1. Emerging roles of purinergic signaling in gastrointestinal epithelial secretion and hepatobiliary function 
Richard M. Roman, J.Gregory Fitz 
Gastroenterology 
Volume 116, Issue 4, Pages (April 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Purinergic signaling pathways. ATP and UTP are released from cells in response to many physiological stimuli. Once in the extracellular space, nucleotides are available to stimulate membrane P2 receptors (P2R). These effects are tightly regulated by membrane-associated nucleotidases that rapidly dephosphorylate nucleotides. Adenosine, the end product of nucleotide hydrolysis, may simulate P1 receptors (P1R) or be taken up by the cell via specific nucleoside transport pathways (NT).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Classification of known P1 and P2 receptors. P1 and P2Y receptors are typical GPCRs, and P2X receptors are ligand-gated ion channels. P1 receptors function primarily by stimulation or inhibition of adenylyl cyclase. Agonist preferences for P2Y receptors are shown; most of these proteins are capable of activating phospholipase C (PLC) but modulate many other effector pathways as well.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Transmembrane topology of GPCRs and ligand-gated P2X receptors (P2XR). Adenosine receptors (AR) and P2Y receptors (P2YR) contain 7 transmembrane-spanning domains, with extracellular amino (N) and cytoplasmic carboxy (C) termini. With only two membrane-spanning domains, conformational changes in P2X proteins (P2XR) after stimulation exposes a cation-selective pore that allows influx of Na+ and Ca2+.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Mechanisms of transmembrane ATP transport. In neurons, blood cells, and endocrine cells, ATP copackaged in secretory granules with neurotransmitters, hormones, and other substances is released during exocytosis. In epithelial cells, a large (~10,000-fold) transmembrane gradient favors movement of cytosolic ATP molecules into the extracellular space, and one mechanism is likely to involve electrodiffusional transport through membrane channel proteins. Whether carrier-mediated mechanisms contribute to ATP release has not yet been determined.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Measurement of cellular ATP release. (A) With whole-cell patch clamp techniques (top), a single cell is dialyzed and transmembrane currents carried by charged ATP molecules are measured (100 mmol/L [MgATP]2− present on both sides of the membrane as the only charge carrier). This graph depicts the current-voltage relation of an ATP conductance in human biliary cells, which reverses near 0 mV. Compared with basal (isotonic) conditions (●), exposure to hypotonic buffer (20% less NaCl) to increase cell volume markedly enhances ATP currents (▾). (B) Using the luciferase-luciferin assay, ATP released from cell monolayers into media catalyzes the oxidation of luciferin and generates bioluminescence, which is detected and quantified as arbitrary light units (top). Relative concentrations of ATP are determined by generating ATP standard curves. Graded dilutions of media to induce cell swelling (arrows corresponding to 20%–40% dilutions) increase ATP permeability in rat hepatoma cells (●) compared with control studies in which isotonic buffer was added instead of water (▾).141 Addition of the ATP scavenger apyrase (2 U/mL) eliminates bioluminescence.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Ectoenzyme-associated nucleotide degradative pathways. ATP released into the extracellular space is rapidly hydrolyzed by either ecto-ATPase or ecto-ATPDase (the latter also hydrolyzes ADP). In addition, ecto–nucleoside diphosphokinase (NDP kinase) may use an ATP phosphate to convert UDP to UTP. AMP is hydrolyzed to adenosine by ecto-5'-nucleotidase, which associates as a disulfide-linked dimer linked to the membrane by glycosyl phosphatidylinositol (GPI). These pathways also apply to uridine nucleotides.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 7
ATP stimulates transepithelial secretion across cholangiocyte monolayers. In this example, polarized cell monolayers are mounted in an Ussing chamber, and the transepithelial movement of Cl− ions from the basolateral to apical space is measured as short-circuit current (Isc, A). Under basal conditions, Isc is low (B). However, addition of ATP (bar) activates P2Y2 receptors (P2R) in the apical membrane and elicits a brisk Cl−-secretory response as demonstrated by a marked increase in Isc. Apical Cl− secretion occurs via opening of apical membrane Cl− channels and is inhibited by application of the selective anion channel blocker NPPB (bar) (adapted and reprinted with permission101).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 8
Autocrine regulation of HTC cell volume by ATP. In model liver cells, ATP is released during increases in cell volume. Subsequently, extracellular ATP enhances channel-mediated Cl− efflux by stimulating membrane P2 receptors (P2R) in an autocrine fashion, which favors passive water loss and RVD (A). (B) Relative changes in rat hepatoma cell volume during exposure to hypotonic buffer (30% decrease in osmolarity, indicated by bar) as measured by a Coulter Multisizer. In control cells (●), volume increases rapidly but then recovers toward basal values despite continued exposure to hypotonic stress; volume recovery is dependent on channel-mediated Cl− efflux (not shown). Addition of apyrase (1 U/mL) to scavenge extracellular ATP prevents RVD (■). However, addition of the nonhydrolyzable P2R agonist ATPγS (20 μmol/L added at 2.5 minutes) restores volume recovery in the presence of apyrase (▾). Values for relative volume represent means ± SE of ~20,000 cells in suspension per time point (adapted and reprinted with permission from Proceedings of the National Academy of Sciences. Copyright 1996 National Academy of Sciences, U.S.A.).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Hepatobiliary coupling by extracellular ATP. In this proposed model, ATP released during changes in cell volume or other stimuli across hepatocyte canalicular and cholangiocyte apical membranes acts as a paracrine luminal signaling molecule. Stimulation of apical cholangiocyte P2 receptors (P2R) enhances channel-mediated release of Cl− into the bile duct lumen, which favors alkalization of bile by Cl− HCO3− exchange and biliary secretion by paracellular movement of water and electrolytes. Purinergic signaling is likely to be rapidly modified by ectoenzyme-associated hydrolysis of nucleotide phosphates and cellular salvage of nucleosides across canalicular (not shown) and apical cholangiocyte membranes. The identity of nucleoside transporters in biliary cells is unknown.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions