Intercellular Ca2 Research Articles

Mitochondria modulate the spatio-temporal properties of intra- and intercellular Ca 2+ signals in cochlear supporting cells

In the cochlea, cell damage triggers intercellular Ca 2+ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca 2+ waves are thought to convey information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca 2+ concentration ([Ca 2+] cyt), and yet little is known about their role during the propagation of such intercellular Ca 2+ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell [Ca 2+] cyt signals that are triggered by ATP or by hair cell damage. ATP application (0.1–50 μM) caused a dose dependent increase in [Ca 2+] cyt which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca 2+ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca 2+ uniporter, enhanced the peak amplitude and duration of ATP-induced [Ca 2+] cyt transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca 2+ waves was significantly increased. Thus, mitochondria function as spatial Ca 2+ buffers during agonist-evoked [Ca 2+] cyt signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca 2+ waves.

Activity-dependent neuronal control of gap-junctional communication in fibroblasts

Intercellular communication through gap junctions plays an important role in fibroblast physiology. Here we report an activity-dependent neuronal control of gap-junctional communication (GJC) in rat dermal fibroblasts, which is associated with N-methyl- d-aspartate (NMDA) glutamate receptor-mediated elevations in intracellular Ca 2+. Both spontaneous and induced activation of dorsal root ganglion (DRG) neurons, manifested by action potentials and TTX-dependent inward currents, significantly reduced fibroblast GJC in Neuron/Fibroblast (N/F) cocultures. Reduced fibroblast GJC by DRG neurons was prevented by blockade of NMDA receptors and decrease of intra- and intercellular Ca 2+. Immunocytochemistry showed that the NR1 subunit of the NMDA receptor coexisted with CX43 at the plasma membrane of fibroblasts. Moreover, glutamate applied to fibroblast cultures triggered NMDA receptor-dependent intracellular Ca 2+ elevations and decrease of GJC. These data demonstrate that NMDA receptor activation contributes to downregulation of GJC in fibroblasts. Since fibroblasts have been shown to facilitate DRG neurite growth and survival, our findings provide a glimpse at the impact that neuronal activation has on fibroblast networks.

Role of K+ Channels in Regulating Spontaneous Activity in Detrusor Smooth Muscle In Situ in the Mouse Bladder

We investigated the functional role of K(+) channels for regulating spontaneous activity in mouse bladder detrusor smooth muscle. The effects of different K(+) channels blockers on spontaneous changes in membrane potential and intracellular Ca(2+) dynamics were examined using intracellular recording techniques and Ca(2+) imaging with fluo-4 fluorescence, respectively. Detrusor smooth muscle generated spontaneous action potentials and Ca(2+) transients. Iberiotoxin (0.1 microM), charybdotoxin (0.1 microM) or tetraethylammonium (1 mM) increased the amplitude of action potentials and prolonged their repolarizing phase without inhibiting their after-hyperpolarization. Tetraethylammonium (10 mM) but not stromatoxin (0.1 microM) suppressed after-hyperpolarization and further increased the amplitude and half duration of action potentials. Apamin (0.1 microM) increased the frequency of action potentials but had no effect on their configuration. Spontaneous Ca(2+) transients were generated in individual detrusor smooth muscle cells and occasionally propagated to neighboring cells to form intercellular Ca(2+) waves. Transmural nerve stimulations invariably initiated synchronous Ca(2+) transients within and across muscle bundles. Charybdotoxin (0.1 microM) increased the amplitude of spontaneous Ca(2+) transients, while the subsequent application of tetraethylammonium (10 mM) increased their half duration. In addition, tetraethylammonium increased the synchronicity of Ca(2+) transients in muscle bundles. These results suggest that large and intermediate conductance Ca(2+) activated K(+) channels contribute to action potential repolarization and restrict the excitability of detrusor smooth muscle in the mouse bladder. In addition, the activation of voltage dependent K(+) channels is involved in repolarization and after-hyperpolarization, and it has a fundamental role in stabilizing detrusor smooth muscle excitability.

ER localized bestrophin1 activates Ca2+ dependent ion channels TMEM16A and SK4

AbstractBestrophins form Ca2+ activated Cl- channels and regulate intercellular Ca2+ signaling1. We demonstrate that bestrophin 1 is localized in the endoplasmic reticulum (ER), where it physically interacts with stromal interacting molecule 1 (Stim1), the ER-Ca2+ sensor2,3. Intracellular Ca2+ transients in HEK293 cells elicited by stimulation of purinergic P2Y2-receptors were augmented but more transient after expression of hBest1, in contrast to dominant negative hBest1-R218C, which attenuated Ca2+ increase. The p21-activated protein kinase Pak2 was found to phosphorylate hBest1, thereby enhancing Ca2+ signaling and activation of Ca2+ dependent Cl- (TMEM16A)4 and K+ (SK4)5 channels. Lack of bestrophin 1 expression in respiratory epithelial cells of mBest1 knockout mice caused expansion of ER cisterns and induced Ca2+ deposits. We propose that hBest1 is important for Ca2+ handling of the ER store, probably by controlling the function of Stim1 and by acting as a counter-ion channel to balance transient membrane potentials occurring through inositol trisphosphate (IP3) induced Ca2+ release and refill of the ER-Ca2+ store. Thus bestrophin 1 controls activation of Ca2+ dependent ion channels by regulation of compartmentalized Ca2+ signaling.

Extracellular ATP Mediates The Intercellular Ca2+ Wave Induced By Mechanical Stimulation In Human Salivary Gland Cells

Intracellular Ca2+ signaling has a central role in regulation of salivary gland cell function. Coordination of Ca2+ signaling between cells contributes to synchronized and effective secretion of saliva. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca2+ waves (ICW) and their propagation in human salivary gland (HSG) cells were investigated using fura-2 fluorescence imaging. While not well understood, mechanical stimulation of a single cell in a cluster with a micropipette induces ICW. The Ca2+ signal is propagated from the stimulated cell to the 7-9th tier of cells or ∼120 μm. The following findings indicate that ICW propagation in HSG cells uses an extracellular and ATP-dependent pathway. The purinergic receptor antagonist suramin significantly decreased ICW propagation. Extracellular ATP or UTP abolished ICW suggestive of receptor desensitization. Gap junction intercellular communication is not involved in ICW in HSG cells because the gap junction inhibitor oleamide did not inhibit ICW. Furthermore, HSG cells showed poor dye coupling upon microinjection of Lucifer Yellow. The Ca2+ transients observed within each cell are dependent on Ca2+ release from the ER as thapsigargin abolished the ICW. The phospholipase C inhibitor U73122 also blocks ICW indicating that these transients are IP3-dependent. Furthermore, store operated Ca2+ entry (SOCE) modulates the amplitude of Ca2+ signal since removal of extracellular Ca2+ or a SOCE inhibitor SK&F 96365 decreased the amplitude of Ca2+ signal. Inhibition of mitochondrial Ca2+ uptake with FCCP/oligomycin or ruthenium red showed similar effects on the amplitude. These results indicate that propagation of this ICW utilizes extracellular ATP, likely through the P2U(P2Y2) receptor in HSG cells. The major Ca2+ mobilization mechanisms are IP3-dependent ER Ca2+ release and SOCE. Finally, mitochondrial energy metabolism and Ca2+ uptake modulated this ICW propagation.

Propagation of Fast and Slow Intercellular Calcium Waves in Primary Cultured Smooth Muscle Cells

Tissue blood flow is controlled by the changes in the diameter of the arteries and arterioles due to the coordinated contraction and relaxation of smooth muscle cells (SMCs) within the vascular wall. The contractile state of SMCs is regulated primarily by the intracellular Ca2+ concentration ([Ca2+]i). The increase in [Ca2+]i in response to hormonal stimuli propagates from cell to cell along the vessel wall as a wave, and activates the process of contraction. The mechanism underlying this phenomenon, however, is not yet fully revealed.In this work, we study the onset and propagation of intercellular calcium waves through gap junctions in primary cultured vascular SMCs. For imaging intercellular Ca2+ waves, SMCs seeded along a collagen line and loaded with the fluorescent Ca2+ indicator Fluo-4 were locally stimulated mechanically or chemically. The stimulation evoked two distinct calcium waves: 1) a fast Ca2+ wave (several mm/s), and 2) a much slower Ca2+ wave (few tens of μm/s); both waves propagated to neighboring cells. The fast Ca2+ wave was caused by the propagation of membrane depolarization and subsequent Ca2+ influx through voltage operated channels. This fast wave facilitated the onset and propagation of a slow, but higher amplitude Ca2+ wave that started from the stimulated cell and propagated to neighboring cells. Our results suggest a possible mechanism for intercellular Ca2+ wave propagation through gap junction channels in SMCs.

Both sides now: multiple interactions of ATP with pannexin-1 hemichannels. Focus on “A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP”

how atp and other nucleotides are released from intact cells is a fundamental question, given the existence of multiple purinergic receptor signaling cascades operative in most vertebrate tissues ([25][1]). It is well-established that neurons and neuroendocrine cells release ATP via classical

Sorting Out Astrocyte Physiology from Pharmacology

A number of exciting findings have been made in astrocytes during the past 15 years that have led many researchers to redefine how the brain works. Astrocytes are now widely regarded as cells that propagate Ca(2+) over long distances in response to stimulation, and, similar to neurons, release transmitters (called gliotransmitters) in a Ca(2+)-dependent manner to modulate a host of important brain functions. Although these discoveries have been very exciting, it is essential to place them in the proper context of the approaches used to obtain them to determine their relevance to brain physiology. This review revisits the key observations made in astrocytes that greatly impact how they are thought to regulate brain function, including the existence of widespread propagating intercellular Ca(2+) waves, data suggesting that astrocytes signal to neurons through Ca(2+)-dependent release of glutamate, and evidence for the presence of vesicular machinery for the regulated exocytosis of gliotransmitters.

Monitoring of IP3 dynamics during the mechanical stimulation-induced intra- and intercellular Ca2+ waves in HSY human parotid cell line

It is well known that mechanical stimulation elicits Ca responses in various cell types. Touch stimulation on a single cell induces an increase in the cytosolic Ca concentration ([Ca]i), which subsequently propagates to neighboring cells as an intercellular Ca wave. In addition to Ca entry through mechanosensitive channels, this Ca response is thought to be induced, at least in part, by inositol 1,4,5-trisphosphate (IP3)-dependent Ca release from intracellular Ca stores (1-3). However, this hypothesis has not been tested directly due to a lack of the quantitative method to measure the cytosolic concentration of IP3 ([IP3]i) in a single living cell. Recently, we developed a FRET-based IP3 biosensor, LIBRAvIII, and a method to measure [IP3]i quantitatively in the single living cell (4). In order to examine the involvement of IP3 in mechanical stimulation-induced Ca responses, we monitored IP3 and Ca responses simultaneously in HSY-EA1 cells, a human parotid cell line, using LIBRAvIII and the Ca indicator fura-2.

ATP Release from Cultured Endothelial Cells and Intercellular Calcium Signaling during Shear Stress Exposure

Intracellular calcium ([Ca2+]i) is a second messenger molecule critical for numerous intracellular signaling pathways in endothelial cells (ECs). Direct mechanical stimulation imposed on single ECs by a microprobe has been demonstrated to increase [Ca2+]i levels in ECs. After an initial delay time, this Ca2+-signal propagates from the mechanically stimulated cell to its neighboring cells in the form of an intercellular Ca2+-wave, a process termed intercellular communication. Although intercellular communication is a fundamental property of many multicellular systems, it remains unclear as to whether intercellular communication following shear stress of ECs actually occurs, and if so, whether this communication occurs via gap junctions or the release of extracellular mediators including ATP. In the current study, we investigated ATP release from ECs during periods of shear stress and measured intercellular Ca2+-waves using adenosine 5'-triphosphate, P3-(1-(2-nitrophenyl)ethyl)ester and disodium salt (NPE-caged ATP) stimulation. In addition, we investigated intercellular communication in ECs during shear stress using chemical inhibitors of both gap junctions and various components of the ATP paracrine signaling pathway. ECs subjected to shear stress loading released ATP. Using NPE-caged ATP, local increase of extracellular ATP induced a Ca2+-wave. Furthermore, [Ca2+]i responses in ECs under shear stress loading was inhibited by the purinergic receptor blocker, ATPase, and several metabolic inhibitors including FCCP and rotenone. These results suggest that [Ca2+]i communication mediated by ATP exists in ECs under shear stress loading in vitro.

Reduced Intercellular Communication and Altered Morphology of Bovine Corneal Endothelial Cells with Prolonged Time in Cell Culture

Purpose: Mechanical stimulation induces intercellular Ca2 + waves in the corneal endothelium. The extent of the wave propagation is dependent on the activity of gap junctions, hemichannels, and ectonucleotidases. To further establish the use of a cell culture model to investigate intercellular communication, in this study, we have characterized the changes in the Ca2 + wave propagation in bovine corneal endothelial cells with prolonged time in culture. Materials and Methods: Freshly isolated BCEC were cultured for a short term (8 to 14 days; referred to as “short term”) and a long term (21 to 30 days; referred to as “long term”). Cell surface area and size were measured by confocal microscopy and flow cytometry, respectively. Calcium wave propagation was assayed by imaging spread of the Ca2 + waves elicited by mechanical stimulation. ATP release was assayed using Luciferin-Luciferase bioluminescence technique. Results: Cells cultured for a long term showed larger surface area and size compared to those cultured for a short term, but a reduced spread of the Ca2 + wave. Exposure to exogenous apyrases, which can rapidly hydrolyze extracellular ATP, reduced the spread of the Ca2 + wave in both groups. The fractional decrease, however, was smaller in cells cultured for a long term. Exposure to ARL-67156 to inhibit the ectonucleotidases led to a larger enhancement of the active area in cells cultured for a long term. However, the active areas of the two groups were not significantly different in the presence of the drug. Furthermore, ATP release in response to mechanical stimulation was lower in cells cultured for a long term in the absence of ARL-67156 but not in its presence. Conclusions: BCEC cultured for a long term show an increase in cell surface area and cell size similar to the effect of aging in human corneas. Moreover, the cells cultured for a long term showed a reduced ATP-dependent paracrine intercellular communication, largely due to an increase in the activity of the ectonucleotidases.

Gap junctional intercellular communication in the juxtaglomerular apparatus

The juxtaglomerular apparatus (JGA) is a specialized contact region between the glomerulus and the cortical thick ascending limb that plays an active role in the maintenance of ion homeostasis and control of blood pressure. The JGA accommodates several different cell types, including vascular smooth muscle cells, endothelial cells, mesangial cells, macula densa cells, and renin-secreting juxtaglomerular granular cells. These cells, with the exception of the macular densa cells, are tightly coupled by gap junctions. Gap junction-mediated intercellular communication in the JGA provides a pathway for signal transduction and coordination of multicellular functions. Disruption of cell-to-cell communication in the JGA results in altered preglomerular vascular tone and renin secretion. This review summarizes recent data about the roles of gap junctions in the JGA and illustrates how gap junction-mediated intercellular Ca(2+) signals determine physiological responses in the JGA.

59 THE INFLUENCE OF DILUENT EGG YOLK PARTICLES ON THE ACCURACY OF FLOW CYTOMETRIC ANALYSES OF SPERM FUNCTIONAL CHARACTERISTICS

Flow cytometry is an important tool to assess semen quality and sperm function, especially after cryopreservation, but the presence of egg yolk particles in many semen diluents can distort the results. The aims of the present study were to investigate the effect of the presence of egg yolk in the semen diluent on the outcome of flow cytometric analysis of dog spermatozoa and to explore the use of Laser Dye Styryl-751 (LDS-751), a permeant DNA stain with an emission wavelength of 712 nm, to exclude egg yolk particles from the analysis of plasma membrane fluidity (Merocyanine 540 and Yo-Pro-1) and intracellular Ca2+ (Fluo-3 and PI). Flow cytometric analyses were carried out on a Coulter Epics XL flow cytometer (Beckman-Coulter Inc., Fullerton, CA). Data were analyzed using EXPO32TM ADC software (Beckman-Coulter). A paired t-test was used to test whether gating out egg yolk particles using LDS-751 affected the analysis. Method agreement analysis was undertaken to assess the agreement between the 2 staining protocols (i.e. with or without LDS-751). The proportion of spermatozoa with high plasma membrane fluidity was significantly (P < 0.05) greater after gating out egg yolk particles; the measurements of high membrane fluidity spermatozoa were on average 7.7 percentage points greater than without gating out egg yolk as shown by the method agreement analysis (SD = 9.35). Similarly, the proportion of spermatozoa with high intercellular Ca2+ was significantly (P < 0.01) greater after gating out egg yolk particles; the proportions of spermatozoa with high intracellular Ca2+ were 5.95 percentage points greater when egg yolk particles were gated out compared with measurements in the presence of egg yolk particles (SD = 5.43). The method agreement shows that both methods show close agreement with and without egg yolk particles (i.e. results mostly fall within the mean of differences between pairs of repeated measurements ±2 SD). Therefore, the 2 methods are measuring the same phenomena. However, the change in measurement values with exclusion of egg yolk particles is significant. Therefore, accuracy is increased; that is, closer to the true value. In conclusion, using MC540/Yo-Pro-1 and Fluo-3/PI staining alone in the presence of egg yolk particles resulted in underestimation of the proportions of high plasma membrane fluidity and high intracellular Ca2+ spermatozoa, respectively. LDS-751 could be used with other fluorescent stains that target organelles such as mitochondria and acrosomes with multicolor flow cytometric analysis to exclude egg yolk particles.

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear.

Extracellular ATP controls various signaling systems including propagation of intercellular Ca(2+) signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP(3)-generating agonist and evokes Ca(2+) responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP(3) elicited Ca(2+) responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca(2+) signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WT cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Px1), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca(2+) responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca(2+) responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca(2+) signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca(2+)-mobilizing second messengers across coupled cells.

Intercellular Ca 2+ wave propagation involving positive feedback between CRAC channels and cysteinyl leukotrienes

Mast cells are key components of the immune system, where they help orchestrate the inflammatory response. Aberrant mast cell activation is linked to a variety of allergic diseases, including asthma, eczema, rhinitis, and nasal polyposis, which in combination affect up to 20% of the population in industrialized countries. On activation, mast cells release a variety of signals that target the bronchi and vasculature and recruit other immune cells to the inflammatory site. Prominent among such signals are the cysteinyl leukotrienes, a family of potent proinflammatory lipid mediators comprising leukotriene C(4) (LTC(4)), LTD(4), and LTE(4). LTC(4), the parent compound, is secreted from mast cells following Ca(2+) influx through store-operated calcium release-activated calcium (CRAC) channels. Here, we show that activated mast cells release a paracrine signal that evokes Ca(2+) signals in spatially separate resting mast cells. The paracrine signal was identified as a cysteinyl leukotriene because 1) RNAi knockdown or pharmacological block of the 5-lipoxygenase enzyme prevented activated mast cells from stimulating resting cells. 2) Block of cysteinyl leukotriene type I receptors on resting mast cells with the clinically prescribed receptor antagonist montelukast prevented their activation by active mast cells. 3) RNAi knockdown of cysteinyl leukotriene type I receptors on resting cells prevented them from responding to the paracrine signal derived from activated mast cells. 4) Purified LTC(4) evoked Ca(2+) signals in mast cells that were identical to those triggered by the paracrine signal. Low levels of stimulus intensity released sufficient levels of leukotriene to activate resting cells. Leukotriene secretion still occurred tens of minutes after stimulation, suggesting a role as a long-lasting trigger in mast cell activation. Stimulation of the cysteinyl leukotriene receptor activated CRAC channels and evoked prominent store-operated Ca(2+) entry. This resulted in further cysteinyl leukotriene production, triggering a positive feedback cascade. Acutely isolated mast cells from patients with allergic rhinitis exhibited store-operated Ca(2+) influx through CRAC channels and responded to cysteinyl leukotrienes. Histological analysis of samples taken from patients revealed clustering of mast cells, often located within 20 microm of each other, a distance sufficient for paracrine signaling by leukotrienes to operate effectively. We conclude that a positive-feedback cascade involving CRAC channels and cysteinyl leukotrienes constitute a novel mechanism for sustaining mast cell activation.

Cellular diversity promotes intercellular Ca 2+ wave propagation

Calcium ions are an important second messenger in living cells. Calcium signals in form of waves serve as a means of intercellular communication and thus represent a vibrant subject for experimental and theoretical investigations. Here we study the role of cellular variability on the occurrence of Ca 2+ wave propagation in a net of diffusively coupled cells. Dynamics of individual cells is simulated by a mathematical model for Ca 2+ oscillations. Structural diversity of cells is introduced via variations of the bifurcation parameters, which signify cell sensitivity for external stimulation. Remarkably, for sufficient values of variability Ca 2+ waves emerge, which are mostly ordered for intermediate variability strength. We analyze the spatial profile via the autocorrelation function, which confirms a resonance-like response due to the cellular variability. Thus, the reported phenomenon is a novel observation of diversity-induced spatial coherence resonance in a tissue-like media.

Sustained depolarization-induced propagation of [Ca 2+] i oscillations in cultured DRG neurons: The involvement of extracellular ATP and P2Y receptor activation

Recently emerging evidence implicates a number of neuroactive substances and their receptors in mediating complex cell-to-cell communications in the ganglia. In the present study, we characterized the nonsynaptic chemical coupling mediated by extracellular ATP in dorsal root ganglia (DRG) neuron cultures by using the real time imaging of ATP, whole-cell patch clamping, in conjunction with confocal calcium imaging. Sustained depolarization by electrical stimulation evoked intracellular Ca 2+ concentrations ([Ca 2+] i) oscillations in individual DRG neurons, and subsequent ATP-dependent propagation [Ca 2+] i oscillations to surrounding non-stimulated neighbors. [Ca 2+] i oscillations were suppressed by inositol-1,4,5-trisphosphate (IP 3) receptor antagonist 2-APB, but not ryanodine. The propagation of [Ca 2+] i oscillations was prevented by the presence of the ATP-degrading enzyme, apyrase, and completely abolished by the blockase of G protein-coupled purinergic receptors-PLC-IP 3 pathway with suramin, U73122 or 2-APB. In parallel, sustained depolarization elicited robust ATP release and diffusion from the stimulation site. Moreover, exogenous application of ATP to DRG cultures in large concentration elicits the [Ca 2+] i oscillations in most neurons. Taken together, this data demonstrates that sustained membrane depolarization elicited ATP release, acting through a highly sensitive P2Y receptors/IP 3-mediated signaling pathway to mediate the propagation of intercellular Ca 2+ signaling, which suggest a novel signaling pathway for neuronal communication in DRG.

The Myosin II ATPase Inhibitor Blebbistatin Prevents Thrombin-Induced Inhibition of Intercellular Calcium Wave Propagation in Corneal Endothelial Cells

Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility. BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses. Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP. Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.

Damage-Induced Activation of ERK1/2 in Cochlear Supporting Cells Is a Hair Cell Death-Promoting Signal That Depends on Extracellular ATP and Calcium

Acoustic overstimulation and ototoxic drugs can cause permanent hearing loss as a result of the damage and death of cochlear hair cells. Relatively little is known about the signaling pathways triggered by such trauma, although a significant role has been described for the c-Jun N-terminal kinase [one of the mitogen-activated protein kinases (MAPKs)] pathway. We investigated the role of another MAPK family, the extracellularly regulated kinases 1 and 2 (ERK1/2) during hair cell damage in neonatal cochlear explants. Within minutes of subjecting explants to mechanical damage, ERK1/2 were transiently activated in Deiters' and phalangeal cells but not in hair cells. The activation of ERK1/2 spread along the length of the cochlea, reaching its peak 5-10 min after damage onset. Release of extracellular ATP and the presence of functional connexin proteins were critical for the activation and spread of ERK1/2. Damage elicited an intercellular Ca(2+) wave in the hair cell region in the first seconds after damage. In the absence of Ca(2+) influx, the intercellular Ca(2+) wave and the magnitude and spread of ERK1/2 activation were reduced. Treatment with the aminoglycoside neomycin produced a similar pattern of ERK1/2 activation in supporting cells surrounding pyknotic hair cells. When ERK1/2 activation was prevented, there was a reduction in the number of pyknotic hair cells. Thus, activation of ERK1/2 in cochlear supporting cells in vitro is a common damage signaling mechanism that acts to promote hair cell death, indicating a direct role for supporting cells in regulating hair cell death.

Connexin 43 Hemichannels Are Permeable to ATP

Astrocytes are electrically nonexcitable cells that communicate by means of Ca(2+) signaling. Long-distance intercellular Ca(2+) waves are initiated by release of ATP and activation of purinergic receptors on nearby cells. Previous studies have implicated connexin 43 (Cx43) in ATP release, but definitive proof that ATP exits through Cx43 hemichannels does not exist. Here, through several alternative approaches, we show that ATP anions can permeate through Cx43 hemichannels. First, openings of Cx43 hemichannels were detected in both cell-attached and inside-out patch recordings in C6 cells expressing Cx43, but not in C6 cells expressing Cx43-eGFP (enhanced green fluorescent protein) or a C-terminus truncation mutant of Cx43. Second, Cx43 hemichannel openings were inhibited by three structurally different gap-junction channel blockers, but not by the P2X(7) blocker Brilliant blue G. Third, bioluminescence imaging of ATP combined with single-channel recording in the inside-out patch configuration showed that ATP efflux coincided with channel openings and was absent when the Cx43 hemichannel was closed. Fourth, ion replacement experiments confirmed that Cx43 hemichannels are permeable to ATP. In summary, these observations provide the first direct evidence for efflux of ATP through Cx43 hemichannels. Furthermore, a putative Cx43 hemichannel with characteristics identical to the Cx43 hemichannel in C6 cells was identified in the membrane of hippocampal astrocytes in acutely prepared slices.