EDITORIAL FOCUS
Focus on "Multiple functional P2X and P2Y receptors in the luminal and basolateral membranes of pancreatic duct cells"

George R. Dubyak

Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106


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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- 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.

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- 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.

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.


    FOOTNOTES

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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Am J Physiol Cell Physiol 277(2):C202-C204
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