Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca²⁺-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca²⁺. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.     

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.     

Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca²⁺-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca²⁺-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR–lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte–mediated immune responses.     

EAAT2 density at the astrocyte plasma membrane and Ca2 + -regulated exocytosis

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca²⁺ strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca²⁺-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca²⁺-dependent manner, since in the absence of extracellular Ca²⁺ local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.     

CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for Ca²⁺-triggered vesicle exocytosis, but whether vesicles fuse into PIP₂-rich membrane domains in live cells and whether PIP₂ is metabolized during Ca²⁺-triggered fusion were unknown. Ca²⁺-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP₂-binding proteins required for Ca²⁺-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP₂, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP₂-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP₂ for its activity. Munc13 is cytoplasmic, but Ca²⁺-dependent translocation to PIP₂-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP₂-rich membrane domains is facilitated by sequential PIP₂-dependent activation of CAPS and PIP₂-dependent recruitment of Munc13. PIP₂ hydrolysis only occurs under strong Ca²⁺ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca²⁺-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP₂ linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.     

Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca²⁺ is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca²⁺ also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca²⁺-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca²⁺-dependent SNARE binding or in Ca²⁺-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca²⁺-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca²⁺-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca²⁺-dependent membrane insertion and SNARE binding to drive fusion pore expansion.     

Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

Exocytosis is evoked by intracellular signals, including Ca²⁺ and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca²⁺]_i increases induced either through Ca²⁺ influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca²⁺ elevations. Neither this Ca²⁺-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca²⁺-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca²⁺ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca²⁺ elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca²⁺ sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca²⁺-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.     

Disruption-induced mucus secretion: repair and protection

When a cell suffers a plasma membrane disruption, extracellular Ca²⁺ rapidly diffuses into its cytosol, triggering there local homotypic and exocytotic membrane fusion events. One role of this emergency exocytotic response is to promote cell survival: the internal membrane thus added to the plasma membrane acts as a reparative “patch.” Another, unexplored consequence of disruption-induced exocytosis is secretion. Many of the cells lining the gastrointestinal tract secrete mucus via a compound exocytotic mechanism, and these and other epithelial cell types lining the digestive tract are normally subject to plasma membrane disruption injury in vivo. Here we show that plasma membrane disruption triggers a potent mucus secretory response from stomach mucous cells wounded in vitro by shear stress or by laser irradiation. This disruption-induced secretory response is Ca²⁺ dependent, and coupled to cell resealing: disruption in the absence of Ca²⁺ does not trigger mucus release, but results instead in cell death due to failure to reseal. Ca²⁺-dependent, disruption-induced mucus secretion and resealing were also demonstrable in segments of intact rat large intestine. We propose that, in addition to promoting cell survival of membrane disruptions, disruption-induced exocytosis serves also the important protective function of liberating lubricating mucus at sites of mechanical wear and tear. This mode of mechanotransduction can, we propose, explain how lubrication in the gastrointestinal tract is rapidly and precisely adjusted to widely fluctuating, diet-dependent levels of mechanical stress.     

Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca²⁺ influx, which triggers insulin exocytosis. The mitochondrial Ca²⁺ uniporter (MCU) mediates Ca²⁺ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca²⁺ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca²⁺. Suppression of the putative Ca²⁺/H⁺ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca²⁺-induced matrix acidification. These results demonstrate that MCU-mediated Ca²⁺ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.     

Ca2+ dependence and kinetics of cell membrane repair after electropermeabilization

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca²⁺]. We explored the impact of Ca²⁺ on the kinetics of membrane resealing by monitoring the entry of a YO-PRO-1 dye (YP) in BPAE and HEK cells. Ca²⁺ was promptly removed or added after the electric pulse (EP) by a fast-step perfusion. YP entry increased sharply after the EP and gradually slowed down following either a single- or a double-exponential function. In BPAE cells permeabilized by a single 300- or 600-ns EP at 14 kV/cm in a Ca²⁺-free medium, perfusion with 2 mM of external Ca²⁺ advanced the 90% resealing and reduced the dye uptake about twofold. Membrane restoration was accomplished by a combination of fast, Ca²⁺-independent resealing (τ = 13–15 s) and slow, Ca²⁺-dependent processes (τ ~70 s with Ca²⁺ and ~ 110 s or more without it). These time constants did not change when the membrane damage was doubled by increasing EP duration from 300 to 600 ns. However, injury by microsecond-range EP (300 and 600 μs) took longer to recover even when the membrane initially was less damaged, presumably because of the larger size of pores made in the membrane. Full membrane recovery was not prevented by blocking both extra- and intracellular Ca²⁺ (by loading cells with BAPTA or after Ca²⁺ depletion from the reticulum), suggesting the recruitment of unknown Ca²⁺-independent repair mechanisms.     

Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K⁺, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca²⁺ influx through L-type Ca²⁺ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca²⁺ influx and accelerated F-actin breakdown on removal of Ca²⁺.To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca²⁺ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.     

The small chemical vacuolin-1 alters the morphology of lysosomes without inhibiting Ca2+-regulated exocytosis

Ca2+-regulated exocytosis of lysosomes was previously shown to be required for the repair of plasma membrane wounds. The small chemical vacuolin-1 alters the morphology of lysosomes without affecting the ability of cells to reseal their plasma membrane after injury. On the basis of a failure to detect Ca2+-triggered lysosomal exocytosis in vacuolin-1-treated cells, a recent study proposed that lysosomes are dispensable for resealing. Here, we show that vacuolin-1, despite altering lysosome morphology, does not inhibit the exocytosis of lysosomes induced by exposure to a Ca2+ ionophore, or by plasma membrane wounding. Thus, lysosomes cannot be excluded as agents of membrane repair in vacuolin-1-treated cells.     

Fine-tuning of store-operated calcium entry by fast and slow Ca2+-dependent inactivation: Involvement of SARAF

Store-operated Ca²⁺ entry (SOCE) is a functionally relevant mechanism for Ca²⁺ influx present in electrically excitable and non-excitable cells. Regulation of Ca²⁺ entry through store-operated channels is essential to maintain an appropriate intracellular Ca²⁺ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca²⁺-dependent inactivation mediated by two temporally separated mechanisms: fast Ca²⁺-dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca²⁺ with residues in the channel pore while slow Ca²⁺-dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca²⁺]_cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P₂-rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca²⁺ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca²⁺ signals.     

Triggered exocytosis and endocytosis have different requirements for calcium and nucleotides in permeabilized bovine chromaffin cells

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells.In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules.The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca²⁺ and MgATP. Although secretion requires Ca²⁺ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca²⁺ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides.These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca²⁺ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.We are grateful to Mr. John Gibbs for excellent technical assistance, and to the Medical Research Council (UK) for financial support.     

VAMP3 is associated with endothelial Weibel-Palade bodies and participates in their Ca-dependent exocytosis

Weibel-Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca²⁺-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca²⁺-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Assay of dense-core vesicle exocytosis using permeabilized PC12 cells

Exocytosis plays an essential role in fundamental cellular events by secreting neurotransmitters, hormones, and cytokines. Although the minimal molecular components termed SNARE that govern membrane fusion have been identified, the precise mechanisms behind the finely-tuned regulation of exocytosis executed by many molecules in addition to the actions of SNARE remain to be fully identified. Here, we evaluated a model system for assaying catecholamine secretion from permeabilized rat pheochromocytoma PC12 cells, in which the structural integrity required was preserved adequately. Among several chemical reagents used for the cell permeabilization and freezing-thawing procedures, the treatment of cells with digitonin at concentrations of 7.5–15 μM was most suitable for the secretion assay, as it was considered to cause mild disruption of the plasma membrane, enabling free access to small molecules such as Ca²⁺ and ATP to the minimal membrane fusion machinery. No additional cytosolic proteins were required to reconstitute the secretion. In this assay model, ATP was necessary to maintain the priming state before Ca²⁺-triggered exocytosis but was not required for the Ca²⁺-triggered membrane fusion process itself. The present study provides a useful cell model for exploring novel molecules that may be implicated in exocytosis such as those playing regulatory roles in addition to the “minimal membrane fusion machinery for exocytosis”, which does not require any additional special apparatus.     

Synaptotagmin IV Acts as a Multi-Functional Regulator of Ca<Superscript>2+</Superscript>-Dependent Exocytosis

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca²⁺) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca²⁺-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca²⁺-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca²⁺-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca²⁺-dependent exocytosis.     

T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.     

The Calcium-mobilizing Messenger Nicotinic Acid Adenine Dinucleotide Phosphate Participates in Sperm Activation by Mediating the Acrosome Reaction

Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca²⁺ from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn²⁺ quenching of luminal fura-2 and ⁴⁵Ca²⁺ to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca²⁺. We propose a new model for acrosome reaction signaling in which Ca²⁺ influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca²⁺ to drive acrosomal exocytosis.