Does lysosomal rupture evoke Ca2+ release? A question of pores and stores

Lysosomotropic agents have been used to permeabilize lysosomes and thereby implicate these organelles in diverse cellular processes. Since lysosomes are Ca²⁺ stores, this rupturing action, particularly that induced by GPN, has also been used to rapidly release Ca²⁺ from lysosomes. However, a recent study has questioned the mechanism of action of GPN and concluded that, acutely, it does not permeabilize lysosomes but releases Ca²⁺ directly from the ER instead. We therefore appraise these provocative findings in the context of the existing literature. We suggest that further work is required to unequivocally rule out lysosomes as contributors to GPN-evoked Ca²⁺ signals.     

Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats

Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y₂ receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Ca²⁺, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y₂R-PLC-IP3 pathway to evoke Ca²⁺ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca²⁺]_I; this effect was partly inhibited by P2Y₂R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca²⁺]_i was significantly lower than that after treatment with P2Y₂R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca²⁺ triggers Ca²⁺ release from ER Ca²⁺ stores. Baseline [Ca²⁺]_i declined after treatment with the Ca²⁺ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca²⁺ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca²⁺ triggers Ca²⁺ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca²⁺ signals indirectly via the P2Y₂R-PLC-IP3 pathway.     

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.     

Acidic Ca(2+) stores in platelets

Changes in cytosolic free Ca²⁺ concentration play a pivotal role in the regulation of platelet functions, from secretion of autocrine and procoagulant factors to reversible or irreversible aggregation. It has long been recognized that platelet agonists release Ca²⁺ accumulated into the dense tubular system, the analogue of the endoplasmic reticulum. However, current evidence indicates that Ca²⁺ can also be stored and released from a number of acidic organelles, including lysosomes and lysosome-related organelles. Ca²⁺ release from the dense tubular system is mediated through phospholipase C-dependent synthesis of inositol 1,4,5-trisphosphate, whereas Ca²⁺ efflux from the acidic stores seems to be associated to the second messenger nicotinic acid adenine dinucleotide phosphate. The biochemical and biophysical properties of both Ca²⁺ stores in platelets have been reported to show significant differences. Selective discharge of one or both stores depends on the platelet agonist and the concentration used, which further supports the complexity of the Ca²⁺ signals that regulate platelet function. In this paper, we summarize the current knowledge on the role of acidic organelles in agonist-evoked Ca²⁺ mobilization and highlight recent progress in understanding the functional aspects of the acidic Ca²⁺ stores in Ca²⁺ signalling and platelet physiology.     

NAADP influences excitation-contraction coupling by releasing calcium from lysosomes in atrial myocytes

In atrial myocytes, the sarcoplasmic reticulum (SR) has an essential role in regulating the force of contraction as a consequence of its involvement in excitation-contraction coupling (ECC). Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺ mobilizing messenger that acts to release Ca²⁺ from an acidic store in mammalian cells. The photorelease of NAADP in atrial myocytes increased Ca²⁺ transient amplitude with no effect on accompanying action potentials or the L-type Ca²⁺ current. NAADP-AM, a cell permeant form of NAADP, increased Ca²⁺ spark amplitude and frequency. The effect on Ca²⁺ spark frequency could be prevented by bafilomycin A1, a vacuolar H⁺-ATPase inhibitor, or by disruption of lysosomes by GPN. Bafilomycin prevented staining of acidic stores with LysoTracker red by increasing lysosomal pH. NAADP-AM also produced an increase in the lysosomal pH, as detected by a reduction in LysoSensor green fluorescence. These effects of NAADP were associated with an increase in the amount of caffeine-releasable Ca²⁺ in the SR and may be regulated by β-adrenoceptor stimulation with isoprenaline. These observations are consistent with a role for NAADP in regulating ECC in atrial myocytes by releasing Ca²⁺ from an acidic store, which enhances SR Ca²⁺ release by increasing SR load.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Mitochondria have a well-established capacity to detect cytoplasmic Ca²⁺ signals resulting from the discharge of ER Ca²⁺ stores. Conversely, both the buffering of released Ca²⁺ and ATP production by mitochondria are predicted to influence ER Ca²⁺ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca²⁺] directly using trapped fluorescent low-affinity Ca²⁺ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca²⁺ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca²⁺ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca²⁺ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca²⁺ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca²⁺ signals.     

Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

Cross-Talk of the Receptors for Bradykinin, Serotonin, and ATP Shown by Single Cell Ca2+ Responses Indicating Different Modes of Ca2+ Activation in a Neuroblastoma × Glioma Hybrid Cell Line

Abstract: Modes of Ca²⁺ activation by bradykinin, serotonin, and ATP and the possible receptor cross-talk were investigated in mouse neuroblastoma × rat glioma hybrid cells (108CC15) by monitoring fura-2 fluorescence in single cells. A transient rise of cytosolic Ca²⁺ activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca²⁺ stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca²⁺-free medium abolished the Ca²⁺ response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, Induces the release of Ca²⁺ from intracellular stores. Serotonin, in contrast, activates Ca²⁺ entry from the extracellular space. To investigate whether ATP releases Ca²⁺ from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca²⁺. The application of ATP before that of bradykinin reduced the size of a following bradykinin-induced Ca²⁺ response in Ca²⁺-free medium, but not in Ca²⁺-containing medium. This suggests that bradykinin may interact with the ATP-activated mechanism by cross-desensitization. Possibly, bradykinin receptors are coupled to additional Ca²⁺ stores not accessible to ATP that are refilled by extracellular Ca²⁺. Cyclic AMP and cyclic GMP apparently do not affect the Ca²⁺ responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.     

Acidic intracellular Ca stores and caveolae in Ca signaling and diabetes

Acidic Ca²⁺ stores, particularly lysosomes, are newly discovered players in the well-orchestrated arena of Ca²⁺ signaling and we are at the verge of understanding how lysosomes accumulate Ca²⁺ and how they release it in response to different chemical, such as NAADP, and physical signals. Additionally, it is now clear that lysosomes play a key role in autophagy, a process that allows cells to recycle components or to eliminate damaged structures to ensure cellular well-being. Moreover, lysosomes are being unraveled as hubs that coordinate both anabolism via insulin signaling and catabolism via AMPK. These acidic vesicles have close contact with the ER and there is a bidirectional movement of information between these two organelles that exquisitely regulates cell survival. Lysosomes also connect with plasma membrane where caveolae are located as specialized regions involved in Ca²⁺ and insulin signaling. Alterations of all these signaling pathways are at the core of insulin resistance and diabetes.     

Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

Inositol 1,4,5-trisphosphate (IP₃) evokes release of Ca²⁺ from the endoplasmic reticulum (ER), but the resulting Ca²⁺ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A₁, which prevents lysosomal Ca²⁺ uptake by inhibiting H⁺ pumping into lysosomes, increased the amplitude of the initial Ca²⁺ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca²⁺ release from distinct IP₃-sensitive Ca²⁺ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A₁ similarly exaggerated the Ca²⁺ signals evoked by carbachol or carbachol with PTH, indicating that Ca²⁺ released from distinct IP₃-sensitive Ca²⁺ stores is sequestered by lysosomes. The Ca²⁺ signals resulting from store-operated Ca²⁺ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A₁. Using Gd³⁺ (1 mM) to inhibit both Ca²⁺ entry and Ca²⁺ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca²⁺ recycling to the ER after IP₃-evoked Ca²⁺ release. Blocking lysosomal Ca²⁺ uptake with bafilomycin A₁ increased the amplitude of each carbachol-evoked Ca²⁺ signal without affecting the rate of Ca²⁺ recycling to the ER. This suggests that Ca²⁺ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca²⁺ released by IP₃ receptors residing within distinct Ca²⁺ stores, but not the Ca²⁺ entering cells via receptor-regulated, store-operated Ca²⁺ entry pathways.     

Calcium-Induced Ultrastructural Interactions in Adrenocorticocytes

Steroidogenesis in adrenocorticocytes is closely related to intracellular [Ca²⁺]. To detect ultrastructural changes induced by growth in cytosolic [Ca²⁺], we used a rat adrenocortical cell culture, which was examined with electron microscopy and morphometric analysis. We established that either KCl-induced membrane depolarization evoking Ca²⁺ influx into the cell via voltage-operated Ca channels and Ca²⁺ ionophore, A23187, induced remarkable ultrastructural interactions between several cytosolic organelles. Lipid droplets known as key elements for Ca²⁺-induced steroidogenesis directly contacted with organelles containing the enzymes providing steroidogenic reactions (mitochondria, smooth and rough endoplasmic reticulum, nucleus, peroxisomes, and lysosomes). In most cases, the lipid droplets formed a specialized morphological structure at the sites of contact with the partner organelles. These structures are interpreted as a specialized transporting system, which provides contacts between organelles and exchange of intermediate products of the steroidogenesis process between the droplet and organelles.     

Okadaic acid induces the release of Ca from intracellular stores in ECV304 endothelial cells

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca²⁺ ([Ca²⁺]_c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca²⁺]_c both in the presence and absence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ stores with either ATP (1 μM) or thapsigargin (100 nM) prevented any further release of Ca²⁺ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca²⁺), a prior release of Ca²⁺ induced by okadaic acid reduced the magnitude of the response to ATP (1 μM). Taken together these observations indicate that okadaic acid induces Ca²⁺ release from the agonist-sensitive pool. The okadaic acid-induced Ca²⁺ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca²⁺]_c oscillations, which at their peak resulted in 40–100% cells, at any one time, having an elevated [Ca²⁺]_c. The response appeared to propagate between adjacent cells and the elevation of [Ca²⁺]_c. appeared initially in the cell periphery. In adherent cells, the release of Ca²⁺ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca²⁺ from the agonist- and thapsigargin-sensitive intracellular stores.     

Staurosporine maintains the activation of store-operated Ca2+ entry even after the refilling of Ca2+ stores

Store-operated Ca²⁺ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca²⁺ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ entry from the extracellular space. Ca²⁺ release was not affected by treatment with ST, but Ca²⁺ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca²⁺ sequestration into Ca²⁺ stores. The Ca²⁺ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca²⁺ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca²⁺ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La³⁺ and 2-APB. The large increase in Ca²⁺ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca²⁺ stores. These results show that SOCE remains activated even after the refilling of Ca²⁺ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.     

Release of arachidonic acid via Ca2+ increase stimulated by pyrophosphonucleotides and bradykinin in mammary tumour cells

The relationship between the increase of intracellular Ca²⁺ and the release of arachidonic acid by bradykinin and pyrophosphonucleotides was studied in cultured mammary tumour cells, MMT060562. Bradykinin, ATP, UTP and UDP induced an increase of intracellular Ca²⁺ and the release of arachidonic acid from phospholipids into the extracellular fluid. Release of arachidonic acid was also induced by the application of the Ca²⁺ ionophore, A23187. Liberation of arachidonic acid by bradykinin and ATP was reduced by mepacrine, a blocker of phospholipase A₂ and W-7, a calmodulin antagonist. It is suggested that the increase in cytosolic Ca²⁺-induced release of arachidonic acid occurs through activation of calmodulin-dependent phospholipase A₂.     

ATP-induced morphological changes in supporting cells of the developing cochlea

The developing cochlea of mammals contains a large group of columnar-shaped cells, which together form a structure known as Kölliker’s organ. Prior to the onset of hearing, these inner supporting cells periodically release adenosine 5′-triphosphate (ATP), which activates purinergic receptors in surrounding supporting cells, inner hair cells and the dendrites of primary auditory neurons. Recent studies indicate that purinergic signaling between inner supporting cells and inner hair cells initiates bursts of action potentials in auditory nerve fibers before the onset of hearing. ATP also induces prominent effects in inner supporting cells, including an increase in membrane conductance, a rise in intracellular Ca²⁺, and dramatic changes in cell shape, although the importance of ATP signaling in non-sensory cells of the developing cochlea remains unknown. Here, we review current knowledge pertaining to purinergic signaling in supporting cells of Kölliker’s organ and focus on the mechanisms by which ATP induces changes in their morphology. We show that these changes in cell shape are preceded by increases in cytoplasmic Ca²⁺, and provide new evidence indicating that elevation of intracellular Ca²⁺ and IP₃ are sufficient to initiate shape changes. In addition, we discuss the possibility that these ATP-mediated morphological changes reflect crenation following the activation of Ca²⁺-activated Cl⁻ channels, and speculate about the possible functions of these changes in cell morphology for maturation of the cochlea.     

Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K⁺) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca²⁺-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca²⁺ concentration, or increase of extracellular K⁺ to a depolarizing level that opens astrocytic L-channels for Ca²⁺ and thereby also increase intra-astrocytic Ca²⁺ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K⁺ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K⁺ concentrations.     

Agonist-Specific Calcium Signaling and Phosphoinositide Hydrolysis in Human SK-N-MCIXC Neuroepithelioma Cells

Abstract: Fura-2 digital imaging microfluorimetry was used to evaluate the Ca²⁺ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca²⁺_i) but of different magnitudes (ET-1 = ATP> NE). The Ca²⁺ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca²⁺ influx. The Ca²⁺ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca²⁺ signal, which was characteristic of the response to Oxo-M, was observed in <40% of cells stimulated with ET-1 and absent from Ca²⁺ signals elicited after NE addition. Removal of extracellular Ca²⁺ enhanced the rate of decay of Ca²⁺ signals generated by ATP, ET-1, or Oxo-M and, when evident, abolished the sustained phase of Ca²⁺ influx. In the absence of extracellular Ca²⁺, NE elicited asynchronous multiple Ca²⁺ transients. In either the absence or presence of extracellular Ca²⁺,>94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were ～74 and ～48%. Sequential addition of agonists to cells maintained in a Ca²⁺-free buffer indicated that each ligand mobilized Ca²⁺ from a common intracellular pool. When monitored as a release of a total inositol phosphate fraction, all four agonists elicited similar (four- to sixfold) increases in phosphoinositide hydrolysis. However, the addition of ET-1 or ATP resulted in larger increases in the net formation of inositol 1,4,5-trisphosphate than did either Oxo-M or NE. These results indicate that, in SK-N-MCIXC cells, the characteristics of both Ca²⁺ signaling and inositol phosphate production are agonist specific.     

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca<Superscript>2+</Superscript> waves

Intercellular Ca²⁺ waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca²⁺ waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca²⁺ waves are triggered during damage to cochlear explants. Both Ca²⁺ waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca²⁺, their velocity, their extent of spread and the cell type through which they propagate. A slower Ca²⁺ wave (14 μm/s) communicates between Deiters’ cells and is mediated by P2Y receptors and Ca²⁺ release from IP₃-sensitive stores. In contrast, a faster Ca²⁺ wave (41 μm/s) propagates through sensory hair cells and is mediated by Ca²⁺ influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca²⁺ wave is mediated by P2X₄ receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.