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.     

myo-Inositol Trisphosphate Mobilizes Calcium from Fusogenic Carrot (Daucus carota L.) Protoplasts

To determine whether or not inositol trisphosphate (IP₃) mobilizes calcium in higher plant cells, we investigated the effect of IP₃ on Ca²⁺ fluxes in fusogenic carrot (Daucus carota L.) protoplasts. The protoplasts were incubated in ⁴⁵Ca²⁺-containing medium and the ⁴⁵Ca²⁺ associated with the protoplasts was monitored with time. Addition of IP₃ (20 micromolar) caused a 17% net loss of the accumulated ⁴⁵Ca²⁺ within 4 minutes. There was a reuptake of ⁴⁵Ca²⁺ and the protoplasts recovered to their initial value by 10 minutes. Phytic acid (IP₆), also stimulated ⁴⁵Ca²⁺ efflux from the protoplasts. Both the IP_3⁻ and the IP_6⁻induced ⁴⁵Ca²⁺ efflux were inhibited by the calmodulin antagonist, trifluoperazine.     

Exact Stochastic Simulation of a Calcium Microdomain Reveals the Impact of Ca2+ Fluctuations on IP3R Gating

In this study, we numerically analyzed the nonlinear Ca²⁺-dependent gating dynamics of a single, nonconducting inositol 1,4,5-trisphosphate receptor (IP₃R) channel, using an exact and fully stochastic simulation algorithm that includes channel gating, Ca²⁺ buffering, and Ca²⁺ diffusion. The IP₃R is a ubiquitous intracellular Ca²⁺ release channel that plays an important role in the formation of complex spatiotemporal Ca²⁺ signals such as waves and oscillations. Dynamic subfemtoliter Ca²⁺ microdomains reveal low copy numbers of Ca²⁺ ions, buffer molecules, and IP₃Rs, and stochastic fluctuations arising from molecular interactions and diffusion do not average out. In contrast to models treating calcium dynamics deterministically, the stochastic approach accounts for this molecular noise. We varied Ca²⁺ diffusion coefficients and buffer reaction rates to tune the autocorrelation properties of Ca²⁺ noise and found a distinct relation between the autocorrelation time τ_ac, the mean channel open and close times, and the resulting IP₃R open probability P_O. We observed an increased P_O for shorter noise autocorrelation times, caused by increasing channel open times and decreasing close times. In a pure diffusion model the effects become apparent at elevated calcium concentrations, e.g., at [Ca²⁺] = 25 μM, τ_ac = 0.082 ms, the IP₃R open probability increased by ≈20% and mean open times increased by ≈4 ms, compared to a zero noise model. We identified the inactivating Ca²⁺ binding site of IP₃R subunits as the primarily noise-susceptible element of the De Young and Keizer model. Short Ca²⁺ noise autocorrelation times decrease the probability of Ca²⁺ association and consequently increase IP₃R activity. These results suggest a functional role of local calcium noise properties on calcium-regulated target molecules such as the ubiquitous IP₃R. This finding may stimulate novel experimental approaches analyzing the role of calcium noise properties on microdomain behavior.     

Inhibition of mitochondrial calcium uptake rather than efflux impedes calcium release by inositol-1,4,5-trisphosphate-sensitive receptors

Mitochondria modulate cellular Ca²⁺ signals by accumulating the ion via a uniporter and releasing it via Na⁺- or H⁺-exchange. In smooth muscle, inhibition of mitochondrial Ca²⁺ uptake inhibits Ca²⁺ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP₃R). At least two mechanisms may explain this effect. First, localised uptake of Ca²⁺ by mitochondria may prevent negative feedback by cytosolic Ca²⁺ on IP₃R activity, or secondly localised provision of Ca²⁺ by mitochondrial efflux may maintain IP₃R function or SR Ca²⁺ content. To distinguish between these possibilities the role of mitochondrial Ca²⁺ efflux on IP₃R function was examined. IP₃ was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na⁺–Ca²⁺ exchanger inhibited with CGP-37157 (10 μM). Mitochondria accumulated Ca²⁺ during IP₃-evoked [Ca²⁺]_c rises and released the ion back to the cytosol (within 15 s) when mitochondrial Ca²⁺ efflux was active. When mitochondrial Ca²⁺ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca²⁺ occurred after IP₃-evoked Ca²⁺ release. IP₃-evoked [Ca²⁺]_c rises were initially unaffected, then only slowly inhibited by CGP-37157. IP₃R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP₃ release did not inhibit IP₃R. CGP-37157 directly inhibited voltage-gated Ca²⁺ channel activity, however SR Ca²⁺ content was unaltered by the drug. Thus, the gradual decline of IP₃R function that followed mitochondrial Na⁺–Ca²⁺ exchanger inhibition resulted from a gradual overload of mitochondria with Ca²⁺, leading to a reduced capacity for Ca²⁺ uptake. Localised uptake of Ca²⁺ by mitochondria, rather than mitochondrial Ca²⁺ efflux, appears critical for maintaining IP₃R activity.     

Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation

Hydrogen peroxide (H₂O₂) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H₂O₂ are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca²⁺) from intracellular stores or influx of extracellular Ca²⁺. One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP₃) mobilizes intracellular Ca²⁺ by binding to a family of receptors (IP₃Rs) on the endoplasmic–sarcoplasmic reticulum that act as ligand-gated Ca²⁺ channels. IP₃Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP₃R content. In this study we show that IP₃R₁ and IP₃R₃ are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H₂O₂, through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP₃R by H₂O₂ is accompanied by a reduction in calcium efflux induced by IP₃ in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP₃R by activation of NADPH oxidase and that preincubation with H₂O₂ decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP₃ receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H₂O₂. Altogether, these results suggest that H₂O₂ mediates IP₃R down-regulation via proteasome activity.     

Initiation and propagation of a neuronal intracellular calcium wave

The ability to image calcium movement within individual neurons inspires questions of functionality including whether calcium entry into the nucleus is related to genetic regulation for phenomena such as long term potentiation. Calcium waves have been initiated in hippocampal pyramidal cells with glutmatergic signals both in the presence and absence of back propagating action potentials (BPAPs). The dendritic sites of initiation of these calcium waves within about 100 μm of the soma are thought to be localized near oblique junctions. Stimulation of synapses on oblique dendrites leads to production of inositol 1,4,5-trisphosphate (IP₃) which diffuses to the apical dendrite igniting awaiting IP₃ receptors (IP₃Rs) and initiating and propagating catalytic calcium release from the endoplasmic reticulum. We construct a reduced mathematical system which accounts for calcium wave initiation and propagation due to elevated IP₃. Inhomogeneity in IP₃ distribution is responsible for calcium wave initiation versus subthreshold or spatially uniform suprathreshold activation. However, the likelihood that a calcium wave is initiated does not necessarily increase with more calcium entering from BPAPs. For low transient synaptic stimuli, timing between IP₃ generation and BPAPs is critical for calcium wave initiation. We also show that inhomogeneity in IP₃R density can account for calcium wave directionality. Simulating somatic muscarinic receptor production of IP₃, we can account for the critical difference between calcium wave entry into the soma and failure to do so.     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Systems Modeling of Ca2+ Homeostasis and Mobilization in Platelets Mediated by IP3 and Store-Operated Ca2+ Entry

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca²⁺]_cyt) and high dense tubular system calcium ([Ca²⁺]_dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP₃) to trigger calcium mobilization from stores and elevation of [Ca²⁺]_cyt. Another major source of [Ca²⁺]_cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca²⁺]_dts drops. A 34-species systems model employed kinetics describing IP₃-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca²⁺-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP₃-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>−70 mV) and low resting IP₃ concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP₃ caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets’ phenotypes during hemostasis or thrombosis.     

Systems Modeling of Ca Homeostasis and Mobilization in Platelets Mediated by IP3 and Store-Operated Ca Entry

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca²⁺]_cyt) and high dense tubular system calcium ([Ca²⁺]_dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP₃) to trigger calcium mobilization from stores and elevation of [Ca²⁺]_cyt. Another major source of [Ca²⁺]_cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca²⁺]_dts drops. A 34-species systems model employed kinetics describing IP₃-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca²⁺-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP₃-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>−70 mV) and low resting IP₃ concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP₃ caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets’ phenotypes during hemostasis or thrombosis.     

Agonist‐evoked Ca2+ wave progression requires Ca2+ and IP3

Smooth muscle responds to IP₃‐generating agonists by producing Ca²⁺ waves. Here, the mechanism of wave progression has been investigated in voltage‐clamped single smooth muscle cells using localized photolysis of caged IP₃ and the caged Ca²⁺ buffer diazo‐2. Waves, evoked by the IP₃‐generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) over a single though substantial length (～30 µm) of the cell. During regenerative propagation, the wave‐front was about 1/3 the length (～9 µm) of the initiation site. The wave‐front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP₃ may explain the amplitude changes. Ca²⁺ was required for IP₃‐mediated wave progression to occur. Increasing the Ca²⁺ buffer capacity in a small (2 µm) region immediately in front of a CCh‐evoked Ca²⁺ wave halted progression at the site. However, the wave front does not progress by Ca²⁺‐dependent positive feedback alone. In support, colliding [Ca²⁺]_c increases from locally released IP₃ did not annihilate but approximately doubled in amplitude. This result suggests that local IP₃‐evoked [Ca²⁺]_c increases diffused passively. Failure of local increases in IP₃ to evoke waves appears to arise from the restricted nature of the IP₃ increase. When IP₃ was elevated throughout the cell, a localized increase in Ca²⁺ now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP₃ and Ca²⁺ are required for active propagation of the wave front to occur. J. Cell. Physiol. 224: 334–344, 2010. © 2010 Wiley‐Liss, Inc.     

The role of inositol 1,4,5-trisphosphate in mobilizing calcium from intracellular stores in the salivary glands of Amblyomma americanum (L.)

Isolated tick salivary glands, permeabilized with digitonin in the presence of the Ca²⁺ uptake inhibitors, sodium azide and vanadate, released Ca²⁺ in response to 20 μM inositol-1,4,5-trisphosphate (IP₃). Inositol-1-phosphate (IP₁) and inositol-1,4-bisphosphate (IP₂) appeared to stimulate an uptake of Ca²⁺ into whole glands. Inositol-1,4,5-trisphosphate caused release of Ca²⁺ from a 100,000 g microsome enriched pellet; however, IP₁ and IP₂ were ineffective in stimulating an uptake or efflux of Ca²⁺. The combined 900 and 11,500 g pellets showed no significant release of Ca²⁺ in response to addition of IP₃. Inositol-1,4,5-trisphosphate concentrations as low as 1 μM are capable of stimulating a significant release of Ca²⁺ from microsomes. Results suggest that intracellular Ca²⁺ is mobilized from microsomal intracellular stores in response to agonists which increase cytosolic IP₃ in tick salivary glands. Results also suggest a possible role for IP₁ and IP₂ or both in stimulating an uptake of Ca²⁺ into vanadate and azide-insensitive intracellular pools.     

Mitochondrial Calcium Signaling Driven by the IP3 Receptor

Many agonists bring about their effects on cellular functions through a rise incytosolic [Ca²⁺]([Ca²⁺]_c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP₃). Imaging studiesof single cells have demonstrated that [Ca²⁺]_c signals display cell specific spatiotemporalorganization that is established by coordinated activation of IP₃ receptor Ca²⁺ channels.Evidence emerges that cytosolic calcium signals elicited by activation of the IP₃ receptors areefficiently transmitted to the mitochondria. An important function of mitochondrial calciumsignals is to activate the Ca²⁺-sensitive mitochondrial dehydrogenases, and thereby to meetdemands for increased energy in stimulated cells. Activation of the permeability transitionpore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death.Furthermore, mitochondrial Ca²⁺ transport appears to modulate the spatiotemporal organizationof [Ca²⁺]_c responses evoked by IP₃ and so mitochondria may be important in cytosolic calciumsignaling as well. This paper summarizes recent research to elucidate the mechanisms andsignificance of IP₃-dependent mitochondrial calcium signaling.     

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.     

Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca²⁺) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ release channels that mediate Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores. The three IP₃R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP₃R by the endogenous modulators IP₃, Ca²⁺, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP₃R subtype in shaping cytosolic Ca²⁺ oscillations.     

An efficient reduced-lattice model of IP3R for probing Ca2+ dynamics

Numerous cellular processes are regulated by Ca²⁺ signals, and the endoplasmic reticulum (ER) membrane's inositol triphosphate receptor (IP₃R) is critical for modulating intracellular Ca²⁺ dynamics. The IP₃Rs are seen to be clustered in a variety of cell types. The combination of IP₃Rs clustering and IP₃Rs-mediated Ca²⁺-induced Ca²⁺ release results in the hierarchical organization of the Ca²⁺ signals, which challenges the numerical simulation given the multiple spatial and temporal scales that must be covered. The previous methods rather ignore the spatial feature of IP₃Rs or fail to coordinate the conflicts between the real biological relevance and the computational cost. In this work, a general and efficient reduced-lattice model is presented for the simulation of IP₃Rs-mediated multiscale Ca²⁺ dynamics. The model highlights biological details that make up the majority of the calcium events, including IP₃Rs clustering and calcium domains, and it reduces the complexity by approximating the minor details. The model's extensibility provides fresh insights into the function of IP₃Rs in producing global Ca²⁺ events and supports the research under more physiological circumstances. Our work contributes to a novel toolkit for modeling multiscale Ca²⁺ dynamics and advances knowledge of Ca²⁺ signals.     

Dynamic regulation of IP3 receptor clustering and activity by IP3

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels. Their regulation by both IP₃ and Ca²⁺ allows interactions between IP₃Rs to generate a hierarchy of intracellular Ca²⁺ release events. These can progress from openings of single IP₃R, through near-synchronous opening of a few IP₃Rs within a cluster to much larger signals that give rise to regenerative Ca²⁺ waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP₃R3 and shown that low concentrations of IP₃ rapidly and reversibly cause IP₃Rs to assemble into small clusters. In addition to bringing IP₃Rs close enough to allow Ca²⁺ released by one IP₃R to regulate the activity of its neighbors, clustering also retunes the regulation of IP₃Rs by IP₃ and Ca²⁺. At resting cytosolic [Ca²⁺], lone IP₃R are more sensitive to IP₃ and the mean channel open time (~10ms) is twice as long as for clustered IP₃R. When the cytosolic free [Ca²⁺] is increased to 1µM, to mimic the conditions that might prevail when an IP₃R within a cluster opens, clustered IP₃R are no longer inhibited and their gating becomes coupled. IP₃, by dynamically regulating IP₃R clustering, both positions IP₃R for optimal interactions between them and it serves to exaggerate the effects of Ca²⁺ within a cluster. During the course of these studies, we have observed that nuclear IP₃R stably express one of two single channel K ⁺ conductances (γ_K ~120 or 200pS). Here we demonstrate that for both states of the IP₃R, the effects of IP₃ on clustering are indistinguishable. These observations reinforce our conclusion that IP₃ dynamically regulates assembly of IP₃Rs into clusters that underlie the hierarchical recruitment of elementary Ca²⁺ release events.     

Role of secretory granules in inositol 1,4,5-trisphosphate-dependent Ca(2+) signaling: from phytoplankton to mammals

The majority of secretory cell calcium is stored in secretory granules that serve as the major IP₃-dependent intracellular Ca²⁺ store. Even in unicellular phytoplankton secretory granules are responsible for the IP₃-induced Ca²⁺ release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP₃-induced Ca²⁺ release, which accounts for the majority of the IP₃-induced cytoplasmic Ca²⁺ release in neuroendocrine cells, but also to the IP₃ sensitivity of the cytoplasmic IP₃ receptor (IP₃R)/Ca²⁺ channels. Moreover, secretory granules contain the highest IP₃R concentrations and the largest amounts of IP₃Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca²⁺ concentrations. The polyanionic molecules undergo pH- and Ca²⁺-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca²⁺-dependent conformational changes with increased exposure of the structure and increased interactions with Ca²⁺ and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species.     

Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells

Objective

Computational models of calcium (Ca²⁺) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca²⁺ signaling model for RPE based on our experimental data of mechanically induced Ca²⁺ wave in the in vitro model of RPE, the ARPE-19 monolayer.

Methods

We combined six essential Ca²⁺ signaling components into a model: stretch-sensitive Ca²⁺ channels (SSCCs), P₂Y₂ receptors, IP₃ receptors, ryanodine receptors, Ca²⁺ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca²⁺ waves reproduced our control experimental data and data where gap junctions were blocked.

Results

Our model was able to explain Ca²⁺ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca²⁺ through plasma membrane SSCCs and gap junctions conduct the Ca²⁺ and IP³ between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca²⁺ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP₃ receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca²⁺ signal in the distance.

Conclusions

The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P₂Y₂ receptors or accelerate P₂Y₂ receptor phosphorylation, which may partially be the reason for Ca²⁺ wave attenuation by suramin. Being the first RPE Ca²⁺ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.     

Ca2+ release and contraction induced by IP3 and contractile agonists in mammalian gastric smooth muscle

The source, time course and stoichiometry of cytosolic free Ca²⁺ ([Ca²⁺]_i) during contraction were examined in smooth muscle cells isolated from the guinea pig and human stomach. Contraction by receptor-linked agonists (eg, acetylcholine, cholecystokinin octapeptide and Met-enkephalin) was preceded by stoichiometric increases in [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux that were maintained in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ channel blocker (13600). The intracellular Ca²⁺ store could be depleted by repeated stimulation with all agonists in Ca²⁺-free medium or in the presence of 13600 resulting in loss of contractile response; response was restored by re-exposure of the cells to Ca²⁺.The source of intracellular Ca²⁺ an the signal for its release were examined in saponin-permeabilized muscle cells. The cells retained their ability to contract in response to receptor-linked agonists and developed an ability to contract in response to inositol trisphosphate (IP₃). The cells accumulated Ca²⁺ to the same extent as intact muscle cells, but only in the presence of ATP. IP₃ caused a prompt, concentration-dependent increase in contraction, [Ca²⁺]_i and net ⁴⁵Ca²⁺ efflux. These effects were maximally similar to those produced by CCK-8 alone or in combination with IP₃: Depletion of the Ca²⁺ store by repeated stimulation of single muscle cells in Ca²⁺-free medium with IP₃, acetylcholine or CCK-8 separately resulted in loss of contractile response to all three agents; the response was restored by re-exposure of the muscle cell to a cytosol-like perfusate (Ca²⁺ 180 nM).The studies demonstrate that a product of membrane phosphoinositide hydrolysis is capable of mobilizing Ca²⁺ from a depletable, non-mitochondrial intracellular store that is utilized by receptor-linked agonists. The magnitude of IP₃-induced Ca²⁺ release is correlated with contraction.