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.     

Regulated ATP release from astrocytes through lysosome exocytosis

Release of ATP from astrocytes is required for Ca2+ wave propagation among astrocytes and for feedback modulation of synaptic functions. However, the mechanism of ATP release and the source of ATP in astrocytes are still not known. Here we show that incubation of astrocytes with FM dyes leads to selective labelling of lysosomes. Time-lapse confocal imaging of FM dye-labelled fluorescent puncta, together with extracellular quenching and total-internal-reflection fluorescence microscopy (TIRFM), demonstrated directly that extracellular ATP or glutamate induced partial exocytosis of lysosomes, whereas an ischaemic insult with potassium cyanide induced both partial and full exocytosis of these organelles. We found that lysosomes contain abundant ATP, which could be released in a stimulus-dependent manner. Selective lysis of lysosomes abolished both ATP release and Ca2+ wave propagation among astrocytes, implicating physiological and pathological functions of regulated lysosome exocytosis in these cells.     

Activation of transient receptor potential ankyrin 1 evokes nociception through substance P release from primary sensory neurons

To examine mechanisms underlying substance P (SP) release from primary sensory neurons in response to activation of the non-selective cation channel transient receptor potential ankyrin 1 (TRPA1), SP release from cultured rat dorsal root ganglion neurons was measured, using radioimmunoassay, by stimulating TRPA1 with allyl isothiocyanate (AITC), a TRPA1 agonist. AITC-evoked SP release occurred in a concentration- and time-dependent manner. Interestingly, p38 mitogen-activated protein kinase (p38) inhibitor SB203580 significantly attenuated AITC-evoked SP release. The in vivo effect of AITC-evoked SP release from primary sensory neurons in mice was evaluated. Hind paw intraplantar injection of AITC induced nociceptive behaviors and inflammation (edema, thermal hyperalgesia). AITC-induced thermal hyperalgesia and edema were inhibited by intraplantar pre-treatment with either SB203580 or neurokinin-1 receptor antagonist CP96345. Moreover, intrathecal pre-treatment with either CP96345 or SB203580 inhibited AITC-induced nociceptive behaviors and thermal hyperalgesia. Immunohistochemical studies demonstrated that intraplantar AITC injection induced the phosphorylation of p38 in mouse dorsal root ganglion neurons containing SP. These findings suggest that activation of TRPA1 evokes SP release from the primary sensory neurons through phosphorylation of p38, subsequent nociceptive behaviors and inflammatory responses. Furthermore, the data also indicate that blocking the effects of TRPA1 activation at the periphery leads to significant antinociception.     

Glutamate release from adult primary sensory neurons in culture is modulated by growth factors.

The aim of this study was to examine possible modulatory effects of some trophic molecules, i.e. nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF), on potassium (K(+))-, bradykinin (BK)- or capsaicin (CAPS)-evoked release of glutamate (GLU) from dorsal root ganglion (DRG) neurons in vitro. BK (0.5 and 1 microM) induced a dramatic and significant increase in glutamate release. Neither CAPS nor K(+) (60 mM) produced any significant increase of GLU release vs. basal levels during a 5-min stimulation. The BK-evoked release of GLU was almost completely blocked by HOE 140, a selective BK2-receptor antagonist at high doses. Basal release of GLU was significantly reduced in cultures grown in the presence of bFGF, whereas BDNF and NGF had no significant effect. Incubation with growth factors generally decreased the BK-stimulated GLU release, an effect most pronounced for bFGF, which completely blocked BK-stimulated release. The rise in intracellular [Ca(2+)] following stimulation with BK (100 nM-1 microM), potassium (60 mM) or ATP (10 microM) was also studied using a Ca(2+)-sensitive indicator, Fura-2, in cultures grown in basal medium with or without bFGF. None of the bFGF-treated cells exhibited strong Ca(2+) responses to BK or ATP stimulation, while 10-20% of the responding cells grown in basal medium exhibited strong responses. The K(+)-induced increase of [Ca(2+)] did not vary between the different groups.The present findings suggest that sensory neurotransmission involving glutamate may be modulated by growth factors and that regulation of intracellular Ca(2+) homeostasis may be a contributing factor.     

Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells

Extracellular ATP is a potent autocrine/paracrine signal that regulates a broad range of liver functions through activation of purinergic receptors. In biliary epithelium, increases in cell volume stimulate ATP release through a phosphoinositide 3-kinase (PI3-kinase)-dependent mechanism. Because PI3-kinase also regulates vesicular exocytosis, the purpose of these studies was to determine whether volume-stimulated vesicular exocytosis contributes to cellular ATP release. In a human cholangiocarcinoma cell line, exocytosis was measured by using the plasma membrane marker FM1-43, whereas ATP release was assessed by using a luciferase-luciferin assay. Under basal conditions, cholangiocytes exhibited constitutive exocytosis at a rate of 1.6%/min, and low levels of extracellular ATP were detected at 48.2 arbitrary light units. Increases in cholangiocyte cell volume induced by hypotonic exposure resulted in a 10-fold increase in the rate of exocytosis and a robust 35-fold increase in ATP release. Both vesicular exocytosis and ATP release were proportional to cell volume, and both exhibited similar regulatory properties including: 1) dependence on intact PI3-kinase, 2) attenuation by inhibition of PKC, and 3) potentiation by activation of PKC before hypotonic exposure. These findings demonstrate that increases in cholangiocyte cell volume stimulate ATP release and vesicular exocytosis through similar regulatory paradigms. Functional interactions among cell volume, PKC, and PI3-kinase modulate exocytosis, thereby regulating ATP release and purinergic signaling in cholangiocytes. It is hypothesized that PKC is involved in the recruitment of a volume-sensitive vesicular pool to a readily releasable state.     

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.     

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

Extracellular ATP regulates many important cellular functions in the liver by stimulating purinergic receptors. Recent studies have shown that rapid exocytosis of ATP-enriched vesicles contributes to ATP release from liver cells. However, this rapid ATP release is transient, and ceases in ~30 s after the exposure to hypotonic solution. The purpose of these studies was to assess the role of vesicular exocytosis in sustained ATP release. An exposure to hypotonic solution evoked sustained ATP release that persisted for more than 15 min after the exposure. Using FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide) fluorescence to measure exocytosis, we found that hypotonic solution stimulated a transient increase in FM1-43 fluorescence that lasted ~2 min. Notably, the rate of FM1-43 fluorescence and the magnitude of ATP release were not correlated, indicating that vesicular exocytosis may not mediate sustained ATP release from liver cells. Interestingly, mefloquine potently inhibited sustained ATP release, but did not inhibit an increase in FM1-43 fluorescence evoked by hypotonic solution. Consistent with these findings, when exocytosis of ATP-enriched vesicles was specifically stimulated by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), mefloquine failed to inhibit ATP release evoked by NPPB. Thus, mefloquine can pharmacologically dissociate sustained ATP release and vesicular exocytosis. These results suggest that a distinct mefloquine-sensitive membrane ATP transport may contribute to sustained ATP release from liver cells. This novel mechanism of membrane ATP transport may play an important role in the regulation of purinergic signaling in liver cells.     

Nonsynaptic and nonvesicular ATP release from neurons and relevance to neuron-glia signaling

Studies on the release of ATP from neurons began with the earliest investigations of quantal neurotransmitter release in the 1950s, but in contrast to ATP release from other cells, studies of ATP release from neurons have been narrowly constrained to one mechanism, vesicular release. This is a consequence of the prominence of synaptic transmission in neuronal communication, but nonvesicular mechanisms for ATP release from neurons are likely to have a broader range of functions than synaptic release. Investigations of activity-dependent communication between axons and myelinating glia have stimulated a search for mechanisms that could release ATP from axons and other nonsynaptic regions in response to action potential firing. This has identified volume-activated anion channels as an important mechanism in activity-dependent ATP release from axons, and renewed interest in micromechanical changes in axons that accompany action potential firing.     

Carbonic anhydrase activity in primary sensory neurons

Summary Subpopulations of primary sensory neurons in mammalian dorsal root ganglion (DRG) exhibit carbonic anhydrase (CA) activity. To identify these subpopulations in DRG cells of mouse and chicken, the reliability of the cytochemical localization of the enzyme requires that several conditions be fulfilled:(1) Preservation of the enzyme activity in glutaraldehyde-containing fixative; (2) accessibility of the cytoenzymatic reaction throughout 20-m thick Vibratome sections; (3) retention of the reaction product in situ during OsO₄ post-fixation; (4) specificity of the cytoenzymatic reaction for CA activity as corroborated by the immunocytochemical detection with antibodies anti-CA II in mouse DRG; (5) strict correlation between the CA activity and the cytological characteristics in a given subclass of neurons. On the basis of these criteria, it is concluded that the CA activity may be used as a cell marker to identify cytologically defined neuronal subpopulations and their axons in mouse DRG. In chicken DRG, CA activity is not consistently expressed in a given subclass of ganglion cells and their axons. Hence, it is assumed that the expression of CA activity by DRG cells in chicken is modulated by functional or environmental conditions.     

Carbonic anhydrase activity in primary sensory neurons

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.     

Characterization of ATP release from cultures enriched in cholinergic amacrine-like neurons.

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of alpha-amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca(2+) dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and omega-Agatoxin IVA, but not by omega-Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca(2+) influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca(2+) channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A(1) receptor antagonist, showing that the release is tonically inhibited by the adenosine A(1) receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation.     

Induced neurotransmitter release from primary cultures of rat brain neurons

Neural cells from fetal rat brain were grown in tissue culture in the absence of serum and maintained for 4–5 weeks without medium renewal. Over 80% of the embryonic cells in the culture had a neuronal appearance and formed intercellular synaptic connections. When mature, a definite population of the neuronal cells accumulated ³H-dopamine in a sodium-dependent, benztropine inhibited process. The mature cells were also able to release ³H-dopamine in a potassium evoked, calcium-dependent process, with half maximal dopamine release achieved at a Ca²⁺ concentration of 120μM. In the maturing cells the capacity for potassium evoked, calcium-dependent dopamine release increased from an undetectable level in the first three days to a plateau level after 10–11 days $\text{in vitro}$. The fully expressed release capacity (20–30% of the neurotransmitter retained in the cells) was maintained thereafter. These results demonstrate that primary brain neurons develop a functional neurosecretion apparatus in a chemically defined medium in the absence of animal serum. This extends the utility of primary cultures of brain neurons for developmental structural and biochemical studies of neurotransmission.     

Localization of SSeCKS in unmyelinated primary sensory neurons

Background

RhoA and Rho kinase inhibitors overcome the inhibition of axonal regeneration posed by central nervous system (CNS) substrates.

Methods

To investigate if inhibition of the Rho pathway augments the neurite extension that naturally occurs in the peripheral nervous system (PNS) following nerve damage, dorsal root ganglion neurons and Schwann cell co-cultures were incubated with culture medium, C3 fusion toxin, and the Rho kinase (ROCK) inhibitors Y27632 and H1152. The longest neurite per neuron were measured and compared. Incubation with Y27632 and H1152 resulted in significantly longer neurites than controls when the neurons were in contact with Schwann cells. When separated by a porous P.E.T. membrane, only the group incubated with H1152 developed significantly longer neurites. This work demonstrates that Rho kinase inhibition augments neurite elongation in the presence of contact with a PNS-like substrate.     

Axotomy-induced apoptosis in adult rat primary sensory neurons

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.     

Changes in ionic movements across rabbit polymorphonuclear leukocyte membranes during lysosomal enzyme release. Possible ionic basis for lysosomal enzyme release.

Changes in the movements of Na+, K+, and Ca+2 across rabbit neutrophils under conditions of lysosomal enzyme release have been studied. We have found that in the presence of cytochalasin B, the chemotactic factor formyl methionyl leucyl phenylalanine (FMLP) induces within 30 s large enhancements in the influxes of both 22Na+ and 45Ca+2 and an increase in the cellular pool of exchangeable calcium. The magnitude of the changes induced by cytochalasin B and FMLP exceeds that induced by FMLP or cytochalasin B alone, and cannot be explained on the basis of an additive effect of the two agents. However, these compounds either separately or together produce much smaller enhancements in 45Ca efflux. The divalent cation ionophore A23187 also produces a rapid and large increase in the influxes of both 22Na and 45Ca+2 in the presence and absence of cytochalasin B. We have also found an excellent correlation between calcium influx and lysosomal enzyme release. 42K influx is not significantly affected by any of these compounds. On the other hand, a large and rapid increase of 42K efflux is observed under conditions which give rise to lysosomal enzyme release. A flow diagram of the events that are thought to accompany the stimulation of polymorphonuclear leukocytes (PMNs) by chemotactic or degranulating stimuli is presented.     

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.     

Characterization of ATP release from cultures enriched in cholinergic amacrine‐like neurons

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine‐like neurons. Depolarization of the cells with KCl, or activation of α‐amino‐3‐hydroxy‐ 5‐methyl‐4‐isoxazole‐propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca²⁺ dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and ω‐Agatoxin IVA, but not by ω‐Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca²⁺ influx necessary to trigger the release of ATP occurs in part through the L‐ and the P/Q types of voltage‐sensitive Ca²⁺ channels (VSCC), but not through N‐type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist, showing that the release is tonically inhibited by the adenosine A₁ receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 340–348, 1999     

Fibronectin stimulates TRPV1 translocation in primary sensory neurons

Extracellular matrix (ECM) molecules are highly variable in their composition and receptor recognition. Their ubiquitous expression profile has been linked to roles in cell growth, differentiation, and survival. Recent work has identified certain ECM molecules that serve as dynamic signal modulators, versus the more-recognized role of chronic modulation of signal transduction. In this study, we investigated the role that fibronectin (FN) plays in the dynamic modulation of transient receptor potential family V type 1 receptor (TRPV1) translocation to the plasma membrane in trigeminal ganglia (TG) sensory neurons. Confocal immunofluorescence analyses identify co-expression of the TRPV1 receptor with integrin subunits that bind FN. TG neurons cultured upon or treated with FN experienced a leftward shift in the EC₅₀ of capsaicin-stimulated neuropeptide release. This FN-induced increase in TRPV1 sensitivity to activation is coupled by an increase in plasma membrane expression of TRPV1, as well as an increase in tyrosine phosphorylation of TRPV1 in TG neurons. Furthermore, TG neurons cultured on FN demonstrated an increase in capsaicin-mediated Ca²⁺ accumulation relative to neurons cultured on poly- d -lysine. Data presented from these studies indicate that FN stimulates tyrosine-phosphorylation-dependent translocation of the TRPV1 receptor to the plasma membrane, identifying FN as a critical component of the ECM capable of sensory neuron sensitization.     

Noise enhances subthreshold oscillations in injured primary sensory neurons

Noise can play a constructive role in the detection of weak signals in various kinds of peripheral receptors and neurons. What the mechanism underlying the effect of noise is remains unclear. Here, the perforated patch-clamp technique was used on isolated cells from chronic compression of the dorsal root ganglion (DRG) model. Our data provided new insight indicating that, under conditions without external signals, noise can enhance subthreshold oscillations, which was observed in a certain type of neurons with high-frequency (20-100 Hz) intrinsic resonance from injured DRG neurons. The occurrence of subthreshold oscillation considerably decreased the threshold potential for generating repetitive firing. The above effects of noise can be abolished by blocking the persistent sodium current (I(Na, P)). Utilizing a mathematical neuron model we further simulated the effect of noise on subthreshold oscillation and firing, and also found that noise can enhance the electrical activity through autonomous stochastic resonance. Accordingly, we propose a new concept of the effects of noise on neural intrinsic activity, which suggests that noise may be an important factor for modulating the excitability of neurons and generation of chronic pain signals.