Orally tolerant CD4 T cells respond poorly to antigenic stimulation but strongly to direct stimulation of intracellular signaling pathways

The response of splenic CD4 T cells from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic mice after long-term feeding of a diet containing this antigen was examined. These CD4 T cells exhibited a decreased response to OVA peptide stimulation, in terms of proliferation, interleukin-2 secretion, and CD40 ligand expression, compared to those from mice fed a control diet lacking OVA, demonstrating that oral tolerance of T cells had been induced through oral intake of the antigen. We investigated the intracellular signaling pathways, which were Ca/CN cascade and Ras/MAPK cascade, of these tolerant CD4 T cells using phorbol-12-myristate-13-acetate (PMA) and ionomycin, which are known to directly stimulate these pathways. In contrast to the decreased response to TCR stimulation by OVA peptide, it was shown that the response of splenic CD4 T cells to these reagents in the state of oral tolerance was stronger. These results suggest that splenic CD4 T cells in the state of oral tolerance have an impairment in signaling, in which signals are not transmitted from the TCR to downstream signaling pathways, and have impairments in the vicinity of TCR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Post-tetanic changes in intracellular response to direct cortical stimulation

Changes in responses to single stimulations of the cortical surface after tetanization (frequency 50/sec, duration 1–10 sec) were studied in sensorimotor cortical neurons of an unanesthetized rabbit on intracellular and "quasiintracellular" recordings. After tetanization insufficient to generate epileptiform after-discharges, an increase was observed in the amplitude and duration of exciting postsynaptic potentials (EPSP) induced by a single test stimulus. This increase is considered as post-tetanic potentiation (PTP). Its duration did not exceed 1 min. The amplitude of inhibitory postsynaptic potentials (IPSP) showed a considerably smaller increase or did not change or even decreased. The PTP increased with an increase in the strength and duration of the tetanization, reaching especially high values during tetanization sufficiently intensive to evoke epileptiform after-discharges. In this case the response to a single test stimulus was identical to an epileptiform intracellular discharge. The data obtained confirm the important role of PTP of the exciting synapses in the generation of epileptiform after-discharges. A simple model of a neuron network with exciting and inhibiting feedbacks which accounts for the generation of epileptiform activity is examined.Institute of the Brain, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 2, No. 6, pp. 601–610, November–December, 1970.     

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca²⁺]_i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca²⁺ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca²⁺]_i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca²⁺]_i after expoxure to 50 mM K⁺ which was of the same magnitude as the increase in [Ca²⁺]_i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca²⁺ channels verapamil had no effect on the hyposmolarity induced increase in [Ca²⁺]_i. On the contrary, this increase in [Ca²⁺]_i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca²⁺ channels Mg²⁺ and La³⁺. Dantrolene which prevents release of Ca²⁺ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca²⁺ which represents an influx of Ca²⁺ rather than a release of Ca²⁺ from internal, dantrolene sensitive stores.     

GL261 glioma tumor cells respond to ATP with an intracellular calcium rise and glutamate release

Necrotizing enterocolitis (NEC) is one of the most severe and unpredictable complications of prematurity. There are two possible mechanisms involved in the pathogenesis of NEC: individual inflammatory response and impaired blood flow in mesenteric vessels with secondary ischemia of the intestine. The aim of this study was to evaluate the possible relationship between polymorphisms: Il-1β 3953C>T, Il-6 ?174G>C and ?596G>A, TNFα ?308G>A, and 86 bp variable number tandem repeat polymorphism of interleukin-1 receptor antagonist (Il-1RN VNTR 86 bp) and three polymorphisms that may participate in arteries tension regulation and in consequence in intestine blood flow impairment: eNOS (894G>T and ?786T>C) and END-1 (5665G>T) and NEC in 100 infants born from singleton pregnancy, before 32 + 0 weeks of gestation, exposed to antenatal steroids therapy, and without congenital abnormalities. In study population, 22 (22%) newborns developed NEC. Surgery-requiring NEC was present in 7 children. Statistical analysis showed 20-fold higher prevalence of NEC in infants with the genotype TT [OR 20 (3.71–208.7); p = 0.0004] of eNOS 894G>T gene polymorphism. There was a higher prevalence of allele C carriers of eNOS 786T>C in patients with surgery-requiring NEC [OR 4.881 (1.33–21.99); p = 0.013]. Our investigation did not confirm any significant prevalence for NEC development in another studied genotypes/alleles. This study confirms the significant role of polymorphisms that play role in intestine blood flow. Identifying gene variants that increase the risk for NEC development may be useful in screening infants with inherent vulnerability and creating strategies for individualized care.     

Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

To study the effects of -opioid receptor stimulation onintracellular Ca²⁺ concentration([Ca²⁺]_i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca²⁺]_iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca²⁺]_itransient, which results from the influx ofCa²⁺ and the subsequentmobilization of Ca²⁺ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca²⁺]_iand inhibited the[Ca²⁺]_itransient induced by caffeine, which mobilizesCa²⁺ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa²⁺ from its intracellular poolinto the cytoplasm. The Ca²⁺responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient were significantly attenuated when the extracellular pH(pH_e) was loweredto 6.8, which itself reduced intracellular pH(pH_i) and increased[Ca²⁺]_i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa⁺/H⁺exchange blocker, or 0.2 mM Ni²⁺,a putativeNa⁺/Ca²⁺exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa⁺/H⁺andNa⁺/Ca²⁺exchanges. When glucose at 50 mM, known to activate theNa⁺/H⁺exchange, was added, both the resting[Ca²⁺]_iand pH_i increased. Interestingly,the effects of U-50488H on [Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solutionwas superfused, the resting[Ca²⁺]_iincreased; the increase was abolished by 0.2 mMNi²⁺, but thepH_i remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca²⁺]_iand electrically induced[Ca²⁺]_itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa²⁺ from SR. Activation of bothNa⁺/H⁺andNa⁺/Ca²⁺exchanges, leading to an elevation of[Ca²⁺]_i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

     

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A₂ (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219–225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced a significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA₂ and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA₂ activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMLP was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMLP-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA₂ rather than secretory PLA₂. Inhibition of PLA₂ with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA₂-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.     

Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina

A sustained high voltage-activated (HVA), nifedipine- and cadmium- sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na- acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout- dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium- dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis.     

Human osteoblast-like cells respond not only to the extracellular calcium concentration but also to its changing rate

The effect of extracellular calcium on human osteoblast-like cells (HOS) has been demonstrated. An experimental setup was used for applying defined rates of change in the extracellular calcium concentration. The intracellular calcium concentration was monitored using the fluorescence dye fura-2. HOS cells showed qualitatively different responses of the intracellular calcium concentration to changes of the extracellular calcium concentration depending on its changing rate. A small rate caused only a small and slow increase of the intracellular calcium concentration, whereas faster changes are able to cause a rapid transient increase followed by a sustained elevation of the internal calcium level. Surprisingly, both an increasing as well as a decreasing external calcium concentration is able to cause cellular responses. These signals could be reduced by the IP₃-inhibitor neomycin. We propose that the G-protein dependent signalling pathway of HOS cells can not only sense the extracellular calcium concentration but also its time derivative. Received: 9 October 1997 / Revised version: 19 February 1998 / Accepted: 22 February 1998     

Phosphorylation-dependent modulation of cardiac calcium current by intracellular free magnesium

We compared the effects of cytosolic free magnesium (Mg(2+)(i)) on L-type Ca(2+) current (I(Ca,L)) in patch-clamped guinea pig ventricular cardiomyocytes under basal conditions, after inhibition of protein phosphorylation, and after stimulation of cAMP-mediated phosphorylation. Basal I(Ca,L) density displayed a bimodal dependence on the concentration of Mg(2+)(i) ([Mg(2+)](i); 10(-6)-10(-2) M), which changed significantly as cell dialysis progressed due to a pronounced and long-lasting rundown of I(Ca,L) in low-Mg(2+) dialysates. Ten minutes after patch breakthrough, I(Ca,L) density (at +10 mV) in Mg(2+)(i)-depleted cells ([Mg(2+)](i) approximately 1 microM) was elevated, increased to a maximum at approximately 20 microM [Mg(2+)](i), and declined steeply at higher [Mg(2+)](i). Treatment with the broad-spectrum protein kinase inhibitor K252a (10 microM) reduced I(Ca,L) density and abolished these effects of Mg(2+)(i) except for a negative shift of I(Ca,L)-voltage relations with increasing [Mg(2+)](i). Maximal stimulation of cAMP-mediated phosphorylation occluded the Mg(2+)(i)-induced stimulation of I(Ca,L) and prevented inhibitory effects of the ion at [Mg(2+)](i) <1 mM but not at higher concentrations. These results show that the modulation of I(Ca,L) by Mg(2+)(i) requires protein kinase activity and likely originates from interactions of the ion with proteins involved in the regulation of protein phosphorylation/dephosphorylation. Stimulatory effects of Mg(2+)(i) on I(Ca,L) seem to increase the cAMP-mediated phosphorylation of Ca(2+) channels, whereas inhibitory effects of Mg(2+)(i) appear to curtail and/or reverse cAMP-mediated phosphorylation.     

Serum modulates the intracellular calcium response of primary cultured bone cells to shear flow

We investigated the effect of newborn bovine serum on the intracellular calcium [Ca²⁺]_i response of primary cultured bone cells stimulated by fluid flow. As it has been previously established that these cells exhibit [Ca²⁺]_i responses to fluid flow shear stress in saline media without growth factors or other chemically stimulatory factors, we hypothesized that the addition of serum to the flow medium would enhance the mechanosensitivity of the cells. We examined the effect of a short-term (10–15 min) exposure of the cells to 2 and 10% serum prior to flow stimulation (pretreated) compared to not exposing the cells prior to flow stimulation (unpretreated). The cells were subjected to a well-defined, 90-s flow stimulus with shear stress levels ranging from 0.02 to 3.5 Pa in a laminar flow chamber using a saline medium supplemented with 2 or 10% serum. For pretreatment, the serum concentration was the same from pre-flow to flow exposure. We observed a differential effect in the magnitude of the peak [Ca²⁺]_i response modulated by the concentration of serum in the pre-flow medium. Additionally, ATP-supplemented flow was examined as a comparison to the serum-supplemented flow and exhibited a similar trend in the peak [Ca²⁺]_i flow response that was dependent on ATP concentration and pre-flow exposure conditions. These findings demonstrate that under the conditions of this study, chemical agonist exposure can modulate the [Ca²⁺]_i response in bone cells subjected to fluid flow-induced shear stress.     

Gonadotropin-releasing hormone stimulation of pituitary gonadotrope cells produces an increase in intracellular calcium

Gonadotropin-releasing hormone (GnRH) stimulates pituitary gonadotrope cells to release luteinizing hormone (LH). Previous studies have indicated a role for Ca+2 in this process; however, the present study provides the first measurements of an increased intracellular Ca+2 concentration. Pituitary cell cultures enriched for gonadotropes were loaded with quin 2, a fluorescent Ca+2-sensitive molecule. Subsequent addition of GnRH to these cells produced a rapid (within 10 sec) increase in fluorescence (indicating an increase in intracellular free Ca+2). In contrast, two GnRH analogs, des1 GnRH (a very low-affinity binder to the GnRH receptor) and Ac[D-pCl-Phe1,2] DTrp3 DLys6 DAla10-GnRH (a pure GnRH antagonist) produced no such Ca+2 change, thus showing a correlation between increased intracellular Ca+2 and LH release. A functional relationship between increased Ca+2 and LH release was suggested by experiments in which LH release was inhibited from cells loaded with high levels of intracellular quin 2 (in order to chelate intracellular Ca+2). Since this inhibition was completely reversed by addition of the Ca+2 ionophore A23187, quin 2 was not toxic at the concentrations used and apparently inhibited LH release by buffering intracellular Ca+2. The results presented here are consistent with the hypothesis that GnRH-stimulated LH release is mediated by increased intracellular Ca+2 and support the notion that the rate-limiting step in GnRH-stimulated LH release is distal to Ca+2 mobilization.     

Response of rabbit visual cortex neurons to direct stimulation by current of different intensities

Response was investigated in 47 rabbit visual cortex neurons to direct intracortical electrical stimulation consisting of single biphasic pulses of increasing intensities within the 150–2,700 µA range. Minimum level for generating all components of response to electrical stimulation remained similar from one neuron to the next, ranging between 150 and 600 µA. Three different patterns of dependence between numbers of spikes at different stages of response and current amplitude were identified: stable, gradual and selective patterns. Percentages of stable dependences were lower for after-discharges in comparison with initial firing (at 11 and 26% respectively) and the proportion of selective dependences were somewhat higher (at 32 and 21%). The selective dependence observed could imply the existence of optimum current levels at which peak neuronal response occurs. At the same time, duration of inhibitory pauses lengthened uniformly in the response of all test neurons during increasing intensity of the electrical stimuli. Findings obtained during this study may have a practical application in matters connected with visual prosthetics in the blind.M. V. Lomonosov State University, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 239–247, March–April, 1989.     

The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels

Autophagy regulates cell survival (or cell death in several cases), whereas apoptosis regulates cell death. However, the relationship between autophagy and apoptosis and the regulative mechanism is unclear. We report that steroid hormone 20-hydroxyecdysone (20E) promotes switching from autophagy to apoptosis by increasing intracellular calcium levels in the midgut of the lepidopteran insect Helicoverpa armigera. Autophagy and apoptosis sequentially occurred during midgut programmed cell death under 20E regulation, in which lower concentrations of 20E induced microtubule-associated protein 1 light chain 3–phosphatidylethanolamine (LC3–II, also known as autophagy-related gene 8, ATG8) expression and autophagy. High concentrations of 20E induced cleavage of ATG5 to NtATG5 and pro-caspase-3 to active caspase-3, which led to a switch from autophagy to apoptosis. Blocking autophagy by knockdown of ATG5, ATG7, or ATG12, or with the autophagy inhibitor 3-methyladenine, inhibited 20E-induced autophagy and apoptosis. Blocking apoptosis by using the apoptosis inhibitor Ac-DEVD-CHO did not prevent 20E-induced autophagy, suggesting that apoptosis relies on autophagy. ATG5 knockdown resulted in abnormal pupation and delayed pupation time. High concentrations of 20E induced high levels of intracellular Ca²⁺, NtATG5, and active caspase-3, which mediated the switch from autophagy to apoptosis. Blocking 20E-mediated increase of cellular Ca²⁺ caused a decrease of NtATG5 and active caspase-3 and repressed the transformation from autophagy to apoptosis, thereby promoting cell survival. 20E induces an increase in the concentration of intracellular Ca²⁺, thereby switching autophagic cell survival to apoptotic cell death.     

Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells

ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP-induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.     

Calcium ion propagation in cultured keratinocytes and other cells in skin in response to hydraulic pressure stimulation

We have previously suggested that a variety of environmental factors might be first sensed by epidermal keratinocytes, which represent the frontier of the body. To further examine this idea, in the present study, we examined the intracellular calcium responses of cultured keratinocytes to external hydraulic pressure. First, we compared the responses of undifferentiated and differentiated keratinocytes with those of fibroblasts, vascular endothelial cells (VEC), and lymphatic endothelial cells. Elevation of intracellular calcium was observed after application of pressure to keratinocytes, fibroblasts, and VEC. The calcium propagation extended over a larger area and continued for a longer period of time in differentiated keratinocytes, as compared with the other cells. The response of the keratinocytes was dramatically reduced when the cells were incubated in medium without calcium. Application of a non‐selective transient receptor potential (TRP) channel blocker also attenuated the calcium response. These results suggest that differentiated keratinocytes are sensitive to external pressure and that TRP might be involved in the mechanism of their response. J. Cell. Physiol. 224:229–233, 2010 © 2010 Wiley‐Liss, Inc.     

Transcytosis of calcium from bone by osteoclast-like cells evidenced by direct visualization of calcium in cells

'Transcytosis' of calcium (Ca) from bone by osteoclasts was identified by using a newly developed method that uses fixed or living osteoclast-like cells previously differentiated in vitro, a Ca-specific cell-membrane-impermeable fluorescent dye, and confocal laser scanning microscopy. This method, called the cell-membrane-impermeable dye method, revealed that in fixed osteoclast-like cells, a large quantity of Ca was confined within vacuoles and transported toward the apical cell membrane in the cells. These Ca-confined vacuoles were co-localized with marker proteins of both ruffled border and lysosome. The vacuoles were disrupted when treated with an inhibitor of ruffled border ATPase. In living osteoclast-like cells, Ca-confined vacuoles were again preferentially located at the central region and near the apical cell membrane. These results suggest actual transcytosis of Ca from bone by osteoclasts, and are the first direct evidence of the significant role of osteoclasts in the entire process of Ca metabolism in bone.     

Induction of filopodia by direct local elevation of intracellular calcium ion concentration.

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from approximately 60 nM to approximately 1 microM within the illuminated zone, and then returned to resting level in approximately 10-15 s. New filopodia arose in this area within 1-5 min, and persisted for approximately 15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.