Regulation of cellular ATP release

Abstract

Epithelial cells exhibit regulated release of ATP. Once outside of the cell, ATP in nanomolar concentrations functions as an autocrine/paracrine signal modulating a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. The mechanisms responsible for ATP release have not been defined. In liver cells, there is evidence for ATP translocation through a conductive, channel-mediated pathway. In addition, indirect observations support a second potential mechanism involving exocytosis of ATP-enriched vesicles. Notably, stimuli that increase ATP release are associated with a five- to ten-fold increase in the rate of exocytosis; and inhibition of the exocytic response impairs cellular ATP release. More recent evidence suggests that these vesicles can be visualized, supporting the concept that in liver cells, ATP release is mediated in part by exocytosis of a pool of vesicles enriched in ATP, which can be mobilized within seconds in response to changing physiologic demands.

Fig. 1

Overview of epithelial purinergic signaling cascade. Extracellular ATP functions as a potent autocrine/paracrine signal in epithelia. The basic elements include 1) regulated release of ATP (and UTP) through ATP-permeable channels and/or exocytosis of ATP-containing vesicles, under active investigation, 2) rapid hydrolysis by ectonucleotidases to degrade ATP and generate metabolites which themselves have agonist properties, 3) binding to a broad range of P1, P2X and P2Y receptors in the plasma membrane, and 4) related signaling events according to the specific receptor involved.

Fig. 2

Detection of extracellular ATP by the luciferin-luciferase reaction. Schwiebert et al (10) developed an assay based on luciferin-luciferase wherein increasing concentrations of ATP are detected as an increase in photon generation in Arbitrary Light Units (ALU). In the example shown, coverslips containing cells were mounted in a luminometer with luciferin-luciferase in the bathing solution as described (11,23). Addition of isotonic media control for mechanical stimulation had small effects. Addition of an equal volume of hypotonic media to cause cell volume increase was associated with an increase in ALU, consistent with volume-stimulated ATP release.

Fig. 3

Theoretical mechanisms of cellular ATP release include opening of ATP-permeable ion channels; exocytosis of vesicles enriched in ATP; and/or exocytosis of ion channel-containing vesicles.

Fig. 4

Detection of exocytosis through FM1-43 fluorescence. FM1-43 fluorescence provides a measure in real time of exocytosis as new membrane-containing vesicles come in contact with FM1-43 in the media (24). In this model biliary cell, images were obtained before and ∼2 min after exposure to hypotonic media (−30%) to increase cell volume. The increase in plasma membrane fluorescence occurred as a result of vesicular exocytosis. Reproduced from Am. J. Physiol. 286:G538–546, 2004 with permission.

Fig. 5

Effect of cell volume increases on exocytosis and ATP release. In model biliary cells as described in Figure 4, exposure to hypotonic media to increase cell volume caused within minutes an increase in exocytosis to values sufficient to replace ∼15% of the plasma membrane min⁻¹ as assessed by FM1-43 fluorescence; and caused a parallel increase in ATP release. Inhibition of exocytosis by blockade of protein kinase C or PI 3-kinase causes a parallel inhibition, suggesting that exocytosis and ATP release are linked. Data redrawn from Am. J. Physiol. 286:G538–546, 2004.