Structure Dependence of the Calcium Dynamics in Purkinje Neuron Dendrites during Generation of Bursting Discharges: a Simulation Study

In the model of a cerebellar Purkinje neuron with reconstructed active dendrites, we investigated the impact of the ratio between volumes of the endoplasmic reticulum (organellar calcium store) and cytosol on the Ca²⁺ dynamics in asymmetrical parts of the dendritic arborization during generation of different structure-dependent patterns of bursting activity. Tonic synaptic excitation homogeneously distributed over the dendrites (a spatially homogeneous stationary input signal) caused spatially heterogeneous variations of the dendritic membrane potential (MP) accompanied by periodical or nonperiodical bursts of action potentials at the cell output. The MP waveforms recorded from the segments of asymmetrical dendrites were then applied to the membrane of selected dendrite segments as command voltages in a dynamic clamp mode. In these segments, the relative size of the stores was varied. This provided equal to each other local calcium currents and influxes into the cytosol of the segment differently filled with the organellar store. Regardless of the impulse pattern, microgeometry of the segment and the store modulated calcium transients exactly in the same way as in previous studies of electrical and concentration responses to local phasic synaptic excitation of the modeled neuron. Peak values of depolarization-induced elevations of the cytosolic Ca²⁺ concentration increased with the portion of the intracellular volume occupied by the store. The most important factor defining this dependence was the ratio of the membrane area vs the organelle-free cytosol volume of the dendritic segment. Concentrations of Са²⁺ deposited in equal-sized segments of asymmetrical parts of the dendritic arborization where asynchronous unequal variations of the MP were observed during generation of nonperiodical bursting at the output demonstrated considerable specificity. A greater amount of calcium was deposited in the segments staying, on average, in a high-depolarization state for a longer time (this intensified activation of calcium channels and amplified the corresponding Ca²⁺ influx into the cytosol). Hence, local dynamics of the Ca²⁺ concentration depend directly on local microgeometry and indirectly on global macrogeometry of the dendrite arborization, as the latter determines spatial asymmetry-related unequal transients in different parts of the dendritic arborization having active membrane properties.     

Simulation of Spontaneous Ca Oscillations in Astrocytes Mediated by Voltage-Gated Calcium Channels

The purpose of this computational study was to investigate the possible role of voltage-gated Ca²⁺ channels in spontaneous Ca²⁺ oscillations of astrocytes. By incorporating different types of voltage-gated Ca²⁺ channels and a previous model, this study reproduced typical Ca²⁺ oscillations in silico. Our model could mimic the oscillatory phenomenon under a wide range of experimental conditions, including resting membrane potential (−75 to −60 mV), extracellular Ca²⁺ concentration (0.1 to 1500 μM), temperature (20 to 37°C), and blocking specific Ca²⁺ channels. By varying the experimental conditions, the amplitude and duration of Ca²⁺ oscillations changed slightly (both <25%), while the frequency changed significantly (∼400%). This indicates that spontaneous Ca²⁺ oscillations in astrocytes might be an all-or-none process, which might be frequency-encoded in signaling. Moreover, the properties of Ca²⁺ oscillations were found to be related to the dynamics of Ca²⁺ influx, and not only to a constant influx. Therefore, calcium channels dynamics should be used in studying Ca²⁺ oscillations. This work provides a platform to explore the still unclear mechanism of spontaneous Ca²⁺ oscillations in astrocytes.     

Calcium content and binding in synaptosomal subfractions during chronic morphine treatment

Chronic exposure to morphine in mice produced an increase in Ca²⁺ content of synaptosomes, synaptic plasma membranes (SPM), and synaptic vesicles. Ca²⁺ binding capacity was significantly reduced in tolerant SPM fractions. Naloxone significantly reversed the increased calcium content and reduced binding capacity of SPM when administered to 72-h-treated mice. Scatchard analysis of binding curves reveals three distinct classes of Ca²⁺ binding sites. During tolerance, the high- and low-affinity sites exhibit a reduced capacity to bind calcium, which may be reversed by in vivo and in vitro administration of naloxone. The increase in SPM and synaptic vesicle calcium content may reflect adaptive changes in the cell membrane during tolerance development, which may contribute to changes in neurotransmitter and second messenger function.     

Calcium extrusion mechanisms in dendrites of mouse hippocampal CA1 inhibitory interneurons

Local circuit GABAergic inhibitory interneurons control the integration and transfer of information in many brain regions. Several different forms of plasticity reported at interneuron excitatory synapses are triggered by cell- and synapse-specific postsynaptic calcium (Ca²⁺) mechanisms. To support this function, the spatiotemporal dynamics of dendritic Ca²⁺ elevations must be tightly regulated. While the dynamics of postsynaptic Ca²⁺ signaling through activation of different Ca²⁺ sources has been explored, the Ca²⁺ extrusion mechanisms that operate in interneuron dendrites during different patterns of activity remain largely unknown. Using a combination of whole-cell patch-clamp recordings and two-photon Ca²⁺ imaging in acute mouse hippocampal slices, we characterized the Ca²⁺ extrusion mechanisms activated by Ca²⁺ transients (CaTs) associated with backpropagating action potentials (bAPs) in dendrites of hippocampal CA1 stratum radiatum interneurons. Our data showed that Ca²⁺ clearance increased as a function of activity, pointing to an activity-dependent recruitment of specific Ca²⁺ extrusion mechanisms. bAP-CaTs were significantly prolonged in the presence of the plasma membrane Ca²⁺ ATPase (PMCA) and Na⁺/Ca²⁺ exchanger (NCX) inhibitors as well as the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) and the mitochondria Ca²⁺ uniporter (MCU) blockers. While PMCA, NCX and SERCA pumps cooperated in the cytosolic Ca²⁺ removal at a wide range of concentrations, the MCU was only activated at higher Ca²⁺ loads produced by repetitive interneuron firing. These results identify a division of labor between distinct Ca²⁺ extrusion mechanisms shaping dendritic Ca²⁺ dynamics and possibly contributing to activity-dependent regulation of synaptic inputs in interneurons. In addition, the MCU activated by larger Ca²⁺ levels may be involved in the activity-dependent ATP production or interneuron-selective vulnerability associated with cytosolic Ca²⁺ overloads under pathological conditions.     

Motility processes in Acantharia (protozoa) III. Calcium regulation of the contraction - relaxation cycles of in vivo myonemes

The myonemes in the marine pelagic protozoa Acantharia are contractile organelles involved in buoyancy regulation. It was previously shown that they can perform three kinds of movement: rapid contraction, slow undulation and slow relaxation. They consist of a periodically striated bundle of 2–4 nm nonactin filaments that are twisted in pairs and shortened by a coiling mechanism. After permeabilization or demembranation, contraction and relaxation can still be performed by varying Ca²⁺ concentration and ATP is not needed. In the present paper, we have studied the role of Ca²⁺ and inhibitors of energy production in intact cells. Our data suggest that; (i) the in vivo rapid contraction subsequent to mechanical or electrical stimulation is triggered by Ca²⁺ influx across the cell membrane; (ii) the slow contraction that takes place during the undulating movement depends on Ca²⁺ release provided by internal calcium stores; (iii) the rapid contraction as well as the progressive shortening that occurs during the slow undulating movement are caused by Ca²⁺ binding to the myoneme filaments; (iv) ATP is not directly involved in the saturation by Ca²⁺ of Ca²⁺ sensitive sites located along the myoneme microstrands; (v) regulation of the movements of Ca²⁺ within the cytoplasm depends mainly upon the alternative pathway of ATP production; (vi) calmodulin is presumably involved in this regulation. A tentative cytophysiologic interpretation of the mechanism of contractility is proposed.     

Phospholemman Modulates the Gating of Cardiac L-Type Calcium Channels

Ca²⁺ entry through L-type calcium channels (Ca_V1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of Ca_V1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with Ca_V1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed Ca_V1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca²⁺-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca²⁺ influx via Ca_V1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of Ca_V1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca²⁺ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca²⁺ influx during the cardiac action potential waveform plateau phase.     

Studies on Capacitative Calcium Entry in Vascular Smooth Muscle Cells by Measuring 45Ca2+ Influx

Abstract

Capacitative calcium entry was studied in the A7r5 vascular smooth muscle cell line by measuring ⁴⁵Ca²⁺ influx. Entry was induced by depletion of the Ca²⁺ pools by either the receptor agonist [Arg]⁸vasopressin (AVP) or the SR-Ca²⁺-ATPase inhibitor thapsigargin (TG). TG showed a higher efficacy for calcium influx than AVP. This is probably due to a larger Ca²⁺ release from the pools induced by TG compared to AVP and the irreversible inhibition of the SR-Ca²⁺-ATPase by TG causing influx to persist for a longer period of time. At maximally effective concentrations signals induced by AVP and TG were synergistic in the absence but not in the presence of the intracellular calcium chelator, 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Depolarisation with 55 mM KCl completely inhibited ⁴⁵Ca²⁺ influx induced by TG but only slightly the one induced by AVP, both effects being less pronounced in the presence of BAPTA. [Ca²⁺]_c signals induced by AVP and TG were both inhibited by depolarisation.

In conclusion, although our results show differences between AVP- and TG-induced Ca²⁺ influx, they can be explained by their different mechanism of action and are in accordance with an activation of the same capacitative entry pathway by both agents.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons

Abstract: A large body of evidence suggests that disturbances of Ca²⁺ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca²⁺]_i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage-sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca²⁺]_i but via different mechanisms. By using the drug dantrolene, it has been shown that 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) apparently exclusively stimulates Ca²⁺ influx through agonist-operated calcium channels and voltage-operated calcium channels. Increased [Ca²⁺]_i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca²⁺]_i may be attributed to a release of Ca²⁺ from intracellular stores. Quisqualate (QA) stimulates Ca²⁺ release from an intracellular store that is independent of Ca²⁺ influx; presumably this store is activated by inositol phosphates. The increase in [Ca²⁺]_i due to exposure to glutamate or N-methyl-d -aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca²⁺ release from internal stores. Only one of the latter stores is dependent on Ca²⁺ influx with regard to release of Ca²⁺, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca²⁺ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca²⁺ pool(s). By using this drug it has been possible to show to what extent these Ca²⁺ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca²⁺ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca²⁺ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.