Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
WHY WOULD A
CELL want to release its ATP? For physiologists and cell
biologists studying extracellular purinergic signaling, this is the
question that keeps us awake at night. Those scientists who doubt that
a cell would want to do such a horrible thing suggest that cell lysis,
cell damage, or some pathophysiological condition would cause ATP to
appear in the extracellular milieu. Give up its own intracellular ATP
and compromise metabolism and enzyme kinetics: ludicrous!
Those scientists who favor physiological reasons for ATP release from
cells answer with the fact that most, if not all, cells express
multiple purinergic receptor subtypes, often of different varieties.
Moreover, many innovative methods have been developed recently to study
regulated and physiological ATP release from cells and monolayers in
real time (see below). Purinergic signaling experts might answer
skeptics with another question: Why would a particular cell express
multiple P2Y G protein-coupled ATP receptors and multiple P2X ATP-gated
receptor channels, unless the cell had a very good reason to sense and
receive extracellular ATP in the external environment?
The study by Sauer et al., the current article in focus (Ref. 14, see
p. C295 in this issue), provides a physiological reason for ATP release
and autocrine and paracrine ATP signaling in the extracellular medium.
With the invention of the Ca2+-sensitive dye fura 2, Ca2+ sparks in individual cells and Ca2+ waves
across cell monolayers were soon discovered (6,
19). As pointed out by Sauer et al., initial attention was
focused on phospholipid mediators such as inositol trisphosphate
[Ins(1,4,5)P3] as a mediator of Ca2+ waves. In monolayers of cells that
maintained gap junctional communication,
Ins(1,4,5)P3
increased intracellular Ca2+ (Cai2+) in the
cell that was stimulated, but it also diffused intercellularly through
gap junctional channels and emptied intracellular Ca2+
stores in neighboring cells until the critical
Ins(1,4,5)P3
concentration was diluted beyond effect. For that matter,
Cai2+ itself could also diffuse laterally between cells
via these gap junctional channels as a wave.
This paper shows that Ca2+ wave propagation can occur
without gap junctional communication between cells. Sauer et al. used a
human prostate cancer cell line to show elegantly and with beautiful fura 2-based fluorescence imaging technique that ATP released from a
mechanically stimulated cell diffused to neighboring cells and
stimulated P2 purinergic receptor-mediated increases in cytosolic free
Ca2+ in neighboring cells without evidence of gap
junctions. Ca2+ wave propagation in these monolayers was
inhibited by four independent maneuvers to circumvent extracellular ATP
signaling: 1) purinergic receptor antagonists, 2)
pretreatment with agonists to both P2Y G protein-coupled receptors and
P2X receptor channels, 3) depletion of intracellular ATP
pools with 2-deoxyglucose, and 4) the ATP scavenger apyrase.
Moreover, both the Ca2+ wave and mechanically induced ATP
release, measured by bioluminescence, were inhibited by a panel of
anion channel-blocking drugs.
Figure 1 illustrates these findings and
provides some basic information as to gradients, mechanisms of release,
and purinergic receptors that may be important in this human prostate
cancer cell model system studied by Sauer et al. First and foremost, there is a large gradient for ATP efflux, transport, or secretion out
of cells. Intracellular ATP concentrations are millimolar (range:
1-10 mM). Whereas extracellular ATP concentrations rely on the
balance between release and degradation, recent assays designed to
measure this concentration estimate a range from nanomolar to
micromolar (maximum extracellular concentration
measured with 10 µM). ATP could be released by any of
three different mechanisms: conductive transport, nonconductive
transport (permease, transporter, flippase), or exocytosis (Fig. 1).
The fact that anion channel inhibitors blocked mechanosensitive ATP
release suggests that an ATP-permeable anion channel is essential;
however, additional mechanisms may be involved. Once released, ATP
diffuses in a paracrine manner and binds to and stimulates either P2Y G
protein-coupled receptors or P2X receptor channels, or both. The fact
that pretreatment with both P2Y-selective agonists (UTP, UDP) and a
P2X-selective agonist (benzoylbenzoyl-ATP) attenuated Ca2+
waves suggests the involvement of both P2Y G protein-coupled receptors
(coupled to phospholipases and increases in cytosolic Ca2+)
and Ca2+-permeable, ATP-gated P2X receptor channels in
Ca2+ wave propagation.
ARTICLE
TOP
ARTICLE
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Paracrine ATP-dependent Ca2+ wave
propagation. Sauer et al. show that ATP, likely through a
mechanosensitive ATP-permeable anion channel, is released in the
extracellular milieu and diffuses to a neighboring cell to propagate a
Ca2+ wave via purinergic receptors expressed by the
neighboring cell in a monolayer devoid of gap junctions. P2YR, a P2Y G
protein-coupled purinergic receptor that couples to phospholipases and
inositol phosphates and that increases in cytosolic Ca2+
(Cai2+) from intracellular stores; P2XR, a P2X
purinergic ATP-gated and Ca2+-permeable nonselective cation
channel that increases Cai2+ directly from
extracellular stores; N, amino terminus; C, carboxy terminus.
This novel paper by Sauer et al. establishes a new role for extracellular ATP signaling in monolayers of cells. In more polarized monolayers that have tight junctions and gap junctions, both extracellular autocrine and paracrine purinergic signaling and gap junctional communication may integrate to propagate Ca2+ waves. Indeed, in 1997, Frame and de Feijter (4) showed that paracrine ATP signaling as well as gap junctional communication was important for Ca2+ wave propagation in rat liver epithelial cell lines that lacked or maintained gap junctions. It was the conclusion of these authors, however, that the ATP was released due to cell injury or damage. It is also important to note that the potential applicability of this paracrine ATP-dependent Ca2+ wave propagation to epithelial cells, endothelial cell, vascular smooth muscle, cardiac myocytes, and neurons is intriguing.
In addition to this highlighted study, other studies have shown the cell biological and physiological importance of the release of ATP, other nucleotides, and nucleosides. ATP release during hypotonic challenge has been shown to be essential for autocrine control of cell volume regulation in hepatocytes (20) and airway epithelial cells (18). Extracellular ATP potentiates ciliary beat frequency in ciliated epithelial cells, possibly through stimulation of P2X receptors in the cilia membrane (10, 17). ATP and ADP released by platelets at the clotting zone promote self-aggregation of platelets in the clot (2). Purinergic signaling also regulates ion and fluid transport (9, 12, 13), leukocyte degranulation (11), and vascular tone (5). Extracellular ATP even acts as a mitogen for vascular smooth muscle cells (3), astrocytes (8), and mesangial cells (16). Taken together, the critical mass of this list demonstrates that extracellular purinergic signaling is essential for the normal cell biology and physiology of many cells and tissues.
It is likely, however, that extracellular purinergic signaling is most
effective in microenvironments. To study ATP release, many laboratories
have developed innovative techniques to measure ATP release in real
time. Taylor et al. (18) adapted the luciferase-luciferin assay system to study ATP release from epithelial monolayers in real
time by lowering an epithelial monolayer into a luminometer and
studying ATP release as it occurred. Upon stimulation in the epithelial
cell models that demonstrated the most ATP release, a maximal ATP
concentration of 5-10 µM was measured (18,
21). Beigi et al. (1) developed a reagent
that linked the luciferase enzyme to protein A, a protein that binds to
IgG antibodies. This construct could then be linked to an IgG that
recognized an extracellular epitope on a transmembrane glycoprotein to
measure the local ATP concentration on the extracellular surface of the
plasma membrane. Hazama et al. (7) developed a biosensor
technique that involves two steps. First, a whole cell recording on a
PC-12 cell is obtained that expresses a P2X receptor channel. Second,
the PC-12 cell that is recorded from is then moved by micromanipulation
to an island or culture of the cell of interest that is hypothesized to
release ATP. When ATP release was measured from pancreatic -cells
following stimulation with Ca2+ agonists, a concentration
that reached 10 µM was assayed. Finally, Schneider et al.
(15) used atomic force microscopy with commercially available probes coated with myosin subfragment S1, which has a high
affinity for ATP and changes shape upon ATP hydrolysis. The tips are
placed near to the cell membrane and, as the ATP is released, the
myosin on the tip changes shape, and the probe vibrates at a frequency
that correlates to the amount of ATP substrate released. They used this
technique to show significant probe vibration only in cystic fibrosis
airway epithelial cells that were corrected by transfection with the
wild-type cystic fibrosis transmembrane regulator gene. In short, many
different methods have successfully measured biologically relevant ATP release.
In closing, the work by Sauer et al. has only enhanced further the field of extracellular purinergic signaling. Moreover, the role of this extracellular ATP-dependent Ca2+ wave propagation in a human prostate cancer cell line begs the following question: Does this paracrine purinergic signaling system play a role in cancer cell biology, especially with regard to proliferation of cancer cells, in tumor formation, and in metastasis? The only other study, found in many different literature searches, that showed ATP agonist-dependent Ca2+ wave propagation was also done on immortalized rat liver epithelial cell lines (4). It is possible that paracrine purinergic signaling may be enhanced or dampened in immortalized vs. primary cell cultures. A comparison of normal vs. immortalized or cancer cells may be warranted and may also yield fruitful results with regard to a role of extracellular ATP signaling in cancer.
![]() |
ACKNOWLEDGEMENTS |
---|
I especially thank graduate students Amanda Taylor and Gavin Braunstein for performing many thorough literature searches for this editorial and other reviews.
![]() |
FOOTNOTES |
---|
This work is funded by grants from the Cystic Fibrosis Foundation and the American Heart Association Southern Research Consortium, a grant from the Polycystic Kidney Research Foundation, and National Institutes of Health Grants R01-DK/HL-54367 and R01-HL-63934.
Address for reprint requests and other correspondence: E. M. Schwiebert, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, MCLM 740, 1918 University Blvd., Birmingham, AL 35294-0005 (E-mail: eschwiebert{at}physiology.uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
1.
Beigi, R,
Kobatake E,
Masuo A,
and
Dubyak GR.
Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase.
Am J Physiol Cell Physiol
276:
C267-C278,
1999
2.
Boarder, MR,
and
Hourani SMO
The regulation of vascular function by P2 receptors: multiple sites and multiple receptors.
Trends Pharmacol Sci
19:
99-107,
1998[ISI][Medline].
3.
Erlinge, D.
Extracellular ATP: a growth factor for vascular smooth muscle cells.
Gen Pharmacol
31:
1-8,
1998[Medline].
4.
Frame, MK,
and
de Feijter AW.
Propagation of mechanically induced intercellular calcium waves via gap junctions and ATP receptors in rat liver epithelial cells.
Exp Cell Res
230:
197-207,
1997[ISI][Medline].
5.
Gordon, JL.
Extracellular ATP: effects, sources, and fates.
Biochem J
233:
309-319,
1986[ISI][Medline].
6.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
7.
Hazama, A,
Hayashi S,
and
Okada Y.
Cell surface measurement of ATP release from single pancreatic cells using a novel biosensor technique.
Pflügers Arch
437:
31-35,
1998[ISI][Medline].
8.
Hindley, S,
Herman MA,
and
Rathbone MP.
Stimulation of reactive astrogliosis in vivo by extracellular adenosine diphosphate or an adenosine A2 receptor agonist.
Neurosci Res
38:
399-406,
1994.
9.
Hwang, T-H,
Schwiebert EM,
and
Guggino WB.
Apical and basolateral ATP stimulates tracheal epithelial chloride secretion via multiple purinergic receptors.
Am J Physiol Cell Physiol
270:
C1611-C1623,
1996
10.
Korngreen, A,
Ma W,
Priel Z,
and
Silberberg SD.
Extracellular ATP directly gates a cation-selective channel in rabbit airway ciliated epithelial cells.
J Physiol (Lond)
508:
703-720,
1998
11.
Kunapuli, SP,
and
Daniel JL.
P2 receptor subtypes in the cardiovascular system.
Biochem J
336:
513-523,
1998[ISI][Medline].
12.
McCoy, DE,
Taylor AL,
Kudlow BA,
Karlson KH,
Slattery MJ,
Schwiebert LM,
Schwiebert EM,
and
Stanton BA.
Nucleotides regulate NaCl transport across mIMCD-K2 cells via P2X and P2Y purinergic receptors.
Am J Physiol Renal Physiol
277:
F552-F559,
1999
13.
Roman, RM,
Feranchak AP,
Salter KD,
Wang Y,
and
Fitz JG.
Endogenous ATP regulates Cl secretion in cultured human and rat biliary epithelial cells.
Am J Physiol Gastrointest Liver Physiol
276:
G1391-G1400,
1999
14.
Sauer, H,
Hescheler J,
and
Wartenberg M.
Mechanical strain-induced Ca2+ waves are propagated via ATP release and purinergic receptor activation.
Am J Physiol Cell Physiol
279:
C295-C307,
2000
15.
Schneider, SW,
Egan ME,
Jena BP,
Guggino WB,
Oberleitner H,
and
Geibel JP.
Continuous detection of extracellular ATP on living cells by using atomic force microscopy.
Proc Natl Acad Sci USA
96:
12180-12185,
1999
16.
Schulze-Lohoff, E,
Zanner S,
Ogilvie A,
and
Sterzel RB.
Extracellular ATP stimulates proliferation of cultured mesangial cells via P2-purinergic receptors.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F374-F383,
1992
17.
Tarasiuk, A,
Bar-Shimon M,
Gheber L,
Korngreen A,
Grossman Y,
and
Priel Z.
Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.
Biophys J
68:
1163-1169,
1995[Abstract].
18.
Taylor, AL,
Kudlow BA,
Marrs KL,
Gruenert DC,
Guggino WB,
and
Schwiebert EM.
Bioluminescence detection of ATP release mechanisms in epithelia.
Am J Physiol Cell Physiol
275:
C1391-C1406,
1998
19.
Tsien, RW,
and
Tsien RY.
Calcium channels, stores, and oscillations.
Annu Rev Cell Biol
6:
715-760,
1990[ISI].
20.
Wang, Y,
Roman R,
Lidofsky SD,
and
Fitz JG.
Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation.
Proc Natl Acad Sci USA
93:
12020-12025,
1996
21.
Wilson, PD,
Hovater JS,
Casey CC,
Fortenberry JA,
and
Schwiebert EM.
ATP release mechanisms in primary cultures of epithelia derived from the cysts of polycystic kidneys.
J Am Soc Nephrol
10:
218-229,
1999