Synchronous Calcium Oscillations Research Articles

PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells.

Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases such as thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 G-protein coupled receptors-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and transient receptor potential canonical 3 (TRPC3) channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells, contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.

Emergent dynamics in an astrocyte-neuronal network coupled via nitric oxide

In the brain, both neurons and glial cells work in conjunction with each other during information processing. Stimulation of neurons can induce calcium oscillations in astrocytes which in turn can affect neuronal calcium dynamics. The ‘glissandi’ effect is one such phenomenon, associated with a decrease in infraslow fluctuations, in which synchronized calcium oscillations propagate as a wave in hundreds of astrocytes. Nitric oxide molecules released from the astrocytes contribute to synaptic functions based on the underlying astrocyte–neuron interaction network. In this study, by defining an astrocyte–neuronal (A–N) calcium unit as an integrated circuit of one neuron and one astrocyte, we developed a minimal model of neuronal stimulus-dependent and NO-mediated emergence of calcium waves in astrocytes. Incorporating inter-unit communication via NO molecules, a coupled network of 1000 such A–N calcium units is developed in which multiple stable regimes were found to emerge in astrocytes. We examined the ranges of neuronal stimulus strength and the coupling strength between A–N calcium units that give rise to such dynamical behaviors. We also report that there exists a range of coupling strength, wherein units not receiving stimulus also start showing oscillations and become synchronized. Our results support the hypothesis that glissandi-like phenomena exhibiting synchronized calcium oscillations in astrocytes help in efficient synaptic transmission by reducing the energy demand of the process.

Phenotypic screening using waveform analysis of synchronized calcium oscillations in primary cortical cultures.

At present, in vitro phenotypic screening methods are widely used for drug discovery. In the field of epilepsy research, measurements of neuronal activities have been utilized for predicting efficacy of anti-epileptic drugs. Fluorescence measurements of calcium oscillations in neurons are commonly used for measurement of neuronal activities, and some anti-epileptic drugs have been evaluated using this assay technique. However, changes in waveforms were not quantified in previous reports. Here, we have developed a high-throughput screening system containing a new analysis method for quantifying waveforms, and our method has successfully enabled simultaneous measurement of calcium oscillations in a 96-well plate. Features of waveforms were extracted automatically and allowed the characterization of some anti-epileptic drugs using principal component analysis. Moreover, we have shown that trajectories in accordance with the concentrations of compounds in principal component analysis plots were unique to the mechanism of anti-epileptic drugs. We believe that an approach that focuses on the features of calcium oscillations will lead to better understanding of the characteristics of existing anti-epileptic drugs and allow to predict the mechanism of action of novel drug candidates.

In vivo imaging of calcium dynamics in zebrafish hepatocytes.

Hepatocytes were the first cell type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a noninvasive manner because of the optical inaccessibility of the mammalian liver. Here, we aimed to take advantage of the transparency of the zebrafish larvae to image hepatocyte calcium dynamics in vivo at cellular resolution. We developed a transgenic model expressing a calcium sensor, GCaMP6s, specifically in zebrafish hepatocytes. Using this, we provide a quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress, and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo , which are lost upon starvation. Starvation induces lipid accumulation in the liver. Feeding recommences calcium waves in the liver, but in a spatially restricted manner, as well as resolves starvation-induced hepatic steatosis. By using a genetically encoded scavenger for calcium, we show that dampening of calcium signaling accelerates the accumulation of starvation-related lipid droplets in the liver. Furthermore, ethanol treatment, as well as cell ablation, induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, whereas the latter leads to a single calcium spike. We demonstrate the presence of oscillations, waves, and spikes in vivo . Calcium waves are present in response to nutrition and negatively regulate starvation-induced accumulation of lipid droplets.

Dopamine controls neuronal spontaneous calcium oscillations via astrocytic signal

Dopamine is a neuromodulator and neurotransmitter responsible for a number of physiological processes. Dysfunctions of the dopamine metabolism and signalling are associated with neurological and psychiatric diseases. Here we report that in primary co-culture of neurons and astrocytes dopamine-induces calcium signal in astrocytes and suppress spontaneous synchronous calcium oscillations (SSCO) in neurons. Effect of dopamine on SSCO in neurons was dependent on calcium signal in astrocytes and could be modified by inhibition of dopamine-induced calcium signal or by stimulation of astrocytic calcium rise with ATP. Ability of dopamine to suppress SSCO in neurons was independent on D1- or D2- like receptors but dependent on GABA and alpha-adrenoreceptors. Inhibitor of monoaminoxidase bifemelane blocked effect of dopamine on astrocytes but also inhibited the effect dopamine on SSCO in neurons. These findings suggest that dopamine-induced calcium signal may stimulate release of neuromodulators such as GABA and adrenaline and thus suppress spontaneous calcium oscillations in neurons.

Contribution of NAADP to Glutamate-Evoked Changes in Ca2+ Homeostasis in Mouse Hippocampal Neurons.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.

Ultradian Rhythms in the Hypothalamic Arcuate Nucleus Kisspeptin Neurons and Developmental Processes.

Numerous physiological processes in nature have multiple oscillations within 24 h, that is, ultradian rhythms. Compared to the circadian rhythm, which has a period of approximately one day, these short oscillations range from seconds to hours, and the mechanisms underlying ultradian rhythms remain largely unknown. This review aims to explore and emphasize the implications of ultradian rhythms and their underlying regulations. Reproduction and developmental processes show ultradian rhythms, and these physiological systems can be regulated by short biological rhythms. Specifically, we recently uncovered synchronized calcium oscillations in the organotypic culture of hypothalamic arcuate nucleus (ARN) kisspeptin neurons that regulate reproduction. Synchronized calcium oscillations were dependent on voltage-gated ion channel-mediated action potentials and were repressed by chemogenetic inhibition, suggesting that the network within the ARN and between the kisspeptin population mediates the oscillation. This minireview describes that ultradian rhythms are a general theme that underlies biological features, with special reference to calcium oscillations in the hypothalamic ARN from a developmental perspective. We expect that more attention to these oscillations might provide insight into physiological or developmental mechanisms, since many oscillatory features in nature still remain to be explored.

Influence of tetramethylenedisulfotetramine on synchronous calcium oscillations at distinct developmental stages of hippocampal neuronal cultures

The spatial and temporal patterns of spontaneous synchronous Ca2+ oscillations (SCOs) regulate physiological pathways that influence neuronal development, excitability, and health. Hippocampal neuronal cultures (HN) and neuron/glia co-cultures (HNG) produced from neonatal mice were loaded with Fluo-4/AM and SCOs recorded in real-time using a Fluorescence Imaging Plate Reader at different developmental stages in vitro. HNG showed an earlier onset of SCOs, with low amplitude and low frequency SCOs at 4days in vitro (DIV), whereas HN were quiescent at this point. SCO amplitude peaked at 9 DIV for both cultures. SCO network frequency peaked at 12 DIV in HN, whereas in HNG the frequency peaked at 6 DIV. SCO patterns were associated with the temporal development of neuronal networks and their ratio of glutamatergic to GABAergic markers of excitatory/inhibitory balance. HN and HNG exhibited differential responses to the convulsant tetramethylenedisulfotetramine (TETS) and were highly dependent on DIV. In HN, TETS triggered an acute rise of intracellular Ca2+ (Phase I response) only in 14 DIV and a sustained decrease of SCO frequency with increased amplitude (Phase II response) at all developmental stages. In HNG, TETS decreased the SCO frequency and increased the amplitude at 6 and 14 but not 9 DIV. There was no acute Ca2+ rise (Phase I response) in any age of HNG tested with TETS. These data demonstrated the importance of glia and developmental stage in modulating neuronal responses to TETS. Our results illustrate the applicability of the model for investigating how caged convulsants elicit abnormal network activity during the development of HN and HNG cultures in vitro.

NAD causes dissociation of neural networks into subpopulations of neurons by inhibiting the network synchronous hyperactivity evoked by ammonium ions

The mechanisms of hyperexcitability of neuronal networks by ammonium ions and inhibition of this activity by coenzyme NAD were investigated on mixed neuro-glial cultures of rat hippocampus. Ammonium ions cause activation of silent or spontaneously active neuronal networks inducing a bursting electrical activity of neurons and high-frequency synchronous calcium oscillations. In control conditions NAD completely inhibits spontaneous activity of the neuronal network. NAD added after NH4Cl disrupts synchronous oscillation in neurons and splits the network into five populations of neurons. In 4% of cells NAD decreased the amplitude of Ca2+ oscillations, preserving initial mode of oscillations. In 32% of cells, a transient suppression of the neuronal oscillations was observed: inhibition was followed by restoration of the synchronous periodic activity. In 10% of cells, NAD produced a gradual decrease of Ca2+ oscillations down to a complete termination of the initial periodic activity induced by ammonium. Fast and total inhibition of Ca2+ oscillations by NAD was observed in two small groups of neurons. First group (A) participated in the initial spontaneous network activity (5% of cells) with a period of 66–100 s. Second group (B), on the contrary, did not participate in the spontaneous activity. This group of neurons began to pulse with a high frequency (with a period of 6–8 s) synchronously with other neurons in the network after the addition of NH4Cl. Based on the comparison of calcium responses of different cell groups to the depolarization caused by KCl and NH4Cl and to the application of domoic acid, as well as on the results obtained in experiments with fluorescent antibodies against GAD 65/67, parvalbumin, calretinin, and calbindin, we propose that neurons of populations (A) and (B) may belong to GABAergic neurons containing calbindin and parvalbumin, respectively. Further analysis of specificity of the NAD effect on these neuronal groups may allow identification of the main targets of the ammonium toxic action in the brain. Thus, we have shown that NAD selectively inhibits neuronal activity and high-frequency synchronous Ca2+ oscillations in GABAergic neurons containing calcium-binding proteins. The inhibition is accompanied by desynchronization of oscillations and dissociation of neuronal network into several populations.

Closing the Gap between Glia and Neuroblast Proliferation

Reporting in this issue of Developmental Cell, Spéder and Brand (2014) show that gap junctions are required in blood-brain barrier glial cells to reactivate proliferation of quiescent neuroblasts. Gap junctions allow synchronous Ca(2+) waves and control insulin-like protein Dipl6 expression and secretion to trigger neuroblast division.

Effect of Gambierol and Its Tetracyclic and Heptacyclic Analogues in Cultured Cerebellar Neurons: A Structure–Activity Relationships Study

The polycyclic ether class of marine natural products has attracted the attention of researchers due to their complex and large chemical structures and diverse biological activities. Gambierol is a marine polycyclic ether toxin, first isolated along with ciguatoxin congeners from the dinoflagellate Gambierdiscus toxicus. The parent compound gambierol and the analogues evaluated in this work share the main crucial elements for biological activity, previously described to be the C28=C29 double bond within the H ring and the unsaturated side chain [Fuwa, H., Kainuma, N., Tachibana, K., Tsukano, C., Satake, M., and Sasaki, M. (2004) Diverted total synthesis and biological evaluation of gambierol analogues: Elucidation of crucial structural elements for potent toxicity. Chem. Eur. J. 10, 4894-4909]. With the aim to gain a deeper understanding of the cellular mechanisms involved in the biological activity of these compounds, we compared its activity in primary cultured neurons. The three compounds inhibited voltage-gated potassium channels (Kv) in a concentration-dependent manner and with similar potency, caused a small inhibition of voltage-gated sodium channels (Nav), and evoked cytosolic calcium oscillations. Moreover, the three compounds elicited a "loss of function" effect on Kv channels at concentrations of 0.1 nM. Additionally, both the tetracyclic and the heptacyclic derivatives of gambierol elicited synchronous calcium oscillations similar to those previously described for gambierol in cultured cerebellar neurons. Neither gambierol nor its tetracyclic derivative elicited cell toxicity, while the heptacyclic analogue caused a time-dependent decrease in cell viability. Neither the tetracyclic nor the heptacyclic analogues of gambierol exhibited lethality in mice after ip injection of 50 or 80 μg/kg of each compound. Altogether, the results presented in this work support an identical mechanism of action for gambierol and its tetracyclic and heptacyclic analogues and indicate a "loss of function" effect on potassium channels even after administration of the three compounds at subnanomolar concentrations. In addition, because gambierol is known to stabilize the closed state of Kv3 channels, the results presented in this paper may have implications for understanding of channel functions and for future development of therapies against ciguatera poisoning and potassium channel-related diseases.

Modulation of synchronous calcium oscillations in hippocampal neurons by photostimulation of astrocytes with femtosecond laser

A large body of evidence indicates that astrocytes play an important role in a range of brain functions through calcium (Ca2+) signaling. Experimentally evoking Ca2+ signaling is a useful technique for investigating the functions of astrocytes. However, conventional stimulation methods typically have poor spatio-temporal precision, and some are invasive. Our group has developed a technique to overcome these problems, in which astrocytes are photostimulated with a femtosecond laser. In the current study, we applied this method to a hippocampal neural network to explore astrocytic functions in detail. The results revealed that applying photostimulation to astrocytes in a cultured hippocampal astrocyte-neuron network caused the following changes: (i) Synchronous Ca2+ oscillations in neurons were induced; (ii) spontaneous Ca2+ synchrony instantaneously emerged; and (iii) high-frequency spontaneous Ca2+ synchrony was regulated. Thus, astrocytic Ca2+ signaling evoked by photostimulation was found to modulate synchronous Ca2+ oscillations in hippocampal neurons. We propose that photostimulation with a femtosecond laser will serve as a powerful tool in investigating astrocytic functions at the network level.

Inhibition of neuronal calcium oscillations by cell surface APP phosphorylated on T668

Adenoviral expression of human APP (hAPP), but not of hAPP deleted from its C-terminal intracellular domain, in rat cortical neurons abolishes spontaneous synchronous calcium oscillations. The intracellular domain of APP695 contains several residues that can be phosphorylated. Contrary to non-neuronal cells, a very high phosphorylation of APP on T668 is observed in neurons, which is mediated by JNK, GSK3 and Cdk5 protein kinases. JNK activity, modulated by GSK3, enhances the traffic of phosphorylated APP to nerve terminals, contrary to Cdk5. Here we show that inhibition of GSK3 and JNK restores calcium oscillations in an hAPP expressing neuronal network, whereas inhibition of Cdk5 does not. Expression of mutant hAPPT668A does not inhibit calcium oscillations, and the proportion of hAPPT668A at the plasma membrane is reduced by more than 50%. Altogether, these results indicate that the intracellular domain of APP is needed to inhibit neuronal calcium oscillations because GSK3/JNK phosphorylation of T668 controls APP trafficking at the plasma membrane.

Neuronal Function of the Amyloid Precursor Protein of Alzheimer's Disease

The amyloid precursor protein (APP) is a type I transmembrane protein with several characterized biochemical domains. However, since APP -/- mice show a normal phenotype, the function of APP remains elusive. Neuronal function of APP was investigated by expression of human APP (hAPP) or inhibition of endogenous APP production in primary cultures of cortical neurons. Primary cultures of neurons were prepared from rat or mouse embryos. Mouse embryonic fibroblasts (MEFs) were maintained in culture as cell lines. hAPP or shRNA expression was performed using recombinant adeno- or lentiviruses. Calcium oscillations and calcium currents were measured using FURA2 and patch clamp, respectively. APP target genes, and HMGCoA reductase mRNA expressions were quantified by qRT-PCR, while the corresponding proteins were quantified by Western blotting. Focal expression of hAPP in some neurons of a network interrupted spontaneous synchronous calcium oscillations in the entire network. hAPP expressing neurons showed increased activity of L-type voltage-dependent calcium channels. This in turn activated calcium-activated K+ channels, responsible for an increased medium afterhyperpolarisation (mAHP) phase following action potentials, preventing propagation of calcium oscillations (1). Neuronal expression of hAPP decreased HMGCoA reductase expression and cholesterol synthesis. This inhibition resulted from the impairment of SREBP processing in the Golgi compartment, where a direct interaction between SREPB and APP was demonstrated by co-immunoprecipitation. Expression of aquaporin 1 (AQP1) was strongly decreased in MEFs lacking APP. AQP1 expression was restored in these cells by expression of hAPP. The transcriptional activity of AQP1 gene promoter was identical in APP+/+ and APP-/- MEFs, as was the stability of AQP1 mRNA. Regulation of AQP1 expression by APP was sensitive to histone acetylation, and HDAC activity co-immunoprecipitated with APP. Efficient expression of hAPP in primary cultures of cortical neurons, as well as inhibition of endogenous APP expression allowed to identify important neuronal functions of APP, including control of neuronal excitability, cholesterol homeostasis, and epigenetic control of gene expression. (1) Ferrao Santos et al. (2009) J. Neurosci. 29, 4708-4718.

Gap Junction Coupling and Calcium Waves in the Pancreatic Islet

The pancreatic islet is a highly coupled, multicellular system that exhibits complex spatiotemporal electrical activity in response to elevated glucose levels. The emergent properties of islets, which differ from those arising in isolated islet cells, are believed to arise in part by gap junctional coupling, but the mechanisms through which this coupling occurs are poorly understood. To uncover these mechanisms, we have used both high-speed imaging and theoretical modeling of the electrical activity in pancreatic islets under a reduction in the gap junction mediated electrical coupling. Utilizing islets from a gap junction protein connexin 36 knockout mouse model together with chemical inhibitors, we can modulate the electrical coupling in the islet in a precise manner and quantify this modulation by electrophysiology measurements. We find that after a reduction in electrical coupling, calcium waves are slowed as well as disrupted, and the number of cells showing synchronous calcium oscillations is reduced. This behavior can be reproduced by computational modeling of a heterogeneous population of β-cells with heterogeneous levels of electrical coupling. The resulting quantitative agreement between the data and analytical models of islet connectivity, using only a single free parameter, reveals the mechanistic underpinnings of the multicellular behavior of the islet.

Hb9 Versus Type 2 Interneurons

to the editor: In a recent issue of the Journal of Neurophysiology , [Wilson, Cowan, and Brownstone (2007)][1] investigated the incidence of electrical coupling between different spinal interneurons. Both Wilson et al. and our group use the Hb9:GFP transgenic mouse line that was generated in the

Gonadotropin-releasing hormone-1 neuronal activity is independent of cyclic nucleotide-gated channels.

Pulsatile release of GnRH-1 is essential for secretion of gonadotropin hormones. The frequency of GnRH-1 pulses is regulated during the reproductive cycle by numerous neurotransmitters. Cyclic nucleotide-gated (CNG) channels have been proposed as a mechanism to integrate the cAMP signal evoked by many neurotransmitters. This study reports the expression of the CNGA2 subunit in GnRH-1 neurons obtained from mouse nasal explants and shows the ability of GnRH-1 neurons to increase their activity in response to forskolin (activator of adenylyl cyclases), or 3-isobutyl-1-methylxanthine (inhibitor of phosphodiesterases) even after removal of gamma-aminobutyric acid (A)-ergic input. Next, the endogenous activity of adenylyl cyclases was evaluated as a component of the oscillatory mechanism of GnRH-1 neurons. Inhibition of endogenous activity of adenylyl cyclases did not alter GnRH-1 activity. The potential involvement of CNGA2 subunit in basal or induced activity was tested on GnRH-1 neurons obtained from CNGA2-deficient mice. Without up-regulation of CNGA1 or CNGA3, the absence of functional CNGA2 did not alter either the endogenous GnRH-1 neuronal activity or the response to forskolin, negating CNG channels from cAMP-sensitive mechanisms leading to changes in GnRH-1 neuronal activity. In addition, the potential role of CNGA2 subunit in the synchronization of calcium oscillations previously described was evaluated in GnRH-1 neurons from CNGA2-deficient explants. Synchronized calcium oscillations persisted in CNGA2-deficient GnRH-1 neurons. Taken together, these results indicate that CNGA2 channels are not necessary for either the response of GnRH-1 neurons to cAMP increases or the basal rhythmic activity of GnRH-1 neurons.

Indirect activation of adenosine A1 receptors in cultured rat hippocampal neurons by volatile anaesthetics

Volatile anaesthetics depress excitatory signal transmission by potentiating the inhibitory action of GABAA receptors and there is strong evidence that this is related with anaesthesia. Using primary hippocampal cultures we analyzed the possibility that the volatile anaesthetics enflurane and sevoflurane depress excitatory signal transmission by activation of adenosine A1 receptors. Primary rat hippocampal cultures on 4 cm poly-L-lysine coated glass coverslips were loaded with the Ca2+-indicator fluo-3 and incorporated in a gastight, temperature-controlled perfusion chamber. The intracellular Ca2+-concentration was monitored with a confocal laser-scanning microscope (BioRad) using the 488 nm laser line of a krypton-argon laser for excitation and the Lasersharp Acquisition software for analysis. Continuous perfusion in Mg2+-free medium generated spontaneous synchronized calcium oscillations, which were dose dependently depressed by the volatile anaesthetics enflurane and sevoflurane (0.25-1 minimum alveolar concentration). Addition of 100 nmol of 2-chloro-N6-cyclopentyladenosine, a specific adenosine A1 receptor antagonist, partly reversed the anaesthetic-induced inhibition of the oscillation amplitude of the oscillating cells. The effect of the anaesthetics was mimicked by the addition of S-(p-nitrobenzyl)-6-thioguanosine, an adenosine transport inhibitor and by the addition of 5-amino-5-deoxyadenosine, an inhibitor of adenosine kinase. The volatile anaesthetics sevoflurane and enflurane activate adenosine A1 receptors in primary rat hippocampal cultures. This effect is mediated by liberation of adenosine most likely by an interaction of the volatile anaesthetics with adenosine transport or key enzymes in adenosine metabolism.

Emergent properties of electrically coupled smooth muscle cells

Asynchronous and synchronous calcium oscillations occur in a variety of cells. A well-established pathway for intercellular communication is provided by gap junctions which connect adjacent cells and can mediate electrical and chemical coupling. Several experimental studies report that cells presenting only a transient increase when freshly dispersed may oscillate when they are coupled. Such observations suggest that the role of gap junctions is not only to coordinate calcium oscillations of adjacent cells. Gap junctions may also be important to generate oscillations. Here we illustrate the emergent properties of electrically coupled smooth muscle cells using a model that we recently proposed. A bifurcation analysis in the case of two cells reveals that synchronous and asynchronous calcium oscillations can be induced by electrical coupling. In a larger population of smooth muscle cells, electrical coupling may result in the creation of groups of cells presenting synchronous calcium oscillations. The elements of one group may be distant from each other. Moreover, our results highlight a general mechanism by which gap junctional electrical coupling can give rise to out of phase calcium oscillations in smooth muscle cells that are non-oscillating when uncoupled. All these observations remain true in the case of non-identical cells, except that the solution corresponding to synchronous calcium oscillations disappears and that the formation of groups is sensitive to the degree of heterogeneity.

Role of the Endothelium on Arterial Vasomotion

It is well-known that cyclic variations of the vascular diameter, a phenomenon called vasomotion, are induced by synchronous calcium oscillations of smooth muscle cells (SMCs). However, the role of the endothelium on vasomotion is unclear. Some experimental studies claim that the endothelium is necessary for synchronization and vasomotion, whereas others report rhythmic contractions in the absence of an intact endothelium. Moreover, endothelium-derived factors have been shown to abolish vasomotion by desynchronizing the calcium signals in SMCs. By modeling the calcium dynamics of a population of SMCs coupled to a population of endothelial cells, we analyze the effects of an SMC vasoconstrictor stimulation on endothelial cells and the feedback of endothelium-derived factors. Our results show that the endothelium essentially decreases the SMCs calcium level and may move the SMCs from a steady state to an oscillatory domain, and vice versa. In the oscillatory domain, a population of coupled SMCs exhibits synchronous calcium oscillations. Outside the oscillatory domain, the coupled SMCs present only irregular calcium flashings arising from noise modeling stochastic opening of channels. Our findings provide explanations for the published contradictory experimental observations.