Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.     

ATP-stimulated interleukin-6 synthesis through P2Y receptors on human osteoblasts

We investigated the effect of extracellular adenosine triphosphate (ATP) on the production of interleukin (IL)-6, whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as are involved in signal transduction systems in human osteoblastic SaM-1 cells. These human osteoblasts constitutively expressed P2X4, P2X5, P2X6, P2Y2, P2Y5, and P2Y6 purinergic receptors. ATP increased gene- and protein-expression of IL-6 in SaM-1 cells. The expression of the IL-6 mRNA was maximal at 1h, and the increase in IL-6 synthesis in response to ATP (10-100 microM) occurred in a concentration-dependent manner. Over the same concentration range of the nucleotide that was effective for IL-6 synthesis, ATP caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which increase was inhibited by pretreatment with suramin, a P2Y receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker, but not by the extracellular Ca(2+)-chelating agent EGTA. The pretreatment of SaM-1 cells with suramin or 2-APB also inhibited the increase in IL-6 synthesis in response to ATP. These findings suggest that extracellular ATP-induced IL-6 synthesis occurs through P2Y receptors and mobilization of Ca(2+) from internal stores in human osteoblastic cells.     

Mitochondrial calcium, oxidative stress and apoptosis in a neurodegenerative disease model induced by 3-nitropropionic acid

Intracellular calcium homeostasis is important for cell survival. However, increase in mitochondrial calcium (Ca2+m) induces opening of permeability transition pore (PTP), mitochondrial dysfunction and apoptosis. Since alterations of intracellular Ca2+ and reactive oxygen species (ROS) generation are involved in cell death, they might be involved in neurodegenerative processes such as Huntington's disease (HD). HD is characterized by the inhibition of complex II of respiratory chain and increase in ROS production. In this report, we studied the correlation between the inhibitor of the complex II, 3-nitropropionic acid (3NP), Ca2+ metabolism, apoptosis and behavioural alterations. We showed that 3NP (1 mm) is able to release Ca2+m, as neither Thapsigargin (TAP, 2 microm) nor free-calcium medium affected its effect. PTP inhibitors and antioxidants inhibited this process, suggesting an increase in ROS generation and PTP opening. In addition, 3NP (0.1 mm) also induces apoptotic cell death. Behavioural changes in animals treated with 3NP (20 mg/kg/day for 4 days) were also attenuated by pre- and co-treatment with vitamin E (VE, 20 mg/kg/day). Taken together, our results show that complex II inhibition could involve Ca2+m release, oxidative stress and cell death that may precede motor alterations in neurodegenerative processes such as HD.     

MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter

The mitochondrial uniporter is a selective Ca²⁺ channel regulated by MICU1, an EF hand‐containing protein in the organelle's intermembrane space. MICU1 physically associates with and is co‐expressed with a paralog, MICU2. To clarify the function of MICU1 and its relationship to MICU2, we used gene knockout (KO) technology. We report that HEK‐293T cells lacking MICU1 or MICU2 lose a normal threshold for Ca²⁺ intake, extending the known gating function of MICU1 to MICU2. Expression of MICU1 or MICU2 mutants lacking functional Ca²⁺‐binding sites leads to a striking loss of Ca²⁺ uptake, suggesting that MICU1/2 disinhibit the channel in response to a threshold rise in [Ca²⁺]. MICU2's activity and physical association with the pore require the presence of MICU1, though the converse is not true. We conclude that MICU1 and MICU2 are nonredundant and together set the [Ca²⁺] threshold for uniporter activity.     

Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy

BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis.     

Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death

J. Neurochem. (2012) 122, 1118-1128. ABSTRACT: P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.     

Increased mitochondrial calcium uptake and concomitant mitochondrial activity by presenilin loss promotes mTORC1 signaling to drive neurodegeneration

Metabolic dysfunction and protein aggregation are common characteristics that occur in age‐related neurodegenerative disease. However, the mechanisms underlying these abnormalities remain poorly understood. We have found that mutations in the gene encoding presenilin in Caenorhabditis elegans, sel‐12, results in elevated mitochondrial activity that drives oxidative stress and neuronal dysfunction. Mutations in the human presenilin genes are the primary cause of familial Alzheimer''s disease. Here, we demonstrate that loss of SEL‐12/presenilin results in the hyperactivation of the mTORC1 pathway. This hyperactivation is caused by elevated mitochondrial calcium influx and, likely, the associated increase in mitochondrial activity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL‐12 function. Consistent with high mTORC1 activity, we find that SEL‐12 loss reduces autophagosome formation, and this reduction is prevented by limiting mitochondrial calcium uptake. Moreover, the improvements of proteostasis and neuronal defects in sel‐12 mutants due to mTORC1 inhibition require the induction of autophagy. These results indicate that mTORC1 hyperactivation exacerbates the defects in proteostasis and neuronal function in sel‐12 mutants and demonstrate a critical role of presenilin in promoting neuronal health.     

Yeast mitochondrial calcium uptake: Regulation by polyamines and magnesium ions

Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca²⁺ transport system ofEndomyces magnusii mitochondria. Mg²⁺ at concentrations up to 5 mM partially inhibits Ca²⁺ transport with a half-maximal inhibiting concentration of 0.5 mM. In the presence of 2 mM MgCl₂, theS _0.5 value of the Ca²⁺ transport system increases from 220 to 490 µM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 µM, respectively. In the case of spermine, theS _0.5 value falls to 50–65 µM, which implies an increase in the transport system affinity for Ca²⁺. Both Mg²⁺ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca²⁺ from the media completely. In contrast, Mg²⁺ lowers the mitochondrial buffer capacity. When both Mg²⁺ and spermine are present in the medium, their effects on theS _0.5 value and the free extramitochondrial Ca²⁺ concentration are additive. The ability of spermine and Mg²⁺ to regulate yeast mitochondrial Ca²⁺ transport is discussed.     

Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca²⁺ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling.     

P2Y1 and P2Y12 receptor cross-talk in calcium signalling: Evidence from nonstarved and long-term serum-deprived glioma C6 cells

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y₁, P2Y₂ and P2Y₁₂. Two of them, P2Y₁ and P2Y₂, directly stimulate calcium response. The protein level of the P2Y₂ receptor did not change during the serum starvation, while P2Y₁ protein level fell dramatically. Observed changes in the calcium response generated by P2Y₁ are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y₁₂, did not directly evoke calcium response, however it is activated by the same ligand as P2Y₁. The experiments with AR-C69941MX, the P2Y₁₂-specific antagonist, indicated that in control and serum-starved cells, calcium response evoked by P2Y₁ receptor is potentiated by the activity of P2Y₁₂-dependent signaling pathways. This potentiation may be mediated by P2Y₁₂ inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y₁ and P2Y₁₂ receptor activity directly depends on the capacitative calcium entrance mechanism.     

Different patterns of Ca2+ signals are induced by low compared to high concentrations of P2Y agonists in microglia

Brain-resident macrophages (microglia) are key cellular elements in the preservation of tissue integrity. On the other hand, they can also contribute to the development of pathological events by causing an extensive and inappropriate inflammatory response. A growing number of reports indicate the involvement of nucleotides in the control of microglial functions. With this study on P2Y receptors in rat microglia, we want to contribute to the definition of their expression profile and to the characterisation of their signalling mechanisms leading to Ca²⁺ movements. Endogenous nucleotides, when applied at a concentration of 100 μM, elicited robust Ca²⁺ transients, thanks to a panel of metabotropic receptors comprising mainly P2Y₂, P2Y₆ and P2Y₁₂ subtypes. The involvement of P2Y₁₂ receptors in Ca²⁺ responses induced by adenine nucleotides was confirmed by the pharmacological and pertussis toxin sensitivity of the response induced by adenosine diphosphate (ADP). Beside the G protein involved, Gi and Gq respectively, adenine and uracil nucleotides differed also for induction by the latter of a capacitative Ca²⁺ plateau. Moreover, when applied at low (sub-micromolar) concentrations with a long-lasting challenge, uracil nucleotides elicited oscillatory Ca²⁺ changes with low frequency of occurrence (≤1 min⁻¹), sometimes superimposed to an extracellular Ca²⁺-dependent sustained Ca²⁺ rise. We conclude that different patterns of Ca²⁺ transients are induced by low (i.e., oscillatory Ca²⁺ activity) compared to high (i.e., fast release followed by sustained raise) concentrations of nucleotides, which can suggest different roles played by receptor stimulation depending not only on the type but also on the concentration of nucleotides.     

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.     

P2Y2 receptors and water transport in the kidney

The kidneys play a critical role in the maintenance of water homeostasis. This is achieved by the inherent architecture of the nephron along with the expression of various membrane transporters and channels that are responsible for the vectorial transport of salt and water. The collecting duct has become a focus of attention by virtue of its ability to transport water independent of solutes (free-water transport), and its apparent involvement in various water balance disorders. It was originally believed that the water transport capability of the collecting duct was solely under the influence of the circulating hormone, arginine vasopressin (AVP). However, during the past decade, locally produced autocrine and/or paracrine factors have emerged as potent modulators of transport of water by the collecting duct. Recently, much attention has been focused on the purinergic regulation of renal water transport. This review focuses on the role of the P2Y₂ receptor, the predominant purinergic receptor expressed in the collecting duct, in the modulation of water transport in physiological and pathophysiological conditions, and its therapeutic potential as a drug target to treat water balance disorders in the clinic. Studies carried out by us and other investigators are unravelling potent interactions among AVP, prostanoid and purinergic systems in the medullary collecting duct, and the perturbations of these interactions in water balance disorders such as acquired nephrogenic diabetes insipidus. Future studies should address the potential therapeutic benefits of modulators of P2Y₂ receptor signalling in water balance disorders, which are extremely prevalent in hospitalised patients irrespective of the underlying pathology.     

The role of P2 receptors in controlling infections by intracellular pathogens

A growing number of studies have demonstrated the importance of ATP_e-signalling via P2 receptors as an important component of the inflammatory response to infection. More recent studies have shown that ATP_e can also have a direct effect on infection by intracellular pathogens, by modulating membrane trafficking in cells that contain vacuoles that harbour intracellular pathogens, such as mycobacteria and chlamydiae. A conserved mechanism appears to be involved in controlling infection by both of these pathogens, as a role for phospholipase D in inducing fusion between lysosomes and the vacuoles has been demonstrated. Other P2-dependent mechanisms are most likely operative in the cases of pathogens, such as Leishmania, which survive in an acidic phagolysosomal-like compartment. ATP_e may function as a “danger signal” that alerts the immune system to the presence of intracellular pathogens that damage the host cell, while different intracellular pathogens have evolved enzymes or other mechanisms to inhibit ATP_e-mediated signalling, which should, thus, be viewed as virulence factors for these pathogens.     

SLP‐2 is required for stress‐induced mitochondrial hyperfusion

Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin‐related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress‐induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L‐OPA1, MFN1, and the mitochondrial inner membrane protein SLP‐2. In the absence of SLP‐2, L‐OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro‐survival response against stress.     

线粒体电压依赖性阴离子通道及其调控功能

电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)是存在于线粒体外膜上的31kDa膜蛋白,能在膜上形成亲水性通道,调控阴离子、阳离子、ATP以及其他代谢物进出线粒体,在调节细胞代谢、维持胞内钙稳态,调节细胞凋亡和坏死等过程中发挥重要功能。现就VDAC的结构、特性、活性调节及对细胞功能的调控作一综述。     

Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition

F‐ATP synthases convert the electrochemical energy of the H⁺ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca²⁺‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca²⁺ binding site and the mechanism(s) through which Ca²⁺ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca²⁺‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca²⁺‐ATP hydrolysis, confer resistance to Ca²⁺‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.     

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y₂ receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y₂ receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y₂ receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y₂ receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y₂ receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y₂ receptors. While ATP and UTP activated the murine and human P2Y₂ receptors with similar potencies (EC₅₀ values were 1.5-5.8 M), ATP was ~ 10-fold less potent (IC₅₀ = 9.1-21.2 M) than UTP (IC₅₀ = 0.7-2.9 M) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.