Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca2+ oscillations

Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca(2+) concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca(2+)/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca(2+) signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca(2+)-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca(2+)-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca(2+), induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca(2+)-releasing activity and activated with either CA-CaMKII cRNA or by SrCl(2), similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca(2+) changes that occur following fertilization.     

Starting a new life: sperm PLC-zeta mobilizes the Ca2+ signal that induces egg activation and embryo development: an essential phospholipase C with implications for male infertility

We have discovered that a single sperm protein, phospholipase C-zeta (PLCζ), can stimulate intracellular Ca(2+) signalling in the unfertilized oocyte ('egg') culminating in the initiation of embryonic development. Upon fertilization by a spermatozoon, the earliest observed signalling event in the dormant egg is a large, transient increase in free Ca(2+) concentration. The fertilized egg responds to the intracellular Ca(2+) rise by completing meiosis. In mammalian eggs, the Ca(2+) signal is delivered as a train of long-lasting cytoplasmic Ca(2+) oscillations that begin soon after gamete fusion and persist beyond the completion of meiosis. Sperm PLCζ effects Ca(2+) release from egg intracellular stores by hydrolyzing the membrane lipid PIP(2) and consequent stimulation of the inositol 1,4,5-trisphosphate (InsP(3) ) receptor Ca(2+) -signalling pathway, leading to egg activation and early embryogenesis. Recent advances have refined our understanding of how PLCζ induces Ca(2+) oscillations in the egg and also suggest its potential dysfunction as a cause of male infertility.     

Calcium signaling differentiation during Xenopus oocyte maturation

Ca(2+) is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca(2+) signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca(2+) signal during oocyte maturation. However, the mechanisms regulating Ca(2+) signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca(2+) (Ca(2+)(cyt)) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca(2+) transport effectors are tightly modulated. The plasma membrane Ca(2+) ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca(2+) out of the cell. Furthermore, IP(3)-dependent Ca(2+) release is required for the sustained Ca(2+)(cyt) rise in eggs, showing that Ca(2+) that is pumped into the ER leaks back out through IP(3) receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca(2+) despite the limited available Ca(2+) in intracellular stores. Therefore, Ca(2+) signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca(2+)(cyt) transient at fertilization, which defines the egg's competence to activate and initiate embryonic development.     

Oscillatory CaMKII activity in mouse egg activation

Fertilization-induced intracellular calcium (Ca(2+)) oscillations stimulate the onset of mammalian development, and little is known about the biochemical mechanism by which these Ca(2+) signals are transduced into the events of egg activation. This study addresses the hypothesis that transient increases in Ca(2+) similar to those at fertilization stimulate oscillatory Ca(2+)/calmodulin-dependent kinase II (CaMKII) enzyme activity, incrementally driving the events of egg activation. Since groups of fertilized eggs normally oscillate asynchronously, synchronous oscillatory Ca(2+) signaling with a frequency similar to fertilization was experimentally induced in unfertilized mouse eggs by using ionomycin and manipulating extracellular calcium. Coanalysis of intracellular Ca(2+) levels and CaMKII activity in the same population of eggs demonstrated a rapid and transient enzyme response to each increase in Ca(2+). Enzyme activity increased 370% during the first Ca(2+) rise, representing about 60% of maximal activity, and had decreased to basal levels within 5 min from the time Ca(2+) reached its peak value. Single fertilized eggs monitored for Ca(2+) had a mean increase in CaMKII activity of 185%. One and two ionomycin-induced Ca(2+) transients resulted in 39 and 49% mean cortical granule (CG) loss, respectively, while CG exocytosis and resumption of meiosis were inhibited by a CaMKII antagonist. These studies demonstrate that changes in the level of Ca(2+) and in CaMKII activity can be studied in the same cell and that CaMKII activity is exquisitely sensitive to experimentally induced oscillations of Ca(2+) in vivo. The data support the hypothesis that CaMKII activity oscillates for a period of time after normal fertilization and temporally regulates many events of egg activation.     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.     

CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs

Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs--fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Interactions between calcium release pathways: multiple messengers and multiple stores

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles.In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.     

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.     

Analysis of subcellular calcium signals in T-lymphocytes

Subcellular Ca(2+) signals were analysed in Jurkat and peripheral human T-lymphocytes by confocal Ca(2+) imaging employing an off-line deconvolution method. Stimulation of the TCR/CD3 complex in T-lymphocytes resulted in a series of subcellular pacemaker Ca(2+) signals preceding the first global Ca(2+) signal. The pacemaker signals occurred in a cytosolic "trigger" zone, which is localised close to the plasma membrane. The pacemaker signals were almost independent of extracellular Ca(2+) as shown by measurements in the absence of extracellular Ca(2+), or in the presence of the Ca(2+) channel blocker SK-F 96365. Analysis of the confocal Ca(2+) images revealed characteristic amplitudes of 82 +/- 30 to 109 +/- 21 nM, signal diameters between 2.5 +/- 0.9 and 3.5 +/- 1.5 microm and frequencies between 0.235 and 0.677 s(-1). Taken together, our data constitute the first analysis of subcellular Ca(2+) signals in T cells and indicate that the pacemaker Ca(2+) release events, which are necessary for the development of the global Ca(2+) signal, are composed of Ca(2+) release both from inositol 1,4,5-trisphosphate- and ryanodine receptors.     

Signal transduction pathways leading to Ca2+ release in a vertebrate model system: lessons from Xenopus eggs

At fertilization, eggs unite with sperm to initiate developmental programs that give rise to development of the embryo. Defining the molecular mechanism of this fundamental process at the beginning of life has been a key question in cell and developmental biology. In this review, we examine sperm-induced signal transduction events that lead to release of intracellular Ca(2+), a pivotal trigger of developmental activation, during fertilization in Xenopus laevis. Recent data demonstrate that metabolism of inositol 1,4,5-trisphosphate (IP(3)), a second messenger for Ca(2+) release, is carefully regulated and involves phospholipase C (PLC) and the tyrosine kinase Src. Roles of other potential regulators in this pathway, such as phosphatidylinositol 3-kinase, heterotrimeric GTP-binding protein, phospholipase D (PLD) and phosphatidic acid (PA) are also discussed. Finally, we address roles of egg lipid/membrane microdomains or 'rafts' as a platform for the sperm-egg membrane interaction and subsequent signaling events of egg activation.     

A bimodal pattern of InsP(3)-evoked elementary Ca(2+) signals in pancreatic acinar cells

InsP(3)-evoked elementary Ca(2+) release events have been postulated to play a role in providing the building blocks of larger Ca(2+) signals. In pancreatic acinar cells, low concentrations of acetylcholine or the injection of low concentrations of InsP(3) elicit a train of spatially localized Ca(2+) spikes. In this study we have quantified these responses and compared the Ca(2+) signals to the elementary events shown in Xenopus oocytes. The results demonstrate, at the same concentrations of InsP(3), Ca(2+) signals consisting of one population of small transient Ca(2+) release events and a second distinct population of larger Ca(2+) spikes. The signal mass amplitudes of both types of events are within the range of amplitudes for the elementary events in Xenopus oocytes. However, the bimodal Ca(2+) distribution of Ca(2+) responses we observe is not consistent with the continuum of event sizes seen in Xenopus. We conclude that the two types of InsP(3)-dependent events in acinar cells are both elementary Ca(2+) signals, which are independent of one another. Our data indicate a complexity to the organization of the Ca(2+) release apparatus in acinar cells, which might result from the presence of multiple InsP(3) receptor isoforms, and is likely to be important in the physiology of these cells.     

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.     

Thirty years of calcium signals at fertilization

It was discovered about 30 years ago that a dramatic increase in intracellular calcium ion concentration ([Ca(2+)](i)) occurs at fertilization and that this increase acts as the pivotal signal for egg activation. Later, the Ca(2+) signal at fertilization turned out to be ubiquitous among animal species. Extensive advance has been brought during these 30 years in research on spatiotemporal aspects and signaling mechanisms of the [Ca(2+)](i) increase, sperm factors that induce the Ca(2+) response, and cell cycle resumption caused by the [Ca(2+)](i) rise. I provide a historical account of these advances in mammals, sea urchins, and a few other models.     

Calcium-binding proteins: intracellular sensors from the calmodulin superfamily.

In all eukaryotic cells, and particularly in neurons, Ca(2+) ions are important second messengers in a variety of cellular signaling pathways. In the retina, Ca(2+) modulation plays a crucial function in the development of the visual system's neuronal connectivity and a regulatory role in the conversion of the light signal received by photoreceptors into an electrical signal transmitted to the brain. Therefore, the study of retinal Ca(2+)-binding proteins, which frequently mediate Ca(2+) signaling, has given rise to the important discovery of two subfamilies of these proteins, neuronal Ca(2+)-binding proteins (NCBPs) and calcium-binding proteins (CaBPs), that display similarities to calmodulin (CaM). These and other Ca(2+)-binding proteins are integral components of cellular events controlled by Ca(2+). Some members of these subfamilies also play a vital role in signal transduction outside of the retina. The expansion of the CaM-like protein family reveals diversification among Ca(2+)-binding proteins that evolved on the basis of the classic molecule, CaM. A large number of NCBP and CaBP subfamily members would benefit from their potentially specialized role in Ca(2+)-dependent cellular processes. Pinpointing the role of these proteins will be a challenging task for further research.     

Calcium signals and mitochondria at fertilisation

At fertilisation, Ca(2+) signals activate embryonic development by stimulating metabolism, exocytosis and endocytosis, cytoskeletal remodelling, meiotic resumption and recruitment of maternal RNAs. Mitochondria present in large number in eggs have long been thought to act as a relay in Ca(2+) signalling at fertilisation. However, only recently have studies on ascidians and mouse proven that sperm-triggered Ca(2+) waves are transduced into mitochondrial Ca(2+) signals that stimulate mitochondrial respiration. Mitochondrial Ca(2+) uptake can substantially buffer cytosolic Ca(2+) concentration and the concerted action of heterogeneously distributed mitochondria in the mature egg may modulate the spatiotemporal pattern of sperm-triggered Ca(2+) waves. Regulation of fertilisation Ca(2+) signals could also be achieved through mitochondrial ATP production and mitochondrial oxidant activity but these hypotheses remain to be explored. A critically poised dynamic interplay between Ca(2+) signals and mitochondrial metabolism is stimulated at fertilisation and may well determine whether the embryo can proceed further into development. The monitoring of Ca(2+) signals and mitochondrial activity during fertilisation in living zygotes of diverse species should confirm the universality of the role for sperm-triggered Ca(2+) waves in the activation of mitochondrial activity at fertilisation.     

Extracellular Ca2+ influx is crucial for the early embryonic development of the sea urchin Echinometra lucunter

The involvement of Ca(2+) in the activation of eggs and in the first steps of the embryonic development of several species is a well-known phenomenon. An association between Ca(2+) sources with the fate of the blastopore during embryonic development has been investigated by several authors. Ca(2+) influx mediated by voltage-gated channels and Ca(2+) mobilization from intracellular stores are the major sources of Ca(2+) to egg activation and succeeding cell divisions. Studies on sea urchins embryonic development show that intracellular Ca(2+) stores are responsible for egg activation and early embryogenesis. In the present work we investigated the involvement of extracellular Ca(2+) in the first stages of the embryonic development of the sea urchin Echinometra lucunter. Divalent cation chelators EDTA and EGTA strongly blocked the early embryonic development. Adding to this, we demonstrated the involvement of voltage-gated Ca(2+) channels in E. lucunter embryogenesis since Ca(2+) channel blockers powerfully inhibited the early embryonic development. Our data also revealed that Ca(2+) influx is crucial for embryonic development during only the first 40?min postfertilization. However, intracellular Ca(2+) remains mandatory to embryonic development 40?min postfertilization, seen that both the intracellular Ca(2+) chelator BAPTA-AM and calmodulin antagonists trifluoperazine and chlorpromazine inhibited the first stages of development when added to embryos culture 50?min postfertilization. Our work highlights the crucial role of extracellular Ca(2+) influx through voltage-gated Ca(2+) channels for the early embryonic development of the sea urchin E. lucunter and characterizes an exception in the phylum Echinodermata.     

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Selected contribution: effect of volatile anesthetics on cADP-ribose-induced Ca(2+) release system.

Volatile anesthetics have multiple actions on intracellular Ca(2+) homeostasis, including activation of the ryanodine channel (RyR) and sensitization of this channel to agonists such as caffeine and ryanodine. Recently it has been described that the nucleotide cADP-ribose (cADPR) is the endogenous regulator of the RyR in many mammalian cells, and cADPR has been proposed to be a second messenger in many signaling pathways. I investigated the effect of volatile anesthetics on the cADPR signaling system, using sea urchin egg homogenates as a model of intracellular Ca(2+) stores. Ca(2+) uptake and release were monitored in sea urchin egg homogenates by using the fluo-3 fluorescence technique. Activity of the ADP-ribosyl cyclase was monitored by using a fluorometric method using nicotinamide guanine dinucleotide as a substrate. Halothane in concentrations up to 800 microM did not induce Ca(2+) release by itself in sea urchin egg homogenates. However, halothane potentiates the Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine. Furthermore, other volatile anesthetics such as isoflurane and sevoflurane had no effect. Halothane also potentiated the activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased about three times by addition of 800 microM halothane. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to halothane. In contrast, all the volatile anesthetics used had no effect on the activity of the enzyme that synthesizes cADPR. I propose that the complex effect of volatile anesthetics on intracellular Ca(2+) homeostasis may involve modulation of the cADPR signaling system.