Geneviève Dupont

One-pool model for Ca2+ oscillations involving Ca2+ and inositol 1,4,5-trisphosphate as co-agonists for Ca2+ release

Experimental observations indicate that Ca(2+)-induced Ca2+ release (CICR) may underlie Ca2+ oscillations in a variety of cells. In its original version, a theoretical model for signal-induced Ca2+ oscillations based on CICR assumed the existence of two types of pools, one sensitive to inositol 1,4,5-trisphosphate (IP3) and the other one sensitive to Ca2+. Recent experiments indicate that Ca2+ channels may sometimes be sensitive to both IP3 and Ca2+. Such a regulation may be viewed as Ca(2+)-sensitized IP3-induced Ca2+ release or, alternatively, as a form of IP3-sensitized CICR. We show that sustained oscillations can still occur in a one-pool model, provided that the same Ca2+ channels are sensitive to both Ca2+ and IP3 behaving as co-agonists. This model and the two-pool model based on CICR both account for a number of experimental observations but differ in some respects. Thus, while in the two-pool model the latency and period of Ca2+ oscillations are of the same order of magnitude and correlate in a roughly linear manner, latency in the one-pool model is always brief and remains much shorter than the period of oscillations. Moreover, the first Ca2+ spike is much larger than the following ones in the one-pool model. These distinctive properties might provide an explanation for the differences in Ca2+ oscillations observed in various cell types.

Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations: a simple model.

The rules that govern the activation and autophosphorylation of the multifunctional Ca2+-calmodulin kinase II (CaMKII) by Ca2+ and calmodulin (CaM) are thought to underlie its ability to decode Ca2+ oscillations and to control multiple cellular functions. We propose a simple biophysical model for the activation of CaMKII by Ca2+ and calmodulin. The model describes the transition of the subunits of the kinase between their different possible states (inactive, bound to Ca2+-CaM, phosphorylated at Thr(286), trapped and autonomous). All transitions are described by classical kinetic equations except for the autophosphorylation step, which is modeled in an empirical manner. The model quantitatively reproduces the experimentally demonstrated frequency sensitivity of CaMKII [Science 279 (1998) 227]. We further use the model to investigate the role of several characterized features of the kinase--as well as some that are not easily attainable by experiments--in its frequency-dependent responses. In cellular microdomains, CaMKII is expected to sense very brief Ca2+ spikes; our simulations under such conditions reveal that the enzyme response is tuned to optimal frequencies. This prediction is then confirmed by experimental data. This novel and simple model should help in understanding the rules that govern CaMKII regulation, as well as those involved in decoding intracellular Ca2+ signals.

Calcium dynamics: spatio-temporal organization from the subcellular to the organ level.

Many essential physiological processes are controlled by calcium. To ensure reliability and specificity, calcium signals are highly organized in time and space in the form of oscillations and waves. Interesting findings have been obtained at various scales, ranging from the stochastic opening of a single calcium channel to the intercellular calcium wave spreading through an entire organ. A detailed understanding of calcium dynamics thus requires a link between observations at different scales. It appears that some regulations such as calcium-induced calcium release or PLC activation by calcium, as well as the weak diffusibility of calcium ions play a role at all levels of organization in most cell types. To comprehend how calcium waves spread from one cell to another, specific gap-junctional coupling and paracrine signaling must also be taken into account. On the basis of a pluridisciplinar approach ranging from physics to physiology, a unified description of calcium dynamics is emerging, which could help understanding how such a small ion can mediate so many vital functions in living systems.

Connexin channels provide a target to manipulate brain endothelial calcium dynamics and blood-brain barrier permeability.

The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5’ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.

Signal-induced CA2+ oscillations : properties of a model based on Ca2+-induced CA2+ release

We consider a simple, minimal model for signal-induced Ca2+ oscillations based on Ca(2+)-induced Ca2+ release. The model takes into account the existence of two pools of intracellular Ca2+, namely, one sensitive to inositol 1,4,5 trisphosphate (InsP3) whose synthesis is elicited by the stimulus, and one insensitive to InsP3. The discharge of the latter pool into the cytosol is activated by cytosolic Ca2+. Oscillations in cytosolic Ca2+ arise in this model either spontaneously or in an appropriate range of external stimulation; these oscillations do not require the concomitant, periodic variation of InsP3. The following properties of the model are reviewed and compared with experimental observations: (a) Control of the frequency of Ca2+ oscillations by the external stimulus or extracellular Ca2+; (b) correlation of latency with period of Ca2+ oscillations obtained at different levels of stimulation; (c) effect of a transient increase in InsP3; (d) phase shift and transient suppression of Ca2+ oscillations by Ca2+ pulses, and (e) propagation of Ca2+ waves. It is shown that on all these counts the model provides a simple, unified explanation for a number of experimental observations in a variety of cell types. The model based on Ca(2+)-induced Ca2+ release can be extended to incorporate variations in the level of InsP3 as well as desensitization of the InsP3 receptor; besides accounting for the phenomena described by the minimal model, the extended model might also account for the occurrence of complex Ca2+ oscillations.

Complex intracellular calcium oscillations. A theoretical exploration of possible mechanisms.

Intracellular Ca(2+) oscillations are commonly observed in a large number of cell types in response to stimulation by an extracellular agonist. In most cell types the mechanism of regular spiking is well understood and models based on Ca(2+)-induced Ca(2+) release (CICR) can account for many experimental observations. However, cells do not always exhibit simple Ca(2+) oscillations. In response to given agonists, some cells show more complex behaviour in the form of bursting, i.e. trains of Ca(2+) spikes separated by silent phases. Here we develop several theoretical models, based on physiologically plausible assumptions, that could account for complex intracellular Ca(2+) oscillations. The models are all based on one- or two-pool models based on CICR. We extend these models by (i) considering the inhibition of the Ca(2+)-release channel on a unique intracellular store at high cytosolic Ca(2+) concentrations, (ii) taking into account the Ca(2+)-activated degradation of inositol 1,4,5-trisphosphate (IP(3)), or (iii) considering explicity the evolution of the Ca(2+) concentration in two different pools, one sensitive and the other one insensitive to IP(3). Besides simple periodic oscillations, these three models can all account for more complex oscillatory behaviour in the form of bursting. Moreover, the model that takes the kinetics of IP(3) into account shows chaotic behaviour.

CaM kinase II as frequency decoder of Ca2+ oscillations

In many cell types, Ca2+ signals are organized in the form of repetitive spikes. The frequency of these intracellular Ca2+ oscillations increases with the level of stimulation, suggesting the existence of a frequency encoding phenomenon. The question arises as to how the frequency of Ca2+ oscillations can be decoded inside the cell. Ca2+/calmodulin kinase II has long been proposed as an attractive candidate, as it is a key target of Ca2+ signals. By immobilizing the Ca2+/calmodulin kinase II and subjecting it to pulses of Ca2+ of variable amplitude, duration, and frequency, De Koninck and Schulman have shown for the first time that the autonomous activity of Ca2+/calmodulin kinase II is highly sensitive to the temporal pattern of Ca2+ oscillations.

Mechanism of receptor-oriented intercellular calcium wave propagation in hepatocytes

Intercellular calcium signals are propagated in multicellular hepatocyte systems as well as in the intact liver. The stimulation of connected hepatocytes by glycogenolytic agonists induces reproducible sequences of intracellular calcium concentration increases, resulting in unidirectional intercellular calcium waves. Hepatocytes are characterized by a gradient of vasopressin binding sites from the periportal to perivenous areas of the cell plate in hepatic lobules. Also, coordination of calcium signals between neighboring cells requires the presence of the agonist at each cell surface as well as gap junction permeability. We present a model based on the junctional coupling of several hepatocytes differing in sensitivity to the agonist and thus in the intrinsic period of calcium oscillations. In this model, each hepatocyte displays repetitive calcium spikes with a slight phase shift with respect to neighboring cells, giving rise to a phase wave. The orientation of the apparent calcium wave is imposed by the direction of the gradient of hormonal sensitivity. Calcium spikes are coordinated by the diffusion across junctions of small amounts of inositol 1,4,5‐trisphos phate (InsP3). Theoretical predictions from this model are confirmed experimentally. Thus, major physiological insights may be gained from this model for coordination and spatial orientation of intercellular signals.—Dupont, G., Tordjmann, T., Clair, C., Swillens, S., Claret, M., Combettes, L. Mechanism of receptororiented intercellular calcium wave propagation in hepatocytes. FASEB J. 14, 279–289 (2000)

Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network

During blastocyst formation, inner cell mass (ICM) cells differentiate into either epiblast (Epi) or primitive endoderm (PrE) cells, labeled by Nanog and Gata6, respectively, and organized in a salt-and-pepper pattern. Previous work in the mouse has shown that, in absence of Nanog, all ICM cells adopt a PrE identity. Moreover, the activation or the blockade of the Fgf/RTK pathway biases cell fate specification towards either PrE or Epi, respectively. We show that, in absence of Gata6, all ICM cells adopt an Epi identity. Furthermore, the analysis of Gata6+/− embryos reveals a dose-sensitive phenotype, with fewer PrE-specified cells. These results and previous findings have enabled the development of a mathematical model for the dynamics of the regulatory network that controls ICM differentiation into Epi or PrE cells. The model describes the temporal dynamics of Erk signaling and of the concentrations of Nanog, Gata6, secreted Fgf4 and Fgf receptor 2. The model is able to recapitulate most of the cell behaviors observed in different experimental conditions and provides a unifying mechanism for the dynamics of these developmental transitions. The mechanism relies on the co-existence between three stable steady states (tristability), which correspond to ICM, Epi and PrE cells, respectively. Altogether, modeling and experimental results uncover novel features of ICM cell fate specification such as the role of the initial induction of a subset of cells into Epi in the initiation of the salt-and-pepper pattern, or the precocious Epi specification in Gata6+/− embryos.

Simulations of the effects of inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase activities on Ca2+ oscillations

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is responsible for Ca2+ mobilization in response to external stimulation in many cell types. The latter phenomenon often occurs as repetitive Ca2+ spikes. In this study, the effect of the two Ins-1,4,5-P3 metabolizing enzymes (Ins-1,4,5-P3 3-kinase and 5-phosphatase) on the temporal pattern of Ca2+ oscillations has been investigated. On the basis of the well-documented Ins-1,4,5-P3 3-kinase stimulation by the Ca2+/calmodulin complex and of the experimentally-determined kinetic characteristics of these enzymes, we predict that 5-phosphatase primarily controls the levels of Ins-1,4,5-P3 and, thereby, the occurrence and frequency of Ca2+ oscillations. Consequently, the model reproduces the experimental observation performed in Chinese hamster ovary cells that 5-phosphatase overexpression has a much more pronounced effect on the pattern of Ca2+ oscillations than 3-kinase overexpression. We also investigated, in more detail, under which conditions a similar effect could be observed in other cell types expressing various Ins-1,4,5-P3 3-kinase activities.