Cross-Talk between Signaling Pathways Can Generate Robust Oscillations in Calcium and cAMP

Background

To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli.

Methodology/Principal Findings

Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP) and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate ‘bursting’ oscillations of calcium and may enable better filtering of noise.

Conclusion

We postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.     

A model of the interaction between autoregulation and neural activation in the brain

In this paper a model is proposed that predicts the response of the cerebral vasculature to changes in arterial blood pressure, arterial CO2 concentration and neural stimulation. Cerebral blood flow (CBF) is assumed to be controlled through changes in arterial compliance, and hence arterial resistance and volume, through three feedback mechanisms, which act in a linear additive manner, based on CBF, arterial CO2 and neural stimulus. Together with arterial, capillary and venous compartments, a tissue compartment is included, which contributes partly to the initial rise found in the deoxyhaemoglobin response to neural activation. Dynamic simulations of the model under different conditions show that there is significant interaction between the autoregulation and activation processes, and that the level of autoregulation has a strong influence on the CBF and deoxyhaemoglobin responses to neural activation. Overshoot in the deoxyhaemoglobin response is eliminated completely in the absence of this regulation. The feedback mechanism time constants significantly affect the CBF and deoxyhaemoglobin responses. Changes in arterial blood pressure (ABP) are found to have a strong influence on the neural activation response, with the amplitude of the response decreasing significantly at high baseline ABP. Dynamic changes in ABP also have a significant and potentially confounding impact on the measured deoxyhaemoglobin response to neural activation.     

Mechanisms of Propagation of Intercellular Calcium Waves in Arterial Smooth Muscle Cells

In rat mesenteric arteries, smooth muscle cells exhibit intercellular calcium waves in response to local phenylephrine stimulation. These waves have a velocity of ∼20 cells/s and a range of ∼80 cells. We analyze these waves in a theoretical model of a population of coupled smooth muscle cells, based on the hypothesis that the wave results from cell membrane depolarization propagation. We study the underlying mechanisms and highlight the importance of voltage-operated channels, calcium-induced calcium release, and chloride channels. Our model is in agreement with experimental observations, and we demonstrate that calcium waves presenting a velocity of ∼20 cells/s can be mediated by electrical coupling. The wave velocity is limited by the time needed for calcium influx through voltage-operated calcium channels and the subsequent calcium-induced calcium release, and not by the speed of the depolarization spreading. The waves are partially regenerated, but have a spatial limit in propagation. Moreover, the model predicts that a refractory period of calcium signaling may significantly affect the wave appearance.     

Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.     

Antigen receptor-induced cell cycle arrest in WEHI-231 B lymphoma cells depends on the duration of signaling before the G1 phase restriction point.

Stimulation of antigen receptors on WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-immunoglobulin M [IgM]) causes irreversible growth arrest. This may be a model for antigen-induced tolerance to self components in the immune system. Antigen receptor stimulation also causes inositol phospholipid hydrolysis, producing diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which causes release of calcium from intracellular stores. To better understand the nature of the antigen receptor-induced growth arrest of WEHI-231 cells, we have examined the basis for it. WEHI-231 cells in various phases of the cell cycle were isolated by centrifugal elutriation, and their response was evaluated following treatment with either anti-IgM or pharmacologic agents that raise intracellular free calcium levels and activate protein kinase C. Treatment with anti-IgM or the pharmacologic agents did not lengthen the cell cycle. Instead, growth inhibition was solely the result of arrest in the G1 phase. The efficiency of G1 arrest increased with the length of time during which the cells received signaling before reaching the G1 phase arrest point. Maximum efficiency of arrest was achieved after approximately one cell cycle of receptor signaling. These results imply that anti-IgM causes G1 arrest of WEHI-231 cells by slowly affecting components required for S phase progression, rather than by rapidly inhibiting such components or by rapidly activating a suicide mechanism. Antigen receptor stimulation was twice as effective as stimulation via the mimicking reagents phorbol dibutyrate and ionomycin. Thus, although the phosphoinositide second messengers diacylglycerol and calcium probably play roles in mediating the effects of anti-IgM on WEHI-231 cells, other second messengers may also be involved.     

Regulation of ciliary activity in the mammalian respiratory tract

A computer-assisted transillumination, photoelectronic technique has been used to measure the beat frequency of cilia of rabbit tracheal cells grown in culture. When ciliated cells are mechanically stimulated with a microprobe the cells respond rapidly by increasing the beat frequency of their cilia. This mechanosensitive response is not limited to the stimulated cell, but is communicated in all directions to neighboring cells. To characterize the progression of this communicated response we used an automated computer-assisted imaging system to examine high-speed films of responding cells. The time it takes for the response to be transmitted between cells is slow (1-3 sec) with each cell responding after a lag-time that is proportional to the distance of the cell from the stimulated cell. We have confirmed that gap junctions are present between cells and that adjacent or non-adjacent ciliated, as well as non-ciliated, cells are electrically coupled. To correlate the mechanosensitive response with intracellular calcium fluxes we have used fura-2, a calcium-specific fluorescent dye, and digital video microscopy. Mechanical stimulation of the cultured ciliated cells, in the presence of extracellular calcium, resulted in an initial increase in intracellular calcium, which was communicated to neighboring cells. Without extracellular calcium, mechanosensitivity of cultured cells was lost and a small decrease in intracellular calcium was observed in the stimulated cell. However, neighboring cells still displayed an increase in intracellular calcium. The time course and general pattern of calcium increase in adjacent cells was similar to the responses in ciliary activity produced by mechanical stimulation. Ciliary beat frequency is also elevated by beta-adrenergic drugs independently of mechanosensitivity. These responses are important because they could provide a dual regulatory mechanism for the control of mucus transport. Adrenergic agonists could provide non-specific control by increasing ciliary activity throughout the airways while mechanosensitivity could provide local control by increasing activity in those regions of heavy mucus load.     

Intracellular cyclic AMP produces effects opposite to those of cyclic GMP and calcium on shape and motility of neuroblastoma cells.

We have directly evaluated the effects of various intracellular second messengers including cyclic nucleotides, calcium ion, and inositol polyphosphates on shape and motility of differentiating mouse neuroblastoma cells. The messengers were microinjected into cells and the responses of the soma, neurite, and growth cone were monitored using time-lapse video microscopy. Each messenger altered cell shape and motility in a characteristic manner. Cyclic AMP promoted lamellipodial expansion, neurite outgrowth, and motility. The other injected messengers opposed motility. Cyclic GMP caused motile structures to freeze and to retract permanently, while the inhibitory effects of calcium injection were concentration-dependent. Small calcium injections affected specifically actin-containing motile structures which froze and retracted temporarily. Intermediate calcium injections caused a strong contraction at the site of injection in all cells. With large injections, cells retracted long neurites, rounded up, and frequently began vigorous blebbing that continued to cell death. Injections of the inositol polyphosphates IP3(1,4,5) and IP4(1,4,5,6) mimicked the effects of small calcium injections, as did electrical stimulation that elicited action potentials. The results suggest that in mouse neuroblastoma cells, intracellular cAMP elevation increases cytoskeletal organization and promotes neurite extension perhaps through an enhancement of cell-substratum adhesion. On the other hand, a rise of intracellular cGMP or intracellular calcium interferes directly with the function and organization of the actin-microfilament system. The integrated action of these second messenger systems may, therefore, operate in vivo to allow substances released from neighboring cells to regulate neuronal architecture.     

xMLP is an early response calcium target gene in neural determination in Xenopus laevis

In vertebrates, neural induction occurs during gastrulation when ectodermal cells choose between two fates, neural and epidermal. In Xenopus, neural induction has been regarded as a default pathway as it occurs, in dorsal ectoderm, when ventralizing signals (mainly Bone Morphogenesis Proteins, BMPs, potent epidermal inducers) are inhibited by dorsalizing signals, including factors such as noggin, chordin, and follistatin. However, our previous studies demonstrated that an instructive signal triggered by the activation of L-type voltage-sensitive calcium channels, resulting in a transient increase in intracellular free calcium, appears to be a necessary and sufficient requirement to induce the competent ectoderm toward the neural pathway. Here we further explore the relationship between the Ca2+ transient signals observed and the expression of early neural genes. We have performed a subtractive approach to identify the genes which are transcribed early after the calcium signal and involved in neural determination. We have analyzed a candidate gene (xMLP) which encodes a MARCKS-like protein, a substrate for PKC. We show that this gene is activated by a calcium transient signals and induced by noggin overexpression. xMLP is expressed at the right time in presumptive neural territories. The putative role of xMLP in the process of neural induction is discussed.     

Production of inositol-phospholipid-derived second messengers in a rat natural killer cell line exposed to susceptible tumor targets

It is currently believed that natural killer (NK) cells kill bound target cells by exocytosis of cytotoxic granules via a calcium-dependent process. After confirming that NK-mediated killing was indeed dependent upon extracellular calcium, we investigated the production of inositol-phospholipid-derived second messengers in a rat NK cell line, RNK, upon exposure to susceptible target cells. These messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG), are associated with calcium-dependent secretory processes in a number of cell types. When RNK cells were exposed to susceptible YAC-1 tumor targets significant amounts of both IP3 and DAG were produced. The levels of the membrane phospholipid parent molecules of these second messengers declined in similarly stimulated RNK cells over a comparable time period. Using three different target cell lines, it was found that the levels of DAG that RNK produced in response to the different targets followed the same rank order as their susceptibility to RNK-mediated lysis. These data suggest that IP3 and DAG are produced in NK cells in response to tumor target cells, and these second messengers may have a functional role in NK-mediated killing.     

Cytosolic calcium microdomains by arachidonic acid and nitric oxide in endothelial cells

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released after proangiogenic stimulation (bFGF, VEGF), arachidonic acid (AA), nitric oxide (NO) and their metabolites play a key role and their effects are strictly related to calcium homeostasis. Recently, we showed that AA and NO are able to stimulate the opening of store-independent calcium-permeable channels in the plasmamembrane of bovine aortic endothelial cells (BAECs). Here, we studied the intracellular spatiotemporal dynamics of AA- and NO-induced calcium increases following store-independent calcium entry from extracellular medium. Using confocal calcium imaging, we show that calcium entry is preferentially restricted to peripheral cytosolic microdomains and does not necessarily invade the nuclear region. These results support the existence of local mitogen-activated calcium signals. Several factors could account for this spatial restriction, including the geometry of the cells and the clusterization of calcium channels and other signalling molecules. Intracellular calcium fingerprints could contribute to the specificity of endothelial cell responses to angiogenic factors.     

Spatial Analysis of 3′ Phosphoinositide Signaling in Living Fibroblasts: I. Uniform Stimulation Model and Bounds on Dimensionless Groups

Fluorescent protein probes now permit spatial distributions of specific intracellular signaling molecules to be observed in real time. Mathematical models have been used to simulate molecular gradients and other spatial patterns within cells, and the output of such models may be compared directly with experiments if the binding of the fluorescent probe and the physics of the imaging technique are each incorporated. Here we present a comprehensive model describing the dynamics of 3′ phosphoinositides (PIs), lipid second messengers produced in the plasma membrane in response to stimulation of the PI 3-kinase signaling pathway, as monitored in the cell-substratum contact area using total internal reflection fluorescence microscopy. With this technique it was previously shown that uniform stimulation of fibroblasts with platelet-derived growth factor elicits the formation of axisymmetric 3′ PI gradients, which we now characterize in the context of our model. We find that upper and lower bounds on the relevant dimensionless model parameter values for an individual cell can be calculated from four well-defined fluorescence measurements. Based on our analysis, we expect that the key dimensionless group, the ratio of 3′ PI turnover and diffusion rates, can be estimated within ~20% or less.     

Satellite Glial Cells and Astrocytes,a Comparative Review

Astroglia are neural cells, heterogeneous in form and function, which act as supportive elements of the central nervous system; astrocytes contribute to all aspects of neural functions in health and disease. Through their highly ramified processes, astrocytes form close physical contacts with synapses and blood vessels, and are integrated into functional syncytia by gap junctions. Astrocytes interact among themselves and with other cells types (e.g., neurons, microglia, blood vessel cells) by an elaborate repertoire of chemical messengers and receptors; astrocytes also influence neural plasticity and synaptic transmission through maintaining homeostasis of neurotransmitters, K⁺ buffering, synaptic isolation and control over synaptogenesis and synaptic elimination. Satellite glial cells (SGCs) are the most abundant glial cells in sensory ganglia, and are believed to play major roles in sensory functions, but so far research into SGCs attracted relatively little attention. In this review we compare SGCs to astrocytes with the purpose of using the vast knowledge on astrocytes to explore new aspects of SGCs. We survey the main properties of these two cells types and highlight similarities and differences between them. We conclude that despite the much greater diversity in morphology and signaling mechanisms of astrocytes, there are some parallels between them and SGCs. Both types serve as boundary cells, separating different compartments in the nervous system, but much more needs to be learned on this aspect of SGCs. Astrocytes and SGCs employ chemical messengers and calcium waves for intercellular signaling, but their significance is still poorly understood for both cell types. Both types undergo major changes under pathological conditions, which have a protective function, but an also contribute to disease, and chronic pain in particular. The knowledge obtained on astrocytes is likely to benefit future research on SGCs.

     

Rundown of secretion after depletion of intracellular calcium stores in bovine adrenal chromaffin cells

In this study, the relationship between intracellular calcium stores and depolarization-evoked stimulation was examined in bovine chromaffin cells, using changes in membrane capacitance to monitor both exocytosis and endocytosis. Cells were voltage-clamped using the perforated whole-cell patch configuration to minimize alterations in intracellular constituents. Control cells exhibited reproducible secretory responses each time the cell was stimulated. However, the same stimulation protocol elicited progressively smaller secretory responses in cells where their intracellular calcium store was emptied by thapsigargin. Transient elevation of the intracellular calcium concentration with a brief histamine treatment enhanced subsequent secretory responses in control but not in thapsigargin-treated cells. A series of depolarizations to -20 mV, which allowed small amounts of Ca(2+) influx but which by itself did not trigger catecholamine secretion, enhanced subsequent exocytosis in both control and thapsigargin-treated cells. Caffeine-pretreated cells exhibited a rundown in the secretory response that was similar to that produced by thapsigargin. These results suggest that brief elevations of [Ca(2+)](i) could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation.     

A novel benzodiazepine increases the sensitivity of B cells to receptor stimulation with synergistic effects on calcium signaling and apoptosis

Bz-423 is a 1,4-benzodiazepine with selective lymphotoxic properties and potent therapeutic activity against lupus-like disease in autoimmune mice. In NZB/W lupus-prone mice, Bz-423 specifically kills germinal center B cells, which are the cells that drive disease both in this model and in human systemic lupus erythematosus. In this report, the mechanistic basis for the selective action of Bz-423 is investigated. We show that Bz-423-induces superoxide as an immediate early response and that this reactive oxygen species is more effective as a second messenger death signal in B cells activated by B cell receptor stimulation compared with resting cells. As a result, low [Bz-423] that are not cytotoxic to non-stimulated cells kill stimulated cells in synergy with anti-immunoglobulin M antibodies. Subsequent experiments demonstrated that Bz-423 extends the rise in intracellular calcium that accompanies anti-immunoglobulin M stimulation, and this effect mediates the synergistic death response. Because B cell hyperactivation and altered calcium signaling is a distinguishing feature of autoreactive lymphocytes in lupus, the mechanism by which Bz-423 induces apoptosis preferentially targets disease-causing cells on the basis of their activation state. Thus, molecules like Bz-423 could form the basis for new and selective anti-lupus agents.     

Calcium Independence of Phosphoinositide Hydrolysis-Induced Increase in Cyclic AMP Accumulation in SK-N-SH Human Neuroblastoma Cells

Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.     

Front Cover: J. Biophotonics 10/2014

If gold nanorods are used as photoabsorbers, then light in the near infrared tissue window can generate a strong photothermal effect. In this issue, C. Paviolo et al. show that near infrared light can be used to evoke a calcium response in neuronal cells treated with gold nanorods. This suggests new opportunities for peripheral nerve regeneration and infrared neural stimulation. (Picture: C. Paviolo et al., pp. 761–765 in this issue)     

Contributions of memory B cells to secondary immune response

The secondary immune response is one of the most important features of immune systems. During the secondary immune response, the immune system can eliminate the antigen, which has been encountered by the individual during the primary invasion, more rapidly and efficiently. Both T and B memory cells contribute to the secondary response. In this paper, we only concentrate on the functions of memory B cells. We explore a model describing the memory contributed by the specific long-lived clone which is maintained by continued stimulation with a small amount of antigens sequestered on the surfaces of the follicular dendritic cells (FDC). The behavior of the secondary response provided by the model can be compared with experimental observations. The model shows that memory B cells indeed play an important role in the secondary response. It is found that a single memory cell in a long-lived clone may not be long-lived. In the present note, the influences of relevant parameters on the secondary response are also explored.     

Evidence That Receptor-Linked G Protein Inhibits Exocytosis by a Post-Second-Messenger Mechanism in AtT-20 Cells

In AtT-20 cells somatostatin inhibits the secretion of adrenocorticotropic hormone (ACTH) through the activation of GTP binding proteins (G proteins) linked to second messengers such as calcium and cyclic AMP (cAMP). Recently, it has been proposed that there may be G proteins that regulate directly the exocytotic machinery. We have investigated whether somatostatin could inhibit secretion at a step distal to second messengers through a GTP binding protein. For these studies two experimental paradigms were used: (1) intact cells stimulated by calcium ionophores and (2) digitonin-permeabilized cells exposed to buffers of increasing Ca2+ concentrations. Somatostatin inhibited by 70% the ACTH release caused by the calcium ionophore ionomycin without modifying the ionophore-induced elevation in cytosolic [Ca2+]. This effect was cAMP independent because (1) it was observed in the presence of high concentrations of membrane-permeant cAMP analogues, and (2) it was not accompanied by a change in cAMP levels. The effect was also independent of the levels of activators of protein kinase C because it could be produced in the presence of high concentrations of phorbol esters. The action of somatostatin was prevented by pertussis toxin. In digitonin-permeabilized AtT-20 cells somatostatin inhibited release induced by calcium buffers in a GTP-dependent manner. These two observations indicate the involvement of a G protein. It is proposed that a G protein coupled to somatostatin receptors inhibits the intracellular machinery of secretion at a step distal to second messengers, perhaps at the exocytotic site.