Spatiotemporal analysis of Ca(2+) waves in relation to the sperm entry site and animal-vegetal axis during Ca(2+) oscillations in fertilized mouse eggs

Fertilized mouse eggs exhibit repetitive rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca(2+)](i) rise were investigated by high-speed Ca(2+) imaging during early development of monospermic eggs. Every [Ca(2+)](i) rise involved a Ca(2+) wave. In the first Ca(2+) transient, [Ca(2+)](i) increased in two steps separated by a "shoulder" point, suggesting two distinct Ca(2+) release mechanisms. The first step was a Ca(2+) wave that propagated from the sperm-fusion site to its antipode in 4-5 s (velocity, approximately 20 microm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca(2+)](i) rise of 12-15 s. A slight cytoplasmic movement followed the Ca(2+) wave in the same direction and recovered in 25-35 s. These characteristics changed as follows, as Ca(2+) oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation ( approximately 3 h after fertilization). (1) The duration of Ca(2+) transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca(2+)](i) rise became greater and wave speeds increased up to 80-100 microm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca(2+) wave, although its displacement became smaller. (5) The Ca(2+) wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.     

The BSA-induced Ca(2+) influx during sperm capacitation is CATSPER channel-dependent

Background  

Serum albumin is a key component in mammalian sperm capacitation, a functional maturation process by which sperm become competent to fertilize oocytes. Capacitation is accompanied by several cellular and molecular changes including an increased tyrosine phosphorylation of sperm proteins and a development of hyperactivated sperm motility. Both of these processes require extracellular calcium, but how calcium enters sperm during capacitation is not well understood.     

Arachidonic acid in astrocytes blocks Ca(2+) oscillations by inhibiting store-operated Ca(2+) entry,and causes delayed Ca(2+) influx

ATP-elicited oscillations of the concentration of free intracellular Ca(2+) ([Ca(2+)](i)) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca(2+) store refilling. Short-term application of AA, but not ETYA, blocked Ca(2+) influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca(2+)](i) oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca(2+)](i) increase. This AA-induced [Ca(2+)](i) rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 microM Gd(3+), indicative for the influx of extracellular Ca(2+). Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca(2+)](i), firstly, a rapid reduction of capacitative Ca(2+) entry thereby suppressing [Ca(2+)](i) oscillations, and secondly inducing a delayed activation of Ca(2+) entry, also sensitive to low Gd(3+) concentration.     

Ca(2+) influx and neurotransmitter release at ribbon synapses

Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis.     

A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium

Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.     

Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.     

Evidence that phospholipase C from the sperm is not responsible for initiating Ca(2+) release at fertilization in mouse eggs

Release of Ca(2+) from intracellular stores at fertilization of mammalian eggs is mediated by inositol 1,4,5-trisphosphate (IP3), but the mechanism by which the sperm initiates IP3 production is not yet understood. We tested the hypothesis that phospholipase C (PLC) activity introduced into the mouse egg as a consequence of sperm-egg fusion is responsible for causing Ca(2+) release. We demonstrated that microinjecting purified, recombinant PLCgamma1 protein into mouse eggs caused Ca(2+) oscillations like those seen at fertilization. However, the PLC activity in the minimum amount of purified PLCgamma1 protein needed to elicit Ca(2+) release when injected into eggs was approximately 500-900 times the PLC activity contained in a single sperm. This indicates that a single mouse sperm does not contain enough PLC activity to be responsible for causing Ca(2+) release at fertilization. We also examined whether phosphatidylinositol 3-kinase (PI3K) could have a role in this process, and found that several inhibitors of PI3K-mediated signaling had no effect on Ca(2+) release at fertilization.     

Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.     

Caffeine alleviates the deterioration of Ca(2+) release mechanisms and fragmentation of in vitro-aged mouse eggs

The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others have found that in these eggs, the intracellular calcium ([Ca(2+)](i)) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca(2+) release is not well known. Here, we investigated if the function of IP(3)R1, the major Ca(2+) release channel at fertilization, was undermined in in vitro-aged mouse eggs. We found that in aged eggs, IP(3)R1 displayed reduced function as many of the changes acquired during maturation that enhance IP(3)R1 Ca(2+) conductivity, such as phosphorylation, receptor reorganization and increased Ca(2+) store content ([Ca(2+)](ER)), were lost with increasing postovulatory time. IP(3)R1 fragmentation, possibly associated with the activation of caspase-3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP(3)R(1) function and maintained [Ca(2+)](ER) content. Caffeine also maintained mitochondrial membrane potential, as measured by JC-1 fluorescence. We therefore conclude that [Ca(2+)](i) responses in aged eggs are undermined by reduced IP(3)R1 sensitivity, decreased [Ca(2+)](ER) , and compromised mitochondrial function, and that addition of caffeine ameliorates most of these aging-associated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs.     

Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs

Mammalian eggs and embryos rely upon mitochondrial ATP production to survive and proceed through preimplantation development. Ca(2+) oscillations at fertilization have been shown to cause a reduction of mitochondrial NAD+ and flavoproteins, suggesting they might also cause changes in cytosolic ATP levels. Here, we have monitored intracellular Ca(2+) and ATP levels in fertilizing mouse eggs by imaging the fluorescence of a Ca(2+) dye and luminescence of firefly luciferase. At fertilization an initial increase in ATP levels occurs with the first Ca(2+) transient, with a second increase occurring about 1 h later. The increase in cytosolic ATP was estimated to be from a prefertilization concentration of 1.9 mM to a peak value of 3 mM. ATP levels returned to prefertilization values as the Ca(2+) oscillations terminated. An increase in ATP also occurred with other stimuli that increase Ca(2+) and it was blocked when Ca(2+) oscillations were inhibited by BAPTA injection. Additionally, an ATP increase was not seen when eggs were activated by cycloheximide, which does not cause a Ca(2+) increase. These data suggest that mammalian fertilization is associated with a sudden but transient increase in cytosolic ATP and that Ca(2+) oscillations are both necessary and sufficient to cause this increase in ATP levels.     

Ca(2+) oscillations promote APC/C-dependent cyclin B1 degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs

Cyclin B1, the regulatory component of M phase-promoting factor (MPF), is degraded during the metaphase-anaphase transition in an anaphase-promoting complex/cyclosome (APC/C)-dependent process. MPF activity is stable in eggs, and a sperm-triggered Ca(2+) signal is needed to promote cyclin degradation. In frogs, a single Ca(2+) spike promotes cell cycle resumption, but, in mammals, the Ca(2+) signal is more complex, consisting of a series of spikes that stop several hours after sperm fusion. Using dual imaging in mouse eggs, we have examined how the Ca(2+) signal generates cyclin B1 destruction using destructible and nondestructible GFP-tagged constructs. APC/C activity was present in unfertilized eggs, giving cyclin B1 a half-life of 1.15 +/- 0.28 hr. However, APC/C-dependent cyclin degradation was elevated 6-fold when sperm raised cytosolic Ca(2+) levels above 600 nM. This activation was transitory since cyclin B1 levels recovered between Ca(2+) spikes. For continued cyclin degradation at basal Ca(2+) levels, multiple spikes were needed. APC/C-mediated degradation was observed until eggs had completed meiosis with the formation of pronuclei, and, at this time, Ca(2+) spikes stopped. Therefore, the physiological need for a repetitive Ca(2+) signal in mammals is to ensure long-term cyclin destruction during a protracted exit from meiosis.     

An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump,ECA1, supports plant growth and confers tolerance to Mn(2+) stress

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity.     

Sperm extract injection into ascidian eggs signals Ca(2+) release by the same pathway as fertilization

Injection of eggs of various species with an extract of sperm cytoplasm stimulates intracellular Ca(2+) release that is spatially and temporally like that occurring at fertilization, suggesting that Ca(2+) release at fertilization may be initiated by a soluble factor from the sperm. Here we investigate whether the signalling pathway that leads to Ca(2+) release in response to sperm extract injection requires the same signal transduction molecules as are required at fertilization. Eggs of the ascidian Ciona intestinalis were injected with the Src-homology 2 domains of phospholipase C gamma or of the Src family kinase Fyn (which act as specific dominant negative inhibitors of the activation of these enzymes), and the effects on Ca(2+) release at fertilization or in response to injection of a sperm extract were compared. Our findings indicate that both fertilization and sperm extract injection initiate Ca(2+) release by a pathway requiring phospholipase C gamma and a Src family kinase. These results support the hypothesis that, in ascidians, a soluble factor from the sperm cytoplasm initiates Ca(2+) release at fertilization, and indicate that the activating factor from the sperm may be a regulator, directly or indirectly, of a Src family kinase in the egg.     

Role of elementary Ca(2+) puffs in generating repetitive Ca(2+) oscillations

Inositol (1,4,5)-trisphosphate (IP(3)) liberates intracellular Ca(2+) both as localized 'puffs' and as repetitive waves that encode information in a frequency-dependent manner. Using video-rate confocal imaging, together with photorelease of IP(3) in Xenopus oocytes, we investigated the roles of puffs in determining the periodicity of global Ca(2+) waves. Wave frequency is not delimited solely by cyclical recovery of the cell's ability to support wave propagation, but further involves sensitization of Ca(2+)-induced Ca(2+) release by progressive increases in puff frequency and amplitude at numerous sites during the interwave period, and accumulation of pacemaker Ca(2+), allowing a puff at a 'focal' site to trigger a subsequent wave. These specific 'focal' sites, distinguished by their higher sensitivity to IP(3) and close apposition to neighboring puff sites, preferentially entrain both the temporal frequency and spatial directionality of Ca(2+) waves. Although summation of activity from many stochastic puff sites promotes the generation of regularly periodic global Ca(2+) signals, the properties of individual Ca(2+) puffs control the kinetics of Ca(2+) spiking and the (higher) frequency of subcellular spikes in their local microdomain.     

Intracellular Ca(2+) and Ca(2+)/calmodulin-dependent kinase II mediate acute potentiation of neurotransmitter release by neurotrophin-3

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve-muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca(2+) influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca(2+) influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca(2+). Depletion of intracellular Ca(2+) stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca(2+) release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.     

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.     

Stimulation of neutrophils by prenylcysteine analogs: Ca(2+) release and influx.

Farnesylthiosalicylic acid (FTS), a synthetic analog of the terminal prenylcysteine present in signaling proteins induces generation of superoxide ions, phospholipase C-driven hydrolysis of inositol lipids and calcium elevation in human neutrophils and DMSO-differentiated HL60 cells. These effects were ascribed to an interaction of the analog with elements responsible for recognition of specific prenylated proteins. The present study demonstrated that in addition to the release of intracellular calcium stores, FTS enhanced entry of Ca(2+) and Mn(2+) from the medium. The biphasic dependence of the influx on the concentration of FTS, as well as its insensitivity to inhibition by PMA and La(3+) suggest that the influx pathway activated by FTS is distinct from the previously described store-operated calcium channels of neutrophils. Consistent with the participation of a cellular membrane component in the interaction, FTS enhanced (45)Ca uptake in neutrophils and neutrophil cell membranes, but not in multilamellar vesicles. To establish specificity of the farnesyl moiety of FTS (C(15)), effects of three other analogs, geranylthiosalicylate, GTS (C(10)), geranylgeranylthiosalicylate, GGTS (C(20)), as well as the carboxymethyl ester FTS-Me on calcium homeostasis and superoxide production were investigated. GGTS dose-dependently elevated [Ca(2+)](i), induced quenching of the 360 nm Fura-2-calcium fluorescence by Mn(2+) and stimulated superoxide release, while GTS and FTS-Me were inactive. These results defined specific structural requirements for the functional interaction of prenylcysteine analogs with myeloid cells.     

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.     

17 beta-estradiol increases Ca(2+) influx and down regulates interleukin-2 receptor in mouse thymocytes

The influx of Ca(2+) across the T lymphocyte membrane is an essential triggering signal for activation and proliferation by an antigen. The aim of this study was to determine if Ca(2+) influx through estradiol receptor (ER) operated channels of Ca(2+) entry induced activation of lymphoid cells. Mouse thymocytes were incubated with 17 beta-estradiol (E) and in the presence or absence of the mitogen, phytohemagglutinin (PHA). Despite evidence of an enhanced binding of E to ER on thymocyte membranes, and an E dose-related influx of Ca(2+), there was a consistent down regulation of IL-2 receptor expression (P < 0.001). Incubation of thymocytes with PHA enhanced IL-2 receptor expression although the down regulatory effect of E was still evident. The results suggest that the Ca(2+) channel activated by E may have a down regulatory effect on the IL-2 receptor in thymus cells leading to the dampening of cell activation process.     

2-aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca(2+) discharge and store-operated Ca(2+) influx.

We have investigated Ca(2+) release and receptor- and store-operated Ca(2+) influxes in Chinese hamster ovary-K1 (CHO) cells, SH-SY5Y human neuroblastoma cells and RBL-1 rat basophilic leukemia cells using Fura-2 and patch-clamp measurements. Ca(2+) release and subsequent Ni(2+)-sensitive, store-operated influx were induced by thapsigargin and stimulation of G protein-coupled receptors. The alleged noncompetitive IP3 receptor inhibitor,2-aminoethoxydiphenyl borate (2-APB) rapidly blocked a major part of the secondary influx response in CHO cells in a reversible manner. It also reduced Mn(2+) influx in response to thapsigargin. Inhibition of Ca(2+) release was also seen but this was less complete, slower in onset, less reversible, and required higher concentration of 2-APB. In RBL-1 cells, I(CRAC) activity was rapidly blocked by extracellular 2-APB whereas intracellular 2-APB was less effective. Store-operated Ca(2+) influxes were only partially blocked by 2-APB. In SH-SY5Y cells, Ca(2+) influxes were insensitive to 2-APB. Ca(2+) release in RBL-1 cells was partially sensitive but in SH-SY5Y cells the release was totally resistant to 2-APB. The results suggest, that 2-APB (1) may inhibit distinct subtypes of IP3 receptors with different sensitivity, and (2) that independently of this, it also inhibits some store-operated Ca(2+) channels via a direct, extracellular action.