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.     

Calcium Current Inactivation Rather than Pool Depletion Explains Reduced Exocytotic Rate with Prolonged Stimulation in Insulin-Secreting INS-1 832/13 Cells

Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca²⁺-dependent insulin exocytosis with respect to pool depletion and Ca²⁺-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔC_m), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔC_m and Q suggests that Ca²⁺-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca²⁺-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.     

Ca2+ influx and clearance at hyperpolarized membrane potentials modulate spontaneous and stimulated exocytosis in neuroendocrine cells

Neuroendocrine adrenal chromaffin cells release neurohormones catecholamines in response to Ca²⁺ entry via voltage-gated Ca²⁺ channels (VGCCs). Adrenal chromaffin cells also express non-voltage-gated channels, which may conduct Ca²⁺ at negative membrane potentials, whose role in regulation of exocytosis is poorly understood. We explored how modulation of Ca²⁺ influx at negative membrane potentials affects basal cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in metabolically intact voltage-clamped bovine adrenal chromaffin cells. We found that in these cells, Ca²⁺ entry at negative membrane potentials is balanced by Ca²⁺ extrusion by the Na⁺/Ca²⁺ exchanger and that this balance can be altered by membrane hyperpolarization or stimulation with an inflammatory hormone bradykinin. Membrane hyperpolarization or application of bradykinin augmented Ca²⁺-carrying current at negative membrane potentials, elevated basal [Ca²⁺]_i, and facilitated synchronous exocytosis evoked by the small amounts of Ca²⁺ injected into the cell via VGCCs (up to 20 pC). Exocytotic responses evoked by the injections of the larger amounts of Ca²⁺ via VGCCs (> 20 pC) were suppressed by preceding hyperpolarization. In the absence of Ca²⁺ entry via VGCCs and Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger, membrane hyperpolarization induced a significant elevation in [Ca²⁺]_i and asynchronous exocytosis. Our results indicate that physiological interferences, such as membrane hyperpolarization and/or activation of non-voltage-gated Ca²⁺ channels, modulate basal [Ca²⁺]_i and, consequently, segregation of exocytotic vesicles and their readiness to be released spontaneously and in response to Ca²⁺ entry via VGCCs. These mechanisms may play role in homeostatic plasticity of neuronal and endocrine cells.     

Localization and properties of a high-affinity (Ca2+ + Mg2+)-ATPase in isolated kidney cortex plasma membranes

Surface membrane fractions from Paramecium tetraurelia cells contain a calmodulin-stimulated Ca²⁺-ATPase responding to low levels of free Ca²⁺ and with features characteristic of a membrane-bound ATPase responding to low levels of free Ca²⁺ and with features characteristics of a membrane-bound ATPase. Among the different strains analyzed this enzyme was practically absent selectively from the ‘non-discharge” mutant nd9—28°C (from J. Beisson); if cultured at a permissive temperature (18°C), this strain showed identical values of calmodulin-stimulated Ca²⁺-ATPase activity as wild-type cells (7S) or strains with mutations which do not affect exocytosis performance. We conclude that this calmodulin-stimulated Ca²⁺-activated ATPase might be a prerequisite for membrane fusion in the course of exocytosis performance.     

CRAC channels in secretory epithelial cell function and disease

The receptor-evoked Ca²⁺ signal in secretory epithelia mediate many cellular functions essential for cell survival and their most fundamental functions of secretory granules exocytosis and fluid and electrolyte secretion. Ca²⁺ influx is a key component of the receptor-evoked Ca²⁺ signal in secretory cell and is mediated by both TRPC and the STIM1-activated Orai1 channels that mediates the Ca²⁺ release-activated current (CRAC) I_crac. The core components of the receptor-evoked Ca²⁺ signal are assembled at the ER/PM junctions where exchange of materials between the plasma membrane and internal organelles take place, including transfer of lipids and Ca²⁺. The Ca²⁺ signal generated at the confined space of the ER/PM junctions is necessary for activation of the Ca²⁺-regulated proteins and ion channels that mediate exocytosis with high fidelity and tight control. In this review we discuss the general properties of Ca²⁺ signaling, PI(4,5)P₂ and other lipids at the ER/PM junctions with regard to secretory cells function and disease caused by uncontrolled Ca²⁺ influx.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

The role of calcium in depigmentation of iris epithelial cells during cell-type conversion

During the conversion of newt iris epithelial cells into lens cells, melanosomes disappear from the cytoplasm. In this “depigmentation,” exocytosis of melanosomes is involved. The role of Ca²⁺ in this process has been the subject of this work. The intracellular Ca²⁺ concentration of cultured iris epithelial cells was increased by three methods: microinjection of 10^?3, M CaCl₂ into the cytoplasm, fusion of phospholipid vesicles containing 10^?3, M CaCl₂ with the cell membrane, and exposure to the calcium ionophore A23187. Each of these treatments caused an increase in the release of melanosomes. Further experiments suggest that cAMP stimulates exocytosis probably by liberating Ca²⁺ from intracellular stores.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

On depolarization-evoked exocytosis as a function of calcium entry: possibilities and pitfalls

Secretion from many endocrine cells is a result of calcium-regulated exocytosis due to Ca²⁺ influx. Using the patch-clamp technique, voltage pulses can be applied to the cells to open Ca²⁺ channels, resulting in a measurable Ca²⁺ current, and evoke exocytosis, which can be seen as an increase in membrane capacitance. A common tool for evaluating the relation between Ca²⁺ influx and exocytosis is to plot the increase in capacitance (ΔC_m) as a function of the integral of the measured Ca²⁺ current (Q). When depolarizations of different lengths are imposed, the rate of exocytosis is typically higher for shorter than for longer pulses, which has been suggested to result from depletion of a granule pool or from Ca²⁺ current inactivation. It is here demonstrated that ΔC_m as a function of Q can reveal whether Ca²⁺ current inactivation masquerades as pool depletion. Moreover, it is shown that a convex, cooperativity-like, relation between ΔC_m and Q surprisingly cannot occur as a result of cooperative effects, but can result from delays in the exocytotic process or in Ca²⁺ dynamics. An overview of expected ΔC_m-versus-Q relations for a range of explicit situations is given, which should help in the interpretation of data of depolarization-evoked exocytosis in endocrine cells.     

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.     

Calcium channels and signal transduction in plant cells

An increasing number of studies indicate that changes in cytosolic free Ca²⁺ ([Ca²⁺]_c) mediate specific types of signal transduction in plant cells. Modulation of [Ca²⁺]_c is likely to be achieved through changes in the activity of Ca²⁺ channels, which catalyse passive influx of Ca²⁺ to the cytosol from extracellular and intracellular compartments. Voltage-sensitive Ca²⁺ channels have been detected in the plasma membranes of algae, where they control membrane electrical properties and cell turgor. These channels are sensitive to 1,4-dihydropyridines, which in animal cells specifically affect one class of voltage-regulated plasma membrane Ca²⁺ channel. Ca²⁺-permeable channels with different pharmacological properties have been found in the plasma membrane of higher plants. Recent evidence suggests the existence of two discrete classes of Ca²⁺ channel co-resident in the vacuolar membrane (tonoplast) of higher plants. The first is gated by inositol 1,4,5-trisphosphate, and bears a number of similarities to its animal counterpart which is located in the endoplasmic reticulum (ER). The second tonoplast Ca²⁺ channel is voltage-operated. However, the specific roles of these tonoplast channels in signal transduction have yet to be elucidated.     

Evidence for Mechanistic Alterations of Ca2+ Homeostasis in Type 2 Diabetes Mellitus

Altered cytosolic Ca²⁺ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca²⁺ regulation. Using human lymphocytes, we studied cytosolic calcium (Ca_i) and various Ca²⁺ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca²⁺-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca²⁺ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), was used to study Ca²⁺- store dependent Ca²⁺ fluxes. Significant (P < 0.05) elevation of basal Ca_i levels was observed in lymphocytes from diabetic subjects. Ca_i levels were positively correlated with fasting, plasma glucose and HbAlc. There was also a significant (P < 0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca²⁺ influx (as measured both by Fluo-3 fliorescence and C45a assays) as a consequence of of thapsigargin-mediated Ca²⁺ store depletion. Upon addition of Mn²⁺ (a surrogate of Ca²⁺), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P < 0.05) difference in the Na⁺/Ca²⁺ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca²⁺ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca²⁺ entry. We conclude that cellular Ca²⁺ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na⁺/Ca²⁺ exchange and (3) increased Ca²⁺ influx across the plasma membrane.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.     

Identifying the targets of the amplifying pathway for insulin secretion in pancreatic beta-cells by kinetic modeling of granule exocytosis

A kinetic model for insulin secretion in pancreatic β-cells is adapted from a model for fast exocytosis in chromaffin cells. The fusion of primed granules with the plasma membrane is assumed to occur only in the “microdomain” near voltage-sensitive L-type Ca²⁺-channels, where [Ca²⁺] can reach micromolar levels. In contrast, resupply and priming of granules are assumed to depend on the cytosolic [Ca²⁺]. Adding a two-compartment model to handle the temporal distribution of Ca²⁺ between the microdomain and the cytosol, we obtain a unified model that can generate both the fast granule fusion and the slow insulin secretion found experimentally in response to a step of membrane potential. The model can simulate the potentiation induced in islets by preincubation with glucose and the reduction in second-phase insulin secretion induced by blocking R-type Ca²⁺-channels (Ca_V2.3). The model indicates that increased second-phase insulin secretion induced by the amplifying signal is controlled by the “resupply” step of the exocytosis cascade. In contrast, enhancement of priming is a good candidate for amplification of first-phase secretion by glucose, cyclic adenosine 3′:5′-cyclic monophosphate, and protein kinase C. Finally, insulin secretion is enhanced when the amplifying signal oscillates in phase with the triggering Ca²⁺-signal.     

Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis

Cytoplasmic calcium concentration ([Ca²⁺]_i) and extracellular calcium (Ca²⁺_o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β-D-Glc)₃). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (β-D-Glc)₃, which binds arabinogalactan proteins (AGPs). Application of (β-D-Glc)₃ inhibited growth but not secretion. Ratiometric imaging of [Ca²⁺]_i revealed an initial spread in the locus of the apical [Ca²⁺]_i gradient and substantial elevations in basal [Ca²⁺]_i followed by the establishment of new regions of elevated [Ca²⁺]_i on the flanks of the tip region. Areas of elevated [Ca²⁺]_i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca²⁺_o influx at the tip of (β-D-Glc)₃-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca²⁺]_i, probably resulting from Ca²⁺_o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.     

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.     

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca²⁺-dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (Ca_V1.2 and Ca_V1.3), Ca_V1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using Ca_V1 .3^-/- KO mice and the AP-clamp it has been possible to resolve the time course of Ca_V1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells Ca_V1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca²⁺ currents and activate BK channels confers to Ca_V1.3 the unique feature of driving Ca²⁺ loading during long interspike intervals and, possibly, to control the Ca²⁺-dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

Cytoplasm calcium-binding proteins of germ cells and embryos of the sea urchin

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca²⁺ in cells ([Ca²⁺]_i).Ca²⁺ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984).The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca²⁺- and calmodulin-dependent protein phosphatase, Ca²⁺-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca²⁺- and calmodulin control of sperm motility in sea urchins.     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

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.