Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106
WHEN RELEASED TO EXTRACELLULAR SPACES, ATP and other
nucleotides can participate in multiple types of intercellular
communication (4). This can involve classical mechanisms of
intercellular signaling (similar to those used in neurotransmission and
many types of endocrine regulation) that involve exocytotic release of
ATP that is copackaged with biogenic amines (or other
neurotransmitters) within specialized secretory vesicles or granules.
Well-characterized examples include synaptic signaling by vesicular ATP
released at nerve junctions or paracrine regulation of endothelial
cells and other platelets by the ATP/ADP released from the dense
granules of activated platelets. Most physiologists can readily
appreciate how such specialized secretory cells can be sources of the
extracellular nucleotides used in intercellular communication. It may
stretch the credulity of some to consider that all cell types have the potential to release ATP from a store usually considered as sacrosanct, i.e., the cytoplasmic pool of ATP that is used to fuel or regulate virtually all critical intracellular functions. However, diverse types
of environmental stress, such as mechanical shear forces (11), stretch
(7), and changes in extracellular osmolarity (15), have now been shown
to induce the release of nucleotides from cells via mechanisms that do
not involve cellular lysis or obvious exocytosis of specialized
secretory granules. In turn, the locally released nucleotides can
stimulate P2 nucleotide receptors in nearby cells, or the releasing
cell itself, with the consequent activation of adaptive or protective
responses to the particular environmental stress. This type of
autocrine or paracrine regulation based on locally released nucleotides
has been observed in several cell types including endothelial cells
(13), glial cells (11), and vascular smooth muscle (7, 13).
Recent studies suggest that localized increases in extracellular
nucleotides also play particularly critical roles in the autocrine or
paracrine regulation of cellular fluid and ion homeostasis in
epithelial cells derived from the airway (2, 9), intestine (18), liver
(15), cervix (6), and other tissues. A noteworthy example is the
regulatory volume decrease response of hepatocytes that can be analyzed
at the single cell level (15). These cells respond to hypotonic
stimulation with a signaling cascade that serially involves a
cell-swelling-induced release of ATP, autocrine stimulation of a G
protein-coupled nucleotide receptor, and activation of a
Cl Characterizing P2 receptors is not a trivial issue for several reasons.
First, the genes encoding more than a dozen distinct P2 nucleotide
receptors (discussed below) have been cloned during the past six years.
To make matters more complex, the receptors comprise two major
families: the G protein-coupled P2Y receptors (8) and the ATP-gated ion
channels of the P2X group (5). Second, all of the identified P2Y and
P2X receptors can trigger rapid changes in cytosolic
Ca2+ due either to mobilization of
inositol trisphosphate-sensitive Ca2+ stores (P2Y subtypes) or to
Ca2+ influx across the plasma
membrane (P2X and P2Y subtypes). Thus most P2 receptor subtypes can
trigger similar integrated cellular responses in epithelial cells, such
as the activation of
Ca2+-regulated ion channels or
Ca2+-dependent kinase cascades.
Finally, as for any membrane proteins in a polarized epithelium,
particular P2 receptors might be selectively localized to the apical or
basolateral surfaces or be expressed on both sides of the cell.
To date, most investigations of epithelial P2 receptors have utilized
tissue-cultured cells (primary isolates or immortalized lines) plated
on permeable filters that permit the establishment of polarized
monolayers with appropriate transcellular resistances. Such studies of
tissue-cultured epithelia have demonstrated that some epithelial cells
can express multiple P2 receptor subtypes and that different P2
receptors can be localized on apical, basolateral, or both cellular
surfaces (2, 9). However, there is always concern that the phenotype of
tissue-cultured cells may differ from that of the cells within their in
vivo tissue of origin. This is a relevant issue with regard to P2
receptors since the expression of these receptors can be very plastic
and subject to upregulation or downregulation by growth conditions,
hormones, and cytokines. Indeed, comparison of the P2 receptors present in certain cells, such as vascular smooth muscle cells (12) and
salivary gland cells (19), immediately following isolation or after
several days of tissue culture, reveals marked differences in the
distribution of P2 receptor subtypes.
Luo et al. (11a) have sidestepped this concern by performing all
studies on rat pancreatic duct cells on the day of isolation. Intact
ductal tubules were microdissected away from contaminating blood
vessels, connective tissue, and pancreatic acini and then used for
either functional studies or RT-PCR analyses of P2 receptor subtype
mRNA expression. The functional experiments involved luminal microperfusion of individual ductal tubules to provide access to apical
P2 receptors and simultaneous superfusion of the surrounding bath
medium for activation of any basolateral receptors. Changes in the
cytosolic Ca2+ within the cells
comprising the tubule were recorded during stimulation of either cell
surface with nucleotide analogs that show varying degrees of
selectivity for different P2 receptor subtypes. The studies of
microperfused tubules were complemented by whole cell patch-clamp
analyses of visually identified single ductal cells. The same
nucleotide analogs were tested for activation of
1) nonselective inward cation
currents that are characteristic of the intrinsic ATP-gated channels of
P2X family receptors, 2)
Ca2+-dependent
Cl What are the physiological implications for a single cell type that can
express at least six nucleotide receptor subtypes? Before the molecular
identification of these multiple receptor genes, P2 receptors were
categorized as "purinergic" receptors and ATP or ADP were assumed
to be the sole physiological agonists. However, three of the five P2Y
receptor subtypes (P2Y2,
P2Y4, and
P2Y6) are activated by uridine
nucleotides at submicromolar levels. Although ATP and UTP are
equipotent agonists for P2Y2 receptors, P2Y4 and
P2Y6 receptors can exhibit high
(1-3 log units) selectivity for uridine nucleotides over adenine
nucleotides, with P2Y4 acting
essentially as a UTP-preferring receptor and P2Y6 as a UDP-selective receptor
(8). These findings strongly suggest that uridine nucleotides are the
physiological agonists for these latter receptors and that uridine
nucleotides may be also released from cells in response to different
extrinsic stresses (1, 11). Given that Luo et al. observed a selective
expression of the uridine nucleotide-sensitive receptors
(P2Y2 and
P2Y4) in the apical membranes of
the ductal epithelial cells, it is interesting to speculate whether UTP
might be preferentially released into the luminal compartment under
some conditions. It should be noted that extracellular nucleotide tri-
and diphosphates can be rapidly metabolized by CD39 family ectoapyrases
to nucleotide monophosphates (20). In turn, extracellular
monophosphates can be hydrolyzed to their respective nucleosides (AMP
to adenosine and UMP to uridine) by the CD73 5'-ectonucleotidase
that is highly expressed in the apical membranes of many epithelial
cells (21). Localized release of uridine vs. adenine nucleotides into
the lumen could facilitate the selective stimulation of
Ca2+-mobilizing P2Y receptors
while minimizing the generation of adenosine and de facto activation of
adenylyl cyclase-coupled adenosine receptors that also are expressed in
many epithelia (17). However, P2Y receptor subtypes can also
differentially activate phospholipase A2 effector enzymes, with
consequent release of arachidonic acid and sundry eicosanoids such as
prostaglandins and leukotrienes. Thus local responses to particular
nucleotides may be further amplified or attenuated by the tertiary
signaling cascades regulated by released eicosanoids. For example, in
renal epithelial cells (which also express multiple P2Y subtypes),
uridine nucleotide-sensitive P2Y2
receptors are more effective than adenine nucleotide-selective P2Y1 receptors in triggering a
tertiary autocrine cascade that involves stimulation of cytosolic
phospholipase A2, release of PGE2, and activation of adenylyl
cyclase (14).
That Luo et al. observed expression of multiple P2X family receptors in
(primarily) the basolateral membrane of the pancreatic duct cells also
raises intriguing issues. In contrast to the various P2Y subtypes, all
P2X receptor subtypes exhibit very high selectivity for ATP over all
other physiological nucleotides. However, the relative agonistic
potency of ATP (as indexed by EC50
values) ranges from 1 µM (P2X1
and P2X3) to 10 µM
(P2X2, P2X4, P2X5,
P2X6) to 300 µM (P2X7). The various P2X
receptors also exhibit large differences in their rates and extents of
desensitization (5). The three subtypes expressed in the ductal
epithelia span the range of desensitization extremes, with
P2X1 being characterized by rapid
complete desensitization, P2X4
showing intermediate desensitization, and
P2X7 acting as a noninactivating
channel. Thus the ductal epithelial cells appear to express ATP-gated
ion channel subtypes that might be activated in a hierarchic manner,
depending on the magnitude and duration of localized changes in
extracellular ATP at the basolateral surface.
The findings of Luo et al. set the stage for many interesting
questions. How do the various P2Y and P2X receptors work together to
coordinate fluid and electrolyte transport in the endocrine pancreas?
What are the cellular sources of the nucleotides required for
activation of this plethora of related receptors? What are the stimuli
that induce release of these nucleotide stores? Are adenine nucleotides
and uridine nucleotides released by common or distinct mechanisms?
Finally, the expression of multiple P2 receptor subtypes has been
observed in many nonepithelial cell types (4), including endothelial
cells (3), blood cells (10), and smooth muscle (12). Thus highly
specialized and highly localized actions of particular nucleotides and
their cognate P2 receptor subtypes may be general features of
integrated tissue functions that require rapid and local fine tuning at
the cellular level. Autocrine and paracrine signaling loops based on
specific extracellular nucleotides may play heretofore unsuspected
roles in many localized tissue responses such as hemostasis,
thrombosis, inflammation, and vasodilation.
ARTICLE
TOP
ARTICLE
REFERENCES
current that ultimately
facilitates loss of intracellular KCl and subsequent shrinkage.
Although the precise mechanisms that underlie facilitated release of
ATP from epithelial cells remain undefined, pharmacological studies
suggest that ATP-binding cassette family membrane proteins are somehow
involved in this process (16). These known or hypothesized effects of
extracellular nucleotides on epithelial function have also prompted
interest in defining the nature and location of the receptors that
actually bind the released nucleotides and thereby trigger adaptive
responses of the particular epithelial tissue. In the current article
in focus (Ref. 11a, see page C205 in this issue), Luo et
al. present an elegant and informative analysis of P2 nucleotide
receptor expression and function in pancreatic duct cells. Their
studies show that these cells express a remarkable diversity of
nucleotide receptors in both their apical and basolateral membranes.
currents that can be
activated by any receptor coupled to
Ca2+ mobilization or influx, and
3)
Ca2+-independent
Cl
currents. By performing
these electrophysiological studies with or without guanosine
5'-O-(2-thiodiphosphate) in the
electrode solution used for intracellular dialysis, Luo et al. could
further discriminate those currents that were indirectly regulated by G
protein-coupled P2Y receptor subtypes vs. those regulated by the
directly ionotropic P2X receptor subtypes. These carefully designed
experiments revealed that pancreatic duct epithelial cells express
three (P2Y1,
P2Y2, and
P2Y4) of the five functionally characterized P2Y receptor subtypes encoded by distinct genes expressed
in mammalian cells. The P2Y2 and
P2Y4 receptors are selectively
targeted to the apical membrane, whereas the ADP-selective P2Y1 receptors could be detected
when ADP was presented to either the apical or basolateral surfaces.
The cells also express three (P2X1,
P2X4, and
P2X7) of the seven known P2X
family receptor subtypes. This class of receptors also showed
asymmetric localization, with P2X1
and P2X4 receptors being limited
to basolateral membranes, whereas
P2X7 receptor function could be
assayed at both cell surfaces.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. R. Dubyak, Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106 (E-mail:gxd3{at}po.cwru.edu).
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