A theoretical study of effects of cytosolic Ca2+ oscillations on activation of glycogen phosphorylase

Taking into account the Ca(2+)-stimulated degradation of inositol 1,4,5-trisphosphate (IP(3)) by a 3-kinase, we have theoretically explored the effects of both simple and complex Ca(2+) oscillations on the regulation of a phosphorylation-dephosphorylation cycle process involved in glycogen degradation by glycogen phosphorylase a-form, respectively. For the case of simple Ca(2+) oscillations, the roles of cytosolic Ca(2+) oscillations in the regulation of active phosphorylase depend upon the maximum rate of IP(3) degradation by the 3-kinase, V(M5). In particular, the smaller the values of V(M5) are, the lower the effective Ca(2+) threshold for the activation of glycogen phosphorylase will be. For the case of complex Ca(2+) oscillations, the average level of fraction of active phosphorylase is nearly independent from the level of stimulation increasing in the bursting oscillatory domain. Both simple and complex Ca(2+) oscillations can contribute to increase the efficiency and specificity of cellular signalling, and some theoretical results of activation of glycogen phosphorylase regulated by Ca(2+) oscillations are close to the experimental results for gene expression in lymphocytes.     

Modeling stochastic Ca2+ release from a cluster of IP3-sensitive receptors

We focused our attention on Ca(2+) release from the endoplasmic reticulum through a cluster of inositol(1,4,5)-trisphosphate (IP(3)) receptor channels. The random opening and closing of these receptors introduce stochastic effects that have been observed experimentally. Here, we present a stochastic version of Othmer-Tang model (OTM) for IP(3) receptor clusters. We address the average behavior of the channels in response to IP(3) stimuli. In our stochastic simulation we found that the fraction of open channels versus [IP(3)] follows a Hill curve, whose associate Hill coefficient increases when intracellular Ca(2+) level increase. This finding suggests that feedback from cytosolic Ca(2+) plays a key role in the channel response to IP(3). We also study several aspects of the stochastic properties of Ca(2+) release and we compare with experimental observations.     

钙离子振荡对肝糖磷酸化酶激活的随机效应研究

在复杂生化系统的研究过程中,仿真与建模变得越来越重要.对于参与分子数量比较大的生化系统,通常可以采用常微分方程来解决这一问题.对于分子数量比较小的系统,离散粒子基础上的随机模拟方法更精确.然而目前还没有明确的理论方法来确定,对于实际问题用哪种方法能得到更合理的结果.因此需要在一个普遍研究的体系中,通过Ca~(2+)振荡传导信号来研究从随机行为到确定行为的过渡过程.本文以肝细胞中Ca~(2+)振荡对肝糖磷酸化酶激活随机效应为例,讨论了利用随机微分方程来解决分子数量比较小的生化系统的仿真与建模问题,利用细胞内Ca~(2+)有关的Li-Rinzel随机模型,研究了在磷酸化酶降解肝糖的磷酸化-去磷酸化作用循环过程中,三磷酸肌醇受体通道(IP_3R)释放Ca~(2+)的调控作用.结果表明,肝糖磷酸化酶的激活率随受体通道IP_3R的总数增大而减弱,而且三磷酸肌醇浓度比较小时出现相干共振.     

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.     

Dual regulation of calcium mobilization by inositol 1,4, 5-trisphosphate in a living cell

Changes in cytosolic free calcium ([Ca(2+)](i)) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca(2+)](i) oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca(2+)](i) oscillates at a particular frequency or becomes a sustained response is poorly understood. We find that [Ca(2+)](i) oscillations in rat megakaryocytes, as in other cells, results from a Ca(2+)-dependent inhibition of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. Moreover, we find that this inhibition becomes progressively less effective with higher IP(3) concentrations. We suggest that disinhibition, by increasing IP(3) concentration, of Ca(2+)-dependent inhibition is a common mechanism for the regulation of [Ca(2+)](i) oscillations in cells containing IP(3)-sensitive Ca(2+) stores.     

Lateral inhibition of inositol 1,4,5-trisphosphate receptors by cytosolic Ca(2+).

Ryanodine and inositol 1,4,5-trisphosphate (IP(3)) receptors - two related families of Ca(2+) channels responsible for release of Ca(2+) from intracellular stores [1] - are biphasically regulated by cytosolic Ca(2+) [2] [3] [4]. It is thought that the resulting positive feedback allows localised Ca(2+)-release events to propagate regeneratively, and that the negative feedback limits the amplitude of individual events [5] [6]. Stimulation of IP(3) receptors by Ca(2+) occurs through a Ca(2+)-binding site that becomes exposed only after IP(3) has bound to its receptor [7] [8]. Here, we report that rapid inhibition of IP(3) receptors by Ca(2+) occurs only if the receptor has not bound IP(3). The IP(3) therefore switches its receptor from a state in which only an inhibitory Ca(2+)-binding site is accessible to one in which only a stimulatory site is available. This regulation ensures that Ca(2+) released by an active IP(3) receptor may rapidly inhibit its unliganded neighbours, but it cannot terminate the activity of a receptor with IP(3) bound. Such lateral inhibition, which is a universal feature of sensory systems where it improves contrast and dynamic range, may fulfil similar roles in intracellular Ca(2+) signalling by providing increased sensitivity to IP(3) and allowing rapid graded recruitment of IP(3) receptors.     

Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria

Cytosolic Ca(2+) ([Ca(2+)](i)) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP(3)R) driven through cycles of activation/inactivation by local Ca(2+) feedback. Consequently, modulation of the local Ca(2+) gradients influences IP(3)R excitability as well as the duration and amplitude of the [Ca(2+)](i) oscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP(3)-dependent [Ca(2+)](i) oscillations in intact hepatocytes, apparently by altering the local Ca(2+) gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP(3) sensitivity for Ca(2+) release from intracellular stores. These effects on IP(3)-dependent [Ca(2+)](i) signals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca(2+) fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca(2+) by sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), lowering basal and inter-spike [Ca(2+)](i). In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (DeltaPsi(m)), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca(2+) uptake and retention by the mitochondria during a rise in [Ca(2+)](i). We suggest that CSA suppresses local Ca(2+) feedback by enhancing mitochondrial and endoplasmic reticulum Ca(2+) uptake, these actions of CSA underlie the lower IP(3) sensitivity found in permeabilized cells and the impaired IP(3)-dependent [Ca(2+)](i) signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca(2+)](i) signals, which, in turn, may adjust the output of downstream Ca(2+)-sensitive pathways.     

Distinct roles of inositol 1,4,5-trisphosphate receptor types 1 and 3 in Ca2+ signaling

Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca(2+) release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca(2+) signaling, such as Ca(2+) oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca(2+) signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca(2+) signals and terminates Ca(2+) oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca(2+) oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (>90% of total IP(3)R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca(2+)-oscillatory unit without contributing to peak amplitude of Ca(2+) signals, irrespective of its relative expression level. Therefore, differential expression of the IP(3)R subtype is critical for various forms of Ca(2+) signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca(2+) oscillations.     

The probability of triggering calcium puffs is linearly related to the number of inositol trisphosphate receptors in a cluster

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through concerted opening of tightly clustered inositol trisphosphate receptor/channels (IP(3)R). They serve both local signaling functions and trigger global Ca(2+) waves. The numbers of functional IP(3)R within clusters differ appreciably between different puff sites, and we investigated how the probability of puff occurrence varies with cluster size. We imaged puffs in SH-SY5Y cells using total internal fluorescence microscopy, and estimated cluster sizes from the magnitude of the largest puff observed at each site relative to the signal from a single channel. We find that the initial triggering rate of puffs following photorelease of IP(3), and the average frequency of subsequent repetitive puffs, vary about linearly with cluster size. These data accord well with stochastic simulations in which opening of any individual IP(3)R channel within a cluster triggers a puff via Ca(2+)-induced Ca(2+) release. An important consequence is that the signaling power of a puff site (average amount of Ca(2+) released per puff × puff frequency) varies about the square of cluster size, implying that large clusters contribute disproportionately to cellular signaling and, because of their higher puff frequency, preferentially act as pacemakers to initiate Ca(2+) waves.     

Effects of gap junction to Ca(2+) and to IP(3) on the synchronization of intercellular calcium oscillations in hepatocytes

The frequency of free cytosolic calcium concentration ([Ca(2+)]) oscillations elicited by a given agonist concentration differs between individual hepatocytes. However, in multicellular systems of rat hepatocytes and even in the intact liver, [Ca(2+)] oscillations are synchronized and highly coordinated. In this paper, we have investigated theoretically the gap junction permeable to calcium and to IP(3) on intercellular synchronization by means of a mathematical model, respectively. It is shown that gap junction permeable to calcium and to IP(3) are effective on synchronizing calcium oscillations in coupled hepatocytes. Our theoretical results are similar either for the case of Ca(2+) acting as coordinating messenger or for the case of IP(3) as coordinating messenger. There exists an optimal coupling strength for a pair of connected hepatocytes. Appropriate coupling strength and IP(3) level can induce various harmonic locking of intercellular [Ca(2+)] oscillations. Furthermore, a phase diagram in two-dimensional parameter space of the coupling strength and IP(3) level (or the velocity of IP(3) synthesis) has been predicted, in which the synchronization region is similar to Arnol'd tongue.     

Stochastic properties of Ca(2+) release of inositol 1,4,5-trisphosphate receptor clusters

Intracellular Ca(2+) release is controlled by inositol 1,4,5-trisphosphate (IP(3)) receptors or ryanodine receptors. These receptors are typically distributed in clusters with several or tens of channels. The random opening and closing of these channels introduces stochasticity into the elementary calcium release mechanism. Stochastic release events have been experimentally observed in a variety of cell types and have been termed sparks and puffs. We put forward a stochastic version of the Li-Rinzel model (the deactivation binding process is described by a Markovian scheme) and a computationally more efficient Langevin approach to model the stochastic Ca(2+) oscillation of single clusters. Statistical properties such as Ca(2+) puff amplitudes, lifetimes, and interpuff intervals are studied with both models and compared with experimental observations. For clusters with tens of channels, a simply decaying amplitude distribution is typically observed at low IP(3) concentration, while a single peak distribution appears at high IP(3) concentration.     

Analysis of puff dynamics in oocytes: interdependence of puff amplitude and interpuff interval

Puffs are localized Ca(2+) signals that arise in oocytes in response to inositol 1,4,5-trisphosphate (IP(3)). They are analogous to the sparks of myocytes and are believed to be the result of the liberation of Ca(2+) from the endoplasmic reticulum through the coordinated opening of IP(3) receptor/channels clustered at a functional release site. In this article, we analyze sequences of puffs that occur at the same site to help elucidate the mechanisms underlying puff dynamics. In particular, we show a dependence of the interpuff time on the amplitude of the preceding puff, and of the amplitude of the following puff on the preceding interval. These relationships can be accounted for by an inhibitory role of the Ca(2+) that is liberated during puffs. We construct a stochastic model for a cluster of IP(3) receptor/channels that quantitatively replicates the observed behavior, and we determine that the characteristic time for a channel to escape from the inhibitory state is of the order of seconds.     

Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization

In neurons, voltage-gated Ca(2+) channels and nuclear Ca(2+) signaling play important roles, such as in the regulation of gene expression. However, the link between electrical activity and biochemical cascade activation involved in the generation of the nuclear Ca(2+) signaling is poorly understood. Here we show that depolarization of Aplysia neurons induces the translocation of ADP-ribosyl cyclase, a Ca(2+) messenger synthesizing enzyme, from the cytosol into the nucleus. The translocation is dependent on Ca(2+) influx mainly through the voltage-dependent L-type Ca(2+) channels. We report also that specific nucleoplasmic Ca(2+) signals can be induced by three different calcium messengers, cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), both produced by the ADP-ribosyl cyclase, and inositol 1,4,5-trisphosphate (IP(3)). Moreover, our pharmacological data show that NAADP acts on its own receptor, which cooperates with the IP(3) and the ryanodine receptors to generate nucleoplasmic Ca(2+) oscillations. We propose a new model where voltage-dependent L-type Ca(2+) channel-induced nuclear translocation of the cytosolic cyclase is a crucial step in the fine tuning of nuclear Ca(2+) signals in neurons.     

A minimal generic model of bacteria-induced intracellular Ca2+ oscillations in epithelial cells

The toxin alpha-hemolysin expressed by uropathogenic Escherichia coli bacteria was recently shown as the first pathophysiologically relevant protein to induce oscillations of the intracellular Ca(2+) concentration in target cells. Here, we propose a generic three-variable kinetic model describing the Ca(2+) oscillations induced in single rat renal epithelial cells by this toxin. Specifically, we take into account the interplay between 1), the cytosolic Ca(2+) concentration; 2), IP(3)-sensitive Ca(2+) channels located in the membrane separating the cytosol and endoplasmic reticulum; and 3), toxin-related activation of production of IP(3) by phospholipase C. With these ingredients, the predicted response of cells exposed to the toxin is in good agreement with the results of experiments.     

Inositol trisphosphate receptors in smooth muscle cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of tetrameric intracellular calcium (Ca(2+)) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP(3)R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca(2+) signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP(3)R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP(3)R channel activity. SMC IP(3)Rs communicate with nearby ryanodine-sensitive Ca(2+) channels and mitochondria to influence SR Ca(2+) release and reactive oxygen species generation. IP(3)R-mediated Ca(2+) release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca(2+) channels. SMC IP(3)Rs also signal to other proteins via SR Ca(2+) release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca(2+)-activated potassium channels. IP(3)R-mediated Ca(2+) release generates a wide variety of intracellular Ca(2+) signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP(3)R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP(3)R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP(3)R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP(3)R signaling contributing to disease.     

Molecular properties of inositol 1,4,5-trisphosphate receptors.

The receptors for the second messenger inositol 1,4,5-trisphosphate (IP3) constitute a family of Ca2+ channels responsible for the mobilization of intracellular Ca2+ stores. Three different gene products (types I-III) have been isolated, encoding polypeptides which assemble as large tetrameric structures. Recent molecular studies have advanced our knowledge about the structure, regulation and function of IP3 receptors. For example, several Ca(2+)-binding sites and a Ca(2+)-calmodulin-binding domain have been mapped within the type I IP3 receptor, and studies on purified cerebellar IP3 receptors propose a second Ca(2+)-independent calmodulin-binding domain. In addition, minimal requirements for the binding of immunophilins and the formation of tetramers have been identified. Overexpression of IP3 receptors has provided further clues to the regulation of individual IP3 receptor isoforms present within cells, and the role that they play in the generation of IP3-dependent Ca2+ signals. Inhibition of IP3 receptor function and expression, and analysis of mutant IP3 receptors, suggests that IP3 receptors are involved in such diverse cellular processes as proliferation and apoptosis and are thus, necessary for normal development. Our understanding of the complex spatial and temporal nature of cytosolic Ca2+ increases and the role that these Ca2+ signals play in cell function depend upon our knowledge of the structure and the regulation of IP3 receptors. This review focuses on the molecular properties of these ubiquitous intracellular Ca2+ channels.     

Sequential opening of IP(3)-sensitive Ca(2+) channels and SOC during alpha-adrenergic activation of rabbit vena cava

Alpha(1)-aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca(2+) oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) release channels (IP(3)R channels) can prevent the initiation and abolish ongoing alpha(1)-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP(3)R channels, not to its slight inhibition of the L-type voltage-gated Ca(2+) channel and the sarco(endo)plasmic reticulum Ca(2+) ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca(2+) release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP(3)R-channel-mediated SR Ca(2+) release or store depletion. This is in accordance with its sensitivity to Ni(2+) and La(3+) (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP(3)R channels and the SOC are required for PE-mediated [Ca(2+)](i) oscillations and constriction of the rabbit IVC.     

IP3 receptors and their regulation by calmodulin and cytosolic Ca2+

Inositol 1,4,5-trisphosphate (IP(3)) receptors are tetrameric intracellular Ca(2+) channels, the opening of which is regulated by both IP(3) and Ca(2+). We suggest that all IP(3) receptors are biphasically regulated by cytosolic Ca(2+), which binds to two distinct sites. IP(3) promotes channel opening by controlling whether Ca(2+) binds to the stimulatory or inhibitory sites. The stimulatory site is probably an integral part of the receptor lying just upstream of the pore region. Inhibition of IP(3) receptors by Ca(2+) probably requires an accessory protein, which has not yet been unequivocally identified, but calmodulin is a prime candidate. We speculate that one lobe of calmodulin tethers it to the IP(3) receptor, while the other lobe can bind Ca(2+) and then interact with a second site on the receptor to cause inhibition.     

The number and spatial distribution of IP3 receptors underlying calcium puffs in Xenopus oocytes

Calcium puffs are local Ca(2+) release events that arise from a cluster of inositol 1,4,5-trisphosphate receptor channels (IP(3)Rs) and serve as a basic "building block" from which global Ca(2+) waves are generated. Important questions remain as to the number of IP(3)Rs that open during a puff, their spatial distribution within a cluster, and how much Ca(2+) current flows through each channel. The recent discovery of "trigger" events-small Ca(2+) signals that immediately precede puffs and are interpreted to arise through opening of single IP(3)R channels-now provides a useful yardstick by which to calibrate the Ca(2+) flux underlying puffs. Here, we describe a deterministic numerical model to simulate puffs and trigger events. Based on confocal linescan imaging in Xenopus oocytes, we simulated Ca(2+) release in two sequential stages; representing the trigger by the opening of a single IP(3)R in the center of a cluster for 12 ms, followed by the concerted opening of some number of IP(3)Rs for 19 ms, representing the rising phase of the puff. The diffusion of Ca(2+) and Ca(2+)-bound indicator dye were modeled in a three-dimensional cytosolic volume in the presence of immobile and mobile Ca(2+) buffers, and were used to predict the observed fluorescence signal after blurring by the microscope point-spread function. Optimal correspondence with experimental measurements of puff spatial width and puff/trigger amplitude ratio was obtained assuming that puffs arise from the synchronous opening of 25-35 IP(3)Rs, each carrying a Ca(2+) current of approximately 0.4 pA, with the channels distributed through a cluster 300-800 nm in diameter.