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)P 3] as a mediator of Ca2+ waves. In monolayers of cells that maintained gap junctional communication, Ins(1,4,5)P 3increased intracellular Ca2+ (Cai 2+) 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)P 3concentration was diluted beyond effect. For that matter, Cai 2+ 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.
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 β-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.
I especially thank graduate students Amanda Taylor and Gavin Braunstein for performing many thorough literature searches for this editorial and other reviews.
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:).
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