The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel CaV1.2

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.     

Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines

Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.     

Live Free or Die: An Immature T Cell Decision Encoded in Distinct Bcl-2 Sensitive and Insensitive Ca2+ Signals

In the developing thymus, strong T cell receptor (TCR) activation by self-antigensinduces negative selection and weak TCR activation induces positive selection. Bothprocesses are mediated by Ca2+ signals, raising the question of how a single secondmessenger like Ca2+ can mediate such diverse cell fates. Recent findings indicate thatgraded TCR activation signals are encoded in distinct patterns of Ca2+ elevation. Theanti-apoptotic protein Bcl-2 discriminates between these Ca2+ signaling patterns,selectively inhibiting pro-apoptotic Ca2+ signals induced by strong TCR activationwithout suppressing pro-survival Ca2+ signals induced by weak TCR activation.     

Ca2+ signal summation and NFATc1 nuclear translocation in sympathetic ganglion neurons during repetitive action potentials

NFATc-mediated gene expression constitutes a critical step during neuronal development and synaptic plasticity. Although considerable information is available regarding the activation and functionality of specific NFATc isoforms, in neurons little is known about how sensitive NFAT nuclear translocation is to specific patterns of electrical activity. Here we used high-speed fluo-4 confocal imaging to monitor action potential (AP)-induced cytosolic Ca2+ transients in rat sympathetic neurons. We have recorded phasic and repetitive AP patterns, and corresponding Ca2+ transients initiated by either long (100-800 ms) current-clamp pulses, or single brief (2 ms) electrical field stimulation. We address the functional consequences of these AP and Ca2+ transient patterns, by using an adenoviral construct to express NFATc1-CFP and evaluate NFATc1-CFP nuclear translocation in response to specific patterns of electrical activity. Ten Hertz trains stimulation induced nuclear translocation of NFATc1, whereas 1 Hz trains did not. However, 1 Hz train stimulation did result in NFATc1 translocation in the presence of 2 mM Ba2+, which inhibits M-currents and promotes repetitive firing and the accompanying small (approximately 0.6 DeltaF/F0) repetitive and summating Ca2+ transients. Our results demonstrate that M-current inhibition-mediated spike frequency facilitation enhances cytosolic Ca2+ signals and NFATc1 nuclear translocation during trains of low frequency electrical stimulation.     

Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites

Increases in cytosolic Ca2+ concentration ([Ca2+]i) mediated by NMDA-sensitive glutamate receptors (NMDARs) are important for synaptic plasticity. We studied a wide variety of dendritic spines on rat CA1 pyramidal neurons in acute hippocampal slices. Two-photon uncaging and Ca2+ imaging revealed that NMDAR-mediated currents increased with spine-head volume and that even the smallest spines contained a significant number of NMDARs. The fate of Ca2+ that entered spine heads through NMDARs was governed by the shape (length and radius) of the spine neck. Larger spines had necks that permitted greater efflux of Ca2+ into the dendritic shaft, whereas smaller spines manifested a larger increase in [Ca2+]i within the spine compartment as a result of a smaller Ca2+ flux through the neck. Spine-neck geometry is thus an important determinant of spine Ca2+ signaling, allowing small spines to be the preferential sites for isolated induction of long-term potentiation.     

Small conductance potassium channels cause an activity-dependent spike frequency adaptation and make the transfer function of neurons logarithmic

We made a computational model of a single neuron to study the effect of the small conductance (SK) Ca2+-dependent K+ channel on spike frequency adaptation. The model neuron comprised a Na+ conductance, a Ca2+ conductance, and two Ca2+-independent K+ conductances, as well as a small and a large (BK) Ca2+-activated K+ conductance, a Ca2+ pump, and mechanisms for Ca2+ buffering and diffusion. Sustained current injection that simulated synaptic input resulted in a train of action potentials (APs) which in the absence of the SK conductance showed very little adaptation with time. The transfer function of the neuron was nearly linear, i.e., both asymptotic spike rate as well as the intracellular free Ca2+ concentration ([Ca2+]i) were approximately linear functions of the input current. Adding an SK conductance with a steep nonlinear dependence on [Ca2+]i (. Pflügers Arch. 422:223-232; K?hler, Hirschberg, Bond, Kinzie, Marrion, Maylie, and Adelman. 1996. Science. 273:1709-1714) caused a marked time-dependent spike frequency adaptation and changed the transfer function of the neuron from linear to logarithmic. Moreover, the input range the neuron responded to with regular spiking increased by a factor of 2.2. These results can be explained by a shunt of the cell resistance caused by the activation of the SK conductance. It might turn out that the logarithmic relationships between the stimuli of some modalities (e.g., sound or light) and the perception of the stimulus intensity (Fechner's law) have a cellular basis in the involvement of SK conductances in the processing of these stimuli.     

Heterogeneity among astroglial cells with respect to 5HT-evoked cytosolic Ca2+ responses. A microspectrofluorimetric study on single cells in primary culture

The effect of 5-hydroxytryptamine (5HT) on cytoplasmic Ca2+ concentration was examined at the single cell level in astroglial enriched primary cultures from newborn rat cerebral cortex. Type 1 astroglial cells were identified and the Ca2+ indicator dye fura 2/AM was used in a microspectrofluorimetric system. Pharmacological studies indicated that the Ca2+ responses were mediated by 5HT2 receptors. Four different patterns of 5HT evoked cytosolic Ca2+ responses were identified including two different types of spike patterns and two types of Ca2+ oscillations (low amplitude and base-line spiking behaviour). In addition, cells with spontaneous Ca2+ oscillations were of two types, those responding to 5HT and those not responding to 5HT. The different responses were identified already on day 7 in culture and were followed up to day 21 with a concomitant increase in the number of responding cells, although the response patterns did not differ during culture. The triggering 5HT concentration was 1 microM. The results suggest that subpopulations of astrocytes exist with respect to 5HT2-evoked cytosolic Ca2+ mobilization.     

Recovery of Ins(1,4,5)-trisphosphate-dependent calcium signaling in neonatal gonadotrophs

Pituitary gonadotrophs express non-desensitizing gonadotropin-releasing hormone (GnRH) receptors and their activations leads to inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ mobilization. When added in physiological concentration range GnRH induces baseline Ca2+ oscillations, whereas in higher concentrations it induces a prolonged spike response accompanied with non-oscillatory or oscillatory plateau response. Here, we studied the recovery of calcium signaling during repetitive stimulation with short (10-30 s) GnRH pulses and variable interpulse intervals in neonatal gonadotrophs perfused with Ca2+/Na+ -containing, Ca2+ -deficient/Na+ -containing, and Ca2+ -containing/Na+ -deficient media. In Ca2+/Na+ -containing medium, baseline Ca2+ oscillations recovered without refractory period and with a time constant of approximately 20 s, whereas the recovery of spike response occurred after 25-35 s refractory period and with a time constant of approximately 30 s. During repetitive GnRH stimulation, removal of Ca2+ had only a minor effect on baseline oscillations but abolished spike response, whereas removal of Na+ slightly extended duration of baseline oscillations and considerably prolonged spike response. These results indicate that two calcium handling mechanisms are operative in gonadotrophs: redistribution of calcium within InsP3-sensitive and -insensitive pools and a sodium-dependent calcium efflux followed by calcium influx. Redistribution of Ca2+ within the cell leads to rapid recovery of InsP3-dependent pool, whereas the Na+ -dependent Ca2+ efflux pathway is activated by spike response and limits the time of exposure to elevated cytosolic Ca2+ concentrations.     

Regulatory and spatial aspects of inositol trisphosphate-mediated calcium signals

Hormones that act to release Ca2+ from intracellular stores initiate a signaling cascade that culminates in the production of inositol 1,4,5-trisphosphate (InsP3). The Ca2+ response mediated by InsP3 is not a sustained increase in the cytosolic Ca2+ concentration, but rather a series of periodic spikes that manifest as waves in larger cells. In vitro studies have determined that the key positive feedback parameter driving spikes and waves is a highly localized direct Ca(2+)-activation of InsP3-gated Ca2+ channels. Advances in fluorescent Ca2+ imaging have facilitated the resolution of individual positive feedback units. These studies have revealed that there are several modes of channel coupling underlying global Ca2+ signals; single channel openings or Ca2+ "blips," synchronized clusters of channels or Ca2+ "puffs," and cell wide calcium waves. It appears that the channel clusters that produce Ca2+ puffs are synchronized by the highly localized positive feedback that was predicted by the in vitro studies of channel regulation. Localization of InsP3-induced Ca2+ signals has been shown to be important for activation of several cellular processes including uni-directional salt flow and mitochondrial activation.     

Ca2+-induced Ca2+ release supports the relay mode of activity in thalamocortical cells

Ca2+ ions play an important role during rhythmic bursting of thalamocortical neurons within sleep. The function of Ca2+ during the tonic relay mode of these neurons during wakefulness is less clear. Here, we report that tonic activity in thalamocortical cells results in an increase in the intracellular Ca2+ concentration and subsequent release of Ca2+ from intracellular stores mediated via ryanodine receptors (RyRs). Blockade of Ca2+ release shifted the regular firing of single action potentials toward the generation of spike clusters. Regular spike firing and intracellular Ca2+ release thus appear to be functionally coupled in a positive feedback manner, thereby supporting the relay mode of thalamocortical cells during wakefulness. Regulatory influences may be coupled to this system via the cyclic ADP ribose pathway.     

Amplification of calcium signals at dendritic spines provides a method for CNS quantal analysis

It has been proposed that the small volume of a dendritic spine can amplify Ca2+ signals during synaptic transmission. Accordingly, we have performed calculations to determine whether the activation of N-methyl-D-aspartate (NMDA) type glutamate receptors during synaptic transmission results in significant elevation in intracellular Ca2+ levels, permitting optical detection of synaptic signals within a single spine. Simple calculations suggest that the opening of even a single NMDA receptor would result in the influx of approximately 310 000 Ca2+ ions into the small volume of a spine, producing changes in Ca2+ levels that are readily detectable using high affinity Ca2+ indicators such as fura-2 or fluo-3. Using fluorescent Ca2+ indicators, we have imaged local Ca2+ transients mediated by NMDA receptors in spines and dendritic shafts attributed to spontaneous miniature synaptic activity. Detailed analysis of these quantal events suggests that the current triggering these transients is attributed to the activation of <10 NMDA receptors. The frequency of these miniature synaptic Ca2+ transients is not randomly distributed across synapses, as some synapses can display a >10-fold higher frequency of transients than others. As expected for events mediated by NMDA receptors, miniature synaptic Ca2+ transients were suppressed by extracellular Mg2+ at negative membrane potentials; however, the Mg2+ block could be removed by depolarization.     

The GABAB1b isoform mediates long-lasting inhibition of dendritic Ca2+ spikes in layer 5 somatosensory pyramidal neurons

The apical tuft of layer 5 pyramidal neurons is innervated by a large number of inhibitory inputs with unknown functions. Here, we studied the functional consequences and underlying molecular mechanisms of apical inhibition on dendritic spike activity. Extracellular stimulation of layer 1, during blockade of glutamatergic transmission, inhibited the dendritic Ca2+ spike for up to 400 ms. Activation of metabotropic GABAB receptors was responsible for a gradual and long-lasting inhibitory effect, whereas GABAA receptors mediated a short-lasting (approximately 150 ms) inhibition. Our results suggest that the mechanism underlying the GABAB inhibition of Ca2+ spikes involves direct blockade of dendritic Ca2+ channels. By using knockout mice for the two predominant GABAB1 isoforms, GABAB1a and GABAB1b, we showed that postsynaptic inhibition of Ca2+ spikes is mediated by GABAB1b, whereas presynaptic inhibition of GABA release is mediated by GABAB1a. We conclude that the molecular subtypes of GABAB receptors play strategically different physiological roles in neocortical neurons.     

Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli

The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.     

Integrating cytosolic calcium signals into mitochondrial metabolic responses.

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.     

Possible link of different slow calcium signals generated by membrane potential and hormones to differential gene expression in cultured muscle cells

The study of fluorescent calcium signals from cultured rat myotubes has provided interesting results in the past few years. Both K+ depolarization and tetanic electrical stimulation were shown to produce slow Ca2+ signals, unrelated to contraction and associated to regulation of gene expression in cultured rat myotubes. We studied the effect of IGF-I, insulin and testosterone on intracellular Ca2+ in cultured muscle cells. Insulin produced a fast (< 1 s) and transient [Ca2+] increase lasting less than 10 s. IGF-I induced a transient [Ca2+] increase, reaching a fluorescence peak 6 s after stimulus, to return to basal values after 60 s. Testosterone induced delayed (35 s) and long lasting (100-200 s) signals, frequently associated with oscillations. IGF-I, testosterone and electrical stimulation-induced Ca2+ signals were shown to be dependent on IP3 production. All of these Ca2+ signals were blocked by inhibitors of the IP3 pathway. On the other hand, insulin-induced Ca2+ increase was dependent on ryanodine receptors and blocked by either nifedipine or ryanodine. The different intracellular Ca2+ patterns produced by electrical stimulation, testosterone, IGF-I and insulin, may help to understand the role of intracellular calcium kinetics in the regulation of gene expression by various stimuli in skeletal muscle cells.     

Different patterns of receptor-activated cytoplasmic Ca2+ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2+ buffering.

Agonist-specific cytosolic Ca2+ oscillation patterns can be observed in individual cells and these have been explained by the co-existence of separate oscillatory mechanisms. In pancreatic acinar cells activation of muscarinic receptors typically evokes sinusoidal oscillations whereas stimulation of cholecystokinin (CCK) receptors evokes transient oscillations consisting of Ca2+ waves with long intervals between them. We have monitored changes in the cytosolic Ca2+ concentration ([Ca2+]i) by measuring Ca2(+)-activated Cl- currents in single internally perfused mouse pancreatic acinar cells. With minimal intracellular Ca2+ buffering we found that low concentrations of both ACh (50 nM) and CCK (10 pM) evoked repetitive short-lasting Ca2+ spikes of the same duration and frequency, but the probability of a spike being followed by a longer and larger Ca2+ wave was low for ACh and high for CCK. The probability that the receptor-evoked shortlasting Ca2+ spikes would initiate more substantial Ca2+ waves was dramatically increased by intracellular perfusion with solutions containing high concentrations of the mobile low affinity Ca2+ buffers citrate (10-40 mM) or ATP (10-20 mM). The different Ca2+ oscillation patterns normally induced by ACh and CCK would therefore appear not to be caused by separate mechanisms. We propose that specific receptor-controlled modulation of Ca2+ signal spreading, either by regulation of Ca2+ uptake into organelles and/or cellular Ca2+ extrusion, or by changing the sensitivity of the Ca2(+)-induced Ca2+ release mechanism, can be mimicked experimentally by different degrees of cytosolic Ca2+ buffering and can account for the various cytosolic Ca2+ spike patterns.     

Chronic ethanol exposure enhances AMPA-elicited Ca2+ signals in the somatic and dendritic regions of cerebellar Purkinje neurons.

Intracellular Ca2+ signals produced by the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 5 microM) were measured in the somatic and dendritic regions of cerebellar Purkinje neurons in mature cerebellar control cultures (> or = 20 days in vitro) and cultures chronically treated with 32 mM ethanol (146 mg%; 8-11 days). Recordings were made in physiological saline without ethanol. The mean peak amplitude of the Ca2+ signal elicited by AMPA (applied by brief 1-s microperfusion) in the somatic region was enhanced 38% in chronic ethanol-treated Purkinje neurons compared with control neurons. In contrast, Ca2+ signals evoked by AMPA in the dendritic region were similar in magnitude between control and chronic ethanol-treated Purkinje neurons. When tetrodotoxin (TTX; 500 nM) was included in the bath saline to block spike activity and synaptically-generated events, the mean peak amplitude of the Ca2+ signal elicited by AMPA was enhanced 60% in both the somatic and dendritic regions of chronic ethanol-treated Purkinje neurons compared with control neurons. Thus, TTX-sensitive mechanisms (i.e., spike or synaptic activity) appear to play a role in normalizing neuronal functions involved in Ca2+ signaling in the chronic ethanol-treated neurons. In parallel current clamp experiments, the resting membrane potential of chronic ethanol-treated neurons was slightly depolarized compared with control neurons. However, no differences were found between control and chronic ethanol-treated Purkinje neurons in input resistance or the peak amplitude or duration of the depolarizations or hyperpolarizations elicited by AMPA. AMPA receptors mediate fast excitatory neurotransmission in the majority of neurons in the central nervous system (CNS) and Ca2+ signals in response to AMPA receptor activation contribute to synaptic function. Thus, our results suggest that modulation of Ca2+ signals to AMPA receptor activation (or other cellular inputs) may provide an important mechanism contributing to the actions of prolonged ethanol exposure in the CNS.     

Determinants of postsynaptic Ca2+ signaling in Purkinje neurons

Neuronal integration in Purkinje neurons involves many forms of Ca2+ signaling. Two afferent synaptic inputs, the parallel and the climbing fibers, provide a major drive for these signals. These two excitatory synaptic inputs are both glutamatergic. Postsynaptically they activate alpha-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Unlike most other types of central neurons, Purkinje neurons do not express NMDA (N-methyl-D-aspartate) receptors (NMDARs). AMPARs in Purkinje neurons are characterized by a low permeability for Ca2+ ions. AMPAR-mediated synaptic depolarization may activate voltage-gated Ca2+ channels, mostly of the P/Q-type. The resulting intracellular Ca2+ signals are shaped by the Ca2+ buffers calbindin and parvalbumin. Ca2+ clearance from the cytosol is brought about by Ca2+-ATPases in the plasma membrane and the endoplasmic reticulum, as well as the Na+-Ca2+-exchanger. Binding of glutamate to mGluRs induces postsynaptic Ca2+-transients through two G protein-dependent pathways: involving (1) the release of Ca2+ ions from intracellular Ca2+ stores and (2) the opening of the cation channel TRPC1. Homer proteins appear to play an important role in postsynaptic Ca2+ signaling by providing a direct link between the plasma membrane-resident elements (mGluRs and TRPC1) and their intracellular partners, including the IP3Rs.     

Dynamic aspects of presynaptic calcium currents mediating synaptic transmission

Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers transmitter release. Direct recording of Ca2+ currents from the calyx of Held nerve terminal revealed that presynaptic VGCCs undergo various modulations via presynaptic G protein-coupled receptors (GPCRs), Ca2+-binding proteins and a developmental switch of their alpha1 subunits. Dynamic changes of presynaptic VGCCs alter synaptic efficacy, thereby contributing to a variety of modulations of the CNS function.     

Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils

During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]i increased rapidly from < or = 100 nM to > or = 400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of approximately 200 nM. The initial Ca2+ spike was insensitive acutely to removal of extracellular Ca2+ but was abolished by treatments expected to deplete internal Ca2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lamellipod activity and suppressed the Ca2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca2+ spike on stimulation. Although cell flattening and lamellipod activity were induced by chemoattractant when the normal Ca2+ response was blocked, cells failed to polarize and crawl, indicating that Ca2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.