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Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510
Address all correspondence and requests for reprints to: Dr. Stanko Stojilkovic, Section on Cellular Signaling, ERRB/NICHD, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of all three types of purinergic receptors in anterior pituitary is consistent with a view that ATP plays an important role in the control of Ca2+-signaling and secretion in anterior pituitary. However, it has not been clarified as to which subtypes of P2 receptors are expressed and operative in the pituitary, nor the cell specificity in the expression/coexpression of these receptors. The focus in this study is on expression, distribution, and Ca2+ signaling function of P2YRs and P2XRs within three major subpopulations of secretory anterior pituitary cells, including lactotrophs, gonadotrophs, and somatotrophs. We screened the expression of all eight known P2XRs in cultured and immortalized pituitary cells. To help with the identification of receptor subtypes within these pituitary cells, we also expressed the P2XRs in GT1 hypothalamic neurons, which have a set of plasma membrane channels highly comparable to that of anterior pituitary cells (15, 16), but do not express P2YRs and P2XRs (12). The pharmacological and Ca2+-signaling profiles of recombinant channels were compared with those observed in secretory anterior pituitary cells. The results of these investigations revealed an unusual complexity in the expression pattern and Ca2+-signaling functions of these receptors and channels in the pituitary, especially in the lactotrophs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell purification
Separation of gonadotroph-, somatotroph-, and
lactotroph-enriched populations was performed according to the size and
density of pituitary cells, using the Eppendorf Cell
Separation System. The one-liter disc-shaped chamber was filled from
the bottom opening with about 900 ml of 2% to 4% Ficoll
continuous gradient solution and then completed with 10% Ficoll
solution. Dispersed cells (100 x 106) were
suspended in 50 ml of 1% Ficoll solution and loaded into the chamber
from the top opening. After adding 30 ml of separation medium on the
top, the chamber was oriented to the horizontal position and was
allowed to sediment at unit gravity for 2 h. The chamber was then
reoriented to the inclined position and 15 fractions were collected
from the bottom opening. Cells were washed, counted, and
105 cells from the unfractionated sample and from
each separated fraction were suspended in 10 mM sodium
carbonate, sonicated, and kept frozen at -20 C. Their hormonal
contents were estimated by RIAs for rat LH, FSH, TSH, ACTH, PRL, and
GH, which were done using the kits provided by Dr. Parlow and the
National Hormone Pituitary Program. The highest concentrations of LH,
TSH, GH, ACTH, and PRL were respectively found in fractions 1, 4, 8,
10, and 14. Estimated by the reverse hemolytic plaque assay, the
percentage of purified cells was: 82% for LH-secreting cells, 68% for
GH-secreting cells, and 85% for PRL-secreting cells. Purification of
somatotrophs and lactotrophs was also done by a two-stage Percoll
discontinuous density gradient centrifugation (18). Using
the immunohistochemical staining approach, the purity of the enriched
somatotroph and lactotroph fractions was estimated to be 92% and 64%,
respectively.
Measurements of intracellular calcium ion concentration
For [Ca2+]i
measurements, cells were incubated in Krebs-Ringer buffer, supplemented
with 2 µM fura-2 AM, at 37 C for 60 min. Coverslips with
cells were washed with this buffer and mounted on the stage of an
Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany)
attached to the Attofluor Digital Fluorescence Microscopy System (Atto
Instruments, Rockville, MD). Cells were examined under a 40x oil
immersion objective during exposure to alternating 340 and 380 nm light
beams, and the intensity of light emission at 520 nm was measured. The
ratio of light intensities,
F340/F380, which reflects
changes in Ca2+ concentration, was followed in
several single cells simultaneously at room temperature.
RT-PCR analysis of purinergic receptors and channels
Total RNA was isolated from mixed pituitary cells, or enriched
somatotrophs and lactotrophs using TRIZOL reagent (Life Technologies, Inc.). First strand complementary DNA (cDNA) was
then synthesized using a Superscript preamplification system
(Life Technologies, Inc.). After RT using Superscript II
RT and removal of RNA templates by RNase H digestion, a 5 µl-aliquot
of first strand cDNA was used in subsequent PCR reactions. Primers for
the P2XRs used in experiments are described in Ref (19).
The P2Y2 specific primers corresponded to the rat
P2Y2R sequence: nucleotides 404 to 424 for the
sense primer (5'-AACGGACGCTGAGCATCCAAG-3') and 1611 to 1631 for the
antisense primer (5'-TGAACTGACACCTGACTGAGC-3'). PCR conditions
consisted of an initial denaturation step of 2 min at 94 C, followed by
20–25 cycles of denaturation at 94 C for 1 min, annealing at 60 C for
1 min, and extension at 72 C for 2 min. All PCRs were concluded with a
final extension step of 10 min at 72 C. PCR samples were then
size-fractionated in 1% agarose gel and visualized with ethidium
bromide staining. To check for the integrity of RNA preparation, RT-PCR
of GAPDH was also conducted as an internal control using primers
GAPDH.S (5'-GGCATCCTGGGCTACACTG-3') and GAPDH.AS
(5'-TGAGGTCCACCACCCTGTT-3') according to the PCR conditions reported
previously (20). In these experiments, plasmids with the
coding sequence of P2YR or P2XR cDNAs were used as the positive control
for the respective PCRs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the rise in
[Ca2+]i in response to
GnRH was highly specific for gonadotrophs, as estimated by the
parallelism between the profiles of GnRH responsive cells (B) and LH
content in these cells (C), and by their inability to respond to TRH
(E). On the other hand, there was a close correlation between PRL
content profile (F) with that of TRH-responsive cells identified based
on single cell Ca2+ measurements (E). The
TSH-containing fractions 3–5 also responded to TRH (E). A small
fraction of unidentified cells (1–2%) from fraction 9 responded to
both TRH and GnRH (see also Ref. 21). Thus, these isolation procedures
provide an effective system to enrich gonadotrophs and lactotrophs, to
separate the TRH-sensitive thyrotrophs and lactotrophs, and to identify
single gonadotrophs and lactotrophs with respect to GnRH- and
TRH-induced [Ca2+]i
responses.
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). The majority of
GnRH-responsive cells also responded to ATP when bathed in
Ca2+-containing medium (B), and only 1–2% of
these cells responded to ATP when bathed in
Ca2+-deficient medium (D). In lactotroph
fractions of pituitary cells, however, ATP elevated
[Ca2+]i in about 40% of
cells in Ca2+-deficient medium (D) and in
80–90% of cells in Ca2+-containing medium (A).
A group of unidentified cells elutriated with somatotroph/corticotroph
fractions also responded to ATP with an extracellular
Ca2+-independent rise in
[Ca2+]i (fractions
5–10). These results indicate that
Ca2+-mobilizing P2YRs are predominantly expressed
in lactotrophs, whereas all subpopulations of anterior pituitary cells
expressed P2XRs capable of facilitating Ca2+
influx.
Characterization of pituitary P2YRs
The pharmacological profile of P2YRs expressed in lactotrophs is
shown in Fig. 2. In addition to ATP,
these receptors also responded to uridine triphosphate (UTP), a
specific agonist for P2Y2R and
P2Y4R, and
adenosine-5'-O-(3-thio-triphosphate) (ATP-
-S) when bathed in
Ca2+-deficient medium (Fig. 2A). The relative
potency of ATP agonists for these receptors was ATP > UTP >
ATP--S. We have also examined the effects of
3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a relatively specific
agonist for P2X7R (22), on
[Ca2+]i response in
lactotrophs bathed in Ca2+-deficient medium. As
shown in Fig. 2A, BzATP was unable to initiate
Ca2+ signaling in lactotrophs. Several other ATP
analogs, including 
,ß-methylene ATP (,ß-meATP; Fig. 2A) and
2-methylthio-ATP (2-MeS-ATP; not shown) were also ineffective in
generating Ca2+ signals in lactotrophs bathed in
Ca2+-deficient medium. Such a pharmacological
profile of [Ca2+]i
responses was typical for all responding cells, suggesting that these
cells express P2Y2R subtype. In accordance with
the results of pharmacological characterization, RT-PCR using
P2Y2R primers also detected a PCR fragment with
the expected size of P2Y2R in enriched
lactotrophs. This specific message was also identified in immortalized
GH3 cells (Fig. 2B).
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, lane 1). As described in
(12), these products corresponded to
P2X2aR and its spliced form,
P2X2bR, and their presence in the mixed
population of pituitary cells was confirmed by specific primers. The
mRNAs for these channels were observed in somatotrophs purified by
two-stage Percoll discontinuous density gradient centrifugation (Fig. 3C, lane 2).
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, lane 1). Immortalized GH3
pituitary cells also expressed transcripts for
P2X3R, P2X4R, and
P2X7R, but not P2X2aR and
P2X2bR (Fig. 3A, lane 4). In parallel to GH3
transcript profile, the messages for P2X3R,
P2X4R, and P2X7R were found
in purified lactotrophs (Fig. 3B, lane 4), but not in somatotrophs
(lane 2). Finally, the transcripts for P2X1R,
P2X5R, and P2X6R were not
observed in mixed pituitary cells (Fig. 3A, lane 1) and GH3 cells (Fig. 3A, lane 4).
Calcium signaling by recombinant channels
To help identify functional P2XR subtypes present in anterior
pituitary cells, the recombinant channels were expressed in GT1 neurons
and their pharmacological and
[Ca2+]i signaling
profiles were compared with those observed in lactotrophs,
somatotrophs, and gonadotrophs. As shown in Fig. 4, GT1 cells expressing homomeric
P2X3R responded to ß-methylene ATP
(ß-meATP) with a small amplitude and transient rise in
[Ca2+]i. In accord with
the literature data (19, 23, 24), the heteromeric
P2X2R + P2X3R channels also
responded to this agonist (Fig. 4A). However, the amplitude of
ß-meATP-induced
[Ca2+]i responses in
cells expressing P2X2R +
P2X3R was higher compared with that observed in
P2X3R-expressing cells (Fig. 4A vs.
4B). Cells expressing P2X2aR,
P2X2bR, P2X4R, and
P2X7R were insensitive to ß-meATP (Fig. 4, C–F). These results indicate that ß-meATP can be used in
[Ca2+]i measurements, as
in current measurements (23) as a highly selective agonist
for the identification of functional P2X3R
homomers and P2X2R + P2X3R
heteromers.
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). In
P2X7R-expressing cells, addition of 100
µM ATP in the presence of 100
µM BzATP was ineffective (Fig. 5A, bottom
trace), whereas in all P2X2aR and
P2X2bR-expressing cells ATP was able to further
elevate [Ca2+]i (two top
traces). This suggests that P2X7R is more
sensitive to BzATP, contrary to P2X2aR and
P2X2bR, which are more sensitive to ATP.
Consistent with this, BzATP induced an additional rise in
[Ca2+]i in
P2X7R-expressing cells already stimulated with
ATP, 2-MeS-ATP, or ATP--S (Fig. 5B, right traces) but was
ineffective in P2X2aR-expressing cells stimulated
with these agonists (Fig. 5B, left traces). Such
pharmacological profiles of P2X2R and
P2X7R provide an effective tool to identify cells
expressing these two channels.
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)
is a useful but not sufficient tool in a search for cells expressing
these channels. However, only two cloned channels,
P2X4R and P2X6R, are
insensitive to suramin, a P2XR antagonist (25). As shown
in Fig. 6A, ATP-induced
[Ca2+]i response in
P2X4R-expressing cells was not affected by the
addition of 200 µM suramin for 3 min before the
application of ATP. Because the message for P2X6R
was not observed in pituitary cells (Fig. 3), the insensitivity of
P2X4R channels to suramin was employed in a
search for their expression in pituitary cells.
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ß-meATP,
the specific agonist for P2X3R and
P2X2R + P2X3R, was able to
induce rise in [Ca2+]i
only in TRH-sensitive cells. A fraction of lactotrophs, but not
gonadotrophs and somatotrophs, also responded to ß-meATP with a
rise in [Ca2+]i, the
pattern of which was highly comparable to that observed in GT1 neurons
expressing homomeric P2X3R (Fig. 7A vs. 4B). None of the
ß-meATP-sensitive cells generated
[Ca2+]i signals with the
amplitude and duration comparable to that observed in cells bearing
heteromeric P2X2R + P2X3R.
This is consistent with the absence of P2X2R
transcripts in immortalized GH3 cells (Fig. 3A) and
P2X3R transcripts in purified somatotrophs (Fig. 3B), further suggesting that lactotrophs only express
P2X3R homomers. Table 1 illustrates that about 50% of
lactotrophs express these channels.
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) responded to BzATP with the nondesensitizing
[Ca2+]i response,
typically observed in P2X7R-expressing cells.
Consistent with this conclusion, addition of ATP, ATP--S, and
2-MeS-ATP in the presence of BzATP was ineffective in all lactotrophs
studied. Figure 7B illustrates three such cells. In contrast to
lactotrophs, addition of ATP, ATP--S, and 2-MeS-ATP in the presence
of BzATP, induced further increase in
[Ca2+]i in somatotrophs
and gonadotrophs (Fig. 8), indicating
that these cells express P2X2R. In accordance
with this, BzATP was unable to elevate
[Ca2+]i in the presence
of ATP, ATP--S, or 2-MeS-ATP (Fig. 8, right tracings).
This pattern of response was observed in 84% of gonadotrophs, and 82%
of somatotrophs (Table 1).
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), 100
µM ATP induced suramin-insensitive
[Ca2+]i response, the
pattern of which was highly comparable to that observed in GT1 neurons
expressing homomeric P2X4R. Figure 6B illustrates
a cell responding to ATP in suramin-containing and -deficient medium.
Such a pattern of [Ca2+]i
response was observed in about 33% of lactotrophs (Table 1). These
results indicate that somatotrophs and gonadotrophs exclusively express
P2X2R, whereas lactotrophs express
P2X3R, P2X4R, and
P2X7R, in addition to already characterized
P2Y2R (6).
The coexpression of P2XRs in single lactotrophs was frequently
observed. A fraction of cells expressing the ,ß-meATP-sensitive
P2X3R, also responded to the application of BzATP
and ATP with additional rise in
[Ca2+]i, presumably by
activating P2X7R (Fig. 7A, two bottom
traces). Similarly, about 15% of lactotrophs responded to BzATP
with a nondesensitizing response and ATP with a spike response,
suggesting the expression of both P2X7R and
P2Y2R in the same cells (Fig. 7C, upper
trace). Consistent with this, removal of extracellular
Ca2+ abolished BzATP-induced rise in
[Ca2+]i and the
subsequent addition of ATP in Ca2+-deficient
medium generated a typical Ca2+-mobilizing spike
response. Finally, ATP was able to induce a suramin-insensitive rise in
[Ca2+]i in a small
fraction of lactotrophs already stimulated with BzATP, the profile of
which was comparable to that observed in GT1 neurons expressing
P2X4R (not shown).
In addition to the variable agonist sensitivity, pituitary P2XRs
also differed with respect to their peak amplitude
[Ca2+]i responses and
temporal response patterns. As shown in Table 2, the highest amplitude of
[Ca2+]i response was
observed in pituitary gonadotrophs and somatotrophs expressing
P2X2R. The peak amplitudes generated by
P2X7R in lactotrophs were somewhat smaller,
whereas the peak amplitude of
[Ca2+]i response in
lactotrophs expressing P2X3R and
P2X4R was about 40% of that observed in cells
expressing P2X2R. The native receptors also
desensitized with different rates: P2X3R >
P2X4R > P2X2R >
P2X7R (Table 2).
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Discussion |
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When activated, these receptor-channels generate [Ca2+]i signals of different amplitude and duration. P2X3R generates small amplitude [Ca2+]i signals, which desensitize within 1–2 min. In contrast, P2X7R generates high amplitude [Ca2+]i signals that do not desensitize during the prolonged agonist stimulation. P2X2Rs expressed in gonadotrophs and somatotrophs are able to generate high amplitude [Ca2+]i signals, but they desensitize gradually and lead to the attenuation of Ca2+ signals to about 10–20% of that observed at the beginning of stimulation. In general, such pattern of Ca2+ signals resembles those observed in GT1 neurons transiently expressing recombinant P2XRs. However, in the case of P2X7R, there are differences between the peak amplitude of [Ca2+]i responses in pituitary cells vs. that of GT1 cells when stimulated with BzATP, which may be result of overexpression of these channels in host cells. The recombinant P2X2aR also desensitizes incompletely in GT1 cells, but the time needed to reach the steady-state signaling is prolonged when compared with the native P2X2Rs expressed in pituitary cells. This suggests that the mechanism of controlling Ca2+ influx through P2X2R is different between pituitary cells and GT1 cells used in our expression studies.
The coexpression of P2X2R with
P2X3R provides an effective mechanism to control
Ca2+ influx through P2X2aR
(23, 24). Although the transcripts for
P2X3R are present in pituitary cells; however,
several lines of evidence argue against the existence of
P2X2aR + P2X3R heteromers
in pituitary cells. Purified lactotrophs and immortalized GH3 cells
expressed the transcripts for P2X3R, whereas the
transcripts for P2X2aR and
P2X2bR were identified in somatotrophs. In
parallel to that, functional P2X3R was identified
in lactotrophs, as well as in an unidentified cell type, but not in
gonadotrophs and somatotrophs. Finally, none of the
,ß-meATP-sensitive cells from mixed subpopulations exhibited the
pattern of Ca2+ signals generated by the
recombinant P2X2aR + P2X3R
heteromers. The cell specific expression of P2X2R
and P2X3R in anterior pituitary probably accounts
for the lack of coexpression of such heteromers.
On the other hand, we (12) and others (26, 27) reported recently that the control of Ca2+ influx through P2X2R can be achieved by the coexpression of P2X2aR and P2X2bR. Depending on the assemble mode, such heteromers can desensitize with variable rates (12). The Ca2+-signaling profiles in pituitary cells expressing P2X2R also resemble that of GT1 cells cotransfected with equal amounts of transcripts for P2X2aR and P2X2bR. Thus, it is reasonable to speculate that the control of ATP-induced [Ca2+]i signals in gonadotrophs and somatotrophs is achieved by expressing P2X2aR + P2X2bR heteromers.
In contrast to P2X3R and P2X2R, the native pituitary P2X7R and its recombinant channels expressed in GT1 cells do not exhibit an obvious desensitization. In the majority of cell expressing P2X7R channels, ATP initially induces opening of a channel selective for cations, including Ca2+ (25). During the prolonged agonist stimulation, P2X7R opens large pores allowing permeation of larger molecules and permeabilization of cells (22, 28). This activation step is dependent on the presence of C-terminus of the receptor (22) and on the environmental temperature (29). The physiological significance of this action is still unknown. Because the increased permeability results in larger ion fluxes and leakage of small metabolites, it may cause cell swelling and vacuolization, leading to cell death by necrosis and/or apoptosis (reviewed in Ref. 25). However, whether and to what extent P2X7R is used in the control of cell death in lactotrophs is still premature to discuss.
In conclusion, the results of these investigations indicate that the specificity found in the Ca2+-signaling responses induced by a common agonist is achieved by the capacity of individual receptor subtypes to generate different amplitude and kinetic patterns of Ca2+ responses and by the selective expression of receptors within the pituitary cell subpopulations. The ATP signaling pathway in somatotrophs and gonadotrophs is relatively simple. The majority of these cells express P2X2aR and P2X2bR, and their coexpression provides an effective system for a rapid elevation in [Ca2+]i, which is followed by a sustained plateau Ca2+ response of a much smaller amplitude. We were unable to observe any other P2X receptor subtypes expressed in these cells. The most striking finding shown here is the complexity of purinergic signaling system of lactotrophs. These cells express three types of channels, P2X3R, P2X4R, and P2X7R, as well as the Ca2+-mobilizing P2Y2R. The identification of P2XRs expressed in thyrotrophs and corticotrophs requires further studies. Pituitary P2X receptors can provide a wide range of Ca2+ signaling patterns when expressed individually or in combination. In this regard, the pituitary cells may provide a model to study how multiple receptors when expressed in excitable cells may generate different signaling in response to the same endogenous ligand.
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Acknowledgments |
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Received March 20, 2000.
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T3–1 cells.
Endocrinology 137:248–256[Abstract]
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