Functional ATP receptors in rat anterior pituitary
cells
Carlos
Villalobos,
Sara R.
Alonso-Torre,
Lucía
Núñez, and
Javier
García-Sancho
Instituto de Biología Genética Molecular, Universidad
de Valladolid y Consejo Superior de Investigaciones
Científicas, Departamento de Fisiología y
Bioquímica, Facultad de Medicina, 47005 Valladolid, Spain
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ABSTRACT |
The effects of
ATP and other nucleotides on the cytosolic
Ca2+ concentration
([Ca2+]i)
of single immunocytochemically typed anterior pituitary (AP) cells have
been studied. ATP increased
[Ca2+]i
in a large percentage (60-88%) of all five AP cell types:
lactotropes, somatotropes, corticotropes, gonadotropes, and
thyrotropes. Additivity experiments suggest the presence of at least
two different receptors, one accepting both ATP and UTP (U receptor),
producing Ca2+ release from the
intracellular stores, and the other preferring ATP (A receptor),
producing Ca2+ (and
Mn2+) entry. The characteristics
of the U and A receptors were consistent with those of
P2Y2 and
P2X2, respectively, and their
distribution in the different AP cell types was not homogeneous. The
presence of other ATP receptors such
P2Y1 or
P2X2/P2X3
heteropolymers in a small fraction of the cells cannot be excluded.
Thus functional ionophoric P2X receptors, which are typical of neural
tissue, are also present in the pituitary gland and could contribute to regulation of the gland's function.
P2X receptor; P2Y receptor; UTP; Ca2+ channels
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INTRODUCTION |
ATP PLAYS AN IMPORTANT ROLE as an extracellular
messenger for excitable cells (29). Nerve and muscle cells possess
ionotropic ATP receptors (P2X) whose activation results in
Ca2+ and
Na+ entry and depolarization of
the plasma membrane. Metabotropic ATP receptors (P2Y) are widespread in
different tissues and act through G protein-mediated activation of
phospholipase C, inositol trisphosphate production, and
Ca2+ release from the
intracellular Ca2+ stores. At
least seven members of each family, P2X and P2Y, have been cloned (13).
Regarding agonist preferences, the P2X receptors and the
P2Y1 subtype prefer ATP over UTP,
whereas both nucleotides are similarly able to activate the other P2Y
receptors. 2-Methylthio-ATP (MeSATP) is a good substrate for the P2X
and P2Y1 receptors but a poor
substrate for the other P2Y receptors (13).
Although P2X receptors are generally regarded as typical of excitable
tissues, a strikingly high P2X2
expression was found in the anterior pituitary (AP), and the functional
role of the receptors at this site is unknown (3, 18). ATP
has been shown to increase luteinizing hormone (LH) secretion by
pituitary cells in primary culture (8). In addition, ATP is coreleased
with pituitary hormones, leaving room for paracrine effects in
neighboring cells (7). ATP is able to induce an increase in cytosolic
free Ca2+ concentration
([Ca2+]i)
in rat lactotropes (5) and gonadotropes (8, 11). In both cases, the
effects were attributed to Ca2+
release from the intracellular
Ca2+ stores by stimulation of
P2Y2 receptors. A G
protein-coupled P2Y2 receptor that
may be responsible for this effect has been cloned recently from a rat
pituitary cDNA library (9). More recently, ATP has been reported to
also induce Ca2+ entry in
gonadotropes, probably through a P2X receptor (24). On the basis of the
above observations, it has been suggested that P2 receptors may play a
role in neuroendocrine regulation (10).
Here we combined Ca2+ imaging and
immunocytochemical identification to study the effects of ATP on the
five different AP cell types: lactotropes, somatotropes, corticotropes,
gonadotropes, and thyrotropes. We find that, in addition to lactotropes
and gonadotropes, the other kinds of secretory cells are also
sensitive to ATP. Furthermore, we present evidence for involvement of
both metabotropic and ionotropic receptors in the response to ATP.
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MATERIALS AND METHODS |
AP cells were obtained from 8- to 10-wk-old male Wistar rats, allowed
to attach to 11-mm diameter polylysine-coated glass coverslips, and
cultured for 2-3 days as described previously (28). Measurements
of
[Ca2+]i
were performed in cells loaded with fura 2 (15) by incubation for
60-90 min at room temperature with 5 µM fura 2-acetoxymethyl ester (AM) in standard medium composed of (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-sodium, at pH 7.4. The coverslips were then mounted under the
microscope (Diaphot; Nikon, Tokyo, Japan) in a chamber thermostated at
36°C and epi-illuminated alternately at 340 and 380 nm. Light emitted above 520 nm was recorded by an extended ISIS-M camera (Photonic Science, Robertbridge, East Sussex, UK) and analyzed using an
Applied Imaging magical image processor (Newcastle, UK) with
32-megabyte video random-access memory. Eight video frames of each
wavelength were averaged by hardware that had an overall time
resolution of ~5 s for each pair of images at alternate wavelengths. A pixel-by-pixel ratio of consecutive frames obtained at 340- and
380-nm excitation was obtained, and
[Ca2+]i
was estimated from this ratio by comparison with fura 2 standards. Test solutions were applied by continuous
perfusion at 2-3 ml/min. This allowed >95% exchange of the
medium bathing the cells within 5-10 s. Further details of these
procedures have been provided previously (25, 28).
For identification of single cells according to the hormone they store,
the coverslips were fixed with 4% paraformaldehyde at the end of the
[Ca2+]i
measurements. Indirect immunofluorescence measurements using antibodies
raised against one of the pituitary hormones were then performed. The
field of interest was located by positioning a cross engraved in the
coverslip, as in the
[Ca2+]i
experiment, and the fluorescence image was captured with the image
processor. The image was digitalized, stored, and later moved and
rotated in the computer as required to match exactly the images
obtained in the
[Ca2+]i
experiment. This procedure has been described in detail elsewhere (20,
28).
In some experiments, multiple sequential primary immunocytochemistry
was performed using the abbreviated protocol that follows. Cells were
fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for
10 min, permeabilized with 0.3% Triton X-100 in the aforementioned
solution for 3 min, and washed with PBS for 5 min. Ten percent goat
serum in PBS was then added. After 5 min, the antibody against one of
the AP hormones labeled with Oregon green 488 was added, and the
incubation was continued for 15-30 min. After a washing, a
fluorescence image was captured (excitation, 490 nm; emission, >510
nm) with the image processor. This process was repeated for a second
(and even a third) fluorescent antibody against another AP hormone, and
the resulting image was captured at the same camera gain. Finally,
nuclei were stained with Hoechst 33258 (0.5 µg/ml, for 10 min), and
another fluorescence image was acquired (excitation, 340 nm; emission,
>420 nm). The first image was used as it was obtained for
identification of the cells stained with the first antibody. The image
corresponding to the cells stained with the second antibody was
obtained by subtracting the first image from the second image. The
image corresponding to the cells stained with the third antibody was
obtained by subtracting the second image from the third image. We
prepared fluorescent antibodies from antisera provided by the National
Institute of Diabetes and Digestive and Kidney Diseases using Oregon
green 488-isothiocyanate and following the labeling directions provided by the manufacturer. The antibody was then purified on a protein A
Sepharose column (17) and used as described above (final dilution, 1:20
to 1:50, determined empirically for every antibody). The image from the
fluorescence-stained nuclei facilitated definition of cellular
boundaries in cells that were physically close.
In several experiments, Mn2+ was
used as a Ca2+ surrogate for
Ca2+ channels. This allowed direct
study of plasma membrane channels without the interference of
Ca2+ released from the
intracellular Ca2+ stores (1, 14).
Mn2+ entry assays were performed
either in the whole population of cells or at the single cell level.
For measurements in the whole cell population, the coverslips were
introduced at a fixed angle (45°) into a quartz cuvette placed into
the sample compartment of a fluorescence spectrophotometer that allowed
rapid (30-300 Hz) alternation of up to six different excitation
wavelengths (Cairn Research, Newnham, Sittingbourne, Kent, UK).
Temperature was 30°C. Solutions were changed by simultaneous
perfusion into the bottom and aspiration at the top of the quartz
cuvette through tubing fitted outside of the optical pathway.
Fluorescence emitted above 510 nm was measured and integrated at 1-s
periods. Mn2+ entry was estimated
from the quenching of the fura 2 fluorescence excited at 360 nm, a
wavelength that is not sensitive to changes in
Ca2+ concentration (16). Changes
in
[Ca2+]i
can be monitored simultaneously from the ratio of the fluorescences excited at 340 and 380 nm. This procedure has been described in detail
elsewhere (26).
For Mn2+ entry measurements at the
single cell level, the experiments were performed in the imaging system
as described above for the
[Ca2+]i
measurements. Because finding an isosbestic wavelength for Ca2+ is difficult in this case,
quenching of fura 2 fluorescence by Mn2+ was estimated by the combined
decrease of fluorescence at the two main excitation wavelengths, 340 and 380 nm. For these purposes, a total fluorescence
(Ftotal) value was
computed by adding the fluorescence at 340 nm
(F340), multiplied by a constant
factor K, and the fluorescence at 380 nm (F380). Because
F340 increases with
[Ca2+] and
F380 decreases with
[Ca2+],
Ftotal becomes insensitive to
[Ca2+] when a proper
value is chosen for K (see Ref. 14 for
detailed discussion). Ftotal
images were obtained by adding, pixel by pixel, the consecutive frames
obtained at 340 nm (multiplied by K)
and at 380 nm (2).
Oregon green 488-isothiocyanate and fura 2-AM were obtained from
Molecular Probes (Eugene, OR). Antibodies against rat prolactin (PRL)
(rabbit, AFP425-10-91),
-thyroid-stimulating
hormone (rabbit, AFP1274789), growth hormone (GH) (monkey, AFP4115),
-follicle-stimulating hormone (guinea pig, AFP85GP9691BFSHB), and
anti-human adrenocorticotropic hormone (ACTH) (rabbit, AFP39013082)
were generous gifts from the National Hormone and Pituitary Program,
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institute of Child Health and Human Development, and the US
Department of Agriculture (Rockville, MD). Fluorescein-labeled
anti-rabbit, anti-guinea pig, or anti-monkey immunoglobulins were
obtained from Sigma (London, UK). Nucleotides and their derivatives
were from RBI Biochemicals (Natick, MA). Thapsigargin was from Alomon (Jerusalem, Israel). Other chemicals were either from Sigma or from
Merck (Darmstandt, Germany).
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RESULTS |
ATP increases
[Ca2+]i
in all five AP cell types.
Perfusion with 10-100 µM concentrations of ATP
elicited an increase in
[Ca2+]i
in most of the AP cells present in the microscope field (62 ± 4%;
mean ± SE of 39 experiments,
n = 3,469 cells). Figure
1A illustrates the increase in
[Ca2+]i
elicited by 1 µM ATP in a representative single cell. On removal of
the nucleotide,
[Ca2+]i
returned to the resting level. A second stimulation performed after a
5-min washing period produced a similar
[Ca2+]i
increase. Figure 1B compares the
average responses to different concentrations of ATP. The size of the
[Ca2+]i
peak increased with ATP concentration, the half-maximal effect being
reached at ~1 µM ATP.

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Fig. 1.
Effects of ATP and other nucleotides on cytosolic
Ca2+ concentration
([Ca2+]i)
of anterior pituitary (AP) cells. A:
single cell stimulated twice with 1 µM ATP.
B: average of traces of 120 single
cells stimulated with different concentrations (0.1-100 µM) of
ATP. C: comparison of effects of ATP
and UTP, both at 50 µM; average of traces of 47 single cells.
D: comparison of effects of
, -methylene ATP ( , -MeATP), 2-methylthio-ATP (MeSATP),
adenosine
5'-O-(3-thiotriphosphate)
(ATP S), and ATP, all at 50 µM; average of traces of 148 single
cells. E: single cell from same
experiment. F-H: correlation
between effects of ATP and ATP S
(F), UTP
(G), or MeSATP
(H), all tested at 50 µM. Effects
were quantified as increase of
[Ca2+]i
(in nM) at peak after application of nucleotide. Lines fitted by least
squares procedure. Values for correlation coefficient
(r) were 0.92 (F, n = 102 cells), 0.58 (G,
n = 100 cells), and 0.39 (H, n = 102 cells). Each experiment is representative of 2-5 similar
ones.
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The AP contains five main cell types, each one secreting a different
pituitary hormone. To investigate the presence of ATP-sensitive cells
within each cell type, immunocytochemical identification of the
individual cell type was performed after stimulation with 50 µM ATP
(in the same microscope field used for the
[Ca2+]i
measurements; see MATERIALS AND
METHODS for details). We have studied 1,845 AP cells in
22 independent experiments in which either lactotropes, somatotropes,
corticotropes, gonadotropes, or thyrotropes were identified with the
use of antibodies raised against their respective AP hormones. Results
are summarized in Table 1. The
ATP-sensitive cells were not restricted to a given cell type but
distributed rather homogeneously within all five cell types, the
percentage of sensitive cells ranging between 60 and 88%.
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Table 1.
Response to ATP in the five AP cell types defined by immunocytochemical
identification of the hormone they store
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Effects of nucleotides other than ATP.
Other nucleotides and nucleotide derivatives were also able to produce
a
[Ca2+]i
increase in AP cells. Figure 1C
compares the effects of ATP and UTP averaged from the traces of 47 cells, and Fig. 1D compares the
effects of ATP,
,
-methylene-ATP (
,
-MeATP), MeSATP, and adenosine
5'-O-(3-thiotriphosphate)
(ATP
S), averaged from the traces of 148 cells. Other
nucleotides tested were ADP and 2-chloro-ATP (ClATP) (results not
shown). All the nucleotides and derivatives produced similar effects
except
,
-MeATP, which was scarcely active. The 50% effective
concentration values determined in experiments similar to those of Fig.
1B were 2 µM for UTP and 2.6 µM
for MeSATP.
The poor response to
,
-MeATP in the average traces was because of
the fact that only a few cells (4-10% in different experiments) responded to this nucleotide, but large responses were found in these
sensitive cells (Fig. 1E). Further
complexities in the action of other nucleotides were also disclosed by
the single cell analysis. The correlation between the effects of ATP
and ATP
S in each single cell was always very good (Fig.
1F), suggesting that both
nucleotides are able to activate the same receptors. However, the
correlation between the effects of ATP and either UTP (Fig.
1G) or MeSATP (Fig.
1H) was much worse. A possible
explanation for this outcome would be the presence of more than one P2
receptor with different affinities for the different nucleotides and
derivatives tested.
In the case of the ATP-UTP correlation (Fig.
1G), two dominant subpopulations may
be defined: 1) cells that respond
similarly to both ATP and UTP, which are positioned near the diagonal
of the plot, and 2) cells that
respond preferentially to ATP, which are positioned closer to the
abscissa. This behavior could be rationalized in terms of one receptor
(U receptor) that is equally well activated by both UTP and ATP and
another receptor (A receptor) that is activated only by ATP. Cells in
which U receptors dominate would correspond to the
subpopulation 1 described above,
whereas cells possessing preferentially A receptors would make up
subpopulation 2.
To test this working hypothesis, the additivity between the effects of
UTP and ATP was studied in experiments in which the cells were
stimulated first with UTP for 2 min and then with UTP + ATP for an
additional 1 min. After a 5-min washing with control solution, the
nucleotides were applied again in the reverse order. Results of a
representative experiment are shown in Fig.
2. Figure 2A shows the average behavior of all
the cells present in the microscope field. It is clear that ATP added
on top of UTP was still able to produce an increase in
[Ca2+], whereas the
reverse did not apply. When analyzed at the single cell level, three
main subpopulations could be identified:
1) cells that responded only to ATP
(Fig. 2B),
2) cells that responded to both ATP
and UTP as the first stimulus but to none of the nucleotides as the
second stimulus [i.e., no response to the nucleotide that is
added on top of the first nucleotide (Fig.
2C)], and
3) cells that responded to both ATP
and UTP as the first stimulus but only to ATP as the second stimulus
(Fig. 2D). Figure
2E shows the relative sizes of each of
these subpopulations, averaged in four similar experiments. The
subpopulation 3 (A + U) was always
dominant. A subpopulation of cells responding only to UTP could not be
identified. This outcome is consistent with the working hypothesis that
postulates the existence of the two types of receptors (U and A) given
that subpopulation 1 had only A
receptors, subpopulation 2 had only U
receptors, and subpopulation 3 had
both kinds.

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Fig. 2.
Additivity between effects of UTP and ATP. Concentrations were 50 µM
for both nucleotides. Cells were first stimulated with UTP for 2 min,
and then perfusion was switched to a solution containing both UTP and
ATP for an additional 1 min; after a 5-min washing with control
solution, cells were first stimulated with ATP and then with solution
containing both ATP and UTP. A:
average of traces of 47 single cells.
B-D: traces corresponding to 3 different single cells. E: relative
sizes, expressed as a percentage of all cells responding to
nucleotides, of the following subpopulations: A, responding only to
ATP; U, responding only to nucleotide added first (either ATP or UTP);
A + U, responding to UTP and also to ATP added after UTP; others, cells
that could not be included within above categories. Results from 4 independent experiments (123 single cells responding to nucleotides).
See text for interpretation of results in terms of different types (A
or U) of ATP receptors.
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The interactions of UTP and MeSATP were also studied in experiments of
the same design as those of Fig. 2, and the results are illustrated in
Fig. 3. In the average trace (Fig.
3A), both MeSATP and UTP were able
to increase
[Ca2+]i
after stimulation with the other nucleotide. Cells responding either to
both nucleotides (Fig. 3B, 63% of the
responding cells) or selectively to one of them (Fig. 3,
C and
D; 9 and 22% of the responding cells,
respectively) were identified. In all the cases, the responding cells
were also sensitive to ATP. The results could be rationalized within
our working hypothesis if MeSATP were able to activate A receptors but
not U receptors. The interactions of MeSATP and ATP (Fig.
4) were also consistent with this
interpretation. ATP was able to increase
[Ca2+]i
after treatment with MeSATP in some cells (Fig. 4,
B and
C; 21 and 63% of the responding
cells, respectively), and all the cells that responded to ATP after
stimulation with MeSATP were also sensitive to UTP. This suggests that
the additional response to ATP results from the presence of U
receptors. MeSATP had little effect when applied on top of ATP (Fig.
4A). Only a few cells responded to
MeSATP added on top of ATP (not shown). We refer to the possible
significance of this minor subpopulation in the DISCUSSION.

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Fig. 3.
Additivity between effects of UTP and MeSATP. Concentrations were 50 µM for all nucleotides. Cells were first stimulated with UTP for 2 min, and then perfusion was switched to a solution containing both UTP
and MeSATP for an additional 1 min; after a 5-min washing with control
solution, cells were first stimulated with MeSATP and then with
solution containing both MeSATP and UTP. At end of experiment, cells
were stimulated with ATP. A: average
of traces of 24 single cells.
B-D: traces corresponding to 3 different single cells.
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Fig. 4.
Additivity between effects of ATP and MeSATP. Concentrations were 50 µM for all nucleotides. Cells were first stimulated with MeSATP for 2 min, and then perfusion was switched to solution containing both MeSATP
and ATP for an additional 1 min; after a 5-min washing with control
solution, cells were first stimulated with ATP and then with solution
containing both ATP and MeSATP. At end of experiment, cells were
stimulated with UTP. A: average of
traces of 55 single cells. B and
C: traces corresponding to 2 different
single cells.
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Calcium entry and calcium release from the intracellular stores.
To investigate the relative contributions of
Ca2+ entry and
Ca2+ release from the
intracellular stores and the
[Ca2+]i
increase induced by ATP and other nucleotides, we compared the effects
obtained in Ca2+-free and
Ca2+-containing media. Figure
5A shows
that ATP and UTP elicited a similar
[Ca2+]i
increase in Ca2+-free medium (60 ± 6 and 73 ± 6 nM, respectively; mean ± SE of 10 independent experiments), suggesting that both nucleotides are able to
release Ca2+ from the
intracellular stores. However, the effect in
Ca2+-containing medium was larger
for ATP (see Fig. 5A for a typical experiment; 306 ± 26 nM compared with 125 ± 13 for UTP, mean ± SE of 10 experiments), indicating that the relative contribution of Ca2+ entry is larger for ATP.
The
[Ca2+]i
increase induced by MeSATP was also very much reduced by removal of
external Ca2+ (by 75 ± 2%, mean ± SE of 4 experiments, results not shown).

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Fig. 5.
Effects of Ca2+ removal
(A) and pretreatment with
thapsigargin (B) on
[Ca2+]i
increases induced by ATP and other nucleotides. Concentrations were 50 µM for all nucleotides. A: effects
of UTP and ATP in Ca2+-free medium
(Ca0, containing 500 µM EGTA added 30 s before the nucleotide) and in
regular, Ca2+-containing medium
(Ca1) are compared. Trace shown is average of 49 single cells.
Experiment is representative of 4 similar ones. Six additional
experiments were performed in the Cairn spectrophotometer.
B: cells were first stimulated with
several nucleotides, as shown, then treated with 500 nM thapsigargin
for 5 min and stimulated again. Experiment performed in the Cairn
spectrophotometer, representative of 2 similar ones. ClATP,
2-chloro-ATP.
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The relative contribution of the intracellular
Ca2+ stores was also assessed in
experiments in which the stores were emptied of
Ca2+ by treatment with the
endomembrane adenosinetriphosphatase inhibitor thapsigargin (23). It
has been shown previously that a 5-min treatment with this drug is
enough to achieve full emptying of the intracellular
Ca2+ stores (26). Fig.
5B illustrates representative results.
Thapsigargin treatment strongly inhibited the action of UTP (by 62%),
had a smaller effect on the action of ATP (32% inhibition), and had little effect on the actions of ClATP (24% inhibition) and MeSATP (3%
inhibition).
The effects of ATP and other nucleotides on
Ca2+ entry were assessed directly
by using Mn2+ as a surrogate of
Ca2+ for
Ca2+ channels. It has been shown
previously that Mn2+ is able to
permeate several types of Ca2+
channels, for example, L-type voltage-gated channels (26, 27) and
N-methyl-D-aspartate
receptor-operated channels (21, 28). Figure
6,
A-C, illustrates the effects of
UTP and ATP on Mn2+ entry in a
representative experiment performed with the imaging system. The ratio
of the fluorescences excited at 340 and 380 nm (an index of
[Ca2+]i;
see MATERIALS AND METHODS) was
measured simultaneously and is shown as dotted lines. The average trace
for all the cells (Fig. 6A) shows
that both nucleotides were able to produce an acceleration of
Mn2+ entry, although this effect
was much more marked for ATP. The dominant cell subpopulation was the
one illustrated in Fig. 6B, in which
both UTP and ATP produced an increase in
[Ca2+]i
but only ATP produced an acceleration of
Mn2+ entry. A smaller fraction of
cells responded only to ATP (Fig. 6C), and another fraction, still
smaller, responded to UTP with an acceleration of
Mn2+ entry (not shown). The
categories represented in Fig. 6, B
and C, made up 78% of the cell
population.

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Fig. 6.
Effects of ATP and other nucleotides on entry of
Mn2+. Concentrations were 50 µM
for all nucleotides. A-C:
experiment performed in imaging system. Ratio of fluorescence at 340 nm
to fluorescence at 380 nm
(F340/F380,
dotted lines) is index of changes in
[Ca2+]i,
whereas decrease of total fluorescence
(Ftotal, solid lines) reflects
quenching of fura 2 fluorescence by entering
Mn2+ (see
MATERIALS AND METHODS).
Concentration of Mn2+ (in
Ca2+-free medium) was 0.2 mM.
A: average of traces from 27 single
cells. B and
C: traces from 2 single cells. This
experiment is representative of 4 similar ones. Note that ratio values
may become inaccurate when Ftotal
goes to very low values, as at the end of traces; experiments performed
in the Cairn spectrophotometer. Readings of fluorescences excited at
340, 360, and 380 nm were taken simultaneously. Decrease of
F360 (insensitive to
Ca2+) reflects quenching of fura
2 fluorescence by entering Mn2+
(see MATERIALS AND METHODS). Cells
had been first treated with thapsigargin (500 nM) for 6 min (not
shown). Concentration of Mn2+ (in
Ca2+-free medium) was 0.2 mM.
Effects of ATP (D), MeSATP
(E), and UTP and ClATP
(F) are shown. Experiments are
representative of 2-4 similar ones.
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In many cell types, emptying of the intracellular
Ca2+ stores activates a plasma
membrane pathway for Ca2+ entry
(capacitative Ca2+ entry; see Ref.
22). This mechanism has been shown to operate in
GH3 pituitary cells (26) and in
rat lactotropes (4). It could be argued that the acceleration of
Mn2+ entry induced by ATP may be
secondary to the emptying of the intracellular
Ca2+ stores and the activation of
capacitative Ca2+ entry (see Ref.
26 for a similar mechanism in GH3
cells) rather than to activation of receptor-operated channels. To test
this point, we studied the effect of ATP in cells whose intracellular Ca2+ stores were first emptied of
Ca2+ by treatment with
thapsigargin (Fig. 6D). ATP also
produced an acceleration of Mn2+
entry under these conditions. Figure 6,
E and
F, illustrates the effects of other
nucleotides also in thapsigargin-treated cells. Both MeSATP and ClATP
increased Mn2+ entry, whereas UTP
had little effect under these conditions. These results suggest that
the increase in
Ca2+-Mn2+
entry observed in some cells after treatment with UTP (Fig.
6A) may be a result of the
activation of the capacitative pathway on depletion of the
Ca2+ stores. However, the entry of
Ca2+-Mn2+
induced by ATP, MeSATP, or ClATP cannot be fully explained by this
mechanism.
Distribution of responses to UTP and MeSATP in different AP cell
types and in freshly prepared cells.
To estimate the relative distribution of U and A receptors among the
different cell types, we compared the
[Ca2+]i
responses to sequential stimulation with 20 µM UTP and 10 µM MeSATP. Corticotropes, somatotropes, and lactotropes present in the
same microscope field were identified by multiple sequential immunocytochemistry with the use of the fluorescent primary antibodies and the abbreviated protocol described in MATERIALS
AND METHODS. Figure
7A
illustrates this approach. In the left column, the images corresponding
to ACTH-, GH-, and PRL-containing cells are shown. The bottom panel
shows the nuclei staining in the same field. The right column shows
[Ca2+]i
images from the same experiment taken at rest or during stimulation with either UTP or MeSATP and the contours of the four cells present in
the field. Combining all this information, we can study the responses
to each nucleotide separately in each cell type. Thus, in Fig.
7A, cell
1 was a corticotrope that showed no response to UTP and
a very weak response to MeSATP. Cell 2 was a somatotrope and responded only to MeSATP. Cell
3 was a lactotrope and responded only to UTP. Finally,
cell 4 did not react with any of the
antibodies and responded to both UTP and MeSATP.

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Fig. 7.
Responses to UTP and MeSATP in individual corticotropes, somatotropes,
and lactotropes. A, left
(labeled ABs, for antibodies): immunocytochemical identification of 3 main cell types, with the use of fluorescent primary antibodies,
is illustrated (see MATERIALS AND
METHODS for details and text for further explanations).
Same 30 × 30 µm section of microscope field is shown stained
with different antibodies or with nuclear stain Hoechst 33258. A, right: 3 images of same field taken during
[Ca2+]i
measurements before (Rest) or after stimulation with either 20 µM UTP
or 10 µM MeSATP. Larger Ca2+
concentrations appear as brighter areas of gray.
B: averaged
[Ca2+]i
traces for 9 corticotropes (ACTH cells), 21 gonadotropes (GH cells), 34 lactotropes (PRL cells), and 12 cells that did not stain with any of
the antibobies against the first 3 cell types (Remaining) from the same
experiment as in A.
|
|
Figure 7B shows the average responses
of all the cells of each kind present in the microscope field. The
smallest average responses to both UTP and MeSAP were observed in
corticotropes. The largest response to MeSATP was observed in
lactotropes. Somatotropes showed
[Ca2+]i
increases of intermediate size. All three cell types showed only modest
responses to UTP. The group containing the remaining cells, negative to
all three antibodies, showed the largest response to UTP, and the
response to MeSATP was as large as in lactotropes (Fig.
7B). This group of cells must have
contained gonadotropes and thyrotropes. A study of the traces of
individual cells showed that, as found in nonidentified AP cells (Fig.
2), many of the cells were sensitive to both nucleotides, and a smaller
fraction was sensitive to only one of them.
The cell-by-cell study allowed us to estimate the fraction of cells
within each cell type that was sensitive to each nucleotide and the
typical amplitude of the responses. Results from three independent
experiments are summarized in Table 2. The
fraction of cells responding to MeSATP was 51-53% in
corticotropes and larger (84%) in lactotropes. The fraction of cells
responding to UTP was uniformly small in all three cell types
(24-29%). The nonidentified cell group was the one responding
best to UTP (62%) and also gave a very good response to MeSATP (80%).
The differences in the size of the responses
(
[Ca2+]i),
expressed as the fraction of the average response of all the cells in
Table 2, were even larger. This indicates that the typical size of the
[Ca2+]i
peak observed in the responding corticotropes and somatropes was
smaller than in the other responding cells.
To check that the responses to the nucleotides observed in AP cells
were not an artifact induced by culture, we repeated some experiments
in freshly isolated cells that were allowed to attach to the polylysine
coverslips for 1 h. Stimulation of these cells with nucleotides gave
results similar to those obtained with the primary cultures. Thus
stimulation with 20 µM UTP, 10 µM MeSATP, and 10 µM ATP produced
a
[Ca2+]i
response in 22, 51, and 53% of the cells, respectively (188 cells in 3 different experiments). The size of the responses was also similar to
the size obtained with cultured cells. Therefore, the results obtained
here with primary cultures are likely to represent the physiological
behavior of AP cells.
 |
DISCUSSION |
We show here that functional ATP receptors are present in all five cell
types of AP. The percentage of cells responding to ATP varied between
60 and 88% in the different cell types. Thus our results confirm
previous reports on the presence of ATP receptors in lactotropes (5)
and gonadotropes (8, 11, 24) and extend them to the other cell types.
Because pituitary hormone release is a
Ca2+-triggered process, our
results suggest that ATP may induce secretion of all five pituitary
hormones. ATP has been shown previously to induce secretion of LH (7,
24). In addition, ATP is coreleased with pituitary hormones (7, 24).
This leaves room for paracrine interactions among different pituitary
cells. Taken together, these results suggest that ATP receptors may
play a role in the control of pituitary function.
The results from the additivity experiments shown here (Figs. 2-4)
suggest the presence of at least two different ATP receptors, one that
is activated equally well by UTP and ATP (U receptor) and another that
shows a strong preference for ATP over UTP (A receptor). ATP
S
activates both receptors, whereas MeSATP and ClATP seem to prefer A
receptors. Many of the cells possess both kinds of receptors, although
either A or U receptors may predominate in a smaller subpopulation of
cells (Fig. 2). Regarding the distribution of A and U responses among
the different AP cell types, we find that the distribution of A
receptors is larger in lactotropes and in another subpopulation that
must include gonadotropes and thyrotropes (Table 2).
Somatotropes and corticotropes presented smaller A-type responses both
by the fraction of responding cells and by the typical amplitude of the
responses. The U-type responses were limited to a smaller fraction of
cells (<30%) in lactotropes, somatotropes, and corticotropes. The AP
cell subpopulation not included in the above kinds of cells showed a
much larger U-type response (Fig. 6 and Table 2). This suggests that
either gonadotropes or thyrotropes must have a large density of U-type
receptors. These differences in ATP receptor distribution among
different AP cell types may be relevant for the control of hormone
secretion.
The characteristics of the U receptor described here were consistent
with those of a P2Y2 receptor. The
increase in
[Ca2+]i
observed on stimulation of these receptors in
Ca2+-free medium (Fig.
5A) indicates that the nucleotide is
able to induce release of Ca2+
from the intracellular stores. As a matter of fact, most previous studies attributed the effects of ATP in pituitary to metabotropic (P2Y2) receptors inducing
Ca2+ release from the
intracellular Ca2+ stores (5, 7,
8, 11) mediated by inositol trisphosphate production (12). In the same
line, a P2Y2 receptor has been identified recently within a rat cDNA pituitary library (9). We find
that UTP also induces some Ca2+
(Mn2+) entry (Fig. 6), but a
large part of it may be regarded as secondary to the activation of the
capacitative pathway on emptying of the Ca2+ stores.
It is clear, however, that a large component of the
[Ca2+]i
increase induced by ATP, MeSATP, and ClATP is a result of
Ca2+ entry through a
noncapacitative pathway because 1)
the
[Ca2+]i
increase was largely decreased after removal of external
Ca2+ (Fig.
5A),
2) the increase was still observed
after emptying of the intracellular
Ca2+ stores by treatment with
thapsigargin (Fig. 5B), and
3) direct evidence of the opening of
a plasma membrane pathway for entry was provided by the acceleration of
Mn2+ entry (Fig. 6). This
component of the ATP action was associated with the A receptors defined
above, with low reactivity for UTP. The phenomenological properties of
this component, including low desensitization (Fig.
1A), fit with the properties of
the P2X2 receptor whose mRNA has
been detected recently in the AP (4, 18). ATP has also been reported to
induce Ca2+ entry in gonadotropes,
probably through a P2X receptor (24). Thus AP cells would be an
exception to the rule that P2X receptors are restricted to
"excitable" (nerve and muscle) cells. Alternatively, it might be
reasonable to include AP cells (and perhaps other secretory cells)
within the excitable category, because they possess voltage-dependent
ion channels and are able to generate action potentials and their
activity is modulated by neurotransmitters.
The A component of the response to ATP in pituitary cells could still
contain minor contributions of other P2 receptors. Thus the presence of
P2Y1 receptors with high affinity
for MeSATP (13) cannot be excluded. On the other hand, the presence of
P2X receptors other than P2X2
seems necessary to explain the response to
,
-MeATP found in a
small fraction of cells (Fig. 1E). A
likely candidate is the P2X3
receptor, which is sensitive to this nucleotide (6, 19) and whose mRNA
has been detected in pituitary tissue, although at very low levels (6).
The existence of a few cells responding to MeSATP added on top of ATP
is also consistent with this idea, because
P2X2 and
P2X3 can form heteropolymers that
are sensitive to
,
-MeATP, like
P2X3, but show little
desensitization, like P2X2 (19).
Other P2X receptors, most of which are sensitive to MeSATP (13), may
contribute to the A response. Such a polymorphism may be responsible
for the rather bad correlation found at the single cell level for the
actions of ATP and MeSATP (Fig. 1H).
In summary, we provide evidence for the presence of at least two types,
ionotropic and metabotropic, of functional ATP receptors in all five
types of AP cells. Experiments with freshly isolated cells suggest that
the results obtained with primary cultures are not artifactual but are
representative of the physiological behavior of AP cells. Because ATP
is coreleased with several AP hormones, the presence of ATP receptors
suggests the possibility of paracrine interactions among the different
pituitary hormones secreted. Further studies are required, however, to
assess the physiological relevance of ATP receptors for the regulation
of pituitary function.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Spanish Dirección General de
Investigación Científica y Técnica Grant
PB92-0268.
 |
FOOTNOTES |
Address for reprint requests: J. García-Sancho, Departamento de
Fisiología, Facultad de Medicina, 47005 Valladolid, Spain.
Received 21 April 1997; accepted in final form 6 August 1997.
 |
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