Regulated Extracellular Nucleotide Metabolism and Function at the Mucosa - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 124

barriers, they must effectively deform a tightly sealed cellular barrier. Since the
active paracellular movement of PMN can cause disruptions to epithelial/endothelial
cell layers, intrinsic pathways must exist to “reseal” the disrupted cells in order to
prevent further tissue damage. Original studies revealed that PMN migrating across
an endothelial barrier secreted 5 AMP which resulted in increased barrier func-
tion as demonstrated by a decrease in paracellular permeability [39]. Furthermore,
blockade of CD73 activity using a neutralizing antibody (1E9), or pharmacological
blockade of CD73 using -methylene ADP (APCP) resulted in an impaired abil-

ity for the migrating neutrophils to reseal both epithelial and endothelial barriers
[39]. This observation strongly suggested the necessity for extracellular nucleotide
metabolism in the regulation of cell barrier function . In addition to the regula-
tion of barrier function from neutrophil-derived AMP, autocrine ATP signaling has
been shown to induce leukocyte sequestration in ischemic cerebral tissue through
P2X 7 -mediated upregulation of

α , β

α M β 2 integrin. Interestingly, this phenomenon of
leukosequestration is inhibited by CD39 directed catalysis of ATP [28]. Once neu-
trophils have tethered to the endothelium, they respond to inflammatory damage
by flowing a chemical gradient set up by a number of chemoattractants secreted
at sites of tissue injury or inflammation. One important mediator of neutrophil
chemotaxis has been identified as interleukin-8 (IL-8). ATP has been shown to stim-
ulate production of IL-8 by astrocytes and eosinophils [29, 32]. Furthermore, ATP,
through P2Y activation has been demonstrated to synergistically induce IL-8 medi-
ated chemotaxis in human neutrophils in vitro [37]. Recent studies have identified
the importance of ATP metabolism in regulating the rate of IL-8, C5a and formyl-
methionyl-leucyl-phenylalanine (fMLP)-mediated PMN chemotaxis. Corriden et al
demonstrated the ability for NTPDase1 at the leading edge of migrating neutrophils
to hydrolyze PMN-derived ATP to AMP, where further metabolism to adeno-
sine, potentially by alkaline phosphatase controls the rate of PMN migration [9].
Additionally, PMN from NTPDase knockout mice exhibited ablation of migra-
tion towards IP injected murine fMLP receptor ligand (W-peptide) and a reduced
migration rate in vitro towards an fMLP gradient [9].
More recent studies have revealed that the active fraction originally defined as
PMN-derived 5 AMP may actually be ATP. Indeed, studies directed at understand-
ing vascular barrier function during conditions of inflammation or hypoxia identified
the existence of a soluble fraction derived from activated PMN which amplified the
resealing of vascular barrier function following subjection of cultured endothelial
cells to periods of hypoxia. A screen of HPLC purified fractions identified this activ-
ity as PMN-derived ATP [15]. Like 5 -AMP, this activity was functionally linked to
CD73 and to Ado receptors. This was puzzling, since CD73 was not known to uti-
lize ATP as a substrate. Subsequent studies directed at understanding mechanisms
of ATP release by activated PMN considered several potential mechanisms, includ-
ing exocytosis of ATP containing vesicles, transport via connexin hemichannels,
transport through nucleoside transporters, or direct transport through ATP-binding
cassette (ABC) proteins [46]. Initially, it was determined that ATP does not localize
with known granule markers in PMN. While isolated granules from resting PMNs
contained greater than 95% of proteolytic enzyme activity markers, ATP levels