Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
IN ADDITION TO THEIR
ROLES in intracellular energy metabolism and nucleic acid
synthesis, ATP and other nucleotides play important functions as
extracellular signaling molecules (6, 15).
Burnstock (3) first proposed that extracellular
nucleotides can be used for signal transduction at nerve endings in
diverse tissues. Consistent with this concept of "purinergic"
neurotransmission were observations that neurons and neuroendocrine
cells release ATP via classic mechanisms involving exocytotic release
of nucleotides copackaged with biogenic amines or other
neurotransmitters within specialized secretory vesicles or granules
(19). Research during the past decade has resulted in the
cloning and characterization of at least 14 distinct ATP/nucleotide
receptors (8, 15), 4 adenosine receptors
(15), and at least 9 different ectonucleotidases
(21, 22). Virtually every mammalian cell type appears to
express one or more subtype of nucleotide or nucleoside receptor along with one or more of the ectonucleotidases used for scavenging extracellular nucleotides. However, the vast majority of these cells
lack direct physical proximity to neurons or neuroendocrine cells. Thus
the identification of alternative sources of extracellular nucleotides
and the elucidation of mechanisms underlying this nonsynaptic
nucleotide release have become significant areas of current
investigation. In the studies described in the current article in focus
(Ref. 17; see p. C289 in this issue), Schwiebert et al. provide significant new insights regarding the role of vascular endothelial cells (EC) as both sources of extracellular ATP
and autocrine targets of this released ATP.
Previous studies determined that multiple types of vascular EC,
including human umbilical vein EC (2, 12), EC from rat aorta and caudal artery (18), porcine aortic EC
(14), and guinea pig cardiac EC (20), can
release nucleotides via noncytolytic but otherwise undefined
mechanisms. Two types of stimuli were shown to trigger ATP release from
these various EC types: mechanical stress during changes in flow and
activation of Ca2+-mobilizing receptors for different
vasoactive hormones/neurotransmitters. These nucleotide release
agonists included bradykinin, acetylcholine, thrombin, norepinephrine,
and ATP itself. Schwiebert et al. (17) have now extended
these earlier findings in several ways.
First, these authors utilized on-line, luciferase-based luminometry to
directly record ATP release from human EC maintained as polarized and
confluent monolayers on permeable filter supports. This particular
methodological approach was useful in two ways. Previous studies of ATP
release from EC utilized "off-line" methods that involved removal
of bathing medium at various times following stimulation of the EC with
agonists or mechanical stress, followed by enzymatic or HPLC-based
analyses of nucleotide content within these extracellular samples
(2, 12, 14, 18, 20). Although sensitive, such off-line
assays suffer from a limited capacity for temporal resolution of
transient ATP release events from EC. This can be a critical limitation
because ATP released from EC will be rapidly catabolized to AMP and
adenosine by the combined actions of CD39-family ectoapyrases and the
CD73 ecto-5'-nucleotidase. By including luciferase (an enzyme with
exquisite sensitivity to, and selectivity for, ATP) and luciferin in
the extracellular medium bathing their EC monolayers, Schwiebert et al.
(17) were able to record minute-to-minute variations in
ATP content at the EC extracellular surface. Moreover, by selectively
including luciferase/luciferin in the extracellular medium bathing
either the apical or basolateral EC surfaces, they were able to
document a marked polarization of basal and mechanically stimulated ATP
release to the apical surface. This would suggest a similar polarized
localization of the ATP release mechanism(s), be it exocytosis of
ATP-laden vesicles or facilitated efflux of cytosolic ATP.
Another significant finding was that human EC released ATP at a
significant rate even in the absence of obvious mechanical stimulation
or addition of Ca2+-mobilizing agents. By directly
measuring basal ATP release at the surface of unstimulated EC with high
sensitivity and temporal resolution, Schwiebert et al.
(17) provided a third experimental approach for
complementing similar findings from other investigators working with
different cell models. Previous data from two other experimental
protocols have provided convincing support for this notion of
constitutive ATP release. Several groups observed that treatment of
nominally unstimulated cells with extracellular nucleotide scavenger
enzymes, such as potato apyrase or hexokinase, can reduce the cytosolic
levels of second messengers, such as cAMP and inositol trisphosphate
(10, 13, 16). Ostrom et al. (13) provided particularly salient evidence that this reduction in second messenger levels by nucleotide scavengers indicated that endogenous ATP (and/or
UTP) is released from cells in amounts sufficient to produce low-level
activation of various G protein-coupled P2Y receptors. Schwiebert et
al. similarly found that treatment of EC with extracellular nucleotide
scavengers induced a rapid and reversible decrease in the basal
concentration of cytosolic [Ca2+]. A second type of
experimental support for constitutive ATP release was provided by
Lazarowski et al. (9), who employed conventional isotopic
tracer methods to measure the steady-state rates of extracellular
nucleotide metabolism by four different cell lines. Those authors
observed that the four cell types steadily maintained extracellular ATP
at the 1-10 nM range for many hours under standard, serum-free
tissue culture conditions. The cells were then pulsed with
extracellular [ Schwiebert et al. (17) performed additional ATP release
studies at various temperatures and in the presence of various
pharmacological reagents. This approach provided suggestive evidence
for involvement of an exocytotic mechanism in the ATP release triggered
by Ca2+-mobilizing agents and mechanical stress but not in
the constitutive ATP release process. These findings indicated that
distinctive cellular mechanisms may underlie constitutive ATP release
vs. stress-stimulated release. Although EC contain exocytotic granules in the form of the Weibel-Palade bodies, no studies have evaluated whether these granules compartmentalize and release nucleotides (4). However, even vesicles involved in the constitutive,
Ca2+-independent release of secreted proteins are likely to
contain low levels of nucleotides due to important roles for
intravesicular, nucleotide-dependent enzymes that catalyze the covalent
modification and maturation of secreted proteins (7). The
likely presence of nucleotides within vesicles that comprise the
constitutive exocytotic machinery may have important implications for
understanding mechanisms for basal ATP release and autocrine activation
of P2 receptors in different cell types. Indeed, the recent studies of
Maroto and Hamill (11) on ATP externalization from single Xenopus oocytes suggested that brefeldin A-sensitive
exocytotic pathways were involved in both basal and mechanically
stimulated nucleotide release in that cell type. It will be interesting
to adapt the methods of Schwiebert et al. to determine whether similar brefeldin A-sensitive pathways might play similar roles in either the
basal or stimulated ATP release observed in EC monolayers.
Thus the use of on-line luciferase-based assays can clearly illuminate
(pun intended) a variety of possible pathways for ATP release from
intact cells. It should be stressed that use of this assay system for
measuring ATP release can be compromised by the spurious effects of
pharmacological reagents on the intrinsic ATP sensitivity or catalytic
rate of the luciferase present within the extracellular medium. As
noted by both Schwiebert et al. (17) and other
investigators (1, 11), a variety of P2 receptor antagonists, ectonucleotidase inhibitors, and modulators of various transporters/channels can affect luciferase activity. Thus changes in
luminescent intensity upon addition of such reagents can reflect decreased luciferase activity at a constant level of extracellular ATP.
Care must be taken to discriminate such changes in luciferase activity
from bona fide changes in extracellular ATP concentration. This is a
significant issue because there is disagreement among different
laboratories regarding the effects of certain ATP release blockers,
such as Gd3+, on luciferase activity. Whereas Schwiebert et
al. have observed no significant inhibitory effects of Gd3+
on luciferase activity, others (11) have reported that
Gd3+ can significantly reduce the ATP sensitivity of
luciferase. Whether this reflects differences in particular assay
conditions is an important methodological concern that requires
clarification. The same caveat applies to the known effects of ionic
milieu on intrinsic luciferase activity (5). Schwiebert et
al. stressed that the high Cl
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ARTICLE
REFERENCES
-32P]ATP at tracer levels that did not
significantly change the extracellular ATP concentration.
Significantly, the [
-32P]ATP tracer was rapidly and
completely metabolized. This finding demonstrated that the steady-state
level of extracellular ATP in the cell cultures reflected a
constitutive rate of ATP release that was balanced by ATP hydrolysis
measured at 20-200
fmol · min
1 · cell
1.
Significantly, the basal rate of ATP release from EC estimated by the
direct luciferase-based assay of Schwiebert et al. was similar in magnitude.
concentration in normal
extracellular saline results in reduced catalytic activity of
luciferase. However, the authors did not discuss how the acute
reduction in extracellular Cl
concentration, which
necessarily accompanies their hypotonic stress stimulus, should result
in enhanced luciferase activity and light output even in the presence
of unchanged ATP concentration. Thus the rapid increase in luminescence
triggered by hypotonic stimulation of the EC may reflect on the
combined effects of mechanically induced ATP release and increased
sensitivity of the extracellular luciferase to ambient ATP.
Establishing protocols for deconvoluting such luciferase-based signals
would be a further improvement to such on-line assays of ATP release
and extracellular metabolism. As shown by these informative studies of
Schwiebert et al. on primary endothelial cells, the excellent temporal
resolution and high sensitivity of this method should provide a
powerful approach for testing multiple models of nucleotide efflux or
secretion from diverse eukaryotic cells.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. R. Dubyak, Dept. of Physiology and Biophysics, Case Western Reserve Univ., Cleveland, OH 44106 (E-mail: gxd3{at}po.cwru.edu).
10.1152/ajpcell.00522.2001
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