Predicting Local SR Ca Dynamics during Ca Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca²⁺) wave propagation, and 2), speed of Ca²⁺ diffusion within the SR. Ca²⁺ waves are initiated when a spontaneous local SR Ca²⁺ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca²⁺ diffusion constant in the SR (D_Ca,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca²⁺] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca²⁺] during Ca²⁺ waves. Simulations have investigated how dynamic local changes in SR [Ca²⁺] are influenced by 1), D_Ca,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca²⁺ pumps; 4), SR Ca²⁺ pump dependence on cytosolic [Ca²⁺]; and 5), the rate of transfer between network and junctional SR. Of these factors, D_Ca,SR is the primary determinant of how release from one RyR cluster alters SR [Ca²⁺] in nearby regions. Specifically, our results show that local increases in SR [Ca²⁺] ahead of the wave can potentially facilitate Ca²⁺ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca²⁺] are possible during SR Ca²⁺release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca²⁺]_SR.     

The effect of ion pumps on the speed of travelling waves in the fire-diffuse-fire model of Ca2+ release

The fire-diffuse-fire model provides an idealized model of Ca²⁺ release within living cells. The effect of calcium pumps, which drive Ca²⁺ back into internal stores, is often neglected for mathematical simplicity. Here we show how to explicitly analyse such effects by extending the work of Keizer et al. [Keizer, J. E., G. D. Smith, S. Ponce Dawson and J. Pearson (1998). Saltatory propagation of Ca²⁺ waves by Ca²⁺ sparks. Biophys. J. 75, 595–600.]. For travelling waves, in which release events occur sequentially, we construct the speed of waves in terms of the time-scale at which pumps operate. An immediate consequence of this analysis is that the inclusion of calcium pumps leads to multiple solutions. A linear stability analysis determines those solution branches in parameter space which are stable. Numerical continuation is used to provide explicit examples of the bifurcation diagrams of the speed of waves as a function of physiologically significant system parameters.     

Mechanisms by which cytoplasmic calcium wave propagation and alternans are generated in cardiac atrial myocytes lacking T-tubules-insights from a simulation study

This study investigated the mechanisms underlying the propagation of cytoplasmic calcium waves and the genesis of systolic Ca²⁺ alternans in cardiac myocytes lacking transverse tubules (t-tubules). These correspond to atrial cells of either small mammals or large mammals that have lost their t-tubules due to disease-induced structural remodeling (e.g., atrial fibrillation). A mathematical model was developed for a cluster of ryanodine receptors distributed on the cross section of a cell that was divided into 13 elements with a spatial resolution of 2 μm. Due to the absence of t-tubules, L-type Ca²⁺ channels were only located in the peripheral elements close to the cell-membrane surface and produced Ca²⁺ signals that propagated toward central elements by triggering successive Ca²⁺-induced Ca²⁺ release (CICR) via Ca²⁺ diffusion between adjacent elements. Under control conditions, the Ca²⁺ signals did not fully propagate to the central region of the cell. However, with modulation of several factors responsible for Ca²⁺ handling, such as the L-type Ca²⁺ channels (Ca²⁺ influx), SERCA pumps (sarcoplasmic reticulum (SR) Ca²⁺ uptake), and ryanodine receptors (SR Ca²⁺ release), Ca²⁺ wave propagation to the center of the cell could occur. These simulation results are consistent with previous experimental data from atrial cells of small mammals. The model further reveals that spatially functional heterogeneity in Ca²⁺ diffusion within the cell produced a steep relationship between the SR Ca²⁺ content and the cytoplasmic Ca²⁺ concentration. This played an important role in the genesis of Ca²⁺ alternans that were more obvious in central than in peripheral elements. Possible association between the occurrence of Ca²⁺ alternans and the model parameters of Ca²⁺ handling was comprehensively explored in a wide range of one- and two-parameter spaces. In addition, the model revealed a spontaneous second Ca²⁺ release in response to a single voltage stimulus pulse with SR Ca²⁺ overloading and augmented Ca²⁺ influx. This study provides what to our knowledge are new insights into the genesis of Ca²⁺ alternans and spontaneous second Ca²⁺ release in cardiac myocytes that lack t-tubules.     

Propagation of fast and slow intercellular Ca(2+) waves in primary cultured arterial smooth muscle cells

Smooth muscle contraction is regulated by changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). In response to stimulation, Ca²⁺ increase in a single cell can propagate to neighbouring cells through gap junctions, as intercellular Ca²⁺ waves. To investigate the mechanisms underlying Ca²⁺ wave propagation between smooth muscle cells, we used primary cultured rat mesenteric smooth muscle cells (pSMCs). Cells were aligned with the microcontact printing technique and a single pSMC was locally stimulated by mechanical stimulation or by microejection of KCl. Mechanical stimulation evoked two distinct Ca²⁺ waves: (1) a fast wave (2 mm/s) that propagated to all neighbouring cells, and (2) a slow wave (20 μm/s) that was spatially limited in propagation. KCl induced only fast Ca²⁺ waves of the same velocity as the mechanically induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptors, shows that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca²⁺ influx through voltage-operated Ca²⁺ channels, whereas, the slow wave was due to Ca²⁺ release primarily through IP₃ receptors. Altogether, these results indicate that temporally and spatially distinct mechanisms allow intercellular communication between SMCs. In intact arteries this may allow fine tuning of vessel tone.     

Nonlinear propagation of agonist-induced cytoplasmic calcium waves in single astrocytes

In astrocytes in primary culture, activation of neurotransmitter receptors results in intracellular calcium signals that propagate as waves across the cell. Similar agonist-induced calcium waves have been observed in astrocytes in organotypic cultures in response to synaptic activation. By using primary cultured astrocytes grown on glass coverslips, in conjunction with fluorescence microscopy we have analyzed agonist-induced Ca²⁺ wave initiation and propagation in individual cells. Both norepinephrine and glutamate elicited Ca²⁺ signals which were initiated focally and discretely in one region of the cell, from where the signals spread as waves along the entire length of the cell. Analysis of the wave propagation and the waveform revealed that the propagation was nonlinear with one or more focal loci in the cytoplasm where the wave was regeneratively amplified. These individual loci appear as discrete focal areas 7–15 μm in diameter and having intrinsic oscillatory properties that differ from each other. The wave initiation locus and the different amplification loci remained invariant in space during the course of the experiment and supported an identical spatiotemporal pattern of signalling in any given cell in response to multiple agonist applications and when stimulated with different agonists which are coupled via InsP₃. Cytoplasmic Ca²⁺ concentration at rest was consistently higher (17 ± 4nM, mean ± S.E.M.) in the wave initiation locus compared with the rest of the cytoplam. The nonlinear propagation results from significant changes in signal rise times, amplitudes, and wave velocity in cellular regions of active loci. Analysis of serial slices across the cell revealed that the rise times and amplitudes of local signals were as much as three- to fourfold higher in the loci of amplification. A phenomenon of hierarchy in local amplitudes of the signal in the amplification loci was observed with the wave initiation locus having the smallest and the most distal locus having largest amplitude. By this mechanism locally very high concentrations of Ca²⁺ are achieved in strategic locations in the cell in response to receptor activation. While the average wave velocity calculated over the length of the cell was 10–15 μm/s, in the active loci rates as high as 40 μm/s were measured. Wave velocity was fivefold lower in regions of the cell separating active loci. The differences in the intrinsic oscillatory periods give rise to local Ca²⁺ waves that show the properties of collision and annihilation. It is hypothesized that the wave front provokes regenerative Ca²⁺ release from specialized areas in the cell where the endoplasmic reticulum is endowed with higher density of InsP₃ receptor channels. Thus wave propagation is achieved by a process of diffusion and regenerative Ca²⁺ release in multiple cellular loci provoked by the advancing wave front; in this way, wave propagation is nonlinear and saltatory. Regenerative Ca²⁺ wave propagation from distal atrocytic processes to the cell body and neighboring cells is likely to provide an important signalling mechanism in the nervous system. 1994 John Wiley & Sons, Inc.     

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca<Superscript>2+</Superscript> waves

Intercellular Ca²⁺ waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca²⁺ waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca²⁺ waves are triggered during damage to cochlear explants. Both Ca²⁺ waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca²⁺, their velocity, their extent of spread and the cell type through which they propagate. A slower Ca²⁺ wave (14 μm/s) communicates between Deiters’ cells and is mediated by P2Y receptors and Ca²⁺ release from IP₃-sensitive stores. In contrast, a faster Ca²⁺ wave (41 μm/s) propagates through sensory hair cells and is mediated by Ca²⁺ influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca²⁺ wave is mediated by P2X₄ receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.     

A model for Ca waves in networks of glial cells incorporating both intercellular and extracellular communication pathways

Networks of glial cells, and in particular astrocytes, are capable of sustaining calcium (Ca²⁺) waves both in vivo and in vitro. Experimentally, it has been shown that there are two separate modes of communication: the first by the passage of an agent (inositol 1,4,5-triphosphate, IP₃) through gap junctions (GJs) joining cells; the second by the diffusion of an extracellular agent (adenosine triphosphate, ATP) that binds to receptors on the cells. In both cases, the outcome is the release of Ca²⁺ from internal stores in the glial cells. These two modes of communication are not mutually exclusive, but probably work in conjunction in many cases. We present a model of a two-dimensional network of glial cells that incorporates regenerative intercellular (GJ) and extracellular (ATP) pathways. In the extreme cases of only one type of pathway, the results are in agreement with previous models. Adding an extracellular pathway to the GJ model increased the extent and duration of the Ca²⁺ wave, but did not significantly change the speed of propagation. Conversely, adding GJs to the extracellular model did increase the wave speed. The model was modified to apply to the retina by extending it to include both astrocytes and Müller cells, with GJs the dominant coupling between astrocytes and ATP responsible for most of the remaining communication. It was found that both pathways are necessary to account for experimental results.     

Spatial characteristics to calcium signalling; the calcium wave as a basic unit in plant cell calcium signalling

Many signals that modify plant cell growth and development initiate changes in cytoplasmic Ca²⁺. The subsequent movement of Ca²⁺ in the cytoplasm is thought to take place via waves of free Ca²⁺. These waves may be initiated at defined regions of the cell and movement requires release from a reticulated endoplasmic reticulum and the vacuole. The mechanism of wave propagation is outlined and the possible basis of repetitive reticulum wave formation, Ca²⁺ oscillations and capacitative Ca²⁺ signalling is discussed. Evidence for the presence of Ca²⁺ waves in plant cells is outlined, and from studies on raphides it is suggested that the capabilities for capacitative Ca²⁺ signalling are also present. The paper finishes with an outline of the possible interrelation between Ca²⁺ waves and organelles and describes the intercellular movement of Ca²⁺ waves and the relevance of such information communication to plant development.     

Three-dimensional simulation of calcium waves and contraction in cardiomyocytes using the finite element method

To investigate the characteristics and underlying mechanisms of Ca²⁺ wave propagation, we developed a three-dimensional (3-D) simulator of cardiac myocytes, in which the sarcolemma, myofibril, and Z-line structure with Ca²⁺ release sites were modeled as separate structures using the finite element method. Similarly to previous studies, we assumed that Ca²⁺ diffusion from one release site to another and Ca²⁺-induced Ca²⁺ release were the basic mechanisms, but use of the finite element method enabled us to simulate not only the wave propagation in 3-D space but also the active shortening of the myocytes. Therefore, in addition to the dependence of the Ca²⁺ wave propagation velocity on the sarcoplasmic reticulum Ca²⁺ content and affinity of troponin C for Ca²⁺, we were able to evaluate the influence of active shortening on the propagation velocity. Furthermore, if the initial Ca²⁺ release took place in the proximity of the nucleus, spiral Ca²⁺ waves evolved and spread in a complex manner, suggesting that this phenomenon has the potential for arrhythmogenicity. The present 3-D simulator, with its ability to study the interaction between Ca²⁺ waves and contraction, will serve as a useful tool for studying the mechanism of this complex phenomenon. cardiac muscle cell; excitation-contraction coupling; mechanoelectrical feedback; spiral wave; arrhythmia     

A bidomain threshold model of propagating calcium waves

We present a bidomain fire-diffuse-fire model that facilitates mathematical analysis of propagating waves of elevated intracellular calcium (Ca²⁺) in living cells. Modeling Ca²⁺ release as a threshold process allows the explicit construction of traveling wave solutions to probe the dependence of Ca²⁺ wave speed on physiologically important parameters such as the threshold for Ca²⁺ release from the endoplasmic reticulum (ER) to the cytosol, the rate of Ca²⁺ resequestration from the cytosol to the ER, and the total [Ca²⁺] (cytosolic plus ER). Interestingly, linear stability analysis of the bidomain fire-diffuse-fire model predicts the onset of dynamic wave instabilities leading to the emergence of Ca²⁺ waves that propagate in a back-and-forth manner. Numerical simulations are used to confirm the presence of these so-called ‘tango waves’ and the dependence of Ca²⁺ wave speed on the total [Ca²⁺].      

Agonist-induced calcium waves in oscillatory cells: A biological example of Burgers' equation

Oscillations in cytosolic Ca²⁺ concentrations in living cells are often a manifestation of propagating waves of Ca²⁺. Numerical simulations with a realistic model of inositol 1, 4, 5-trisphosphate (IP₃)-induced Ca²⁺ wave trains lead to wave speeds that increase linearly at long times when (a) IP₃ levels are in the range for Ca²⁺ oscillations, (b) a gradient of phase is established by either an initial ramp or pulse of IP₃, and (c) IP₃ concentrations asymptotically become uniform. We explore this phenomenon with analytical and numerical methods using a simple two-variable reduction of the De Young-Keizer model of the IP₃ receptor that includes the influence of Ca²⁺ buffers. For concentrations of IP₃ in the oscillatory regime, numerical solution of the resulting reaction diffusion equations produces nonlinear wave trains that shows the same asymptotic growth of wave speed. Due to buffering, diffusion of Ca²⁺ is quite slow and, as previously noted, these waves occur without appreciable bulk movement of Ca²⁺. Thus, following Neu and Murray, we explore the behavior of these waves using an asymptotic expansion based on the small size of the buffered diffusion constant for Ca²⁺. We find that the gradient in phase of the wave obeys Burgers' equation asymptotically in time. This result is used to explain the linear increase of the wave speed observed in the simulations.     

Subcellular propagation of calcium waves in Müller glia does not require autocrine/paracrine purinergic signaling

The polarized morphology of radial glia allows them to functionally interconnect different layers of CNS tissues including the retina, cerebellum, and cortex. A likely mechanism involves propagation of transcellular Ca²⁺ waves which were proposed to involve purinergic signaling. Because it is not known whether ATP release is required for astroglial Ca²⁺ wave propagation we investigated this in mouse Müller cells, radial astroglia-like retinal cells in which in which waves can be induced and supported by Orai/TRPC1 (transient receptor potential isoform 1) channels. We found that depletion of endoplasmic reticulum (ER) stores triggers regenerative propagation of transcellular Ca²⁺ waves that is independent of ATP release and activation of P₂X and P₂Y receptors. Both the amplitude and kinetics of transcellular, depletion-induced waves were resistant to non-selective purinergic P₂ antagonists such as pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). Thus, store-operated calcium entry (SOCE) is itself sufficient for the initiation and subcellular propagation of calcium waves in radial glia.     

Assessment of Sarcoplasmic Reticulum Ca Depletion During Spontaneous Ca Waves in Isolated Permeabilized Rabbit Ventricular Cardiomyocytes

In this study, Ca²⁺ release due to spontaneous Ca²⁺ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca²⁺ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca²⁺ signals during spontaneous Ca²⁺ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca²⁺] increase during a Ca²⁺ wave was 98.1% ± 0.3% of that observed during caffeine application. Conversion to total Ca²⁺ release suggested that Ca²⁺ release from a Ca²⁺ wave was not significantly different from that released during caffeine application (104% ± 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca²⁺ wave was 82.5% ± 2.6% of that observed during caffeine application. Assuming a maximum free [Ca²⁺] of 1.1 mM, this translates to a 96.2% ± 0.8% change in intra-SR free [Ca²⁺] and a 91.7% ± 1.6% depletion of the total Ca²⁺. This equates to a minimum intra-SR free Ca²⁺ of 46 ± 7 μM during a Ca²⁺ wave. Reduction of RyR2 Ca²⁺ sensitivity by tetracaine (50 μM) reduced the spontaneous Ca²⁺ release frequency while increasing the Ca²⁺ wave amplitude. This did not significantly change the total depletion of the SR (94.5% ± 1.1%). The calculated minimum [Ca²⁺] during these Ca²⁺ waves (87 ± 19 μM) was significantly higher than control (p < 0.05). A computational model incorporating this level of Ca²⁺ depletion during a Ca²⁺ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca²⁺ release. In conclusion, spontaneous Ca²⁺ release results in substantial but not complete local Ca²⁺ depletion of the SR. Furthermore, measurements suggest that Ca²⁺ release terminates when luminal [Ca²⁺] reaches ∼50 μM.     

Images of Ca flux in astrocytes: evidence for spatially distinct sites of Ca release and uptake

In this study, we have developed a mathematical method to derive the Ca²⁺ fluxes underlying agonist-evoked Ca²⁺ waves in cultured rat cortical astrocytes. Astrocytes were stimulated with norepinephrine (100 nM) to evoke Ca²⁺ waves, which were recorded by measuring FIuo-3 fluorescence changes with high spatial and temporal resolution. Normalized fluorescence (ΔF/F) was analyzed in discrete cellular spaces in a series of successive slices along the length of the cell. From these data, Ca²⁺ flux was then calculated using a one dimensional reaction-diffusion equation which utilizes the temporal and spatial derivatives of the fluorescence data and the diffusion coefficient of Ca²⁺ in the cytosol. This method identified distinct sites of positive flux (Ca²⁺ release into the cytosol) and of negative flux (Ca²⁺ removal from cytosol) and showed that in astrocytes, sites of Ca²⁺ release from stores regularly alternate with sites of Ca²⁺ removal from the cytosol. Cross correlation analysis of the two distribution patterns gave positive correlation at 2 μm out of phase and a negative correlation in phase. Thapsigargin-induced Ca²⁺ waves were analyzed to determine if the negative flux was due to Ca²⁺ uptake via thapsigargin-sensitive Ca²⁺ pumps. Negative flux sites were still found under these conditions, suggesting that multiple mechanisms of Ca²⁺ removal from the cytosol may contribute to negative flux sites. This method of calculation of flux may serve as a means to describe the distribution of functional ion channels and pumps participating in cellular Ca²⁺ signalling.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by Connexin43

Abstract

Intercellular Ca^{2 +} wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca^{2 +} wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca^{2 +} waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca^{2 +} wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca^{2 +} waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca^{2 +} waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

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.     

Control of Ca2+ wave propagation in mouse pancreatic acinar cells

We haveinvestigated control mechanisms involved in the propagation ofagonist-induced Ca²⁺ waves inisolated mouse pancreatic acinar cells. Using a confocal laser-scanningmicroscope, we were able to show that maximal stimulation of cells withacetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initialCa²⁺ release of comparable amountswith both agonists at the luminal cell pole. SubsequentCa²⁺ spreading to the basolateralmembrane was faster with ACh (17.3 ± 5.4 µm/s) than with bombesin(8.0 ± 2.2 µm/s). The speed of bombesin-inducedCa²⁺ waves could be increased upto the speed of ACh-induced Ca²⁺waves by inhibition of protein kinase C (PKC). Activation of PKCsignificantly decreased the speed of ACh-inducedCa²⁺ waves but had only littleeffect on bombesin-evoked Ca²⁺waves. Within 3 s after stimulation, production of inositol1,4,5-trisphosphate [Ins(1,4,5)P₃]was higher in the presence of ACh compared with bombesin, whereasbombesin induced higher levels of diacylglycerol (DAG) than ACh. Thesedata suggest that the slower propagation speed of bombesin-inducedCa²⁺ waves is due to higheractivation of PKC in the presence of bombesin compared with ACh. Thehigher increase in bombesin- compared with ACh-induced DAG productionis probably due to activation of phospholipase D (PLD). Inhibition ofthe PLD-dependent DAG production by preincubation with 0.3% butanolled to an acceleration of the bombesin-induced Ca²⁺ wave. In further experiments,we could show that ruthenium red (100 µM), an inhibitor ofCa²⁺-inducedCa²⁺ release in skeletal muscle,also decreased the speed of ACh-induced Ca²⁺ waves. The effect ofruthenium red was not additive to the effect of PKC activation. Fromthe data, we conclude that, following Ins(1,4,5)P₃-inducedCa²⁺ release in the luminal cellpole, secondary Ca²⁺ release fromstores, which are located in series between the luminal and the basalplasma membrane, modifies Ca²⁺spreading toward the basolateral cell side byCa²⁺-inducedCa²⁺ release. Activation of PKCleads to a reduction in Ca²⁺release from these stores and therefore could explain the slower propagation of Ca²⁺ waves in thepresence of bombesin compared with ACh.

     

Location-dependent photogeneration of calcium waves in HeLa cells

The calcium ion (Ca²⁺) concentrations in a cell are responsible for the control of vital cellular functions and have been widely studied as a means to investigate and control cell activities. Here, we demonstrate Ca²⁺ wave generation in HeLa cells by femtosecond laser irradiation and show unexpected properties of the Ca²⁺ release and propagation. When the laser was focused in the cell cytoplasm, Ca²⁺ release was independent of both external Ca²⁺ influx and the phosphoinositide-phospholipase C (PLC) signaling pathway. The nucleus was not a susceptible target for laser-induced Ca²⁺ release, whereas irradiation of the plasma membrane produced evidence of transient poration, through which the extracellular solution could enter the cell. By chelating extracellular Ca²⁺, we found that laser-induced influx of ethylene glycol tetra-acetic acid (EGTA) can compete with calcium-induced calcium release and significantly delay or suppress the onset of the Ca²⁺ wave in the target cell. Intercellular Ca²⁺ propagation was adenosine triphosphate-dependent and could be observed even when the target cell cytosolic Ca²⁺ rise was suppressed by influx of EGTA. The irradiation effect on overall cell viability was also tested and found to be low (85% at 6h after irradiation by 60 mW average power). Laser-induced Ca²⁺ waves can be reliably generated by controlling the exposure and focal position and do not require the presence of caged Ca²⁺. The technique has the potential to replace other methods of Ca²⁺ stimulation, which either require additional caged molecules in the cell or do not have an interaction that is as well localized.     

A locally-induced increase in intracellular Ca propagates cell-to-cell in the presence of plasma membrane Ca ATPase inhibitors in non-excitable cells

Intercellular Ca²⁺ waves are commonly observed in many cell types. In non-excitable cells, intercellular Ca²⁺ waves are mediated by gap junctional diffusion of a Ca²⁺ mobilizing messenger such as IP₃. Since Ca²⁺ is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca²⁺ to intercellular Ca²⁺ waves is limited. Here, we report that in the presence of plasma membrane Ca²⁺ ATPase inhibitors, locally-released Ca²⁺ from the flash-photolysis of caged-Ca²⁺ appeared to induce further Ca²⁺ release and were propagated from one cell to another, indicating that Ca²⁺ was self-amplified to mediate intercellular Ca²⁺ waves. Our findings support the notion that non-excitable cells can establish a highly excitable medium to communicate local responses with distant cells.     

Intrinsic Islet Heterogeneity and Gap Junction Coupling Determine Spatiotemporal Ca2+ Wave Dynamics

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca²⁺]_i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca²⁺]_i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca²⁺]_i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca²⁺]_i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet.