Cytosolic calcium oscillators

Many cells display oscillations in intracellular calcium resulting from the periodic release of calcium from intracellular reservoirs. Frequencies are varied, but most oscillations have periods ranging from 5 to 60 s. For any given cell, frequency can vary depending on external conditions, particularly the concentration of natural stimuli or calcium. This cytosolic calcium oscillator is particularly sensitive to those stimuli (neurotransmitters, hormones, growth factors) that hydrolyze phosphoinositides to give diacylglycerol and inositol 1,4,5-trisphosphate (Ins1,4,5P3). The ability of Ins1,4,5P3 to mobilize intracellular calcium is a significant feature of many of the proposed models that are used to explain oscillatory activity. Receptor-controlled oscillator models propose that there are complex feedback mechanisms that generate oscillations in the level of Ins1,4,5P3. Second messenger-controlled oscillator models demonstrate that the oscillator is a component of the calcium reservoir, which is induced to release calcium by a constant input of either Ins1,4,5P3 or calcium itself. In the latter case, the process of calcium-induced calcium release might be the basis of oscillatory activity in many cell types. The function of calcium oscillations is still unknown. Because oscillator frequency can vary with agonist concentration, calcium transients might be part of a frequency-encoded signaling system. When an external stimulus arrives at the cell surface the information is translated into a train of calcium spikes, i.e., the signal is digitized. Certain cells may then convey information by varying the frequency of this digital signal.     

Dissecting calcium oscillators in plant cells.

To understand Ca2+ signaling, we need to identify all the Ca2+ transporters and their regulatory components. The first Ca2+ transporters to be cloned from plants and shown to have regulated activity were calmodulin-dependent Ca2+ -pumps. The regulation of these pumps suggests that being able to change the rate of Ca2+ efflux is important for Ca2+ signaling. The identification of pumps and antiporters in different subcellular locations is helping to dissect the complexities of Ca2+ signaling in plants.     

Cytoprotection and intracellular calcium.

It seems that prostacyclin has an increasing effect on gastric mucosal (antral and fundic) calmodulin level in rats. Using either the calcium channel blocker verapamil or anti-calmodulin drugs (diazepam, trifluoperazine,) the cytoprotective effect of prostacyclin can be inhibited. Therefore, it is probable that calcium ions and calcium-activated calmodulin play a role in the effect of prostacyclin.     

Model for receptor-controlled cytosolic calcium oscillations and for external influences on the signal pathway.

The external stimulation of many cells by a hormone, for example, often leads to an oscillating cytosolic calcium concentration. This periodic behavior is now designated the cytosolic calcium oscillator. A theoretical model is presented that describes this behavior on the basis of inositol(1,4,5)trisphosphate-induced calcium oscillations. In contrast to other models only a single positive feedback loop is taken into account to obtain oscillations. The model includes important innovations compared to other approaches. It includes the contribution of extracellular calcium and its modification after the stimulation of the cell. Furthermore, the signal pathway that leads to cytosolic calcium oscillations is described in more detail than in other models. This enables investigations on the influence of additional parameters like external electromagnetic fields on the signal transduction pathway. The model and the calculations are based on the theory of nonlinear self-sustained oscillators.     

Phorbol ester blocks the coupling of GTP-binding protein with receptor-controlled calcium channels

Using the intracellular Ca2+-specific indicator, Quin 2, it was demonstrated that an addition to platelet suspensions of the GTP-binding protein activator, sodium fluoride, stimulates the Ca2+ and Ba2+ influx from the incubation medium into the cytoplasm via receptor-operated Ca2+ channels (Ca-ROC). The fluoride-induced Ca2+ influx is blocked by the protein kinase C activator, phorbol myristate acetate as well as by the platelet adenylate cyclase activator, prostaglandin E1. A two-dimensional electrophoretic analysis of platelet phosphoproteins revealed that the phorbol ester enhances the phosphorylation of proteins with molecular masses of about 20 and 40 kDa. The experimental results suggest that the participation of the GTP-binding protein in the receptor coupling to Ca-ROC. The mechanism of the blocking effect of phorbol esters and prostaglandin E1 on Ca-ROC consists in an impaired coupling of these channels to the GTP-binding protein that activates them.     

Polarity in intracellular calcium signaling.

The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.     

Mechanism of intracellular calcium transients.

Calcium ions mediate extracellular signals on intracellular processes. The signalling system based on transient rises or oscillations of the cytoplasmic calcium concentration has potential advantages. The relevant mechanisms of intracellular concentration changes include calcium-induced calcium release and calcium dependent inactivation of calcium release. A model has been devised based on these processes to generate repetitive transients of the cytoplasmic calcium concentration.     

Spiral waves and intracellular calcium signalling.

Confocal imaging of intracellular Ca2+ brings a new level of resolution to the study of hormonal control of intracellular Ca2+ release. This approach has demonstrated the existence of pulsatile circular and spiral waves of Ca+ release induced by receptor activation. The data obtained by confocal imaging support a new framework for understanding intracellular Ca2+ signalling. The goal of this chapter is to review our data on the complexity of intracellular Ca2+ release in Xenopus oocytes, introduce the concept of Ca2+ excitability as a model for Ca2+ release and discuss the implications for encoding intracellular signal information.     

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca²⁺ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca²⁺]_i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC₅₀ (200 nM). Upon receptor activation, [Ca²⁺]_i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca²⁺]_i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca²⁺ release is higher than other regions of the cell. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ channels by inorganic cations (Cd²⁺ and Ni²⁺) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca²⁺]_i response. In the absence of Ca²⁺ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca²⁺ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca²⁺ entry is not operative. Furthermore, our experiments provide evidence for local Ca²⁺ oscillations in cells which can support both wave propagation as well as spatially discrete Ca²⁺ signalling.     

Enhanced intracellular calcium concentration during poliovirus infection.

The infection of human fibroblasts by poliovirus leads to a notable increase in the intracellular calcium concentration, [Ca2+]i, measured by microfluorimetry or by flow cytometry. [Ca2+]i increases from 2 to 3 h postinfection, and by the fifth hour there is a 5- to 10-fold increase in [Ca2+]i. At this time postinfection there is active viral protein synthesis. The modifications in [Ca2+]i are not observed in the presence of cycloheximide, guanidine, or Ro 09-0179, indicating that virus gene expression is required for the increase in [Ca2+]i. Attempts to identify the source of the intracellular Ca2+ by using different inhibitors of calcium fluxes suggest that calcium enters from the culture medium through voltage-sensitive calcium channels.     

Modelling intracellular H(+) ion diffusion

Intracellular pH, an important modulator of cell function, is regulated by plasmalemmal proteins that transport H(+), or its equivalent, into or out of the cell. The pH(i) is also stabilised by high-capacity, intrinsic buffering on cytoplasmic proteins, oligopeptides and other solutes, and by the extrinsic CO(2)/HCO(3)(-) (carbonic) buffer. As mobility of these buffers is lower than for the H(+) ion, they restrict proton diffusion. In this paper we use computational approaches, based on the finite difference and finite element methods (FDM and FEM, respectively), for analysing the spatio-temporal behaviour of [H(+)] when it is locally perturbed. We analyse experimental data obtained for various cell-types (cardiac myocytes, duodenal enterocytes, molluscan neurons) where pH(i) has been imaged confocally using intracellular pH-sensitive dyes. We design mathematical algorithms to generate solutions for two-dimensional diffusion that fit data in terms of an apparent intracellular H(+) diffusion coefficient, D(H)(app). The models are used to explore how the spatial distribution of [H(+)](i) is affected by membrane H(+)-equivalent transport and by cell geometry. We then develop a mechanistic model, describing spatio-temporal changes of [H(+)](i) in a cardiac ventricular myocyte in terms of H(+)-shuttling on mobile buffers and H(+)-anchoring on fixed buffers. We also discuss how modelling may include the effects of extrinsic carbonic-buffering. Overall, our computational approach provides a framework for future analyses of the physiological consequences of pH(i) non-uniformity.     

Local positive feedback by calcium in the propagation of intracellular calcium waves.

In many types of eukaryotic cells, the activation of surface receptors leads to the production of inositol 1,4,5-trisphosphate and calcium release from intracellular stores. Calcium release can occur in complex spatial patterns, including waves of release that traverse the cytoplasm. Fluorescence video microscopy was used to view calcium waves in single mouse neuroblastoma cells. The propagation of calcium waves was slowed by buffers that bind calcium quickly, such as BAPTA, but not by a buffer with slower on-rate, EGTA. This shows that a key feedback event in wave propagation is rapid diffusion of calcium occurring locally on a scale of < 1 micron. The length-speed product of wavefronts was used to determine that calcium acting in feedback diffuses at nearly the rate expected for free diffusion in aqueous solution. In cytoplasm, which contains immobile Ca2+ buffers, this rate of diffusion occurs only in the first 0.2 ms after release, within 0.4 micron of a Ca2+ release channel mouth. Calcium diffusion from an open channel to neighboring release sites is, therefore, a rate-determining regenerative step in calcium wave propagation. The theoretical limitations of the wave front analysis are discussed.     

Neuropeptide inhibition of voltage-gated calcium channels mediated by mobilization of intracellular calcium.

Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca2+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylglycerol, a product of phosphatidyl-inositol inositol hydrolysis. Here we show that thyrotropin-releasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca2+ channels via the other limb of the phosphatidylinositol signaling system: TRH causes inositol trisphosphate-triggered Ca2+ release from intracellular organelles, thus causing Ca2(+)-dependent inactivation of Ca2+ channels. Elevation of intracellular Ca2+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca2+ buffers into cells and mimicked by a variety of agents that mobilize Ca2+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca2+ channels occurs not only as a result of Ca2+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca2+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca2+ buffers in patch-clamp electrodes.     

Simultaneous measurement of ciliary beating and intracellular calcium.

A novel system for measuring, simultaneously, ciliary beating and intracellular free calcium is presented. The advantages and dynamic nature of the system are demonstrated by measuring the effects of the calcium ionophore lonomycin and of extracellular ATP on ciliated rabbit trachea. The results are discussed with regard to the ciliary and calcium stimulation.     

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.     

Modelling calcium carbonate biomineralisation processes

The structure-directing influence of the organic dicarboxylates malonate, succinate, glutarate and adipate as templating species on the hydrothermal formation of CaCO(3) was investigated at different temperatures (60, 80, 90, 120, 150 and 190 degrees C) and with a range of molar ratios of [Ca(2+)]/[templating species] (20, 14.3, 10, 7.7, 5, 1, 0.5 and 0.33). In the presence of the dicarboxylates, one, two or three polymorphs of CaCO(3) - calcite, aragonite and vaterite - could be formed, depending on the reaction conditions. In addition changes in crystal morphology were observed for the CaCO(3) polymorphs depending on the concentration of the template. In contrast, synthesis under ambient conditions of temperature and pressure resulted only in calcite formation, although template-dependent morphological changes were again observed. Crystalline products were all characterized by powder X-ray patterns and SEM (Scanning Electron Microscopy) micrographs. The ambient reactions with the chelating, dinucleating carboxylato ligands H(3)heidi and H(5)hpdta produce more profound changes in calcite morphology. With H(3)heidi rounded calcite crystals with shapes similar to that of otoliths are formed and with H(5)hpdta the formation of microtrumpets of constructed from bundles of nanocrystals of calcite is observed. The possible mode of action of these ligands on calcite formation is discussed in the context of known coordination chemistry with other metal ions.     

Modelling calcium microdomains using homogenisation

Microdomains of calcium (i.e., areas on the nanometer scale that have qualitatively different calcium concentrations from that in the bulk cytosol) are known to be important in many situations. In cardiac cells, for instance, a calcium microdomain between the L-type channels and the ryanodine receptors, the so-called diadic cleft, is where the majority of the control of calcium release occurs. In other cell types that exhibit calcium oscillations and waves, the importance of microdomains in the vicinity of clusters of inositol trisphosphate receptors, or between the endoplasmic reticulum (ER) and other internal organelles or the plasma membrane, is clear. Given the limits of computational power, it is not currently realistic to model an entire cellular cytoplasm by incorporating detailed structural information about the ER throughout the entire cytoplasm. Hence, most models use a homogenised approach, assuming that both cytoplasm and ER coexist at each point of the domain. Conversely, microdomain models can be constructed, in which detailed structural information can be incorporated, but, until now, methods have not been developed for linking such a microdomain model to a model at the level of the entire cell. Using the homogenisation approach we developed in an earlier paper [Goel, P., Friedman, A., Sneyd, J., 2006. Homogenization of the cell cytoplasm: the calcium bidomain equations. SIAM J. Multiscale Modeling Simulation, in press] we show how a multiscale model of a calcium microdomain can be constructed. In this model a detailed model of the microdomain (in which the ER and the cytoplasm are separate compartments) is coupled to a homogenised model of the entire cell in a rigorous way. Our method is illustrated by a simple model of the diadic cleft of a cardiac half-sarcomere.