Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis.

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.

Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation.

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca(2+)-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.

Melanoma growth and progression after ultraviolet a irradiation: impact of lysosomal exocytosis and cathepsin proteases.

Ultraviolet (UV) irradiation is a risk factor for development of malignant melanoma. UVA-induced lysosomal exocytosis and subsequent cell growth enhancement was studied in malignant melanoma cell lines and human skin melanocytes. UVA irradiation caused plasma membrane damage that was rapidly repaired by calcium-dependent lysosomal exocytosis. Lysosomal content was released into the culture medium directly after irradiation and such conditioned media stimulated the growth of non-irradiated cell cultures. By comparing melanocytes and melanoma cells, it was found that only the melanoma cells spontaneously secreted cathepsins into the surrounding medium. Melanoma cells from a primary tumour showed pronounced invasion ability, which was prevented by addition of inhibitors of cathepsins B, D and L. Proliferation was reduced by cathepsin L inhibition in all melanoma cell lines, but did not affect melano-cyte growth. In conclusion, UVA-induced release of cathepsins outside cells may be an important factor that promotes melanoma growth and progression.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Optical control of calcium-regulated exocytosis.

BACKGROUND: Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis. METHODS: Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx. RESULTS: Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs. CONCLUSION: LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells. GENERAL SIGNIFICANCE: LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.

[The rationale for the application of laserophoresis of biologically active compounds].

Laserophoresis is a technique for the transcutaneous administration of biologically active compounds by means of low-intensity laser radiation (LFLR). It is currently regarded as a most promising method for the integrated application of a pharmaceutical substance and a physical factor. At present laserophoresis of various medicinal preparations is successfully used after preliminary experimental studies of their phoretic properties for the treatment of various inflammatory and dystrophic conditions as well as for the prevention of skin ageing. The most important route for the administration of the majority of drug preparations is through the shunts provided by perspiratory glands and hair follicles. Another essential factor determining the potential possibility of drug penetration through the skin is the characteristic of the substance chosen for the administration, such as its molecular weight, chemical structure, conformation, and hydrophilic properties. However, the most likely mechanism underlying the transport of the substance through the glandular cells of perspiratory glands and epithelial cells of hair follicles is pinocytosis, i.e. the process integrating exocytosis and endocytosis. To-day, the majority of the researchers lay emphasis on thermodynamic triggering of Ca2+-dependent processes as the primary mechanism behind the biological action of low-intensity laser radiation. Both exocytosis and endocytosis being the Ca2+-dependent processes, the liberation of Ca2+-ions under the influence of LFLR causes the activation of pinocytosis as a whole.

Mitochondrial pathway of apoptosis is ancestral in metazoans.

The mitochondrial pathway of apoptosis is the major mechanism of physiological cell death in vertebrates. In this pathway, proapoptotic members of the Bcl-2 family cause mitochondrial outer membrane permeabilization (MOMP), allowing the release of cytochrome c, which interacts with Apaf-1 to trigger caspase activation and apoptosis. Despite conservation of Bcl-2, Apaf-1, and caspases in invertebrate phyla, the existence of the mitochondrial pathway in any invertebrate is, at best, controversial. Here we show that apoptosis in a lophotrochozoan, planaria (phylum Platyhelminthes), is associated with MOMP and that cytochrome c triggers caspase activation in cytosolic extracts from these animals. Further, planarian Bcl-2 family proteins can induce and/or regulate cell death in yeast and can replace Bcl-2 proteins in mammalian cells to regulate MOMP. These results suggest that the mitochondrial pathway of apoptosis in animals predates the emergence of the vertebrates but was lost in some lineages (e.g., nematodes). In further support of this hypothesis, we surveyed the ability of cytochrome c to trigger caspase activation in cytosolic extracts from a variety of organisms and found this effect in cytosolic extracts from invertebrate deuterostomes (phylum Echinodermata).

Survivin is released from cancer cells via exosomes.

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.

Solar radiation induces non-nuclear perturbations and a false start to regulated exocytosis in Cryptosporidium parvum.

Stratospheric ozone depletion, climate warming and acidification of aquatic ecosystems have resulted in elevated levels of solar radiation reaching many aquatic environments with an increased deleterious impact on a wide range of living organisms. While detrimental effects on living organisms are thought to occur primarily through DNA damage, solar UV can also damage cellular proteins, lipids and signalling pathways. Cryptosporidium, a member of the eukaryotic phylum Apicomplexa, contain numerous vesicular secretory organelles and their discharge via regulated exocytosis is essential for the successful establishment of infection. Using flow cytometric techniques we demonstrate that solar UV rapidly induces sporozoite exocytosis resulting in a significant reduction in the ability of sporozoites to attach and invade host cells. We found that solar UV induced sporozoite membrane depolarization, resulting in reduced cellular ATP and increased cytosolic calcium. These changes were accompanied by a reduction in the internal granularity of sporozoites, indicative of apical organelle discharge, which was confirmed by analysis of sporozoites with an exocytosis-sensitive dye. The precise timing of apical organelle discharge in the presence of a compatible host cell is critical for sporozoite attachment and invasion. Our results demonstrate for the first time how solar UV radiation can interfere with exocytosis, a fundamental cellular process in all eukaryotic cells. We contend that not only may the forecast increases in solar radiation in both aquatic and terrestrial environments significantly affect members of the Apicomplexa, solar UV-induced membrane depolarizations resulting in cytosolic calcium perturbation may affect a wider range of eukaryotic organisms through antagonistic effects on a myriad of calcium dependant cellular functions.

Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by sanazole.

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. In this study, we determined whether sanazole enhances the radiation-induced apoptosis of human lymphoma U937 cells. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential (MMP), and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis, and that these effects are associated with modulation of the Fas-mitochondria-caspase-dependent pathway, an increase in [Ca(2+)](i), and a decrease in the Hsp70 expression levels.

Reversible control of exo- and endo-budding transitions in a photosensitive lipid membrane.

We have developed a method for the photomanipulation of lipid membrane morphology in which the shape of a vesicle can be switched by light through the use of a synthetic photosensitive amphiphile containing an azobenzene unit (KAON12). We prepared cell-sized liposomes from KAON12 and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and conducted real-time observations of vesicular transformation in the photosensitive liposome by phase-contrast microscopy. Budding transitions-either budding toward the centre of the liposome (endo-bud) or budding out of the liposome (exo-bud)-could be controlled by light. We discuss the mechanism of this transformation in terms of the change in the effective membrane surface area due to photoisomerization of the constituent molecules.

Tonotopic variation in the calcium dependence of neurotransmitter release and vesicle pool replenishment at mammalian auditory ribbon synapses.

The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (DeltaC(m)) in low-frequency (apical region, approximately 300 Hz) and high-frequency (basal, approximately 30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mm extracellular Ca(2+) and body temperature). With maturation, the Ca(2+) efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca(2+) cooperativity of exocytosis despite the smaller DeltaC(m) in apical cells. After maturation, DeltaC(m) in high-frequency IHCs increased linearly with the Ca(2+) current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca(2+) sensor otoferlin or the spatial coupling between Ca(2+) channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.

Glutamate exocytosis from astrocytes controls synaptic strength.

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

Myosin light chain kinase is not a regulator of synaptic vesicle trafficking during repetitive exocytosis in cultured hippocampal neurons.

The mechanism by which synaptic vesicles (SVs) are recruited to the release site is poorly understood. One candidate mechanism for trafficking of SVs is the myosin-actin motor system. Myosin activity is modulated by myosin light chain kinase (MLCK), which in turn is activated by calmodulin. Ca(2+) signaling in presynaptic terminals, therefore, may serve to regulate SV mobility along actin filaments via MLCK. Previous studies in different types of synapses have supported such a hypothesis. Here, we further investigated the role of MLCK in neurotransmitter release at glutamatergic synapses in cultured hippocampal neurons by examining the effects of two MLCK inhibitors, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine.HCl (ML-7) and wortmannin. Bath application of ML-7 enhanced short-term depression of EPSCs to repetitive stimulation, whereas it reduced presynaptic release probability. However, ML-7 also inhibited action potential amplitude and voltage-gated Ca(2+) channel currents. These effects were not mimicked by wortmannin, suggesting that ML-7 was not specific to MLCK in hippocampal neurons. When SV exocytosis was directly triggered by a Ca(2+) ionophore, calcimycin, to bypass voltage-gated Ca(2+) channels, ML-7 had no effect on neurotransmitter release. Furthermore, when SV exocytosis elicited by electrical field stimulation was monitored by styryl dye, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide], the unloading kinetics of the dye was not altered in the presence of wortmannin. These data indicate that MLCK is not a major regulator of presynaptic SV trafficking during repetitive exocytosis at hippocampal synapses.

Involvement of basal protein kinase C and extracellular signal-regulated kinase 1/2 activities in constitutive internalization of AMPA receptors in cerebellar Purkinje cells.

AMPA receptor (AMPAR) internalization provides a mechanism for long-term depression (LTD) in both hippocampal pyramidal neurons and cerebellar Purkinje cells (PCs). Cerebellar LTD at the parallel fiber (PF)-PC synapse is the underlying basis of motor learning and requires AMPAR activation, a large Ca2+ influx, and protein kinase C (PKC) activation. However, whether these requirements affect the constitutive AMPAR internalization in PF-PC synapses remains unclarified. Tetanus toxin (TeTx) infusion into PCs decreased PF-EPSC amplitude to 60% within 20-30 min (TeTx rundown), without change in paired-pulse facilitation ratio or receptor kinetics. Immunocytochemically measured glutamate receptor 2 (GluR2) internalization ratio decreased at the steady state of TeTx rundown. TeTx rundown did not require AMPAR activity nor an increase in intracellular Ca2+ concentration. TeTx rundown was suppressed partially by the inhibition of either conventional PKC or mitogen-activated protein kinase kinase (MEK) and completely by the inhibition of both kinases. The background PKC activity was shown to be sufficient, because a PKC activator did not facilitate TeTx rundown. The inhibition of protein phosphatase 1/2A (PP1/2A) enhanced TeTx rundown slightly, and both inhibition of PP1/2A and activation of PKC maximized it, but one-half of AMPARs at PF-PC synapses remained in the TeTx-resistant pool. The inhibition of actin depolymerization suppressed TeTx rundown and decreased the GluR2 internalization ratio. In contrast, the inhibition of actin polymerization enhanced TeTx rundown and increased the GluR2 internalization ratio. We suggest that the regulation of actin polymerization is involved in the surface expression of AMPARs and the surface expressing AMPARs are constitutively internalized through both basal PKC and MEK-ERK1/2 (extracellular signal-regulated kinase 1/2) activities at PF-PC synapses.

Mechanisms of signal transduction in photo-stimulated secretion in Phaeocystis globosa.

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca(2+) signal. However, the source of Ca(2+) in these cells has not been established. The present studies indicate that Phaeocystis' secretory granules work as an intracellular Ca(2+) oscillator. Optical tomography reveals that photo-stimulation induces InsP(3)-triggered periodic lumenal [Ca(2+)] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca(2+)] that trigger exocytosis. This Ca(2+) dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca(2+)-sensitive ion channels located on the granule membrane: an InsP(3)-receptor-Ca(2+) channel, and an apamin-sensitive K(+) channel.

Effect of static electric fields in root cells of Vicia faba (Fabaceae).

Serial electron microscopic sections were prepared from half-ripened meristematic root cells of Vicia faba (Fabaceae) which had been exposed gradually to 700, 1000, 2500, 3500, and 5000 V/m static electric fields during seven days with and without Zn and Cd electrodes. At the end of five weeks, wall loosenings and very small nuclei were observed in those root cells which were exposed to static electric currents from the lower side of the medium without electrodes, while abnormalities in cell formation, e.g., two cells with one nucleus, and GER occurrence were present in an electrolytic (Cd upward and Zn downward) medium. The cells exposed to a static current from the upper side of the medium had small nuclei and abnormal cell divisions in the electrolyte, but in a non-electrolyte very large nuclei and thicker cell walls were observed, the cytoplasm was dense with GER, pinocytosis was seen filled with mitochondria, and protoplast formation with big nuclei was seen in exocytosis.

Frequency-dependent depression of exocytosis and the role of voltage-gated calcium channels.

Synaptic vesicle exocytosis in primary cultures of baroreceptor neurons is reduced during high-frequency stimulation. Calcium influx through voltage-gated calcium channels (VGCC) is a key step in neurotransmitter release. With the help of FM2-10, a marker of synaptic vesicle recycling, the present study investigates the differential contribution of several VGCC subtypes to exocytosis in neuronal processes and how this contribution is altered at high frequencies. In control experiments, field stimulation at 0.5 Hz evoked about 66 +/- 5% destaining. Combined blockade of N- and P/Q-subtypes with Ctx-MVIIC was far more effective in reducing exocytosis (11 +/- 8%) than blocking N-type (49 +/- 5%, Ctx-GVIA) or P-type (46 +/- 1%, Agatoxin) alone. The effectiveness of the blockers also varied with the duration of stimulation: Ctx-GVIA attenuating exocytosis significantly in the first 60 s and Agatoxin affecting exocytosis only towards the end of 180 s stimulation period. Field stimulation at 10 Hz evoked exocytosis (36 +/- 18%) comparable to that evoked by 0.5 Hz in the presence of Ctx-GVIA. While blockade with Agatoxin had no effects, Ctx-GVIA, Ctx-MVIIC and L-type blocker Nifedepine had small but similar inhibitory effects on exocytosis at 10 Hz. The data suggest that N-type is the major contributor to exocytosis at 0.5 Hz, and this contribution is reduced during prolonged stimulation periods and at high frequencies.

Secretion in unicellular marine phytoplankton: demonstration of regulated exocytosis in Phaeocystis globosa.

Almost half of the global photosynthetic activity is carried out in the ocean. During blooms, Phaeocystis can fix CO(2) at rates up to 40 g C m(-2) month(-1). Most of this carbon is released as polysaccharides. However, the cellular mechanism whereby this huge amount of organic material is exported into the seawater remains unknown. A vaguely defined process of "exudation" is believed responsible for the release of these biopolymers. Here we report the first demonstration that Phaeocystis globosa does not "exude", but secretes microscopic gels. Secretion is stimulated by blue light (lambda = 470+/-20 nm), and it is transduced by a characteristic intracellular Ca(2+) signal that precedes degranulation. The polysaccharides that form the matrix of these gels remain in condensed phase while stored in secretory vesicles. Upon exocytosis, the exopolymer matrix undergoes a characteristic phase transition accompanied by extensive swelling resulting in the formation of microscopic hydrated gels. Owing to their tangled topology, once released into the seawater, the polymers that make these gels can reptate (axially diffuse), interpenetrate neighboring gels, and anneal them together forming massive mucilage accumulations that are characteristic of Phaeocystis blooms. These gel masses can supply a rich source of microbial substrates, disperse in the seawater, and/or eventually sediment to the ocean floor.

Synaptic stimulation of Aplysia peptidergic neurons can activate hormone secretion in the absence of an afterdischarge.

For over 20 years, the bag cell neurons of the marine mollusk Aplysia have been used to investigate second-messenger pathways that mediate effects of synaptic stimulation on ion currents and membrane excitability, presumably leading to exocytotic release of the neuropeptide egg-laying hormone (ELH). It is widely cited that a train of action potentials, called an afterdischarge, is necessary for activating cellular events leading to ELH secretion. Using a combination of electrophysiology, optical imaging of calcium signaling, and radioimmunoassay of ELH secretion, we show that an afterdischarge is not required for ELH secretion. Electrical stimulation that failed to produce afterdischarges but did lead to prolonged membrane depolarization and a rise in intracellular calcium concentration was sufficient to stimulate significant ELH release.