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.     

Active Generation and Propagation of Ca Signals within Tunneling Membrane Nanotubes

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca²⁺ signaling; however, our simulations indicate that passive diffusion of Ca²⁺ ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP₃)-evoked Ca²⁺ signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca²⁺ signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP₃ receptors along the TNT. We propose that IP₃ receptors may actively propagate intercellular Ca²⁺ signals along TNTs via Ca²⁺-induced Ca²⁺ release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials.     

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.     

Modeling of the Modulation by Buffers of Ca Release through Clusters of IP3 Receptors

Intracellular Ca²⁺ release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca²⁺ and Ca²⁺ buffers, with spatially discrete clusters of stochastic IP₃ receptor channels (IP₃Rs) controlling the release of Ca²⁺ from the endoplasmic reticulum. IP₃Rs are activated by a small rise of the cytosolic Ca²⁺ concentration and inhibited by large concentrations. Buffering of cytosolic Ca²⁺ shapes global Ca²⁺ transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca²⁺ release, but affect the time course of the signals from the fluorescent Ca²⁺ indicator mainly by competing for Ca²⁺. At low [IP₃], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

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.     

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.     

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.     

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer’s Disease-Causing Mutants in Presenilins

Familial Alzheimer’s disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) Ca²⁺ release channels resulting in enhanced IP₃R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP₃R activity is considered to be the main reason behind the upregulation of intracellular Ca²⁺ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca²⁺ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP₃R channel activity records obtained under optimal Ca²⁺ and multiple IP₃ concentrations to gain deeper insights into the enhancement of IP₃R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP₃R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP₃R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP₃ and Ca²⁺ concentrations and quantify the sensitivity of IP₃R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP₃ and Ca²⁺ and that very small amount of IP₃ is required to stimulate IP₃R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP₃ concentrations. We further demonstrate with simulations that the relatively longer time spent by IP₃R in the H mode leads to the observed higher frequency of local Ca²⁺ signals, which can account for the more frequent global Ca²⁺ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca²⁺ signals in cells expressing FAD-causing mutant PS.     

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.     

Cyanide-stimulated inositol 1,4,5-trisphosphate formation: An intracellular neurotoxic signaling cascade

Cyanide-induced neurotoxicity is associated with altered cellular Ca²⁺ homeostasis resulting in sustained elevation of cytosolic Ca²⁺. In order to characterize the effect of cyanide on intracellular signaling mechanisms, the interaction of KCN with the inositol 1,4,5-triphosphate Ca²⁺ signaling system was determined in the PC12 cell line. KCN in the concentration range of 1.0–100 μM produced a rapid rise in intracellular IP₃ levels (peak level occurred within 60 sec); 10 μM KCN elevated intracellular levels of IP₃ to 148% of control levels. This response was mediated by phospholipase C (PLC) since U73122, a specific PLC inhibitor, blocked the response. Removal of Ca²⁺ from the incubation medium and chelation of intracellular Ca²⁺ with BAPTA partially attenuate the cyanide-stimulated IP₃ generation, showing that the response is partially Ca²⁺ dependent. Also, treatment of cells with nifedipine or LaCl₃, Ca²⁺ channel blockers, partially blocked the generation of IP₃. This study shows that cyanide in concentrations as low as 1 μM stimulates IP₃ generation that may be mediated by receptor and nonreceptor IP₃ production since they have differential dependence on Ca²⁺. It is proposed that this response is an early intracellular signaling action that can contribute to altered Ca²⁺ homeostasis characteristic of cyanide neurotoxicity. © 1997 John Wiley & Sons, Inc.     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000     

Abortive and Propagating Intracellular Calcium Waves: Analysis from a Hybrid Model

The functional properties of inositol(1,4,5)-triphosphate (IP₃) receptors allow a variety of intracellular Ca²⁺ phenomena. In this way, global phenomena, such as propagating and abortive Ca²⁺ waves, as well as local events such as puffs, have been observed. Several experimental studies suggest that many features of global phenomena (e.g., frequency, amplitude, speed wave) depend on the interplay of biophysical processes such as diffusion, buffering, efflux and influx rates, which in turn depend on parameters such as buffer concentration, Ca²⁺ pump density, cytosolic IP₃ level, and intercluster distance. Besides, it is known that cells are able to modify some of these parameters in order to regulate the Ca²⁺ signaling. By using a hybrid model, we analyzed different features of the hierarchy of calcium events as a function of two relevant parameters for the calcium signaling, the intercluster distance and the pump strength or intensity. In the space spanned by these two parameters, we found two modes of calcium dynamics, one dominated by abortive calcium waves and the other by propagating waves. Smaller distances between the release sites promote propagating calcium waves, while the increase of the efflux rate makes the transition from propagating to abortive waves occur at lower values of intercluster distance. We determined the frontier between these two modes, in the parameter space defined by the intercluster distance and the pump strength. Furthermore, we found that the velocity of simulated calcium waves accomplishes Luther’s law, and that an effective rate constant for autocatalytic calcium production decays linearly with both the intercluster distance and the pump strength.     

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.     

Inositol 1,4,5-Trisphosphate Receptor in Chromaffin Secretory Granules and Its Relation to Chromogranins

The inositol 1,4,5-trisphosphate (IP₃)-mediated intracellular Ca²⁺ releases in secretory cells play vital roles in controlling not only the intracellular Ca²⁺ concentrations but also the Ca²⁺-dependent exocytotic processes. Of intracellular organelles that release Ca²⁺ in response to IP₃, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP₃-dependent Ca²⁺ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP₃-mediated Ca²⁺ release as well as the presence of the IP₃ receptor (IP₃R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP₃R isoforms, and 58–69% of total cellular IP₃R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin II are high-capacity, low-affinity Ca²⁺ binding proteins, binding 30–93 mol of Ca²⁺/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca²⁺ storage properties of chromogranins secretory granules contain ~40 mM Ca²⁺. Furthermore, chromogranins A and B directly interact with the IP₃Rs and modulate the IP₃R/Ca²⁺ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively. Coupled chromogranins change the IP₃R/Ca²⁺ channels to a more ordered, release-ready state, whereby making the IP₃R/Ca²⁺ channels significantly more sensitive to IP₃.     

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.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release from permeabilized mastocytoma cells

The effect of inositol 1,4,5-trisphosphate (IP₃) on Ca²⁺ release in the transformed murine mast cells, mastocytoma P-815 cells permeabilized with digitonin was studied. Ca²⁺ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca²⁺ concentration was lowered to a new steady-state level. The subsequent addition of IP₃ caused a rapid Ca²⁺ release, which was followed by a slow re-uptake of Ca²⁺. Fifty percent of the sequestered Ca²⁺ was released by 10 μM IP₃. Maximal Ca²⁺ release occurred at 10 μM and half maximal activity was at 1.3 μM. These results indicate that IP₃ may function as a messenger of intracellular Ca²⁺ mobilization in mastocytoma cells.     

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.     

Enhanced ER Ca2+ store filling by overexpression of SERCA2b promotes IP3-evoked puffs

Liberation of Ca²⁺ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP₃R) is modulated by the ER Ca²⁺ content, and overexpression of SERCA2b to accelerate Ca²⁺ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP₃-evoked Ca²⁺ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP₃-evoked puffs to elucidate whether ER [Ca²⁺] may modulate IP₃R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca²⁺ release. SERCA2b and Ca²⁺ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca²⁺ influx and thereby load ER stores. Puffs evoked by photoreleased IP₃ were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca²⁺ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca²⁺ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP₃R gating kinetics.     

Modeling Ca2+ feedback on a single inositol 1,4,5-trisphosphate receptor and its modulation by Ca2+ buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP₃R) is a major regulator of intracellular Ca²⁺ signaling, and liberates Ca²⁺ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP₃ and Ca²⁺. Although the steady-state gating properties of the IP₃R have been extensively studied and modeled under conditions of fixed [IP₃] and [Ca²⁺], little is known about how Ca²⁺ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca²⁺ binding sites. We thus simulated the dynamics of Ca²⁺ self-feedback on monomeric and tetrameric IP₃R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca²⁺ buffers that slow the collapse of the local [Ca²⁺] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca²⁺ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca²⁺ binding site on the IP₃R in relation to the channel pore.