 µM) produced a similar set of
electrical responses. The summary data for a set of ATP/UTP experiments
at 50 µM are presented in Table 1 (the
responses to ATP or UTP are indistinguishable and have been combined).
Table 1 indicates that apical or basal addition of secretagogue
elicited similar responses. Addition of ATP/UTP caused membrane voltage
and resistance changes that are consistent with a conductance increase
of an apical membrane channel whose equilibrium potential is
depolarized with respect to VA.

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Fig. 3.
A: effects of apical UTP (20 µM) on membrane voltages
and resistances in 31EG4 cells. B: effects of basal UTP (20 µM) on membrane voltages and resistances in 31EG4 cells.
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Effects of apical or basal DIDS or suramin on the ATP- and/or
UTP-induced changes in membrane voltage and resistance.
It has been shown that DIDS and suramin can block P2Y receptors
(8, 23, 41). Figure
4A shows the UTP-induced
changes in membrane voltage and resistance first in the absence and
then in the presence of basal DIDS. After addition of 50 µM UTP to the basal bath, VA depolarized more than
VB (TEP increased by 1.5 mV),
RT decreased by 75
· cm2, and
RA/RB decreased from
~3.0 to 1.0. After 16 min in control Ringer, DIDS (500 µM) was
added to the basal bath where it had little effect on
VA, VB,
RA/RB,
RT, or TEP. Subsequent addition of 50 µM
basolateral UTP had little effect on membrane voltage and produced
no significant resistance changes. DIDS blockade of the UTP
responses was equally effective in three other experiments (
TEP =
0.2 ± 0.1 mV,
RT =
1.7 ± 1.5).

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Fig. 4.
A: basal UTP (50 µM)-induced changes in
membrane voltage and resistance blocked by basal DIDS. B:
apical ATP (100 µM)-induced changes in membrane voltage and
resistance blocked by apical DIDS. C, top:
sequential UTP (50 and 25 µM) basal control responses.
Bottom: sequential ATP (100 µM) apical and basal control
responses.
|
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In many epithelia, DIDS has been shown to specifically block
Ca2+-activated Cl channels at 0.5 mM (47).
Figure 4B compares the electrical responses obtained by the
addition of 100 µM ATP to the apical (or basal) bath, first in the
absence (control) and then in the presence of apical DIDS. After the
addition of apical ATP, the tissue was returned to control Ringer for 8 min, and then 500 µM DIDS was added to the apical bath to block
Ca2+-activated Cl channels and P2Y receptors. Again, DIDS
itself produced no noticeable changes in voltage or resistance. In the
presence of apical DIDS, ATP was added to the basal bath and produced
electrical responses much smaller than control (by a factor of >10).
Subsequent addition of ATP to the apical bath was also without
appreciable effect, because both apical membrane Cl channels and P2
purinoceptors were blocked. The control experiment in Fig.
4C shows that, in the absence of DIDS, repeated application
of ATP or UTP produces typical changes in TEP and
RT (and VA,
VB, and
RA/RB; data not shown).
Figure 5A compares the
electrical responses obtained by the addition of 100 µM ATP to the
basal bath, first in the absence and then in the presence of basal
suramin. In Fig. 5A, basal addition of ATP produced typical
control responses (Table 1). After the ATP control response, 100 µM
suramin was added to the basal bath. In its presence, ATP (100 µM)
had no effect on membrane voltage or resistance. This result was also
obtained in three other tissues. Figure 5B (same tissue)
shows that the subsequent addition of suramin to the apical bath
completely blocked the apical ATP responses (n = 4).

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Fig. 5.
A: basal ATP-induced changes in membrane voltage and
resistance are blocked by basal suramin. B: apical
ATP-induced changes in membrane voltage and resistance are blocked by
apical suramin. Continuation of recording in A.
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ATP- and/or UTP-regulated
[Ca2+]in.
The electrical effects of ATP/UTP and the abolition of those effects by
suramin or DIDS suggest the presence of metabotropic (G
protein-coupled) purinoceptors at both the apical and basolateral membranes of the 31EG4 mammary cells. Activation of metabotropic receptors is normally followed by an inositol trisphosphate
(IP3)-mediated increase in
[Ca2+]in (32). Figure
6A, top, shows the
change in [Ca2+]in after multiple additions
of 25 µM ATP to the apical bath, whereas Fig. 6B shows the
[Ca2+]in changes after multiple additions of
100 µM ATP to the basal bath. In six experiments, we found no
significant difference between ATP- and UTP-induced rises in
[Ca2+]in (15-25 µM). However, apical
addition of agonist (ATP or UTP) produced a significantly larger
increase in [Ca2+]in than basal addition (see
DISCUSSION). Apical ATP/UTP
(15-25 µM) increased [Ca2+]in by
427 ± 206 nM from an initial value of 29 ± 12 nM (mean ± SD, n = 6), whereas basal addition (15-25 µM)
increased [Ca2+]in by 158 ± 61 nM from
an initial value of 45 ± 12 nM (mean ± SD,
n = 3). Basal addition of 100 µM ATP increased
[Ca2+]in by 268 ± 96 nM from a baseline
of 35 ± 7 nM (n = 2).

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Fig. 6.
A: apical ATP-induced elevation of
intracellular Ca2+ concentration
([Ca2+]in) of 31EG4 mammary monolayer (25 µM ATP). B: basal ATP-induced elevation of
[Ca2+]in (100 µM).
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Figure 7A shows that basal
H2DIDS, a nonfluorescent DIDS analog, blocked the
UTP-induced increase in Ca2+, presumably by blocking the P2
receptor (41). The control response shows that 25 µM
UTP, added to the basal bath, caused a 6.5-fold increase in free
[Ca2+]in, from 25 to 130 nM. After the
addition of H2DIDS (500 µM) to the basal bath, basal UTP
had no effect on cell Ca2+. In other experiments, suramin
produced similar results (data not shown; n = 3).
Figure 7B compares the ATP-induced rise in cell
Ca2+ in the absence and presence of apical
H2DIDS (100 µM). Addition of 25 µM ATP to the apical
bath caused a 600 nM increase in cell Ca2+. In the presence
of H2DIDS, the ATP-induced rise in
[Ca2+]in was practically abolished.

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Fig. 7.
A: effects of basal DIDS (500 µM) on
UTP-induced increase in [Ca2+]in (25 µM
UTP). B: effects of apical DIDS (100 µM) on ATP-induced
increases in [Ca2+]in (25 µM ATP).
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If the ATP- and/or UTP-induced changes in cell Ca2+ are the
result of IP3-induced release from the ER stores,
then the purinoceptor-linked increase in
[Ca2+]in should be inhibited by depletion of
these stores. Cyclopiazonic acid (CPA), a specific inhibitor of
endoplasmic reticulum (ER) Ca2+-ATPase, was added to the
apical bath to block Ca2+ uptake without affecting
Ca2+ leakage out of the stores (9, 33). Figure
8A shows that apical ATP (25 µM) almost doubled the fura 2 ratio (from 2 to 3.8). After ATP was
washed out, 5 µM CPA was added to the apical bath and caused a
transient increase in cell Ca2+ followed by a decrease to a
sustained, elevated plateau. In the presence of CPA, the ATP-induced
rise in cell Ca2+ was abolished. The electrical responses
(Fig. 8B) followed the same time course as the
Ca2+ changes; ATP addition increased the TEP (1 mV) and
concomitantly decreased RT (~25
· cm2). These electrical
responses were also abolished in the presence of CPA.

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Fig. 8.
Blockade of ATP-induced changes in
[Ca2+]in (given as fura 2 ratio,
F340/380), TEP, and RT by treatment
with cyclopiazonic acid (CPA; 5 µM) (25 µM ATP). A:
[Ca2+]in; B: TEP,
RT.
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Figure 9 shows that the addition of 25 µM ATP to the apical bath increased
[Ca2+]in from 30 to 350 nM. The subsequent
addition to the basal bath produced a much smaller response from 27 to
50 nM, as expected for a basal vs. apical and second vs. first pulse.
The addition of 2.5 µM CPA caused a small, slow increase in
steady-state [Ca2+]in compared with 5 µM
CPA (Fig. 8). In the presence of CPA, apical ATP only increased
[Ca2+]in from ~50 to 75 nM, whereas basal
ATP had no effect on [Ca2+]in. These
experiments strongly suggest that the UTP- and/or ATP-induced increases
in [Ca2+]in originate from the ER stores and
mediate the membrane voltage and resistance changes (TEP and
RT). If the ATP- and/or UTP-induced increases in
cell Ca2+ originate from the ER stores, then these
increases should also be blocked by BAPTA-AM, a cell-permeable
Ca2+ chelator (52), and xestospongin C, an
IP3 receptor blocker (21). Figure 10,
A and B, shows
that BAPTA-AM (n = 3 tissues) and xestospongin C
(n = 1) almost completely block the apical ATP-induced
rise in [Ca2+]in.

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Fig. 10.
A: block of apical ATP-induced changes in
[Ca2+]in by treatment with BAPTA-AM (50 µM)
and 25 µM ATP. B: blockade of apical ATP-induced changes
in [Ca2+]in by treatment with xestospongin C
(10 µM) (25 µM ATP).
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Purinoceptor receptor subtype.
ATP hydrolysis products such as adenosine may have produced the
ATP-induced changes observed in 31EG4 cells. However, the addition of
50 or 100 µM adenosine to either the apical or basal bath produced no
change in membrane voltage or resistance (n = 8; data
not shown). By measuring agonist-induced changes in TEP, we examined
the potency of several different concentrations of ATP or UTP at each
membrane (Fig. 11). We also tested the
efficacy of several other P2Y purinoceptor agonists (ADP, UDP,
2-MeSATP, and
,
-MeATP) on each side of the epithelium. The
selected agonists had the following rank in potency at all
concentrations: ATP = UTP; ATP > > > > > 2-MeSATP; and
ADP, UDP, or
,
-MeATP were without effect, as expected for the
P2Y2 receptor subtype. We fit our
TEP/concentration data using first-order kinetics to estimate EC50, but full saturation was not achieved at the highest
concentration tested. The approximate EC50 for ATP and UTP
is ~24 ± 4 µM (± SE) for the apical membrane and ~30 ± 7 µM for the basolateral membrane.

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Fig. 11.
Dose-response curves; effects of ATP, UTP, ADP, UDP,
, -methylene-ATP ( , -MeATP), and 2-methylthio-ATP
(2-MeSATP) on TEP. A: apically applied agonists.
B: basolaterally applied agonists. The rank order of
affinity to the putative purinoceptor receptor (apical or basolateral)
is as follows: ATP = UTP, > > > > 2-MeSATP and not ADP, UDP,
or , -MeATP.
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The data summarized in Fig. 11 show that the addition of ADP to the
apical or basal baths produced practically no TEP or
RT (data not shown) responses, suggesting that
P2Y1 purinoceptors do not significantly contribute to the
ATP/UTP responses. This notion was tested further using ADP
S, a
P2Y1 purinoceptor agonist (43). Figure
12A shows that, compared
with the same concentration of ATP (25 µM), apical ADP
S produces a
16-fold smaller increase in cell Ca2+ (n = 3). In addition, apical ADP
S produced no change in
JV. Figure 12B shows that basal
application of ADP
S produces no change in cell Ca2+
(n = 2). In addition, the rise in
[Ca2+]in induced by 25 µM apical ATP (Fig.
6A) was completely unaffected by the presence of
P2Y1 blockers [MRS2179
10 µM; A3P5P
100 µM (43)] (not shown). The ATP- and/or UTP-induced changes in
cell physiology could also have been induced by activation of P2X
receptors also located at the apical or basolateral membranes. Figure
13 shows that 25 µM apical BzBz-ATP,
a P2X agonist (43), caused a small increase in cell
Ca2+ relative to the increase caused by an equal
concentration of ATP (n = 2). At higher concentrations
(50 µM; n = 2), the magnitude of the BzBz-ATP-induced
increases in [Ca2+]in were comparable to
those of ATP but produced no change in JV. On
the basal side of the cell, 100 µM BzBz-ATP elicited no change in
cell Ca2+ (data not shown).

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Fig. 12.
A: effect of apical ADP S (25 µM)
compared with ATP (25 µM) on [Ca2+]in.
B: basal ATP-induced elevation of
[Ca2+]in (100 µM) compared with ADP S
(100 µM).
|
|

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Fig. 13.
Apical benzoylbenzoyl-ATP (BzBz-ATP)-induced elevation
of [Ca2+]in of 31EG4 mammary monolayer (25 µM ATP, 25 µM BzBz-ATP).
|
|
Agonist-induced changes on fluid transport.
JV, TEP, and RT were
first measured under control conditions and then after the addition of
ATP/UTP to the apical or basal baths. Figure
14 shows that apical or basal ATP
induced fluid secretion, increased TEP, and decreased
RT. Figure 15
summarizes the results from 10 cultures that exhibited baseline
secretion or absorption that ranged from
5.0 to 2.0 µl · cm
2 · h
1.
In 10 cultures, the apical or basal ATP-induced increase in secretion
ranged from
2.0 to
11.0
µl · cm
2 · h
1;
for apical addition, the mean increase in secretion was 6.2 ± 2.4 µl · cm
2 · h
1
(mean ± SD; n = 6; P < 0.001),
and for basal addition the mean increase was 5.6 ± 3.1 µl · cm
2 · h
1
(n = 4, P < 0.02).

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Fig. 14.
A: apical ATP-induced alteration in steady-state fluid
absorption. Top: rate of transepithelial fluid flow
(JV) in
µl · cm 2 · h 1;
positive values of JV indicate net fluid
absorption and negative values fluid secretion. Arrows indicate that
the probes have been moved away from the fluid surface during solution
composition change. In these time periods, JV
was arbitrarily set to 0. Bottom: continuous trace of TEP
and RT. B: same as A
except that ATP was added to the basal bath (50 µM ATP).
|
|

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Fig. 15.
Summary of fluid transport experiments with apical
(n = 6) or basal (n = 4) addition of
ATP (50 µM). The ATP-induced increase in fluid secretion ranged from
2.0 to 10 µl · cm 2 · h 1.
|
|
Figure 16 shows that the ATP-induced
changes in TEP, RT, and
JV were reduced by 50-75% in the presence
of apical DIDS (500 µM). In two other monolayers, inhibition in all
three parameters was even greater (data not shown). These experiments
show that activation of apical or basolateral P2 purinoceptors
activates fluid secretion across 31EG4 monolayers and that the apical
membrane mechanisms(s) is DIDS sensitive.

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Fig. 16.
Apical UTP-induced increase in fluid secretion partially
blocked by apical DIDS (50 µM UTP). Arrows (and horizontal dashed
line) indicate that the probes have been moved away from the fluid
surface during solution composition changes. In these time periods,
JV was arbitrarily set to 0.
|
|
 |
DISCUSSION |
Purinoceptor-mediated ion and fluid transport in 31EG4 epithelial
cells.
Addition of micromolar amounts of ATP/UTP to either the apical or basal
baths of mammary epithelia caused an increase in free [Ca2+]in followed by large voltage and
resistance changes at the apical membrane and fluid secretion across
the epithelium. These agonist-induced changes are best explained in
terms of purinoceptors located on the apical or basolateral membranes.
Metabotropic purinoceptors increase [Ca2+]in
by phospholipase C
activation and IP3 formation
(43, 57). In the present study, prerelease of ER
Ca2+ stores using CPA, or treatment with BAPTA or
xestospongin C greatly reduced the ATP- and/or UTP-evoked
Ca2+ and electrical responses (Figs. 8-10), providing
a link between the activation of plasma membrane purinoceptors, the
ER-mediated increase in [Ca2+]in, and plasma
membrane voltage and resistance changes. The intracellular data (Table
1 and Figs. 2 and 3) strongly suggest that ATP/UTP increases apical
membrane conductance and activates DIDS-inhibitable, Ca2+-activated Cl channels in the apical membrane. Basal
DIDS (Fig. 4A) or suramin blocked the electrical responses
produced by basal UTP, indicating that changes in apical membrane
voltage and resistance can be mediated by activation of P2
purinoceptors on the basolateral membrane.
Figure 1 shows that 31EG4 mammary epithelial cells contain the message
for P2Y2 receptors. These receptors are equally sensitive to UTP and ATP but not sensitive to 2-MeSATP, ADP, UDP, and
,
-MeATP and are suramin and DIDS inhibitable (4, 24, 32,
43). The ATP/UTP-sensitive receptors that we have examined fit
all of these characteristics but not those of other P2 purinoceptors, as confirmed in Figs. 2-5, 7, and 11. The purinoceptors have an
approximate EC50 of
24 µM for both UTP and ATP at the
apical membrane and
30 µM at the basolateral membrane. Because the
apical and basolateral membrane electrophysiological responses to ATP,
UTP, 2-MeSATP, ADP, UDP, and
,
-MeATP are practically identical,
it appears that the receptor subtype is the same on both membranes. The
basal bath agonist-induced Ca2+ responses are smaller than
those produced by apical addition of secretagogue, probably because of
the relatively slow perfusion rate into the basal bath of the
fluorescence chamber (MATERIALS AND
METHODS).
Previously, we showed that the 31EG4 mammary epithelial cells express
CFTR and ENaC in the apical membrane and that both channels help
determine apical membrane resting potential and net fluid transport
across the monolayer (3). CFTR regulation by protein kinase C has been reported in various cell lines (10, 28, 56) and may play a role in the movement of Cl in mammary
epithelia. Addition of ATP to the basal bath did not evoke any
electrical changes when preceded by the addition of DIDS to the apical
bath to block the Ca2+-activated Cl channel (and apical
purinoceptors; Fig. 4B). This result suggests that
activation of basolateral metabotropic purinoceptors does not cause
observable changes in apical membrane CFTR conductance.
The localization of these transport proteins and the separate pathways
that mediate fluid absorption and secretion are shown in the model in
Fig. 17. We have preliminary evidence
for the presence of Ba2+-sensitive apical membrane K
channels, as shown in other epithelia (1). Thus the apical
membrane resting potential is determined by a combination of Cl, Na,
and probably K channels. In addition, there is evidence for Na/H
exchangers and Na-K-2Cl cotransporters on the basolateral membrane of
mammary epithelia (48, 49). On the basis of present data,
fluid absorption is most likely driven by active NaCl absorption (Na
through the cell, Cl through the paracellular pathway driven by the
TEP), and fluid secretion is driven by active (K + Na) Cl
transport (KCl through the cell and Na through the paracellular pathway
driven by the TEP). Net fluid transport is determined by the balance
between these two pathways. For example, fluid secretion can be induced
either by activating apical CFTR in the secretory pathway or by
inhibiting apical ENaC in the absorption pathway (3).
Fluid secretion is also induced (Fig. 14) by activating apical membrane
Ca2+-dependent Cl channels via activation of
P2Y2-purinoceptors at either the apical or basolateral
membranes.

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Fig. 17.
Model of ion and fluid transport in 31EG4 mammary
epithelia. Shown are apical and basolateral membrane mechanisms and
second messengers involved in the purinoceptor-mediated alterations in
cell Ca2+, membrane potential, and resistance. Also
included are transport proteins identified in previous studies (see
text). ENaC, epithelial Na+ channel; CFTR, cystic fibrosis
transmembrane conductance regulator.
|
|
Mammary gland physiology and function.
The present data provide the first demonstration of functional
purinoceptors on both membranes of the mammary epithelium. The presence
of functional P2Y2 receptors in the apical and basolateral membranes of 31EG4 cells is consistent with previous studies showing the presence of purinoceptors in mammary cell lines and primary cultures (15, 17, 19, 20, 40). The activation of these receptors likely occurs in vivo. ATP has been found in bovine milk
(14, 44), and ATP, UTP, and UDP are released from human mammary epithelia upon mechanical stimulation. In vivo, this mechanical stimulation is provided by myoepithelial cell contraction probably induced by ATP, perhaps released from acinar/duct cells, and oxytocin, released from the posterior pituitary (40). Activation of
fluid secretion across the 31EG4 monolayer suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition.
In addition, P2 purinoceptors may be involved in abnormal mammary cell
growth (11). Several studies have shown the existence of
metabotropic P2 purinoceptors on tumor cell lines (11, 13, 26). It has also been observed that, at physiological
concentrations, extracellular ATP can cause mitogenic activity in a
variety of cell types (27) and can act as a comitogen in
concert with other growth factors to enhance cellular proliferation in
cell culture models (54). In MCF-7 breast cancer cells,
ATP- and UTP-stimulated P2 purinoceptors were found to increase
[Ca2+]in and induce cell proliferation
(possibly by a K current-dependent mechanism; see Refs. 11
and 34). Because breast cyst fluid often consists of several growth
factors, it is possible that the abnormal accumulation of ATP in cyst
fluid could cause cell proliferation over time.
ATP- and/or UTP-induced fluid secretion may also play a role in
fibrocystic disease of the breast, a frequent mammary pathology in premenopausal women that is characterized by the abnormal
accumulation of breast cystic fluid (5, 36). The ATP-
and/or UTP-induced increases in fluid secretion (2.0-10
µl · cm
2 · h
1)
reported here would be equivalent to the addition of ~0.2-1.0 ml
fluid/day for a large cyst (51). These cysts are
characterized by two different electrolyte disturbances. Type I cysts
(lined by epithelia of apocrine morphology) are characterized by
relatively low concentrations of Na (~41 meq/l) and Cl (~15 meq/l)
levels and high concentrations of K (~101 meq/l). In contrast, type
II or transudative cysts (lined with flattened epithelium) contain moderately high Na (~140 meq/l) and Cl levels (~90 meq/l) and relatively low K levels (~9 meq/l; see Refs. 12 and 36).
Type I cysts could result from the activation of the Na absorption
pathway, which consists of apical membrane ENaC and basolateral membrane Na/K pumps. Upregulation of Na-K-ATPase activity could drive K
secretion into the lumen, perhaps mediated by an ATP-induced increase
in Ca2+-activated apical membrane K conductance. Organic
anion or bicarbonate transport may provide the accompanying anions
needed to mediate fluid secretion. In type II cysts, abnormal levels of
extracellular ATP could stimulate Ca2+-activated Cl
channels and help drive fluid into the luminal space. The counterion
for this fluid secretion could be provided by
Ca2+-activated apical membrane K channels or cation
movement through the paracellular path, driven by the ATP-induced
increase in TEP (Fig. 2). Therefore, blockade of apical membrane K
channels may help prevent type I cysts, whereas purinoceptor blockade
may be therapeutically advantageous against type II cyst formation.
 |
FOOTNOTES |
*
S. Blaug and J. Rymer contributed equally to this work.
Address for reprint requests and other correspondence: S. S. Miller, National Eye Institute, National Institutes of Health, Bldg.
31,6A20, 31 Center Dr., Bethesda, MD 20892-2510 (E-mail: millers{at}nei.nih.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.
First published November 27, 2002;10.1152/ajpcell.00238.2002
Received 22 May 2002; accepted in final form 22 November 2002.
 |
REFERENCES |
1.
Bialek, S,
and
Miller SS.
K+ and Cl
transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition.
J Physiol
475:
401-417,
1994[Abstract].
2.
Bialek, S,
Quong J,
Yu K,
and
Miller SS.
Nonsteroidal anti-inflammatory drugs alter chloride and fluid transport in bovine retinal pigment epithelium.
Am J Physiol Cell Physiol
270:
C1175-C1189,
1996[Abstract/Free Full Text].
3.
Blaug, S,
Hybiske K,
Cohn J,
Firestone GL,
Machen TE,
and
Miller SS.
ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia.
Am J Physiol Cell Physiol
281:
C633-C648,
2001[Abstract/Free Full Text].
4.
Boarder, MR,
and
Hourani SM.
The regulation of vascular function by P2 receptors: multiple sites and multiple receptors.
Trends Pharmacol Sci
19:
99-107,
1998[ISI][Medline].
5.
Bodian, CA.
Benign breast diseases, carcinoma in situ, and breast cancer risk.
Epidemiol Rev
15:
177-187,
1993[ISI][Medline].
6.
Bouyer, P,
Paulais M,
Cougnon M,
Hulin P,
Anagnostopoulos T,
and
Planelles G.
Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule.
J Physiol
510:
535-548,
1998[Abstract/Free Full Text].
7.
Brake, AJ,
and
Julius D.
Signaling by extracellular nucleotides.
Annu Rev Cell Dev Biol
12:
519-541,
1996[ISI][Medline].
8.
Christoffersen, BC,
Hug MJ,
and
Novak I.
Different purinergic receptors lead to intracellular calcium increases in pancreatic ducts.
Pflügers Arch
436:
33-39,
1998[ISI][Medline].
9.
Darby, PJ,
Kwan CY,
and
Daniel EE.
Use of calcium pump inhibitors in the study of calcium regulation in smooth muscle.
Biol Signals
2:
293-304,
1993[Medline].
10.
Dechecchi, MC,
Rolfini R,
Tamanini A,
Gamberi C,
Berton G,
and
Cabrini G.
Effect of modulation of protein kinase C on the cAMP-dependent chloride conductance in T84 cells.
FEBS Lett
311:
25-28,
1992[ISI][Medline].
11.
Dixon, JM,
McDonald C,
Elton RA,
and
Miller WR.
Breast cancer risk with cyst type in cystic disease of the breast. Larger study found no association between cyst type and breast cancer.
Br Med J
315:
545-546,
1997[Free Full Text].
12.
Dixon, JM,
McDonald C,
Elton RA,
and
Miller WR.
Risk of breast cancer in women with palpable breast cysts: a prospective study. Edinburgh Breast Group.
Lancet
353:
1742-1745,
1999[ISI][Medline].
13.
Dubyak, GR.
Signal transduction by P2-purinergic receptors for extracellular ATP.
Am J Respir Cell Mol Biol
4:
295-300,
1991[ISI][Medline].
14.
Emanuelson, U,
Olsson T,
Mattila T,
Astrom G,
and
Holmberg O.
Effects of parity and stage of lactation on adenosine triphosphate, somatic cell count and antitrypsin content in cows' milk.
J Dairy Res
55:
49-55,
1988[ISI][Medline].
15.
Enomoto, K,
Furuya K,
Moore RC,
Yamagishi S,
Oka T,
and
Maeno T.
Expression cloning and signal transduction pathway of P2U receptor in mammary tumor cells.
Biol Signals
5:
9-21,
1996[ISI][Medline].
16.
Enomoto, K,
Furuya K,
Yamagishi S,
Oka T,
and
Maeno T.
The increase in the intracellular Ca2+ concentration induced by mechanical stimulation is propagated via release of pyrophosphorylated nucleotides in mammary epithelial cells.
Pflügers Arch
427:
533-542,
1994[ISI][Medline].
17.
Flezar, M,
and
Heisler S.
P2-purinergic receptors in human breast tumor cells coupling of intracellular calcium signaling to anion secretion.
J Pharmacol Exp Ther
265:
1499-1510,
1993[Abstract].
18.
Fulford, GR,
and
Blake JR.
Mucociliary transport in the lung.
J Theor Biol
121:
381-402,
1986[ISI][Medline].
19.
Furuya, K,
Enomoto K,
and
Yamagishi S.
Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells.
Pflügers Arch
422:
295-304,
1993[ISI][Medline].
20.
Furuya, K,
Enomoto K,
Nakano H,
and
Yamagishi S.
Purinergic and mechanical interactions between myo- and secretory epithelial cells in mammary gland.
Jpn J Physiol Suppl
1:
S62,
1997.
21.
Gafni, J,
Munsch JA,
Lam TH,
Catlin MC,
Costa LG,
Molinski TF,
and
Pessah IN.
Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor.
Neuron
19:
723-733,
1997[ISI][Medline].
22.
Gobran, LI,
Xu ZX,
Lu Z,
and
Rooney SA.
P2u purinoceptor stimulation of surfactant secretion coupled to phosphatidylcholine hydrolysis in type II cells.
Am J Physiol Lung Cell Mol Physiol
267:
L625-L633,
1994[Abstract/Free Full Text].
23.
Grobben, B,
Claes P,
Van Kolen K,
Roymans D,
Fransen P,
Sys SU,
and
Slegers H.
Agonists of the P2Y(AC)-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma.
J Neurochem
78:
1325-1338,
2001[ISI][Medline].
24.
Harden, TK,
and
Lazarowski ER.
Release of ATP and UTP from astrocytoma cells.
Prog Brain Res
120:
135-143,
1999[ISI][Medline].
25.
Hughes, BA,
Miller SS,
and
Machen TE.
Effects of cyclic AMP on fluid absorption and ion transport across frog retinal pigment epithelium. Measurements in the open-circuit state.
J Gen Physiol
83:
875-899,
1984[Abstract].
26.
Insel, PA,
Ostrom RS,
Zambon AC,
Hughes RJ,
Balboa MA,
Shehnaz D,
Gregorian C,
Torres B,
Firestein BL,
Xing M,
and
Post SR.
P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides.
Clin Exp Pharmacol Physiol
28:
351-354,
2001[ISI][Medline].
27.
Ishikawa, S,
Kawasumi M,
Kusaka I,
Komatsu N,
Iwao N,
and
Saito T.
Extracellular ATP promotes cellular growth of glomerular mesangial cells mediated via phospholipase C.
Biochem Biophys Res Commun
202:
234-240,
1994[ISI][Medline].
28.
Jia, Y,
Mathews CJ,
and
Hanrahan JW.
Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A.
J Biol Chem
272:
4978-4984,
1997[Abstract/Free Full Text].
29.
Jiang, C,
Finkbeiner WE,
Widdicombe JH,
McCray PB, Jr,
and
Miller SS.
Altered fluid transport across airway epithelium in cystic fibrosis.
Science
262:
424-427,
1993[ISI][Medline].
30.
Joseph, DP,
and
Miller SS.
Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium.
J Physiol
435:
439-463,
1991[Abstract].
31.
Kenyon, E,
Yu K,
La Cour M,
and
Miller SS.
Lactate transport mechanisms at apical and basolateral membranes of bovine retinal pigment epithelium.
Am J Physiol Cell Physiol
267:
C1561-C1573,
1994[Abstract/Free Full Text].
32.
King, BF,
Townsend-Nicholson A,
and
Burnstock G.
Metabotropic receptors for ATP and UTP: exploring the correspondence between native and recombinant nucleotide receptors.
Trends Pharmacol Sci
19:
506-514,
1998[ISI][Medline].
33.
Kirischuk, S,
Scherer J,
Kettenmann H,
and
Verkhratsky A.
Activation of P2-purinoreceptors triggered Ca2+ release from InsP3-sensitive internal stores in mammalian oligodendrocytes.
J Physiol
483:
41-57,
1995[Abstract].
34.
Klimatcheva, E,
and
Wonderlin WF.
An ATP-sensitive K(+) current that regulates progression through early G1 phase of the cell cycle in MCF-7 human breast cancer cells.
J Membr Biol
171:
35-46,
1999[ISI][Medline].
35.
Lin, H,
Kenyon E,
and
Miller SS.
Na-dependent pHi regulatory mechanisms in native human retinal pigment epithelium.
Invest Ophthalmol Vis Sci
33:
3528-3538,
1992[Abstract].
36.
Malatesta, M,
Mannello F,
Sebastiani M,
Cardinali A,
Marcheggiani F,
Reno F,
and
Gazzanelli G.
Ultrastructural characterization and biochemical profile of human gross cystic breast disease.
Breast Cancer Res Treat
48:
211-219,
1998[ISI][Medline].
37.
Mason, SJ,
Paradiso AM,
and
Boucher RC.
Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.
Br J Pharmacol
103:
1649-1656,
1991[Abstract].
38.
Miller, SS,
and
Steinberg RH.
Passive ionic properties of frog retinal pigment epithelium.
J Membr Biol
36:
337-372,
1977[ISI][Medline].
39.
Muraki, K,
Imaizumi Y,
and
Watanabe M.
Effects of UTP on membrane current and potential in rat aortic myocytes.
Eur J Pharmacol
360:
239-247,
1998[ISI][Medline].
40.
Nakano, H,
Furuya K,
Furuya S,
and
Yamagishi S.
Involvement of P2-purinergic receptors in intracellular Ca2+ responses and the contraction of mammary myoepithelial cells.
Pflügers Arch
435:
1-8,
1997[ISI][Medline].
41.
Peterson, W,
Meggyesy C,
Yu K,
and
Miller SS.
Extracellular ATP activates calcium signaling, ion and fluid transport in retinal pigment epithelium.
J Neurosci
17:
2324-2337,
1997[Abstract/Free Full Text].
42.
Quinn, RH,
Quong JN,
and
Miller SS.
Adrenergic receptor activated ion transport in human fetal retinal pigment epithelium.
Invest Ophthalmol Vis Sci
42:
255-264,
2001[Abstract/Free Full Text].
43.
Ralevic, V,
and
Burnstock G.
Receptors for purines and pyrimidines.
Pharmacol Rev
50:
413-492,
1998[Abstract/Free Full Text].
44.
Richardson, T,
McGann TC,
and
Kearney RD.
Levels and location of adenosine 5'-triphosphate in bovine milk.
J Dairy Res
47:
91-96,
1980[ISI][Medline].
45.
Sabater, JR,
Mao YM,
Shaffer C,
James MK,
O'Riordan TG,
and
Abraham WM.
Aerosolization of P2Y(2)-receptor agonists enhances mucociliary clearance in sheep.
J Appl Physiol
87:
2191-2196,
1999[Abstract/Free Full Text].
46.
Schlenker, T,
Romac JM,
Sharara AI,
Roman RM,
Kim SJ,
LaRusso N,
Liddle RA,
and
Fitz JG.
Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes.
Am J Physiol Gastrointest Liver Physiol
273:
G1108-G1117,
1997[Abstract/Free Full Text].
47.
Schultz, BD,
Singh AK,
Devor DC,
and
Bridges RJ.
Pharmacology of CFTR chloride channel activity.
Physiol Rev Suppl
79:
109-144,
1999.
48.
Shennan, DB,
and
Peaker M.
Transport of milk constituents by the mammary gland.
Physiol Rev
80:
925-951,
2000[Abstract/Free Full Text].
49.
Sjaastad, MD,
Zettl KS,
Parry G,
Firestone GL,
and
Machen TE.
Hormonal regulation of the polarized function and distribution of Na/H exchange and Na/HCO3 cotransport in cultured mammary epithelial cells.
J Cell Biol
122:
589-600,
1993[Abstract].
50.
Stutts, MJ,
Fitz JG,
Paradiso AM,
and
Boucher RC.
Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP.
Am J Physiol Cell Physiol
267:
C1442-C1451,
1994[Abstract/Free Full Text].
51.
Takei, H,
Iino Y,
Horiguchi J,
Maemura M,
Yokoe T,
Koibuchi Y,
Oyama T,
Ohwada S,
and
Morishita Y.
Natural history of fibroadenomas based on the correlation between size and patient age.
Japn J Clin Oncol
29:
8-10,
1999[Abstract/Free Full Text].
52.
Taylor, CW,
and
Broad LM.
Pharmacological analysis of intracellular Ca2+ signalling: problems and pitfalls.
Trends Pharmacol Sci
19:
370-375,
1998[ISI][Medline].
53.
Viana, F,
de Smedt H,
Droogmans G,
and
Nilius B.
Calcium signalling through nucleotide receptor P2Y2 in cultured human vascular endothelium.
Cell Calcium
24:
117-127,
1998[ISI][Medline].
54.
Wang, DJ,
Huang NN,
and
Heppel LA.
Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.
J Cell Physiol
153:
221-233,
1992[ISI][Medline].
55.
Watt, WC,
Lazarowski ER,
and
Boucher RC.
Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia.
J Biol Chem
273:
14053-14058,
1998[Abstract/Free Full Text].
56.
Winpenny, JP,
McAlroy HL,
Gray MA,
and
Argent BE.
Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells.
Am J Physiol Cell Physiol
268:
C823-C828,
1995[Abstract/Free Full Text].
57.
Yerxa, BR,
and
Johnson FL.
P2Y2 receptor agonists: structure, activity, and therapeutic utility.
Drugs
24:
759-769,
1999.
58.
Zettl, KS,
Sjaastad MD,
Riskin PM,
Parry G,
Machen TE,
and
Firestone GL.
Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro.
Proc Natl Acad Sci USA
89:
9069-9073,
1992[Abstract].
Am J Physiol Cell Physiol 284(4):C897-C909
0363-6143/03 $5.00
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