Clinical Science (2003) 104, 467–481 (Printed in Great Britain)
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Novel insights into how purines regulate
pituitary cell function
D. Aled REES, Maurice F. SCANLON and Jack HAM
Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K.
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Purine nucleosides and nucleotides are widely distributed substances that exhibit a diverse range
of effects in a number of tissues, acting as important extracellular signalling molecules in addition
to their more established roles in cellular metabolism. They mediate their effects via activation
of distinct cell surface receptors, termed adenosine (or P1) and P2 purinergic receptors.
Although roles for adenosine and adenine nucleotides have been described previously in the
pituitary gland, the distribution of the receptor subtypes and the effects of their activation
on pituitary function are not well defined. Recent evidence, however, has emerged to describe a
complex signalling system for purines in the pituitary gland. Data from a variety of studies have
shown that the expression pattern, number and affinity of adenosine and/or P2 receptors may be
cell-type specific and that non-endocrine in addition to endocrine cells elaborate these receptors.
These variations, along with the diverse range of signalling pathways activated, dictate the
response of individual cell types to extracellular purines, with roles now emerging for these
substances in the regulation of hormone release, pituitary cell proliferation and cytokine/growth
factor expression. In this review, we discuss these advances and examine some implications for
pituitary growth control and the response of the hypothalamic–pituitary–adrenal axis to stress
and inflammation.
INTRODUCTION AND HISTORICAL ASPECTS
Extracellular purines (adenosine, AMP, ADP and ATP)
and pyrimidines (UDP and UTP) are important signalling molecules that mediate their effects via specific
cell surface receptors. The concept of purinergic compounds as signal substances was first introduced as long
ago as 1929 when Drury and Szent-Gyo$ rgyi [1] described
potent effects of AMP and adenosine from heart muscle
extracts on arterial dilatation, blood pressure regulation
and intestinal contraction. In 1934, Gillespie [2] expanded
on these observations and demonstrated different effects
of adenosine and ATP in inducing contraction of guinea
pig ileum and uterus. By implication, this was the first
indication of the existence of more than one type of
purine receptor. Many of the early studies into the effects
of purines on biological function focused on the heart and
the vasculature, but a diverse number of responses have
since been described in a variety of biological systems,
including effects on neurotransmission, immunoregulation, inflammation, platelet aggregation, pain
responses and exocrine and endocrine function [3–6].
Insights into the sources of purines and stimuli for their
release came, in particular, from experiments showing
that adenosine is released from the heart during hypoxia,
in turn inducing reactive hyperaemia [7]. This notion of
Key words : adenosine receptors, cytokine, pituitary, proliferation, purine receptors.
Abbreviations : ACTH, adrenocorticotrophic hormone (corticotropin) ; bFGF, basic fibroblast growth factor ; EGF, epidermal
growth factor ; GH, growth hormone ; GHS, GH secretagogue ; IFN-γ, interferon-γ ; IL, interleukin ; IP , inositol 1,4,5$
trisphosphate ; LH, luteinizing hormone ; LPS, lipopolysaccharide ; MAP kinase, mitogen-activated protein kinase ; NECA, 5h-Nethylcarboxamidoadenosine ; PLC, phospholipase C ; PRL, prolactin ; R-PIA, R-N'-phenylisopropyl adenosine ; RT-PCR, reverse
transcriptase–PCR ; TRH, thyrotrophin-releasing hormone ; TSH, thyroid-stimulating hormone (thyrotrophin) ; VEGF, vascular
endothelial growth factor ; VIP, vasoactive intestinal peptide.
Correspondence : Dr D. A. Rees (e-mail reesda!cf.ac.uk).
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D. A. Rees, M. F. Scanlon and J. Ham
Table 1
Classification of adenosine receptors
Adenosine receptors activate a wide range of signal transduction pathways, in part related to their G-protein specificity. A1 receptors can activate several types of K+
channel (probably via β- and γ-subunits) and couple to inhibition of Ca2+ currents (N-type, P-type and L-type) in a number of cell types. Stimulation or inhibition
of the MAP kinase family of cascades may depend on the cellular context. , decrease ; , increase.
Adenosine receptor subtypes
G-protein coupling
Effects of G-protein coupling
A1
A2A
A2B
A3
Gi/o
cAMP
IP3
K+
Ca2+ currents
MAP kinase
Gs
cAMP
MAP kinase
Gs, Gq
cAMP
IP3
/ MAP kinase
Gi, Gq
cAMP
IP3
MAP kinase
coupling of purine release to metabolic demands by local
control of blood flow has since been extended to other
adenine nucleotides, such as ATP that is released during
skeletal muscle contraction [8,9], and thus forms a
fundamental concept in our understanding of purinergic
secretion. Sensory nerves, adrenal cells, platelets, leucocytes, erythrocytes, mast cells, fibroblasts and endothelial
cells have all now been shown to release purines under
physiological and pathological conditions [6,10–15] ;
these, in turn, can stimulate the purine receptors located
on these or adjacent cells in an autocrine or paracrine
fashion.
There are two principal classes of purinergic receptors,
termed P1 or adenosine receptors and P2 receptors, that
recognize mainly ADP, ATP, UDP and UTP [16]. This
classification was based on a number of observations,
including the relative potencies of adenosine and adenine
nucleotides, antagonism of the effects of adenosine by
methylxanthines, activation of adenylate cyclase by adenosine and induction of prostaglandin synthesis by ADP
and ATP. This division remains a fundamental part of
purine receptor classification, although both classes
are now characterized principally according to their
molecular structures and pharmacological profiles.
Adenosine receptors have been subdivided further into
A , A A, A B and A subtypes, all of which couple to G" #
#
$
proteins. As with other G-protein-coupled receptors,
adenosine receptors contain seven membrane-spanning
domains composed of hydrophobic amino acids. Each of
the adenosine receptor subtypes is able to mediate a
broad range of signalling responses upon agonist binding,
but the affinities of the receptors for adenosine vary
widely. For example, A and A receptors usually bind
"
$
adenosine with high affinity, A A receptors have inter#
mediate-affinity binding and A B receptors are conven#
tionally thought of as low-affinity receptors [17,18]. In
this manner the local concentration of adenosine at the
cell surface under physiological or pathological conditions will be a crucial determinant of which receptor is
activated.
# 2003 The Biochemical Society
The signal transduction pathways activated by each
receptor are, in part, attributable to coupling to different
G-proteins. These responses include principally the
inhibition or stimulation of cAMP and the activation of
phospholipase C (PLC), inositol 1,4,5-trisphosphate
(IP ) and diacylglycerol (‘ DAG ’), and subsequent in$
tracellular Ca#+ mobilization. In addition the A receptor
"
is associated with the activation of K+ channels and the
inhibition of cell-surface Ca#+ currents. All adenosine
receptors are now known to modulate the mitogenactivated protein (MAP) kinase family of signal transduction proteins [19]. The classification of adenosine
receptors and their associated signal transduction systems
are summarized in Table 1.
P2 receptors are divided into two families, either
ligand-gated cation channels or G-protein-coupled receptors, that are termed P2X and P2Y respectively.
Subtypes within each family are classified according to
the molecular structure, and their nomenclature is
identified by a different numerical subscript (i.e. P2Xn or
P2Yn). To date, approximately eight mammalian P2X
receptors (with several splice variants) and 11 P2Y receptors have been cloned and expressed [20]. Of these,
however, only seven P2X receptors (P2X – ) and six
"(
mammalian P2Y receptors (P2Y , P2Y , P2Y , P2Y ,
"
#
%
'
P2Y and P2Y ) can induce functional responses.
""
"#
In this review, we will examine the distribution and
function of P1 (or adenosine) and P2 receptors in the
pituitary gland and consider the implications of these
findings for the regulation of pituitary cell function in
physiological and pathophysiological states.
DISTRIBUTION OF PURINERGIC RECEPTORS
IN THE PITUITARY GLAND
P1/adenosine receptors in the pituitary
gland
Following the discovery of adenosine receptors in the
brain [21], a number of groups demonstrated that
Novel insights into how purines regulate pituitary cell function
adenosine inhibits neurotransmitter release by binding to
presynaptic A adenosine receptors [22,23]. The identi"
fication of adenosine deaminase, which catalyses the
deamination of adenosine to inosine, in the basal hypothalamus of the rat [24] led to the hypothesis that
adenosine might act as a neurotransmitter within the
hypothalamus or alternatively might regulate pituitary
hormone secretion via release into the portal circulation.
Adenosine receptors were first described in the anterior
and later the posterior pituitary gland in the mid 1980s
when Anand-Srivastava and co-workers [25,26] demonstrated the presence of adenosine-sensitive adenylate
cyclase in primary rat pituitary cultures. These receptors
were termed ‘ Ra ’ (now named A ) on the basis that
#
agonists stimulated cAMP with an order of potency
of 5h-N-ethylcarboxamidoadenosine (NECA) L-N'phenylisopropyladenosine (‘ PIA ’) 5h-N-methylcarboxamidoadenosine (‘ MECA ’). This same group later
showed that adenosine, in low micromolar concentrations, stimulates the release of adrenocorticotrophic
hormone (ACTH ; corticotropin) from cultures of primary rat anterior pituitary cells via the A receptor [27].
#
A number of investigators simultaneously examined
the effects of adenosine on pituitary physiology using
rodent cell lines and rat hemipituitaries as suitable models
of pituitary disease. Adenosine had been shown previously [28] to stimulate growth hormone (GH) synthesis
and reverse the apomorphine inhibition of prolactin
(PRL) synthesis in rat hemipituitaries, but these effects
were only apparent at very high concentrations of the
nucleoside (2.5 mM) and were mimicked by guanosine.
Dorflinger and Schonbrunn [29] examined the ability of
adenosine to regulate PRL and GH secretion by the
GH C rat pituitary tumour cell line. Adenosine
% "
( 50 µM) inhibited PRL and GH secretion from these
cells. The abolition of this inhibition by adenosine
deaminase and the secretion of biologically effective
concentrations of adenosine in these cells, as determined
by HPLC, suggested that adenosine produced in culture
tonically inhibited hormone release. They also concluded
that adenosine induced this response by activation of an
A adenosine receptor on the basis that the selective A
"
"
agonist R-N'-phenylisopropyladenosine (R-PIA) inhibited GH and PRL release and vasoactive intestinal
peptide (VIP)-stimulated cAMP production. These
authors also demonstrated that dipyridamole, an adenosine transport inhibitor, failed to reduce adenosine
inhibition of PRL release, suggesting that adenosine was
acting via an external receptor. This was the first
description of functional A adenosine receptors in
"
pituitary cells.
Delahunty and co-workers [30] expanded on these
observations when they described the presence of functional A adenosine receptors in the related GH rat
"
$
pituitary tumour cell line. In addition to inhibiting cAMP
production, adenosine and stable A receptor agonists
"
were shown to modify thyrotrophin-releasing hormone
(TRH)-stimulated IP accumulation in these cells, sug$
gesting that the regulation of TRH-stimulated GH and
PRL release described previously by Dorflinger and
Schonbrunn in GH C cells [29] might be mediated via
% "
the PLC rather than the adenylate cyclase pathway.
Scorziello et al. [31] demonstrated that the A adenosine
"
receptor in rat lactotrophs is coupled to both adenylate
cyclase and PLC. However, they were unable to confirm
the ability of adenosine to modify the TRH-induced
increase in intracellular Ca#+ concentration, which had
been suggested previously in GH cells [32], a dis$
crepancy the authors attributed to variations in sensitivity
to TRH and adenosine among primary lactotrophs and
GH cells. Mollard et al. [33], however, showed that A
$
"
adenosine receptors could directly inhibit Ca#+ currents
in GH cells, but the nature of these Ca#+ channels was
$
not fully defined until their identification as L-type
channels by Zapata and co-workers in 1997 [34]. These
authors also used a range of selective adenosine receptor
agonists and antagonists to show, by displacement of
radiolabelled compounds, that the A receptors in these
"
cells were present in a low-affinity conformation [35].
Reppert and co-workers [36] subsequently demonstrated
that the A receptor gene is expressed in the pituitary
"
gland. Adenosine acting via the A receptor can also
"
modulate A-type K+ currents in frog melanotrophs
[37,38]. These studies indicate that A receptors expressed
"
in primary lactotrophs and lactotroph cell lines are
coupled to adenylate cyclase, PLC and Ca#+ channels.
The coupling of A receptors to A-type K+ channels may,
"
however, be specific to the frog melanotroph.
In contrast with the A adenosine receptor, the dis"
tribution of other adenosine receptor subtypes in the
pituitary gland is less well described. A A receptor gene
#
expression has been reported to occur transiently during
the embryological development of the anterior and
intermediate lobes of the pituitary gland [39]. The
distribution of the A B receptor, cloned by Stehle and co#
workers [40], was examined by Northern-blot analysis
and transcripts were found to be present in the pituitary
gland. There are no reports of A adenosine receptor
$
mRNA or protein expression in the pituitary gland,
although transcripts have been detected by reverse
transcriptase (RT)–PCR in every brain region studied in
the rat [41].
It is clear from these studies that A and, to a certain
"
extent, A adenosine receptors are expressed in the
#
pituitary gland, but their precise location (i.e. which cell
types) had not been defined. We attempted to address
these limitations by studying the distribution of A , A A
" #
and A B receptors in primary rat anterior pituitary cell
#
cultures and rodent pituitary cell lines using a complementary range of methodologies [42]. We were able to
demonstrate the presence of functional A B adenosine
#
receptors in primary anterior pituitary cell cultures and
# 2003 The Biochemical Society
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D. A. Rees, M. F. Scanlon and J. Ham
tionally dominant in the anterior pituitary and that A
"
receptor expression in the cell lines is present in a lowaffinity conformation. These data highlight differential
expression patterns for A and A B adenosine receptors
"
#
within the various pituitary cell types and also suggest
that the physiologically relevant subtype in the anterior
pituitary may be the A B receptor. A summary of the
#
distribution of adenosine receptors and their putative
mechanisms of action in pituitary endocrine and folliculostellate cells is shown in Figure 2.
P2 receptors in the pituitary gland
Figure 1 Production of intracellular cAMP in (A) primary rat
anterior pituitary cell cultures and (B) TtT/GF folliculostellate
cell line stimulated with adenosine, NECA (universal adenosine receptor agonist) and CGS 21680 (selective A2A receptor
agonist)
The compounds stimulate cAMP production with a rank order of potency
appropriate for mediation via the A2B receptor subtype. Data represent
meanspS.E.M. of quadruplicate determinations from a representative experiment.
*P 0.05, **P 0.01, ***P 0.001 when compared with control values.
Reproduced from D. A. Rees, M. D. Lewis, B. M. Lewis, P. Smith, M. F. Scanlon and
J. Ham, Adenosine-regulated cell proliferation in pituitary folliculostellate and
endocrine cells : differential roles for the A1 and A2B adenosine receptors.
Endocrinology 143, 2427–2436 (2002), with permission. # The Endocrine
Society.
in non-endocrine pituitary folliculostellate [42] cell lines
by demonstrating a rank order of potency of NECA
(universal adenosine receptor agonist) adenosine CGS
21680
o2-[ p-(2-carbonyl-ethyl)-phenylethylamino]-5h-N-ethylcarboxamidoadenosine ; selective A A
#
receptor agonistq for stimulating cAMP production
(Figure 1) [43]. We were, however, unable to demonstrate
these responses in the pituitary endocrine cell lines GH
$
or AtT20, suggesting the absence of A receptors in
#
these cells. On the other hand, we confirmed that GH
$
and AtT20 cells expressed the A receptor by showing
"
that adenosine was able to inhibit VIP- or forskolinstimulated cAMP production. These findings were
supported by RT-PCR, immunocytochemistry and
ligand-binding studies [43]. Our data show that A B
#
receptors are expressed mainly in folliculostellate cells
and A receptors in both folliculostellate and endocrine
"
cells. We also concluded that the A B receptor is func-
#
# 2003 The Biochemical Society
ATP and other adenine nucleotides are involved in the
regulation of a diverse range of physiological functions in
a number of tissues, including endocrine glands, such as
the adrenal medulla and endocrine pancreas. In fact, ATP
is co-stored and co-released with the products of adrenal
chromaffin cells [44,45] and pancreatic β-cells [46,47].
Studies demonstrating activation of several second
messengers by extracellular ATP in both these endocrine
cell types [46,48] suggest that the nucleotide can play
a paracrine role in endocrine cell regulation.
Further evidence of such a paracrine pathway in the
neuroendocrine system is indicated by the observations
that ATP induces increases in cytosolic Ca#+ in hypothalamic neurons [49] and stimulates IP production in
$
heterogeneous preparations of sheep pituitary cells
in primary culture [50,51]. Chen and co-workers [52]
elaborated on these findings by using real-time imaging
of fura 2 fluorescence at the single cell level to show that
ATP causes a rapid rise in cytosolic Ca#+ in approx. 30 %
of primary rat anterior pituitary cells in culture. Furthermore, they were able to identify gonadotrophs as one
of the target cells for ATP action and showed that the
intracellular Ca#+ responses were mediated by G-proteincoupled Ca#+-mobilizing membrane ATP receptors of
the P2U subtype (now reclassified as P2Y receptors).
#
The physiological importance of this observation was
highlighted by the subsequent discovery that ATP (and
UTP) could stimulate the release of luteinizing hormone
(LH) from superfused pituitary cells through this class of
receptor [53].
The observation that ATP could be released from
pituitary cells by treatment with a Ca#+ ionophore also
implied that ATP and other nucleotides could have
important paracrine and\or autocrine effects within the
pituitary gland [53]. Using gonadotroph-derived αT3-1
cells, Chen and co-workers [54] also examined the signal
transduction pathways involved in mediating the action
of ATP in gonadotrophs. They were able to demonstrate
that extracellular ATP caused a biphasic increase in
cytosolic Ca#+ with no apparent change in the levels of
cAMP or cGMP. Furthermore, the intracellular Ca#+
increase was mediated via a pertussis toxin-insensitive
and PLC-coupled G-protein and consisted of Ca#+
release from an intracellular pool in addition to
Novel insights into how purines regulate pituitary cell function
Figure 2
Distribution of adenosine receptors in pituitary endocrine and folliculostellate cells
A1 adenosine receptors have been shown to be functionally coupled to PLC (possibly by cross-coupling of Gi/o signalling pathway to PLC by the βγ dimer), L-type Ca2+
channels and adenylate cyclase (AC) in rodent pituitary endocrine cell lines (GH3, GH4, GH4C1 and AtT20). The A1 receptor (A1R) has also been shown to be in a lowaffinity conformation in these cells. A1 receptors may also couple to K+ channels, but this may be specific to the frog melanotroph. The A2B receptor appears to be the
predominant functional adenosine receptor expressed in primary anterior pituitary cell cultures and studies have shown this receptor to be expressed principally in
folliculostellate cells. mRNA and protein expression of this receptor subtype is also detectable in folliculostellate cells. DAG, diacylglycerol ; ERK1/2, extracellular-signalregulated kinase 1/2 ; JNK, c-Jun N-terminal kinase ; PKC, protein kinase C.
Ca#+ influx via high-voltage-gated Ca#+ channels. These
studies also provide the first evidence for protein kinase
C (‘ PKC ’) ε translocation by ATP receptors. A pituitary
ATP receptor gene was subsequently cloned from a rat
pituitary cDNA library and functionally characterized as
a P2Y receptor [55]. The pharmacological characteristics
#
of this receptor closely resembled those of the P2Y
#
receptors described previously in primary rat gonadotrophs [52] and mixed sheep pituitary cells [50].
Other studies have highlighted the complexity of
purinergic signalling in the pituitary gland by demonstrating expression of a number of P2 purinergic
receptors in a variety of pituitary cell types. The ability of
ATP to signal through a P2X receptor in gonadotrophs,
for example, was suggested by Tomic and co-workers
[56] and later confirmed by Villalobos et al. [57]. These
investigators also demonstrated that ATP increased
cytosolic Ca#+ concentration in a large percentage of all
anterior pituitary endocrine cell types, i.e. lactotrophs,
somatotrophs, corticotrophs and thyrotrophs as well as
gonadotrophs. Furthermore, their observations suggested not only that these responses were mediated
by at least two different ATP receptors, the ionotropic (P2X ) and metabotropic (P2Y ) subtypes, but
#
#
also that the receptor distribution was not homogeneous
among the different pituitary cells. In addition, they were
unable to exclude the presence of other ATP receptors,
such as P2Y or P2X \P2X heteromers, in a small
"
#
$
number of cells.
The development of sensitive techniques to detect
mRNA expression in cells has enabled investigators to
confirm and elaborate on the observations outlined
above. The mRNAs for the P2X a receptor and its spliced
#
form, P2X b, for example, have been detected in the
#
anterior pituitary by a number of investigators [58–61].
The complex expression pattern and Ca#+-signalling
functions of these receptors in the pituitary was illustrated further in a comprehensive analysis of pituitary cell
subpopulations [62]. Mixed populations of rat anterior
pituitary cells were found to express mRNA transcripts
for P2X a, P2X b, P2X , P2X , P2X and P2Y receptors.
#
#
$
%
(
#
When enriched subpopulations of primary pituitary
endocrine cells and pituitary cell lines were analysed,
lactotrophs were found to be particularly complex,
expressing mRNA transcripts for P2X , P2X , P2X and
$
%
(
P2Y receptors. Functional P2X and P2Y receptor#
(
#
mediated responses were also detected in the majority of
lactotrophs and co-expression of these receptor subtypes
was common. In contrast, the expression of receptors for
ATP in pituitary somatotrophs and gonadotrophs was
comparatively simple, both cell types elaborating only
P2X a and P2X b receptors. P2X receptors were also
#
#
identified, but not analysed in detail, in corticotrophs and
thyrotrophs [62]. In addition to a cell-type-specific
expression pattern, these receptors also exhibited differences with respect to their peak amplitude and
duration of Ca#+ response, enabling specificity of function to be developed within the pituitary gland in
response to a common agonist.
As with the adenosinergic signalling system in the
pituitary, receptors for adenine nucleotides have also
been described recently in folliculostellate cells [63]. In
primary rat folliculostellate cells, ATP and UTP, but not
adenosine or ADP, induced a biphasic rise in intracellular
Ca#+ by activating PLC. These actions on Ca#+ currents
were probably mediated via a P2Y purinergic receptor as
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D. A. Rees, M. F. Scanlon and J. Ham
Figure 3
Distribution of P2 receptors in pituitary endocrine and folliculostellate cells
ATP elicits Ca2T responses in mixed sheep anterior pituitary cell cultures and induces a rapid rise in intracellular Ca2+ in 30 % of primary rat anterior pituitary cells.
Functional G-protein-coupled P2Y2 purinergic receptors have been identified in gonadotrophs and lactotrophs and mediate the release of LH by ATP in superfused pituitary
cells. Functional P2Y receptors are also present in folliculostellate cells, although the subtype of receptor is not known. In gonadotrophs and somatotrophs, RT-PCR studies
have demonstrated mRNA expression of the P2X2a receptor and its spliced form P2X2b only. In contrast, lactotrophs express mRNA transcripts for P2X3, P2X4 and P2X7,
and studies have confirmed the functional nature of the latter. P2X purinergic receptors have also been identified in corticotrophs and thyrotrophs, although the subtype
of receptor has not been determined.
they were reversed in the presence of a P2Y receptor
antagonist. Although the subtype of P2Y receptor was
not identified in this particular study, the results strengthen the hypothesis that purines exert important paracrine and\or autocrine roles within the anterior pituitary.
A summary of the expression pattern of P2 purinergic
receptors in the pituitary gland is illustrated in Figure 3.
BIOLOGICAL EFFECTS OF PURINES IN THE
PITUITARY GLAND
Effects on pituitary hormone release
Indirect evidence for the existence of adenosine receptors
in pituitary cell types other than those discussed previously has come from in vitro and in vivo experiments
examining hormonal responses to selective adenosine
receptor agonists and antagonists. For example, the
effects of xanthines, such as caffeine, theobromine and
denbufylline, which act as adenosine receptor antagonists, have been examined. These compounds have been
shown to increase ACTH and corticosterone, while
lowering thyroid-stimulating hormone (TSH ; thyrotrophin) and GH concentrations [64–66]. Xanthines,
# 2003 The Biochemical Society
however, also act as potent inhibitors of phosphodiesterases and it is likely that much of their activity in the
pituitary gland relates to inhibition of these enzymes as
much as blockade of adenosine receptors [66].
A number of authors have shown that peripheral [67]
or central [68] administration of adenosine can stimulate
the release of PRL but not LH [68] or TSH [69]. These
data, however, gave little insight into the site or mode of
action of adenosine in the neuroendocrine system, and
could in fact represent non-specific stressful actions.
Adenosine produced locally in the pituitary gland could
have quite different actions. The inhibitory effects of
adenosine on GH and PRL release from cell lines
discussed above [29–31] supported this contention, as did
the inhibition by adenosine of LH [70] and folliclestimulating hormone (‘ FSH ’) [70,71] release from pituitary segments. Further studies using rat pituitary segments showed that adenosine stimulated the release of
ACTH [72], supporting the observations of AnandSrivastava et al. [27], where primary rat pituitary cultures
as opposed to pituitary segments were used. The effects
on PRL release, however, are controversial. In contrast
with earlier studies [29–31], Picanco-Diniz et al. [70] and
Yu et al. [71] described a dose-dependent increase in PRL
secretion in response to low concentrations of adenosine
(10−"! to 10−& M). The latter group concluded that these
Novel insights into how purines regulate pituitary cell function
stimulatory effects were mediated via activation of the A
"
receptor subtype on account of specific inhibition by
selective A receptor antagonists. However, Kumari and
"
co-workers [73], who also used rat anterior pituitary
segments as their chosen experimental model, concluded
that adenosine tonically suppresses PRL (and TSH)
release via the A receptor, but activation of the A A
"
#
receptor enhanced the release of both hormones. These
discrepancies are not easily reconciled, as similar techniques were used in each case. Nevertheless, these studies
highlight the importance of analysing such responses in
the correct context and emphasize the need for a more
detailed description of the expression patterns of adenosine receptors in endocrine and non-endocrine pituitary
cells. The use of specific cell lines as opposed to mixed cell
cultures may provide more precise information on the
localization and function of specific adenosine receptors.
More recently, adenosine was identified as an agonist
of the GH secretagogue (GHS) receptor [74] following
HPLC fractionation of crude hypothalamic extracts and
screening of the fractions for cytosolic Ca#+-generating
activity in cells which expressed recombinant GHS
receptor but were devoid of adenosine receptors. The
authors showed that adenosine as well as the A receptor
"
agonist R-PIA induced Ca#+ responses in these cells.
They showed additionally that radiolabelled adenosine
bound to these cells and the GHS receptor antagonist
inhibited adenosine-, R-PIA- and GHS-induced Ca#+
responses at similar IC . In vitro studies, however,
&!
failed to show any effects of adenosine or R-PIA on
GH secretion either basally or during stimulation by
a GH secretagogue (GH-releasing peptide-6) or GHreleasing hormone (‘ GHRH ’). These findings raise
doubts as to the physiological relevance of adenosine as
a stimulator of GH release in the pituitary.
The effects of P2 purinergic receptor activation on
hormone release in anterior pituitary cells have also been
examined. In addition to the ATP- and UTP-induced
stimulation of LH release from superfused pituitary cells
[53], studies have also shown that ATP is able to function
as a local regulator of PRL secretion [75]. ATP secretion
was shown in the latter study to be induced or inhibited
respectively, following stimulation of primary pituitary
cultures by the lactotroph-selective secretagogues TRH
and bromocriptine. Moreover, ATP was found to stimulate the release of PRL from most lactotrophs and
removal or antagonism of ATP resulted in a reduction in
basal or TRH-stimulated PRL release. These observations suggested that ATP can prolong the PRL secretory
response elicited by traditional hypophyseal signals. The
physiological relevance of this system is highlighted
further by the observation that maximal concentrations
of ATP were as effective in evoking PRL release as
comparable concentrations of TRH. Despite evidence for
expression of P2X receptors in somatotrophs, thyrotrophs and corticotrophs [62], there are no reports to date
of the effects of purine nucleotides on hormone release in
these cells.
Effects on pituitary cell proliferation
In contrast with the effects of adenosine on hormone
release from the anterior pituitary, there are few reports
describing a proliferative role for adenosine in pituitary
cells. Several members of the G-protein-coupled transmembrane spanning cell-surface receptors have been
shown to mediate inhibition of proliferation in pituitary
tumour-derived cells. Among them are somatostatin
receptors [76], TRH receptors [77] and dopamine receptors [78,79]. These hormones modify pituitary cell
proliferation by blocking entry into S phase, as do many
other growth-inhibitory factors such as the transforming
growth factors [80]. Navarro and co-workers [35] studied
the effects of A adenosine receptor activation on the
"
proliferation of cultured pituitary endocrine GH cells.
%
Their results showed that, although the A receptor
"
agonist R-PIA did not affect the number of cells, it did
modify the kinetics of the cell cycle. R-PIA in fact
stimulated synchronized GH cells to progress more
%
rapidly from G \G to S phase and from S phase to G \M
! "
#
phase without accelerating initiation of a new cycle. On
the basis of these findings and their observation that cells
in G \G were more refractory to TRH-induced PRL
! "
release than the overall cell population, these authors
proposed that adenosine acts in the pituitary to permit
cells to remain longer in a state (G /G ) that is more
! "
refractory to secretory signals.
Lewis and co-workers [81] also examined the effects of
adenosine on cell proliferation in GH cells. They showed
$
that high concentrations of adenosine (100 µM or above)
inhibited the proliferation of these cells, an action they
attributed to an intracellular rather than a receptormediated site of action, as the effects were reversible by
the adenosine transport inhibitor dipyridamole. Analysis
of the treated population of cells showed an increase in
DNA laddering in comparison with controls, with no
differences in the proportion of cells in the various phases
of the cell cycle, suggesting that adenosine reduced
proliferation in GH cells by increasing the rate of
$
apoptosis. Epidermal growth factor (EGF), by preventing apoptosis, was found to restore the growth rate of the
adenosine-treated cells to that observed in the control
population. Other researchers have also shown that EGF
can interact with the adenosine signalling system in the
pituitary. For example, EGF has been shown to modify
A adenosine receptor expression in a rat pituitary
"
tumour cell line [82], suggesting that distinct signalling
pathways can interact dynamically to regulate pituitary
cell function. The data from these studies suggest that
adenosine can modify pituitary cell proliferation by at
least two apparently unrelated mechanisms.
Our recent observations [43] have indicated that
adenosine can regulate proliferation in rat pituitary
# 2003 The Biochemical Society
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474
D. A. Rees, M. F. Scanlon and J. Ham
tumour GH cells by another mechanism. In these cells
$
and in murine pituitary AtT20 cells, adenosine and the
selective A adenosine receptor agonist 2-chloro-N'"
cyclopentyladenosine (‘ CCPA ’) at high concentration
inhibited cell growth. We showed that this inhibition was
mediated in GH cells by a slowed progression through
$
the phases of the cell cycle rather than through a cellcycle-stage-specific arrest. In contrast with pituitary
endocrine cells, we showed that adenosine, at physiological concentrations (up to micromolar) and acting
through a different adenosine receptor subtype (A B),
#
caused a dose-dependent stimulation of growth in a
pituitary folliculostellate cell line (TtT\GF). These findings suggest that adenosine may play a significant role in
regulating the trophic activity of folliculostellate cells
within the anterior pituitary.
Effects of adenosine on interleukin (IL)-6
and vascular endothelial growth factor
(VEGF) production within the anterior
pituitary
The above descriptions provide a complex picture of
purinergic signalling pathways in the pituitary gland.
P2 purinergic receptors are widely distributed, but seem
to show cell-type-selectivity both in the pattern and
quantity of receptor expression [62,63]. Similarly, we [43]
and others [29–34] have shown that adenosine receptors
are also preferentially expressed within pituitary cell subpopulations. Our investigations have suggested that the
A B receptor, in particular, is the physiologically domi#
nant adenosine receptor in the pituitary. The demonstration of its preferential expression in folliculostellate
cells raises intriguing questions as to its role in these cells.
Pituitary folliculostellate cells, with their characteristic
star-shaped morphology, were first described in the
pituitary in 1953 [83]. Until recently, they were thought
to be non-secretory and their proposed function remained obscure. A number of investigators, however,
have shown that folliculostellate cells produce bioactive
molecules which are not secreted into the bloodstream as
hormones, but act locally as paracrine and\or autocrine
factors, modulating the function of hormone-secreting
cells and other cell types such as the endothelium
[42,84,85]. These paracrine effects of folliculostellate cells
on neighbouring endocrine cell function have been
studied extensively. They include enhancement of basal
and TRH-stimulated PRL secretion by GH cells [86],
$
mediation of the inhibitory effects of interferon-γ (IFNγ) on the stimulated release of ACTH, PRL and GH in
primary pituitary cultures [87] and release of the proinflammatory cytokine IL-6 in response to VIP [88]
and pituitary adenylate cyclase-activating polypeptide
(‘ PACAP ’) [89]. In addition, IL-6 is well known to
stimulate the release of PRL, GH, LH and folliclestimulating hormone in vitro [90,91] and ACTH in vivo
# 2003 The Biochemical Society
[92]. The ability of folliculostellate cells to respond to
IFN-γ and produce IL-6 highlights the key role that these
cells are now known to play in regulating cross-talk
between the neuroendocrine and immune systems.
In this context, it is interesting to note that adenosine
has been shown to stimulate IL-6 release in primary
anterior pituitary cell cultures [93]. Our recent studies
have examined this relationship in more detail. We have
shown that adenosine, via the A B receptor, potently
#
stimulates IL-6 release from folliculostellate cell cultures,
mediated via adenylate cyclase and PLC coupled to p38
MAP kinase (D. A. Rees, B. M. Lewis, K. Francis, T.
Easter, M. F. Scanlon and J. Ham, unpublished work).
Similar but significantly lower magnitude responses were
observed on VEGF release, but these latter findings may
represent an autocrine effect of IL-6 [94]. The production
of both IL-6 and VEGF was reversible by co-incubation
with dexamethasone.
Double-labelling techniques have shown previously
[95] that glucocorticoid receptors are present in folliculostellate cells, and these studies have been supplemented by cell culture-based analyses confirming the
presence of functional glucocorticoid receptors through
the reversal of dexamethasone effects by the glucocorticoid receptor antagonist RU 486 [96,97]. The immunosuppressive effects of glucocorticoids are often mediated
via transcriptional inhibition of immunologically relevant
genes, but it is interesting to note that glucocorticoids
have also been reported to decrease A B adenosine
#
receptor signalling in both Jurkat cells [98] and DDT
"
MF2 smooth-muscle cells [99]. In the latter, decreased
A B receptor signalling was accompanied by an increase
#
in the number of co-expressed A receptors. Dexametha"
sone, therefore, could be modifying the release of these
substances by a number of separate mechanisms. As both
systemic and locally generated IL-6 can potently stimulate ACTH (and GH, PRL and gonadotrophin) release in
the pituitary gland [90–92], these results, therefore,
suggest that adenosine may have an important role in
activation of the pituitary gland in regulating the response of the hypothalamic–pituitary–adrenal axis to
inflammation. Whether similar effects can be mediated
by P2 purinergic receptors is not known at present,
although the development of a murine folliculostellate
cell line (Tpit\F1) expressing P2Y receptors and able to
#
secrete IL-6 [100] could represent an important model
for studies in this setting.
PHYSIOLOGICAL AND PATHOLOGICAL
CONSIDERATIONS
Co-expression of purinergic receptors
Adenosine and P2 purinergic receptors within the pituitary gland clearly have distinct, but frequently overlapping, tissue distributions. The fact that more than one
Novel insights into how purines regulate pituitary cell function
adenosine or P2 receptor can be expressed by the same
cell raises important questions about the functional
significance of this co-localization. Given the ubiquitous
distribution of purine receptors in the body, it is perhaps
not surprising that more than one subtype is often
expressed in the same tissue and even the same cell. For
example, the expression of more than one subtype of
adenosine receptor on the same cell allows the common
agonist adenosine to activate multiple signalling pathways. Furthermore, the presence of adenylate cyclase as
a common second messenger for all four adenosine
receptor subtypes, with negative and positive coupling to
the A and A , and A A and A B, receptors respectively,
"
$
#
#
enables reciprocal control of this signalling pathway.
Under such circumstances, it would seem likely that the
functional role of higher-affinity adenosine receptors
would predominate over A B receptors. Nevertheless,
#
A B receptors could still play a role under such conditions
#
by modifying the events triggered by other receptor
systems.
The relative importance of A B receptors may be
#
greater in situations characterized by considerable increases in interstitial levels of adenosine, as occurs in
hypoxia. There are, however, cellular systems where the
actions of A B receptors appear to predominate [101],
#
and the discovery in folliculostellate and pituitary endocrine cells of A receptors with a much lower affinity
"
for adenosine and selective agonists compared with those
described elsewhere [17,18] suggests a functional relevance of the A B receptor in the pituitary gland.
#
The co-existence of both adenosine and P2 receptors in
pituitary cells adds another level of complexity. Such coexpression has been identified for a number of cell types,
including mast cells, endothelial cells, smooth-muscle
cells and PC12 phaeochromocytoma cells [20], although
the exact functional significance of such co-localization is
not clear at present. The co-expression of more than one
purine receptor, with their different affinities for the
endogenous agonist, also highlights the importance of
the local concentration of agonist in physiological and
pathological conditions in determining individual receptor activation. The extracellular concentration of
adenosine, for example, in rat phrenic motor nerve
terminals needed to increase transmitter release via
activation of A A receptors is higher than that required to
#
inhibit neurotransmitter release via A receptors [102].
"
Because adenosine is formed as a breakdown product of
ATP released from nerve endings, this suggests that the
concentration of adenosine is critically related to neuronal activity.
The local concentration of extracellular adenosine is
clearly important in determining which adenosine receptor is activated. However, the concentration of
purines in the extracellular environment is controlled not
only by release from the intracellular space, but also by a
series of cell-surface and intracellular enzymes, catalysing
the breakdown and synthesis of these compounds and,
therefore, limiting their spatiotemporal activity. Several
of these enzymes have been characterized and those
involved in the metabolism of adenosine include ecto5h-nucleotidase (which catalyses the hydrolysis of AMP
to adenosine), intracellular and extracellular adenosine
deaminase (catalysing deamination of adenosine to inosine) and adenosine kinase (regulating the phosphorylation of adenosine to AMP and expressed in the
intracellular space only). The fate of adenosine in any
given situation is governed by its concentration, since
adenosine kinase and adenosine deaminase have vastly
different kinetic properties. At physiological (submicromolar) concentrations of adenosine, phosphorylation is
the predominant route of metabolism, whereas adenosine
deaminase in most circumstances functions as a highvolume\low-affinity system that is activated when
adenosine concentrations increase [103]. In a similar
manner, ecto-5h-nucleotidase is ubiquitously expressed,
but often differentially regulated in malignant and benign
tissue [104] and by cytokines [105] and glucocorticoids
[106], suggesting that local concentrations of purines at
the cell surface can be controlled according to the
prevailing physiological or pathophysiological circumstances.
These numerous factors combine to determine which
adenosine or P2 receptor subtype, or combination of
receptors, is activated in any given circumstance and
dictate, in combination with the cell distribution of the
receptor subtypes, which cellular response is activated.
Implications for pituitary tumorigenesis
Pituitary tumorigenesis is a multifactorial process requiring specific favourable environmental conditions in
addition to altered expression of oncogenes or tumour
suppressor genes. Adenosine has been proposed as a
potential contributor to solid tumour growth on the basis
of its cytoprotective, immunomodulatory and growthpromoting properties [103]. It is tempting to speculate,
based on the considerations outlined above, that the
differential distribution, affinities and functional effects
of the A and A B receptors may have a relevance to
"
#
pituitary tumour formation and\or maintenance.
Hypoxia is a major stimulus to the release of adenosine
[7]. Under these circumstances micromolar concentrations of the nucleoside typically accumulate [107]. Solid
tumours of all types are characterized by their hypoxic
cores and it is conceivable that adenosine and other
purines accumulate in the interstitium of such tumours in
high concentration. Pituitary tumours are less vascular
than the normal pituitary gland [108], suggesting that
hypoxia may be a particular feature of these neoplasms.
Under these conditions, the hypoxia-induced release of
adenosine as an adaptive response of the cell to metabolic
demands could indirectly contribute to the proliferation
of transformed pituitary cells. Should the distribution
# 2003 The Biochemical Society
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D. A. Rees, M. F. Scanlon and J. Ham
and the relative affinities of the adenosine receptors
identified in our recent study [43] be mirrored in vivo,
then folliculostellate A B adenosine receptors would be
#
preferentially activated, resulting not only in folliculostellate growth, but also indirectly in vascular and
endocrine cell proliferation induced by VEGF and IL-6
release. The importance of IL-6, in particular, as a
pituitary tumour-progression factor has received much
attention recently. It is known to stimulate hormone
release not only from normal pituitary cells [90–92],
but also from pituitary tumours placed in culture
[109]. Furthermore, IL-6 stimulates the growth of
pituitary tumour cells [110] and IL-6 expression
in pituitary tumours correlates with biological aggression [111], raising the possibility that local IL-6 levels
could be an indirect marker of tumour size, hypoxia
and adenosine concentration.
There are, of course, a number of assumptions made in
the above hypothesis, not least being the relevance of
folliculostellate cells to the pathogenesis of pituitary
adenomas. IL-6 is known to be a growth factor for
TtT\GF folliculostellate cells [94] and GH and MtT\E
$
pituitary tumour cell lines [110,112]. However, whether
the growth stimulatory effect of IL-6 on TtT\GF cells is
specific for transformed folliculostellate cells or also
occurs in normal cells remains to be determined. Nonfunctioning pituitary tumours that arise as a result of the
clonal expansion of mutated folliculostellate cells and
consisting mainly of transformed folliculostellate cells
(TtT\GF) are rare [113] and it may be that the autocrine
stimulatory effect of IL-6 is only relevant in these cells.
With respect to endocrine cells, evidence for the importance of folliculostellate cells in pituitary tumour
formation comes from the induction of lactotroph
adenomas by oestrogen in Fischer rats, where adenoma
formation is accompanied by an activation of folliculostellate cells [114]. Oestrogen has also been shown to
induce the expression of the immediate early gene, c-fos,
in folliculostellate cells and lactotrophs [115], possibly
indicating direct responsiveness of folliculostellate cells
to oestradiol treatment. The observation that folliculostellate cells mediate the transforming-growth-factor(‘ TGF-β3 ’)-induced growth of pituitary lactotrophs
[116] supports an important role for these cells in
lactotroph cell growth. In addition, folliculostellate-like
TtT\GF cells have been shown to be essential to the
formation and growth of MtT\S pituitary tumours in
nude mice [117]. These observations suggest that folliculostellate cells play an important role in pituitary
adenoma formation, perhaps by regulating the production of angiogenic factors, such as VEGF or basic
fibroblast growth factor (bFGF), and\or regulators
of the extracellular matrix, such as tissue inhibitors of
metalloproteinases (‘ TIMPs ’) [118].
The relevance of folliculostellate cells to pituitary
tumour pathogenesis is cast into doubt, however, by the
# 2003 The Biochemical Society
observation that they occur only rarely [119], although it
is interesting to note that, at the border between the
normal pituitary and the adenoma, a transition zone
often exists which is particularly rich in folliculostellate
cells [119,120]. The source of IL-6 in pituitary adenomas
is also an area of controversy. As folliculostellate cells
seem to be rare in adenoma tissue and may be immunonegative for IL-6 [121], it may be that the neoplastic
endocrine cells are the source of IL-6 [122]. However,
this does not exclude an important role for folliculostellate cells in supporting the growth of pituitary
tumours, as the IL-6 produced by tumour cells could
stimulate the proliferation of the normal folliculostellate
cells in the transition zone and increase their production
of growth-supporting substances such as VEGF
and bFGF. The folliculostellate-cell-derived VEGF and
bFGF could then diffuse, or be carried in the blood
supply, to the tumour where it could promote angiogenesis. Therefore, during tumour initiation, the border
of the adenoma could represent the principal site of
initiation and regulation of angiogenesis. This concept is
similar to that proposed by Fukumara and co-workers
[123], who speculated that fibroblasts on the tumour
edge are the most important source of VEGF during
formation of some types of tumours.
Possible roles in inflammatory and stress
responses
It has become increasingly clear that adenosine exerts
important effects in the regulation of immune responses
in a number of cell systems. Numerous reports have
alluded to its immunosuppressive and anti-inflammatory
properties, which include inhibition of pro-inflammatory IL-2, tumour necrosis factor (‘ TNF ’)-α and
macrophage inflammatory protein (‘ MIP ’)-1α synthesis
[124–126], induction of anti-inflammatory IL-10 synthesis [125], general inhibition of T-cell activation and
expansion [127], inhibition of neutrophil superoxide
formation [128] and a reduction in neutrophil accumulation at sites of inflammation [129]. In addition,
many drugs with potent anti-inflammatory activities
often exert their therapeutic benefit by increasing
adenosine concentration. Methotrexate, for example, is
thought to inhibit neutrophil function by stimulating
adenosine release from connective tissue cells [130],
and sulphasalazine increases adenosine secretion at
sites of inflammation [131].
A adenosine receptors, in particular, may be re#
sponsible for mediating many of the anti-inflammatory
properties of adenosine. For example, A A receptor#
deficient mice are more susceptible to endotoxin
[lipopolysaccharide (LPS)]-mediated release of proinflammatory tumour necrosis factor-α, IL-6 and IFN-γ
[132]. In addition, concanavalin A is able to induce death
within 8 h in a number of A A receptor-deficient, but
#
not in wild-type, animals. Our recent demonstration of
Novel insights into how purines regulate pituitary cell function
Figure 4 Postulated mechanisms by which adenosine regulates activation of the pituitary gland in the response of the
hypothalamic–pituitary–adrenal axis to inflammation
Adenosine binds to folliculostellate A2B receptors (A2BR) to stimulate IL-6 release via adenylate cyclase and PLC coupled to p38 MAP kinase. IL-6 induces ACTH secretion
from corticotrophs which, in turn, stimulates corticosteroid release from the adrenal cortex. Glucocorticoids bind to nuclear glucocorticoid receptors (GR), present in
both folliculostellate and corticotroph cells, to regulate this response in a negative feedback pattern. Bacterial LPS stimulates IL-6 release from folliculostellate cells via
the toll-like receptor-4 (Tlr-4). We postulate that this pathway may generate increased extracellular adenosine which could augment the release of IL-6 further.
A , notably A B, receptor expression in pituitary folli#
#
culostellate cells [43] suggests that this receptor may
have an immunomodulatory role in the pituitary gland.
Folliculostellate cells secrete a number of growth factors
and cytokines, such as IL-6, TNF-α, VEGF and bFGF
[85,96,97], which appear to regulate pituitary function by
playing a supportive role for neighbouring endothelial
and endocrine cells. These properties of folliculostellate
cells, coupled with the fact that they possess receptors for
bacterial LPS [97], complement C3a (K. Francis, B. M.
Lewis, H. Akatsu, P. N. Monk, S. A. Cain, M. F. Scanlon,
B. P. Morgan, J. Ham and P. Gasque, unpublished work)
and glucocorticoids [95], suggest that they co-ordinate
the bidirectional exchange of information between the
immune and endocrine systems and can regulate stress
and inflammatory responses [133]. Maintaining this
interaction is critical for homoeostatic regulation and
resistance to disease. IL-6, in particular, may play an
important role as a linking molecule between the folliculostellate and endocrine cells in the anterior pituitary,
since it has been shown to regulate GH, PRL and ACTH
secretion [90–92]. Our observation (D. A. Rees, B. M.
Lewis, K. Francis, T. Easter, M. F. Scanlon and J. Ham,
unpublished work), therefore, that adenosine stimulates
IL-6 secretion via the A B receptor linked to the adenylate
#
cyclase and PLC pathways coupled to p38 MAP kinase
adds to the anti-inflammatory roles for the nucleoside
described elsewhere. The enzymes involved in the p38
MAP kinase pathway are transducers of stress responses
that are activated by ultraviolet light, cytokines and
osmotic shock [134,135]. The coupling of adenosine to
p38 MAP kinase and IL-6 secretion, together with its
suppression in the presence of corticosteroids, indicates a
role of adenosine and folliculostellate cells in inflammation, stress and hypoxia. We hypothesize that folliculostellate cells respond to a variety of immune molecules and endotoxins, resulting in accumulation of
extracellular adenosine [71,136] and an autocrine stimulation of IL-6 and VEGF to modulate inflammatory
processes and stimulate steroidogenesis. These possible
mechanisms are outlined in Figure 4.
CONCLUSION
Extracellular purines mediate a diverse variety of biological responses in a wide range of tissues. The distribution of purine receptors reflects this diversity and is
illustrated particularly well in the pituitary gland, where
recent evidence has emerged to suggest that specificity of
function can be determined by cell-selective expression.
These observations have also revealed a wide diversity of
effects on several aspects of pituitary function, including
trophic effects, actions on hormone release and roles in
cytokine synthesis, with resultant implications for pathological in addition to physiological states. Future research
may expand on these observations and go on to determine
the integrated pattern of events arising from differential
# 2003 The Biochemical Society
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478
D. A. Rees, M. F. Scanlon and J. Ham
activation and desensitization or up-regulation of coexisting receptors under conditions of different concentrations of agonist in the pituitary. Such developments
may pave the way for an improved understanding of the
role of purines in pituitary physiology and disease, and
could offer important advances for future therapies.
18
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Received 3 February 2003; accepted 11 February 2003
Published as Immediate Publication 11 February 2003, DOI 10.1042/CS20030053
# 2003 The Biochemical Society
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