Regulated Extracellular Nucleotide Metabolism and Function at the Mucosa - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 125

within the isolated granules were nearly undetectable. Moreover, PMN cytoso-
lic fractions contained ATP concentrations that were higher than 5 mM, thereby
suggesting that activation-dependent ATP release likely occurs independent of clas-
sical PMN degranulation. Based on these findings, a pharmacologic approach was
employed to examine potential mechanisms. Brefeldin A (BFA), a general vesicular
secretion inhibitor did not influence activated PMN ATP secretion. Likewise, nei-
ther the nucleoside transport inhibitor dipyridamole nor the general ABC transport
inhibitor verapamil significantly influenced PMN ATP secretion [14].
Based on previous reports suggesting that connexin hemichannels may serve as
ATP release channels [23] and the observation that PMN express surface connex-
ins [74], the non-specific gap junction inhibitor 18- α -glycyrrhetinic acid (18 α GA)

was examined. These studies revealed that 18 GA inhibited ATP release in a

concentration-dependent manner. Likewise, connexin-mimetic peptides specifically
directed against Cx43 [23], but not Cx40, significantly blocked ATP liberation from
activated PMN. Cx43 molecules can assemble as hexadimers (so called “connex-
ons”) that form junctional connections between different cell types. In addition to
their role as gap-junction proteins, recent studies indicate that Cx43 connexons
are also active in single plasma membranes and can function in intercellular sig-
naling as ATP release channels [23]. The conductance and permeability of such
Cx43 hemichannels is regulated by modification of their cytoplasm domain, with
phosphorylation of Ser-368 causing a conformational change resulting in decreased
connexon permeability [5]. Studies addressing Cx43 Ser-368 phosphorylation in
intact PMN showed prominent phosphorylation in resting PMN and protein phos-
phatase 2A-dependent dephosphorylation within minutes of PMN activation. As a
proof of principle, PMN were isolated from tamoxifen-inducible Cx43 condition-
ally deleted mice [12] and examined for ATP release. These studies revealed that
ATP release correlated with the degree of Cx43 expression. In total, these studies
provide strong evidence that ATP release occurs through a conformational opening
of membrane Cx43 hemichannels in response to PMN activation [14].
In addition to neutrophil-derived extracellular nucleotides, there is increasing
evidence to suggest that epithelial cells hydrolyze platelet-derived ATP as platelets
migrate alongside neutrophils across an epithelial barrier. In a recent study by
Weissmuller et al., it was revealed that platelets “piggyback” on the surface of
PMN during transmigration in vitro and in biopsies from patients with inflamma-
tory bowel disease [71]. Together, these cells represent a rich source of ATP at
the luminal aspect of the epithelium. Indeed, translocated platelets were observed
to release large quantities of ATP, which is metabolized to adenosine via a 2-step
enzymatic reaction mediated by ectonucleotideases, including CD73 and NTPDases
expressed on the luminal membrane of the epithelium. Measurement of electrogenic
Cl secretion in human epithelial cells demonstrated the ability for platelet/PMN
derived ATP to induce copious amounts of fluid transport, which was blocked by
the pan-NTPDase inhibitor POM-1. Futhermore, blockade of CD73 using APCP
also abrogated ATP-mediated Cl - secretion, as did inhibition of the adenosine A2B
receptor using a specific antagonist, MRS1754. Further studies using a Cd73 − / −
knockout mouse model, it was demonstrated that ATP induced water transport

α