Shape Of Ca2 Research Articles

Calcium oscillations in HEK293 cells lacking SOCE suggest the existence of a balanced regulation of IP3 production and degradation

The concentration of free cytosolic Ca2+ is a critical second messenger in almost every cell type, with the signal often being carried by the period of oscillations, or spikes, in the cytosolic Ca2+ concentration. We have previously studied how Ca2+ influx across the plasma membrane affects the period and shape of Ca2+ oscillations in HEK293 cells. However, our theoretical work was unable to explain how the shape of Ca2+ oscillations could change qualitatively, from thin spikes to broad oscillations, during the course of a single time series. Such qualitative changes in oscillation shape are a common feature of HEK293 cells in which STIM1 and 2 have been knocked out. Here, we present an extended version of our earlier model that suggests that such time-dependent qualitative changes in oscillation shape might be the result of balanced positive and negative feedback from Ca2+ to the production and degradation of inositol trisphosphate.

Glutathione-related substances maintain cardiomyocyte contractile function in hypoxic conditions

Severe hypoxia leads to decline in cardiac contractility and induces arrhythmic events in part due to oxidative damage to cardiomyocyte proteins including ion transporters. This results in compromised handling of Ca2+ ions that trigger heart contractile machinery. Here, we demonstrate that thiol-containing compounds such as N-acetylcysteine (NAC), glutathione ethyl ester (et-GSH), oxidized tetraethylglutathione (tet-GSSG), oxidized glutathione (GSSG) and S-nitrosoglutathione (GSNO) are capable of reducing negative effects of hypoxia on isolated rat cardiomyocytes. Preincubation of cardiomyocytes with 0.1 mM GSNO, 0.5 mM et-GSH, GSSG, tet-GSSG or with 10 mM NAC allows cells 5-times longer tolerate the hypoxic conditions and elicit regular Ca2+ transients in response to electric pacing. The shape of Ca2+ transients generated in the presence of GSNO, et-GSH and NAC was similar to that observed in normoxic control cardiomyocytes. The leader compound, GSNO, accelerated by 34% the recovery of normal contractile function of isolated rat heart subjected to ischemia-reperfusion. GSNO increased glutathionylation of Na,K-ATPase alpha-2 subunit, the principal ion-transporter of cardiac myocyte sarcolemma, which prevents irreversible oxidation of Na,K-ATPase and regulates its function to support normal Ca2+ ion handling in hypoxic cardiomyocytes. Altogether, GSNO appears effective cardioprotector in hypoxic conditions worth further studies toward its cardiovascular application.

Inhibition of spontaneous synchronous activity of hippocampal neurons by excitation of GABAergic neurons

The molecular mechanisms of the neuronal spontaneous synchronous activity (SSA) regulation by population of GABAergic neurons have been investigated in rat hippocampal culture. The neurons from this population contain Ca2+-permeable KA receptors on the presynaptic membrane. Using image analysis, confocal microscopy and immunocytochemistry, we identified by the shape of Ca2+ signal the population of GABAergic neurons with unique charachteristics allowing these neurons to control SSA. The SSA in a neuronal network was suppressed by the KA-receptor mediated [Ca2+]i increase in neurons of this population. Agonists of GluR5/GluK1-containing KA receptors (domoic acid (DA), SYM2081, and ATPA) evoked a fast high-amplitude Ca2+ signal without desensitization only in this population of neurons. This fact points to Ca2+ permeability of KA receptors in these neurons. The GABA(A) receptor antagonist bicuculline increased the activity of AMPA but not KA receptors of these neurons, indicating presynaptical localization of KA receptors. Depolarization of cells induced by KCl (unlike bicuculline-induced depolarization) increased the activity of AMPA and KA receptors twofold, which points to the dependence of the activity on depolarization. A tenfold increase of the SSA frequency in neurons of this population caused an increase in the basal [Ca2+]i level, which was accompanied by inhibition of SSA in another numerous population of neurons, suggesting that an increased GABAergic inhibition takes place. Prolonged high-frequency oscillations causes a global [Ca2+]i increase in the neurons of this population and their subsequent death. Thus, KA receptors in the population of fast GABAergic neurons may implement a negative feedback under hyperexcitation by glutamate enhancing GABA release due to the fast and prolonged [Ca2+]i increase. It has been shown that this mechanism can be used to suppress hyperactivation of a certain population of neurons under high-frequency SSA and ischemia. It is obvious that selective death of inhibitory neurons from this population may lead to hyperexcitability of certain brain regions.

The role of parvalbumin-containing interneurons in the regulation of spontaneous synchronous activity of brain neurons in culture

Subtypes of inhibitory GABAergic neurons containing Ca2+-binding proteins play a pivotal role in the regulation of spontaneous synchronous [Ca2+]i transients in a neuronal network. In this study it is shown that: (1) the interneurons that containing Ca2+-binding proteins at buffer concentration can be identified by the shape of Ca2+-signa1 in response to depolarization or activation of ionotropic glutamate receptors; (2) Ca2+-binding proteins are involved in desynchronization of spontaneous Ca2+ transients. At low frequencies of spontaneous synchronous [Ca2+]i transients (less than 0.2 Hz) neurons show quasi-synchronous pulsations. At higher frequencies, synchronization of spontaneous synchronous [Ca2+]i transients occurs in all neurons; (3) it is established that several synchronous oscillations with different frequencies coexist in the network and the amplitude of their depolarizing pulse also varies. This phenomenon is apparently the mechanism that selectively directs information in separate neurons using the same network; and (4) in one population of interneurons at high frequencies of spontaneous synchronous [Ca2+]i transients the inversion of Cl– concentration gradient is observed. In this case, the inhibition of GABA(A) receptors suppresses the activity of neurons in this population and excites other neurons in the network. Thus, the GABAergic neurons that contain Ca-binding proteins show different mechanisms to regulate the synchronous neuronal activities in cultured rat hippocampal cells.

Modulation of calcium signalling by the endoplasmic reticulum in Carassius neurons

It is known that endoplasmic reticulum (ER), being a calcium store participates in the regulation of intracellular calcium concentration. Ca-ATPase of the ER is one of the crucial agents providing the calcium-accumulating function of this intracellular structure. We studied the role of the ER in modulation of calcium signalling in Carassius neurons using a Ca2+-imaging technique. We tested the role of the ER in the maintenance of a steady state calcium level in the cytoplasm and in modulation of Ca2+ transients evoked by cell depolarizations. The ER calcium stores were depleted using inhibitors of ER Ca-ATPase, which provided blocking of Ca2+ uptake by the ER. Our experiments firstly showed that the ER can significantly modulate the characteristics of intracellular calcium signals in Carassius neurons during their activity. These findings also indicate that the ER modulates the shape of Ca2+ signals rather than the basal level of intracellular Ca2+ in these neurons.

Parvalbumin deficiency in fast‐twitch muscles leads to increased ‘slow‐twitch type’ mitochondria, but does not affect the expression of fiber specific proteins

Parvalbumin (PV), a small cytosolic protein belonging to the family of EF-hand calcium-binding proteins, is highly expressed in mammalian fast-twitch muscle fibers. By acting as a 'slow-onset' Ca2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV-/- mice. Unexpectedly, PV-/- fast-twitch muscles were considerably more resistant to fatigue than the wild-type fast-twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV-/- fast-twitch muscle, extensor digitorum longus, similar to levels observed in the slow-twitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA-encoded cytochrome oxidase c subunit I and nuclear DNA-encoded cytochrome oxidase c subunit Vb and F1-ATPase subunit beta revealed the PV-/- tibialis anterior mitochondria composition to be almost identical to that in wild-type soleus, but not in wild-type fast-twitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post-transcriptional level. Besides the function in energy metabolism, mitochondria in both fast- and slow-twitch muscles act as temporary Ca2+ stores and are thus involved in the shaping of Ca2+ transients in these cells. Previously observed altered spatio-temporal aspects of Ca2+ transients in PV-/- muscles are sufficient to up-regulate mitochondria biogenesis through the probable involvement of both calcineurin- and Ca2+/calmodulin-dependent kinase II-dependent pathways. We propose that 'slow-twitch type' mitochondria in PV-/- fast muscles are aimed to functionally replace the slow-onset buffer PV based on similar kinetic properties of Ca2+ removal.

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes.

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.

Action Currents Generate Stepwise Intracellular Ca2+Patterns in a Neuroendocrine Cell

It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca2+]i) are used to control cellular processes such as gene transcription, cell proliferation, differentiation, and secretion. We recently showed that the Ca2+ oscillations in the neuroendocrine melanotrope cells of Xenopus laevis are built up by a number of discrete Ca2+ rises, the Ca2+ steps. The origin of the Ca2+ steps and their role in the generation of long-lasting Ca2+ patterns were unclear. By simultaneous, noninvasive measuring of melanotrope plasma membrane electrical activity and the [Ca2+]i, we show that numbers, amplitude, and frequency of Ca2+ steps are variable among individual oscillations and are determined by the firing pattern and shape of the action currents. The general Na+ channel blocker tetrodotoxin had no effect on either action currents or the [Ca2+]i. Under Na+-free conditions, a depolarizing pulse of 20 mM K+ induced repetitive action currents and stepwise increases in the [Ca2+]i. The Ca2+ channel blocker CoCl2 eliminated action currents and stepwise increases in the [Ca2+]i in both the absence and presence of high K+. We furthermore demonstrate that the speed of Ca2+ removal from the cytoplasm depends on the [Ca2+]i, also between Ca2+ steps during the rising phase of an oscillation. It is concluded that Ca2+ channels, and not Na+ channels, are essential for the generation of specific step patterns and, furthermore, that the frequency and shape of Ca2+ action currents in combination with the Ca2+ removal rate determine the oscillatory pattern.

Dynamic Regulation of Calcium Influx by G-Proteins, Action Potential Waveform, and Neuronal Firing Frequency

The time course of Ca2+ channel activation and the amplitude and rate of change of Ca2+ influx are primarily controlled by membrane voltage. G-protein-coupled signaling pathways, however, modulate the efficacy of membrane voltage on channel gating. To study the interactions of membrane potential and G-proteins on Ca2+ influx in a physiological context, we have measured N-type Ca2+ currents evoked by action potential waveforms in voltage-clamped chick dorsal root ganglion neurons. We have quantified the effect of varying action potential waveforms and frequency on the shape of Ca2+ current in the presence and absence of transmitters (GABA or norepinephrine) that inhibit N current. Our results demonstrate that both the profile of Ca2+ entry and the time course and magnitude of its transmitter-induced inhibition are sensitive functions of action potential waveform and frequency. Increases in action potential duration enhance total Ca2+ entry, but they also prolong and blunt Ca2+ signals by slowing influx rate and reducing peak amplitude. Transmitter-mediated inhibition of Ca2+ entry is most robust with short-duration action potentials and decreases exponentially with increasing duration. Increases in action potential frequency promote a voltage-dependent inactivation of Ca2+ influx. In channels exposed to GABA or norepinephrine, however, this inactivation is counteracted by a time- and frequency-dependent relief of modulation. Thus, multiple stimuli are integrated by Ca2+ channels, tuning the profile of influx in a changing physiological environment. Such variations are likely to be significant for the control of Ca2+-dependent cellular responses in all tissues.

Alterations in the frequency and shape of Ca2+ fluctuations in GH4C1 cells induced by thyrotropin-releasing hormone and Bay K 8644.

We have examined statistically the actions of thyrotropin-releasing hormone (TRH) and Bay K 8644, an L-type Ca(2+)-channel agonist, on the frequency and shape of cytosolic Ca2+ spikes in individual GH4C1 rat pituitary cells. TRH induced a brief (0-40 s) suppression of Ca2+ spikes followed by a period (40-200 s) of increased spike frequency. TRH treatment reduced the rate of rise and amplitude of Ca2+ spikes, and increased the rate of fall, relative to spontaneous spikes before treatment. TRH had no significant effect on the correlation between spike amplitude and the spike decay time constant tau, suggesting that the increased rate of fall was due to enhanced Ca2+ extrusion and not to decreased Ca(2+)-induced Ca2+ release. Bay K rapidly (t1/2 = 9-13 s) induced a 2-fold increase in the rate of rise of spikes with no change in the total rise time, leading to an increase in spike amplitude. It increased by 2-fold the fall time of spikes, as predicted solely by the previously observed relationship between spike amplitude and fall time. Bay K therefore appeared to increase the number of Ca2+ channels participating in each spike event without altering the kinetics of channel activation or deactivation, and without influencing Ca2+ extrusion. After addition of Bay K, the interval between spikes gradually (t1/2 approximately 100 s) increased, whereas the rate of rise remained constant and maximal. To explain these actions of TRH and Bay K, we postulate that a fraction of L-type Ca2+ channels are inactivated during each spike and must be re-activated in order to participate in a subsequent spike. We conclude further that the changes in spike frequency and profiles induced by these secretagogues are most consistent with a model in which TRH induces increases in both Ca2+ influx and efflux while Bay K induces a large increase in Ca2+ influx but has little effect on efflux.