Mediating ATP Release Research Articles

Losing control over adenosine 5′-triphosphate release: Implications for the red blood cell storage lesion*

Losing control over adenosine 5′-triphosphate release: Implications for the red blood cell storage lesion*

A “Swell” Way to Regulate Pannexin-Mediated ATP Release

TRPV4 and RhoA signaling act upstream of pannexin 1 to mediate ATP release induced by osmotic cell swelling.

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.

The ‘connexin’ between astrocytes, ATP and central respiratory chemoreception

The ‘connexin’ between astrocytes, ATP and central respiratory chemoreception

Purinergic Signalling special issue on “Purinergic Signalling in the Inner Ear”; a commentary by Professor Geoffrey Burnstock, Editor-in-Chief

This special edition—“Purinergic Signalling in the Inner Ear”—assembled under the steerage of Guest Editors Gary Housley and Jonathan Gale, extends the recent series of issues that have highlighted emerging themes of purinergic signalling. This special edition reflects the breadth of understanding of the role of extracellular nucleoside and nucleotide signalling in the cochlea. The inner ear has revealed fascinating processes related to the development and function of hearing and balance with broad relevance to other systems. The ten fully peer-reviewed papers are provided by research leaders who have made sustained contributions to this field and have accepted the invitation to contribute highlights of their current research focus to this special edition. The issue opens with papers describing the dynamics of ATP release mechanisms and related inter-cellular synchronous communication between the sensory and supporting cells and neurons. These studies resolve connexin hemi-channel mediated ATP release, controlled by P2X and P2Y receptors, contributing to the establishment of auditory neurotransmission, and effector pathways, including Ca2+ and nitric oxide second messenger signalling, that sustain the function and integrity of the cochlea. Subsequent papers provide new data on the breadth of P1 and P2 receptor and NTPDase expression in the cochlea and investigate the dynamics of these systems with regard to hearing function with ageing and noise-stress. Of particular note is the final paper, by Vlajkovic et al, which provides an exciting glimpse at the translational potential of the field. This study reports what may be the first effective treatment of hearing loss after noise trauma; systemic treatment of rats with the A1 receptor agonist (adenosine amine congener—ADAC) significantly reduced noise-induced hearing loss. As this issue moves from immediate on-line access, to print, it is pleasing to report that this exciting finding has already attracted considerable media attention.

Pannexin1 is a novel renal ATP release mechanism

Purinergic signaling regulates key functions of the renal tubules, including salt and water transport. Due to ectonucleotidase activity along the nephron, it has been suggested that ATP is locally released by tubular cells. Previously, we have shown that the gap junction protein connexin 30 (Cx30) plays a role in ATP release and purinergic mediation of salt reabsorption in the distal nephron/collecting duct. A new candidate for ATP transport is the pannexin1 hemichannel (Px1) which is sensitive to mechanical stress, allows the passage of ATP and unlike Cxs, is insensitive to [Ca2+]e. Therefore we tested the hypothesis that Px1 hemichannels may also mediate ATP release in the distal nephron. Using immunofluorescence, we observed Px1 in the distal convoluted tubules, thin limbs, and select cells of the cortical collecting duct. Px1 function was tested using a luciferase‐based ATP assay and a dye uptake assay in two renal epithelial cell types that endogenously express Px1: MDCK and M1. Px1 was inhibited with either probenecid or Px1 siRNA. Probenecid significantly inhibited ATP release in both M1 (by ~60%) and MDCK (by ~40%) cell types, while Px1 siRNA significantly reduced ATP release in MDCK cells only (by ~30%). Both treatments failed to significantly alter dye uptake in either cell type. Based on these results, we concluded that Px1 is co‐expressed with other Cx isoforms and may regulate ATP release in the distal nephron.

5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles

Cells release ATP in response to physiologic stimuli. Extracellular ATP regulates a broad range of important cellular functions by activation of the purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure ATP release in HTC rat hepatoma and Mz-Cha-1 human cholangiocarcinoma cells. Exposure to a Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50-300 microM) stimulated a 5-100-fold increase in extracellular ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl(-) channel activity. NPPB also potently stimulated ATP release in HEK293 cells and HEK293 cells expressing a rat P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of ATP release by NPPB. In all cells studied, NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of NPPB-evoked exocytosis and ATP release were similar. Furthermore, the magnitudes of NPPB-evoked exocytosis and ATP release were correlated (correlation coefficient 0.77), indicating that NPPB may stimulate exocytosis of a pool of ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular ATP release and suggest that NPPB can be used as a biochemical tool to specifically stimulate ATP release through exocytic mechanisms.

Mechanisms of ATP release from 1321N1 cells

Astrocytes are an important source of extracellular ATP in the brain. Regulated release of ATP from astrocytes occurs in response to diverse metabolic, mechanical, or inflammatory stimuli. Non‐junctional hemichannels composed of connexin 43 have been shown to be a major conduit for release of ATP from murine astrocytes in response to hypoxia, but the role of non‐junctional connexin hemichannels in G‐protein coupled receptor (GPCR) mediated ATP release is not determined. Previously, using 1321N1 human astrocytes we found that rapid accumulation of active GTP‐bound Rho in response to GPCR activation greatly enhances Ca2+ dependent ATP release. GPCR induced ATP release from 1321N1 cells is attenuated by carbenoxolone, flufenamic acid, and probenecid. These structurally unrelated compounds are non‐selective inhibitors of non‐junctional hemichannels. Here we investigate the contribution of connexin 43 non‐junctional hemichannels to GPCR induced ATP release from 1321N1 astrocytes by connexin 43 overexpression and connexin 43 knockdown.

Voltage-dependent Anion Channel-1 (VDAC-1) Contributes to ATP Release and Cell Volume Regulation in Murine Cells

Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b−) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (−/−)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (−/−) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (−/−) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (−/−) and WT mice after luminal hypotonic challenge, but VDAC-1 (−/−) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (−/−) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.

O-273 - Different P2-receptor subtypes mediating ATP release and contraction by α, β-methylene ATP and OTP in intact and cultured guinea-pig ileal smooth muscles

O-273 - Different P2-receptor subtypes mediating ATP release and contraction by α, β-methylene ATP and OTP in intact and cultured guinea-pig ileal smooth muscles