ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger’ for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg²⁺ and/or H⁺ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP⁴⁻ in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.     

Pancreatic islet beta-cells transiently metabolize pyruvate

Pancreatic beta-cell metabolism was followed during glucose and pyruvate stimulation of pancreatic islets using quantitative two-photon NAD(P)H imaging. The observed redox changes, spatially separated between the cytoplasm and mitochondria, were compared with whole islet insulin secretion. As expected, both NAD(P)H and insulin secretion showed sustained increases in response to glucose stimulation. In contrast, pyruvate caused a much lower NAD(P)H response and did not generate insulin secretion. Low pyruvate concentrations decreased cytoplasmic NAD(P)H without affecting mitochondrial NAD(P)H, whereas higher concentrations increased cytoplasmic and mitochondrial levels. However, the pyruvate-stimulated mitochondrial increase was transient and equilibrated to near-base-line levels. Inhibitors of the mitochondrial pyruvate-transporter and malate-aspartate shuttle were utilized to resolve the glucose- and pyruvate-stimulated NAD(P)H response mechanisms. These data showed that glucose-stimulated mitochondrial NAD(P)H and insulin secretion are independent of pyruvate transport but dependent on NAD(P)H shuttling. In contrast, the pyruvate-stimulated cytoplasmic NAD(P)H response was enhanced by both inhibitors. Surprisingly the malate-aspartate shuttle inhibitor enabled pyruvate-stimulated insulin secretion. These data support a model in which glycolysis plays a dominant role in glucose-stimulated insulin secretion. Based on these data, we propose a mechanism for glucose-stimulated insulin secretion that includes allosteric inhibition of tricarboxylic acid cycle enzymes and pH dependence of mitochondrial pyruvate transport.     

Pancreatic beta-cells: From generation to regeneration

The pancreas is composed of two main compartments consisting of endocrine and exocrine tissues. The majority of the organ is exocrine and responsible for the synthesis of digestive enzymes and for their transport via an intricate ductal system into the duodenum. The endocrine tissue represents less than 2% of the organ and is organized into functional units called islets of Langerhans, comprising alpha-, beta-, delta-, epsilon- and PP-cells, producing the hormones glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide (PP), respectively. Insulin-producing beta-cells play a central role in the control of the glucose homeostasis. Accordingly, absolute or relative deficiency in beta-cells may ultimately lead to type 1 and/or type 2 diabetes, respectively. One major goal of diabetes research is therefore to understand the molecular mechanisms controlling the development of beta-cells during pancreas morphogenesis, but also those underlying the regeneration of adult injured pancreas, and assess their significance for future cell-based therapy. In this review, we will therefore present new insights into beta-cell development with focus on beta-cell regeneration.     

Autophagy-dependent ATP release from dying cells via lysosomal exocytosis

ATP not only represents a key molecule for the intracellular storage and utilization of energy, but also plays a central role in the extracellular milieu, as it both recruits and activates immune cells. The secretion of ATP in the course of immunogenic cell death (ICD), a peculiar type of apoptosis that elicits adaptive immune responses, critically relies on the molecular machinery for autophagy. We have recently demonstrated that autophagy allows for the ICD-associated secretion of ATP as it contributes to the maintenance of lysosomal ATP stores. In addition, we found that ATP release in this setting is mediated by a molecular mechanism that involves lysosomal-associated membrane protein 1 (LAMP1)- and pannexin 1 (PANX1)-dependent lysosomal exocytosis.     

Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes

ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 muM. The OGD-induced ATP release was inhibited by Gd(3+) and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl(-) channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X(7) receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd(3+) and arachidonic acid. The channel was found to be permeable to ATP(4-) with a permeability ratio of P(ATP)/P(Cl) = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions.     

Somatostatin inhibits insulin release via SSTR2 in hamster clonal beta-cells and pancreatic islets

Somatostatin (SST) inhibits pancreatic endocrine secretion. It is generally accepted that SSTR2 and SSTR5 mediate the inhibition of glucagon and insulin release, respectively. The present study was performed to test the hypothesis that SSTR2, but not SSTR5, mediates SST-induced inhibition of insulin release in hamster beta-cells. Both hamster clonal beta-cells HIT-T15 and pancreatic islets were used to test this hypothesis. Both SST and a nonpeptide SSTR2 agonist L-779,976 (1-100 nM) inhibited insulin release from HIT-T15 and islets in a concentration-dependent manner. In contrast, nonpeptide agonists for SSTR1, 3, 4 and 5 at the highest concentration studied (1 microM) failed to inhibit insulin release. PRL-2903, a peptide SSTR2 antagonist (0.1-1 muicroM), antagonized SST-induced inhibition of insulin release in a concentration-dependent manner. Taken together, we conclude that, in hamster beta-cells, SST inhibits insulin release via SSTR2 but not SSTR5.     

Pancreatic beta-cells: Role of glycerol and aquaglyceroporin 7

Pancreatic β-cells originate from gut endoderm during development. Pancreatic endocrine cells represent about 10% of the mature pancreatic cells, and β-cells represent the majority of endocrine cells. β-cells secrete insulin in response to elevation of nutrient concentrations. Insulin maintains glucose homeostasis by stimulating glucose uptake into muscle and adipose tissue. Aquaglyceroporin 7, permeable to water, glycerol and urea, is expressed in pancreatic β-cells and was recently described as being involved in the control of insulin secretion.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.     

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.     

ATP release through pannexon channels

Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut.     

ATP release from non-excitable cells

All cells release nucleotides and are in one way or another involved in local autocrine and paracrine regulation of organ function via stimulation of purinergic receptors. Significant technical advances have been made in recent years to quantify more precisely resting and stimulated adenosine triphosphate (ATP) concentrations in close proximity to the plasma membrane. These technical advances are reviewed here. However, the mechanisms by which cells release ATP continue to be enigmatic. The current state of knowledge on different suggested mechanisms is also reviewed. Current evidence suggests that two separate regulated modes of ATP release co-exist in non-excitable cells: (1) a conductive pore which in several systems has been found to be the channel pannexin 1 and (2) vesicular release. Modes of stimulation of ATP release are reviewed and indicate that both subtle mechanical stimulation and agonist-triggered release play pivotal roles. The mechano-sensor for ATP release is not yet defined.     

ATP-induced ATP release from astrocytes

Propagation of interastrocyte Ca2+ waves is mediated by diffusion of extracellular adenosine triphosphate (ATP), and may require regenerative release of ATP. The ability of ATP to initiate release of intracellular ATP was assessed by labeling adenine nucleotide pools in astrocyte cultures with 14C-adenine. The 14C-purines released during exposure to ATP were then identified by thin-layer chromatography. ATP treatment caused a five-fold increase in release of 14C-ATP but not 14C-ADP or 14C-AMP, indicating selectivity for release of ATP. Other P2 receptor agonists also caused significant 14C-ATP release, and the P2 receptor antagonists suramin, reactive blue-2 and pyridoxalphosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) inhibited ATP-induced 14C-ATP release to varying degrees, suggesting the involvement of a P2 receptor. ATP-induced 14C-ATP release was not affected by chelation of intracellular Ca2+ with BAPTA-AM, or by blockers of Ca2+ release from intracellular stores or of extracellular Ca2+ influx, suggesting a Ca2+-independent response. ATP-induced 14C-ATP release was significantly inhibited by non-selective anion channel blockers but not by blockers of ATP-binding cassette proteins, gap junction hemichannels, or vesicular exocytosis. Release of adenine nucleotides induced by 0 Ca2+ was, in contrast, not selective for ATP, and was susceptible to inhibition by gap junction blockers. These findings indicate that astrocytes are capable of ATP-induced ATP release and support a role for regenerative ATP release in glial Ca2+ wave propagation.     

ATP stimulates MMP-2 release from human aortic smooth muscle cells via JNK signaling pathway

Aortic smooth muscle cell release of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) has been implicated in aortic aneurysm pathogenesis, but proximal modulation of release is poorly understood. Extracellular nucleotides regulate vascular smooth muscle cell metabolism in response to physiochemical stresses, but nucleotide modulation of MMP and/or TIMP release has not been reported. We hypothesized that nucleotides modulate MMP-2 and TIMP-2 release from human aortic smooth muscle cells (HASMCs) via distinct purinergic receptors and signaling pathways. We exposed HASMCs to exogenous ATP and other nucleotides with and without interleukin-1beta (IL-1beta). HASMCs were pretreated in some experiments with apyrase, which degrades ATP, and inhibitors of ERK1/2, JNK, and p38 MAPK. MMP-2 and TIMP-2 released into supernatant were assessed using ELISA and Western blotting. ATP, adenosine, and UTP significantly stimulated MMP-2 release in the presence of IL-1beta (300 nM ATP: 181 +/- 22%, P = 0.003; 30 microm adenosine: 244 +/- 150%, P = 0.001; and 200 microm UTP: 153 +/- 40%, P = 0.015; vs. 100% constitutive). ATP also stimulated MMP-2 release in the absence of IL-1beta (100 microm ATP: 148 +/- 38% vs. 100% constitutive). Apyrase significantly reduced ATP-stimulated MMP-2 release (apyrase + 500 nM ATP: 59 +/- 3% vs. 124 +/- 7% with 500 nM ATP). Rank-order agonist potency for MMP-2 release was consistent with ATP activation of PAY and PAY receptors. ATP induced phosphorylation of intracellular JNK, and inhibition of the JNK pathway blocked ATP-stimulated MMP-2 release, indicating signaling via this pathway. Nucleotides are thus novel stimulants of MMP-2 release from HASMCs and may provide a mechanistic link between physiochemical stress in the aorta and aneurysms, especially in the context of inflammation.     

Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.     

The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas.

The isolated perfused rat pancreas was used to test the hypothesis that total cellular ATP or the ratio of ATP/free ADP plays the primary role in coupling intermediary metabolism to the biophysical events that are the basis of glucose-stimulated insulin release. The pancreas was preperfused for 20 min with 4.0 mM of a physiological mixture of 20 amino acids plus 4.2 mM glucose, and insulin release was then stimulated for 150 s by suddenly increasing the glucose to 8.3 mM. The pancreas was sampled at 24, 48, 72, and 150 s after the switch. The content of total ATP, ADP, AMP, Pi, phosphocreatine, and creatine were measured in beta-cell enriched cores of pancreatic islets microdissected from freeze-dried pancreas cryostat sections. Metabolites were measured by quantitative histochemical enzymatic cycling techniques. Modeling studies were carried out to assess the impact of biochemical analytical results on the membrane potential of the beta-cells. The level of free ADP was calculated using the creatine kinase equilibrium reaction and an intracellular pH of 7.2. First phase insulin release was stimulated at least 10-fold with the maximum reached 45 s after adding high glucose. The biochemical analytical data demonstrate that the total cellular level of the putative coupling factor ATP and of the ratios ATP/free ADP and ATP/free ADP x Pi are not significantly influenced by a glucose level change that causes a more than 10-fold surge of insulin release. The strength and limitations of the present experimental strategy and the implications of the results for our understanding of metabolic coupling in glucose-stimulated insulin release are discussed.     

Short-term intermittent exposure to diazoxide improves functional performance of beta-cells in a high-glucose environment

Prolonged periods of "beta-cell rest" exert beneficial effects on insulin secretion from pancreatic islets subjected to a high-glucose environment. Here, we tested for effects of short-term intermittent rest achieved by diazoxide. Rat islets were cultured for 48 h with 27 mmol/l glucose alone, with diazoxide present for 2 h every 12 h or with continuous 48-h presence of diazoxide. Both protocols with diazoxide enhanced the postculture insulin response to 27 mmol/l glucose, to 200 mumol/l tolbutamide, and to 20 mmol/l KCl. Intermittent diazoxide did not affect islet insulin content and enhanced only K(ATP)-dependent secretion, whereas continuous diazoxide increased islet insulin contents and enhanced both K(ATP)-dependent and -independent secretory effects of glucose. Intermittent and continuous diazoxide alike increased postculture ATP-to-ADP ratios, failed to affect [(14)C]glucose oxidation, but decreased oxidation of [(14)C]oleate. Neither of the two protocols affected gene expression of the ion channel-associated proteins Kir6.2, sulfonylurea receptor 1, voltage-dependent calcium channel-alpha1, or Kv2.1. Continuous, but not intermittent, diazoxide decreased significantly mRNA for uncoupling protein-2. A 2-h exposure to 20 mmol/l KCl or 10 mumol/l cycloheximide abrogated the postculture effects of intermittent, but not of continuous, diazoxide. Intermittent diazoxide decreased islet levels of the SNARE protein SNAP-25, and KCl antagonized this effect. Thus short-term intermittent diazoxide treatment has beneficial functional effects that encompass some but not all characteristics of continuous diazoxide treatment. The results support the soundness of intermittent beta-cell rest as a treatment strategy in type 2 diabetes.     

Light-induced ATP release from the lens

The recent discovery of the photoreceptor melanopsin in lens epithelial cells has opened the possibility of modulating this protein by light stimulation. Experiments carried out on New Zealand white rabbits have demonstrated that the release of ATP from the lens to the aqueous humor can be reduced either when a yellow filter or a melanopsin antagonist is used. Compared to control (1.10?±?0.15 μM ATP), the application of a yellow filter (λ465–480) reduced ATP in the aqueous humor 70%, while the melanopsin antagonist AA92593 reduced the presence of ATP 63% (n?=?5), an effect which was also obtained with the PLC inhibitor U73122. These results indicate that when melanopsin is blocked either by the lack of light, a filter, or an antagonist, the extracellular presence of ATP is significantly reduced. This discovery may be relevant, on the one hand, because many ocular physiological processes are controlled by ATP and, on the other hand, because it is possible to stimulate ATP release with just light and without using any added substance.