Intracellular Ca Oscillations Research Articles

Participation of calcium-permeable AMPA receptors in the regulation of epileptiform activity of hippocampal neurons.

Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites. Later it was shown that these rhythms have a cellular correlate in vitro called paroxysmal depolarization shift (PDS). In 13-15 DIV neuron-glial cell culture, inhibition of the GABA(A) receptors induces bursts of action potential in the form of clasters PDS and oscillations of intracellular Ca2+ concentration ([Ca2+]i). We demonstrate that GABAergic neurons expressing calcium-permeable AMPA receptors (CP-AMPARs) as well as Kv7-type potassium channels regulate hippocampal glutamatergic neurons' excitability during epileptiform activity in culture. A combination of whole-cell patch-clamp in current clamp mode and calcium imaging microscopy was used to simultaneously register membrane potential and [Ca2+]i level. To identify GABAergic cell cultures were fixed and stained with antibodies against glutamate decarboxylase GAD 65/67 and neuron-specific enolase (NSE) after vital [Ca2+]i imaging. It was shown that CP-AMPARs are involved in the regulation of the PDS clusters and [Ca2+]i pulses accompanied them. Activation of CP-AMPARs of GABAergic neurons is thought to cause the release of GABA, which activates the GABA(B) receptors of other GABAergic interneurons. It is assumed that activation of these GABA(B) receptors leads to the release of beta-gamma subunits of Gi protein, which activate potassium channels, resulting in hyperpolarization and inhibition of these interneurons. The latter causes disinhibition of glutamatergic neurons, the targets of these interneurons. In turn, the CP-AMPAR antagonist, NASPM, has the opposite effect. Measurement of membrane potential in GABAergic neurons by the patch-clamp method in whole-cell configuration demonstrated that NASPM suppresses hyperpolarization in clusters and individual PDSs. It is believed that Kv7-type potassium channels are involved in the control of hyperpolarization during epileptiform activity. The blocker of Kv7 channels, XE 991, mimicked the effect of the CP-AMPARs antagonist on PDS clusters. Both drugs increased the duration of the PDS cluster. In turn, the Kv7 activator, retigabine, decreased the duration of the PDS cluster and Ca2+ pulse. In addition, retigabine led to deep posthyperpolarization at the end of the PDS cluster. The Kv7 channel is believed to be involved in the formation of PDS, as the channel blocker reduced the rate of hyperpolarization in the PDS almost three times. Thus, GABAergic neurons expressing CP-AMPARs, regulate the membrane potential of innervated glutamatergic neurons by modulating the activity of postsynaptic potassium channels of other GABAergic neurons.

The role of highly functional β-cell subpopulations in the multicellular islet

The pancreatic islet is a multicellular network comprised of a majority of excitable insulin-producing β-cells. Electrically active β-cells show oscillations in intracellular Ca2+ that lead to insulin release. β-cell electrical activity is coordinated by gap junction channels. β-cell Ca2+ activity is globally elevated at high glucose, whereas it is globally silenced at low glucose due to gap junction communication. Using multicellular computational models of mouse and human islet electrophysiology, we investigated the role highly functional β-cell subpopulations may play in driving islet Ca2+ dynamics.

Behavioural switching during oscillations of intracellular Ca2+ concentration in free-swimming human sperm.

Lay summaryA human sperm must swim to the egg to fertilise it. To do this the sperm uses different types of swimming (behaviours) as they are needed. When we watch sperm swimming we see that they regularly change behaviour, sometimes repeatedly switching between two different types. Calcium ions inside cells are crucial in controlling many cell functions and in sperm they play a key role in regulating their behaviour. Here we have measured the concentration of calcium ions inside swimming human sperm. We found that in 12/35 (34%) of the cells we assessed, the concentration of calcium changed repeatedly, averaging more than one cycle of rise and fall per minute. These changes in the concentration of calcium ions occurred as the sperm switched swimming stroke, suggesting that oscillation of calcium concentration is involved in controlling the switching of sperm behaviour. Impaired sperm motility is an important cause of subfertility in men. Understanding how sperm behaviour is controlled will allow the development of treatments that can rescue the fertility of sperm with impaired motility.

Impact of the Potential Antitumor Agent 2-(4-Hydroxyphenyl) Amino-1,4-Naphthoquinone (Q7) on Vasomotion Is Mediated by the Vascular Endothelium, But Not Vascular Smooth Muscle Cell Metabolism.

Vasomotion is defined as rhythmic oscillations in arterial diameter that regulate the blood flow and blood pressure. Because antitumor treatment may impair vascular functions and increase the blood pressure, we sought to evaluate whether a new naphthoquinone derivative, postulated as an antitumor agent, manifests adverse effects on vascular function. In this article, we evaluated the toxicity of 2-(4-hydroxyphenyl) amino-1,4-naphthoquinone (Q7) and its effects on vascular vasomotion in 3 models of vascular structure: endothelial cells, aortic ring, and smooth muscle cells. Although showing nontoxic effects, Q7 inhibited the formation of capillary-like structures of the EA.hy926 endothelial cell line grown on Matrigel. In exvivo experiments with aortic rings precontracted with phenylephrine (PE, 10-6 M), Q7 (10-5 M) significantly (P < 0.05) reduced vascular rhythmic contractions induced by the acetylcholine (ACh; 10-7-10-5 M), whereas sodium nitroprusside (a nitric oxide donor; 10-8 M) recovered the vasomotion. Furthermore, Q7 (10-5 M) did not decrease KCl-induced vascular rhythmic contractions in the aortic rings precontracted with BaCl2 (a nonselective K+ channel blocker; 10-3 M). Vascular smooth muscle cells (A7r5) preincubated with Q7 (10-5 M) for 3 hours also demonstrated a reduced glucose uptake. However, the Adenosine Triphosphate content was unaffected, suggesting that the rapid reduction in vasomotion observed in vascular reactivity experiments did not involve cellular metabolism but may be due to faster mechanisms involving endothelial nitric oxide and K+ channels leading to oscillations in intracellular Ca2+. In summary, the naphthoquinone derivative Q7 presents low cytotoxicity yet may alter the endothelial cell response and vasomotion in the absence of changes in smooth muscle cell metabolism.

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.

Manipulating Intracellular Ca2+ Signals to Stimulate Therapeutic Angiogenesis in Cardiovascular Disorders.

Endothelial progenitor cells (EPCs) are mobilized in peripheral blood to rescue blood perfusion in ischemic tissues. Several approaches were, therefore, designed to inject autologous EPCs and induce therapeutic angiogenesis in patients affected by cardiovascular disorders. Endothelial colony forming cells (ECFCs) represent the only truly endothelial precursors and are regarded as the most suitable substrate for cell based therapy of ischemic diseases. Intracellular Ca2+ signalling drives ECFC proliferation, migration, homing and neovessel formation. Vascular endothelial growth factor (VEGF) triggers repetitive oscillations in intracellular Ca2+ concentration ([Ca2+]i) in peripheral blood- and umbilical cord blood-derived ECFCs by initiating a dynamic interplay between inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release and store-operated Ca2+ entry (SOCE). SOCE, in turn, is mediated by Stim1, Orai1 and Transient Receptor Potential (TRP) Canonical 1 (TRPC1). Intriguingly, intracellular Ca2+ oscillations are triggered by TRPC3 in umbilical cord blood-derived ECFCs, which display higher proliferative potential. Additionally, stromal cell-derived factor-1α (SDF-1α) triggers a biphasic increase in [Ca2+]i in ECFCs which is mediated by InsP3 receptors (InsP3Rs) and SOCE. Finally, arachidonic acid (AA) and nicotinic acid adenine dinucleotide phosphate (NAADP) stimulate ECFC proliferation by stimulating two-pore channel 1 (TPC1), thereby promoting Ca2+ release from the endolysosomal Ca2+ compartment. AA-evoked Ca2+ signals are further supported by InsP3Rs and TRP Vanilloid 4 (TRPV4). In this article, we describe how genetic manipulation of the Ca2+ toolkit (i.e. TRPC3, SOCE, TPC1) endowed to circulating ECFCs could rejuvenate or restore their reparative phenotype for therapeutic angiogenesis purposes.

The oscillation of intracellular Ca2+ influx associated with the circadian expression of Piezo1 and TRPV4 in the bladder urothelium

We previously showed that bladder functions are controlled by clock genes with circadian rhythm. The sensation of bladder fullness (SBF) is sensed by mechano-sensor such as Piezo1 and TRPV4 in the mouse bladder urothelium. However, functional circadian rhythms of such mechano-sensors remain unknown. To investigate functional circadian changes of these mechano-sensors, we measured circadian changes in stretch-evoked intracellular Ca2+ influx ([Ca2+]i) using mouse primary cultured urothelial cells (MPCUCs). Using Ca2+ imaging, stretch-evoked [Ca2+]i was quantified every 4 h in MPCUCs derived from wild-type (WT) and ClockΔ19/Δ19 mice, which showed a nocturia phenotype. Furthermore, a Piezo1 inhibitor GsMTx4 and a TRPV4 inhibitor Ruthenium Red were applied and stretch-evoked [Ca2+]i in MPCUCs was measured to investigate their contribution to SBF. Stretch-evoked [Ca2+]i showed a circadian rhythm in the WT mice. In contrast, ClockΔ19/Δ19 mice showed disrupted circadian rhythm. The administration of both GsMTx4 and Ruthenium Red eliminated the circadian rhythm of stretch-evoked [Ca2+]i in WT mice. We conclude that SBF may have a circadian rhythm, which is created by functional circadian changes of Piezo1 and TRPV4 being controlled by clock genes to be active during wakefulness and inactive during sleep. Abnormalities of clock genes disrupt SBF, and induce nocturia.

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca2+-calcineurin-NFATc1 signaling pathway

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca2+-calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levels of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca2+ in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca2+ channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca2+-calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca2+-calcineurin-NFATc1 signaling pathway.

Estradiol-modified prolactin secretion independently of action potentials and Ca2+ and blockade of outward potassium currents in GH3 cells

Estrogens facilitate prolactin (PRL) secretion acting on pituitary cells. In GH3 cells, estradiol induces acute action potentials and oscillations of intracellular Ca2+ associated with the secretagogue function. Estradiol modulates several ion channels which may affect the action potential rate and the release of PRL in lactotroph cells, which might depend on its concentration. The aims were to characterize the acute effect of supraphysiological concentrations of estradiol on Ca2+ and noninactivating K+ currents and measure the effect on the spontaneous action potentials and PRL release in the somatolactotroph cell line, GH3. Electrophysiological studies were carried out by voltage- and current-clamp techniques and ELISA determination of PRL secretion. Pharmacological concentrations of estradiol (above 1μM), without a latency period, blocked Ca2+ channels and noninactivating K+ currents, including the large-conductance voltage- and Ca2+-activated K+ channels (BK), studied in whole-cell nystatin perforated and in excided inside-out patches of GH3 and CHO cells, transiently transfected with the human α-pore forming subunit of BK. The effect on BK was contrary to the agonist effect associated with the regulatory β1-subunits of the BK, which GH3 cells lack, but its transient transfection did not modify the noninactivating current blockade, suggesting a different mechanism of regulation. Estradiol, at the same concentration range, acutely decreased the frequency of action potentials, an expected effect as consequence of the Ca2+ channel blockade. Despite this, PRL secretion initially increased, followed by a decrease in long-term incubations. This suggests that, in GH3 cells, supraphysiological concentrations of estradiol modulating PRL secretion are partially independent of extracellular Ca2+ influx.

Phase Analysis of Metabolic Oscillations and Membrane Potential in Pancreatic Islet β-Cells

Metabolism in islet β-cells displays oscillations that can trigger pulses of electrical activity and insulin secretion. There has been a decades-long debate among islet biologists about whether metabolic oscillations are intrinsic or occur in response to oscillations in intracellular Ca2+ that result from bursting electrical activity. In this article, the dynamics of oscillatory metabolism were investigated using five different optical reporters. Reporter activity was measured simultaneously with membrane potential bursting to determine the phase relationships between the metabolic oscillations and electrical activity. Our experimental findings suggest that Ca2+ entry into β-cells stimulates the rate of mitochondrial metabolism, accounting for the depletion of glycolytic intermediates during each oscillatory burst. We also performed Ca2+ clamp tests in which we clamped membrane potential with the KATP channel-opener diazoxide and KCl to fix Ca2+ at an elevated level. These tests confirm that metabolic oscillations do not require Ca2+ oscillations, but show that Ca2+ plays a larger role in shaping metabolic oscillations than previously suspected. A dynamical picture of the mechanisms of oscillations emerged that requires the restructuring of contemporary mathematical β-cell models, including our own dual oscillator model. In the companion article, we modified our model to account for these new data.

CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma.

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC.

Intracellular Ca2+ oscillations generated via the extracellular Ca2+-sensing receptor (CaSR) in response to extracellular Ca2+ or l-phenylalanine: Impact of the highly conservative mutation Ser170Thr

The extracellular Ca2+-sensing receptor (CaSR) is an allosteric protein that responds to changes in the extracellular concentration of Ca2+ ([Ca2+]e) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]e stimulates sinusoidal oscillations in [Ca2+]i whereas aromatic amino acid-induced CaR activation in the presence of a threshold [Ca2+]e promotes transient oscillations in [Ca2+]i. Here, we examined spontaneous and ligand-evoked [Ca2+]i oscillations in single HEK-293 cells transfected with the wild type CaSR or with a mutant CaSR in which Ser170 was converted to Thr (CaSRS170T). Our analysis demonstrates that cells expressing CaSRS170T display [Ca2+]i oscillations in the presence of low concentrations of extracellular Ca2+ and respond to L-Phe with robust transient [Ca2+]i oscillations. Our results indicate that the S170T mutation induces a marked increase in CaSR sensitivity to [Ca2+]e and imply that the allosteric regulation of the CaSR by aromatic amino acids is not only mediated by an heterotropic positive effect on Ca2+ binding cooperativity but, as biased agonists, aromatic amino acids stabilize a CaSR conformation that couples to a different signaling pathway leading to transient [Ca2+]i oscillations.

Intracellular Ca²⁺ Signals to Reconstruct A Broken Heart: Still A Theoretical Approach?

The infusion of autologous stem cells has recently been put forward as an alternative strategy to regenerate infarcted myocardium and restore the contractile functions of diseased hearts. A growing number of cell types have been probed to induce cardiac repair in several animal models of ischemic myocardium, including human cardiac progenitor cells (hCPCs), human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs) and human endothelial progenitor cells (hEPCs). The enthusiasm raised by pre-clinical studies has been dampened by clinical practice, according to which the extent of cardiac repair by cell based therapy is inadequate with respect to animal models. There is no doubt that regenerative medicine of acute myocardial infarction (AMI) will greatly benefit from the full comprehension of the signal transduction pathways which guide stem cell towards the injury site and their subsequent acquisition of a therapeutically relevant phenotype. The present review will focus on the role that oscillations in intracellular Ca(2+) concentration might play to promote the stem cells-dependent regrowth of ischemic myocardium. We will describe how intracellular Ca(2+) spikes may be manipulated to redirect stem cell fate to the most suitable lineage to restore cardiac vascularisation and contractility.

A single and rapid calcium wave at egg activation in Drosophila.

Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca2+ concentration in mammals, ascidians and polychaete worms and a single Ca2+ peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca2+ levels occur. Here, we utilise ratiometric imaging of Ca2+ indicator dyes and genetically encoded Ca2+ indicator proteins to identify and characterise a single, rapid, transient wave of Ca2+ in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca2+ wave requires an intact actin cytoskeleton and an increase in intracellular Ca2+ can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca2+ wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca2+ transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca2+ at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca2+ channels; a single Ca2+ wave then propagates in an actin dependent manner; this Ca2+ wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.

NMDA channel activation regulates Ca2+ permeation and gene transcription during histamine‐stimulated gastric acid secretion (904.5)

Background: Histamine‐stimulated gastric acid secretion is accompanied by oscillations in intracellular Ca2+. The receptor mediating these oscillations and the function of this Ca2+ signal are unknown. We aimed to test the hypothesis that histamine stimulation activates N‐methyl‐D‐aspartate (NMDA) channel‐mediated Ca2+ influx and gene transcription in parietal cells. Methods: Cultured rat parietal cells were stimulated with histamine plus IBMX with or without NMDA channel antagonists. Intracellular Ca2+ was visualized by microscopy using fura‐2 or fluo‐3. Western blots and RNA‐Seq were used to evaluate CREB/ATF‐1 activation and gene transcription, respectively. Results: Histamine stimulation induced Ca2+ oscillations/permeation that were blocked by NMDA channel antagonists. CREB and ATF‐1 were activated by histamine stimulation and blocked by NMDA channel antagonists. Transcriptome analysis identified numerous up‐ and down‐regulated genes linked to histamine stimulation with a subset affected by NMDA channel blockade. The top network function attributed to NMDA channel‐mediated genes was cell death and survival. Most of the highly up‐regulated genes were CREB target genes. Conclusions: Histamine‐stimulated Ca2+ oscillations are regulated by NMDA channels. Our data show that this pool of intracellular Ca2+ transcriptionally regulates cell survival‐associated genes in parietal cells.

Substance P plays an important role in cell adhesion molecule 1-mediated nerve–pancreatic islet α cell interaction

Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca2+ concentration ([Ca2+]i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6siRNA-CADM1 cells) in aggregates, respectively, within 1min after specific SCG nerve stimulation with scorpion venom. In αTC6siRNA-CADM1 cells, the responding rate during 3min after SCG nerve stimulation significantly increased compared with that within 1min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6siRNA-CADM1 cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve–α cell interaction. Pretreatment with CP99,994 (0.1μg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors.

Canonical Transient Receptor Potential 3 Channel Triggers Vascular Endothelial Growth Factor-Induced Intracellular Ca2+Oscillations in Endothelial Progenitor Cells Isolated from Umbilical Cord Blood

Endothelial colony-forming cells (ECFCs) are the only endothelial progenitor cells (EPCs) that are capable of acquiring a mature endothelial phenotype. ECFCs are mainly mobilized from bone marrow to promote vascularization and represent a promising tool for cell-based therapy of severe ischemic diseases. Vascular endothelial growth factor (VEGF) stimulates the proliferation of peripheral blood-derived ECFCs (PB-ECFCs) through oscillations in intracellular Ca(2+) concentration ([Ca(2+)]i). VEGF-induced Ca(2+) spikes are driven by the interplay between inositol-1,4,5-trisphosphate (InsP3)-dependent Ca(2+) release and store-operated Ca(2+) entry (SOCE). The therapeutic potential of umbilical cord blood-derived ECFCs (UCB-ECFCs) has also been shown in recent studies. However, VEGF-induced proliferation of UCB-ECFCs is faster compared with their peripheral counterpart. Unlike PB-ECFCs, UCB-ECFCs express canonical transient receptor potential channel 3 (TRPC3) that mediates diacylglycerol-dependent Ca(2+) entry. The present study aimed at investigating whether the higher proliferative potential of UCB-ECFCs was associated to any difference in the molecular underpinnings of their Ca(2+) response to VEGF. We found that VEGF induces oscillations in [Ca(2+)]i that are patterned by the interaction between InsP3-dependent Ca(2+) release and SOCE. Unlike PB-ECFCs, VEGF-evoked Ca(2+) oscillations do not arise in the absence of extracellular Ca(2+) entry and after pharmacological (with Pyr3 and flufenamic acid) and genetic (by employing selective small interference RNA) suppression of TRPC3. VEGF-induced UCB-ECFC proliferation is abrogated on inhibition of the intracellular Ca(2+) spikes. Therefore, the Ca(2+) response to VEGF in UCB-ECFCs is shaped by a different Ca(2+) machinery as compared with PB-ECFCs, and TRPC3 stands out as a promising target in EPC-based treatment of ischemic pathologies.

The 10 Most Read Articles Published in Circulation Research in 2012

The 10 Most Read Articles Published in <i>Circulation Research</i> in 2012

KCa3.1 and TRPM7 channels at the uropod regulate migration of activated human T cells.

The migration of T lymphocytes is an essential part of the adaptive immune response as T cells circulate around the body to carry out immune surveillance. During the migration process T cells polarize, forming a leading edge at the cell front and a uropod at the cell rear. Our interest was in studying the involvement of ion channels in the migration of activated human T lymphocytes as they modulate intracellular Ca2+ levels. Ca2+ is a key regulator of cellular motility. To this purpose, we created protein surfaces made of the bio-polymer PNMP and coated with ICAM-1, ligand of LFA-1. The LFA-1 and ICAM-1 interaction facilitates T cell movement from blood into tissues and it is critical in immune surveillance and inflammation. Activated human T lymphocytes polarized and migrated on ICAM-1 surfaces by random walk with a mean velocity of ∼6 µm/min. Confocal microscopy indicated that Kv1.3, CRAC, and TRPM4 channels positioned in the leading-edge, whereas KCa3.1 and TRPM7 channels accumulated in the uropod. The localization of KCa3.1 and TRPM7 at the uropod was associated with oscillations in intracellular Ca2+ levels that we measured in this cell compartment. Further studies with blockers against Kv1.3 (ShK), KCa3.1 (TRAM-34), CRAC (SKF-96365), TRPM7 (2-APB), and TRPM4 (glibenclamide) indicated that blockade of KCa3.1 and TRPM7, and not Kv1.3, CRAC or TRPM4, inhibits the T cell migration. The involvement of TRPM7 in cell migration was confirmed with siRNAs against TRPM7. Downregulation of TRPM7 significantly reduced the number of migrating T cells and the mean velocity of the migrating T cells. These results indicate that KCa3.1 and TRPM7 selectively localize at the uropod of migrating T lymphocytes and are key components of the T cell migration machinery.

Calcium influx-mediated signaling is required for complete mouse egg activation

Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.