Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

NGF induces neuronal differentiation by modulating [Ca²⁺]_i. However, the role of the three isoforms of the main Ca²⁺-extruding system, the Na⁺/Ca²⁺ exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca²⁺]_i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca²⁺ content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na⁺ currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na⁺]_i significantly increased in cells overexpressing NCX1.4 as well as ER Ca²⁺ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca²⁺]_i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca²⁺ content and PI3K signaling.     

Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury

Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca²⁺ ([Ca²⁺]_i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca²⁺]_i are linked critically by the ATP-driven plasma membrane Ca²⁺-ATPase (PMCA) important for maintaining low resting [Ca²⁺]_i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca²⁺]_i was measured by fura-2 imaging. An in situ [Ca²⁺]_i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca²⁺ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca²⁺ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca²⁺ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca²⁺ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.     

Role of Phosphoinositide 3-Kinase �� in Glycoprotein VI-mediated Akt Activation in Platelets

Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y₁₂ receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PI3Kβ inhibitor TGX-221 indicating an essential role of PI3Kβ in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca²⁺ mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3Kα inhibitor PIK75, but were not affected by PI3Kγ inhibitor AS252424 and PI3Kδ inhibitor IC87114. Consistently, GPVI-induced integrin α_IIbβ₃ activation of PI3Kγ^−/− and PI3Kδ^−/− platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on G_i stimulation through P2Y₁₂ receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3Kβ plays an essential role in GPVI-mediated platelet aggregation and Akt activation.     

Regulatory Volume Decrease and Intracellular Ca2+ in Murine Neuroblastoma Cells Studied with Fluorescent Probes

The possible role of Ca²⁺ as a second messenger mediating regulatory volume decrease (RVD) in osmotically swollen cells was investigated in murine neural cell lines (N1E-115 and NG108-15) by means of novel microspectrofluorimetric techniques that allow simultaneous measurement of changes in cell water volume and [Ca²⁺]_i in single cells loaded with fura-2. [Ca²⁺]_i was measured ratiometrically, whereas the volume change was determined at the intracellular isosbestic wavelength (358 nm). Independent volume measurements were done using calcein, a fluorescent probe insensitive to intracellular ions. When challenged with ∼40% hyposmotic solutions, the cells expanded osmometrically and then underwent RVD. Concomitant with the volume response, there was a transient increase in [Ca²⁺]_i, whose onset preceded RVD. For hyposmotic solutions (up to ∼−40%), [Ca²⁺]_i increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca²⁺, with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ′,N ′-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca²⁺-independent RVD proceeded even when there was a concomitant decrease in [Ca²⁺]_i below resting levels. Similar results were obtained in cells loaded with calcein. For cells not treated with BAPTA, restoration of external Ca²⁺ during the relaxation of RVD elicited by Ca²⁺-free hyposmotic solutions produced an increase in [Ca²⁺]_i without affecting the rate or extent of the responses. RVD and the increase in [Ca²⁺]_i were blocked or attenuated upon the second of two ∼40% hyposmotic challenges applied at an interval of 30–60 min. The inactivation persisted in Ca²⁺-free solutions. Hence, our simultaneous measurements of intracellular Ca²⁺ and volume in single neuroblastoma cells directly demonstrate that an increase in intracellular Ca²⁺ is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca²⁺ chelation could occur through secondary effects or could indicate that Ca²⁺ is required for optimal RVD responses.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

The Involvement of PI3K-Mediated and L-VGCC-Gated Transient Ca2+ Influx in 17β-Estradiol-Mediated Protection of Retinal Cells from H2O2-Induced Apoptosis with Ca2+ Overload

Intracellular calcium concentration ([Ca²⁺]_i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca²⁺]_i and its control mechanisms in process of hydrogen peroxide (H₂O₂)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca²⁺ indicator to detect [Ca²⁺]_i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H₂O₂-induced retinal cell apoptosis occurred at 4 h after H₂O₂ stress and increased in a time-dependent manner, but [Ca²⁺]_i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H₂O₂ stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca²⁺]_i, which appeared only at 0.5 h after βE2 application; c) increased [Ca²⁺]_i under 100 µM H₂O₂ treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca²⁺ stores; d) importantly, the transiently increased [Ca²⁺]_i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca²⁺ channels (L-VGCC), but the increased [Ca²⁺]_i induced by 100 µM H₂O₂ treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H₂O₂, which was also associated with its transient [Ca²⁺]_i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.     

Glucose Decouples Intracellular Ca2+ Activity from Glucagon Secretion in Mouse Pancreatic Islet Alpha-Cells

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca²⁺]_i) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca²⁺]_i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K_ATP channel activity but not on tetrodotoxin-sensitive Na⁺ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca²⁺]_i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K_ATP channel activity or reduction in α-cell [Ca²⁺]_i. Our results demonstrate that glucose uncouples the positive relationship between [Ca²⁺]_i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.     

Generation of repetitive Ca transients by bombesin requires intracellular release and influx of Ca through voltage-dependent and voltage independent channels in single HIT cells

In the present study, the bombesin-induced changes in cytosolic free Ca²⁺ ([Ca²⁺]_i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca²⁺ transients. The average frequency of the Ca²⁺ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min⁻¹ (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca²⁺ transient followed by a sustained plateau or by a decrease to basal levels. In Ca²⁺- free medium, bombesin caused only one or two Ca²⁺ transients and withdrawal of extracellular Ca²⁺ abolished the Ca²⁺ transients. The voltage-dependent Ca²⁺ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca²⁺ transients and stopped the Ca²⁺ transients in some cells. Thapsigargin caused a sustained rise in [Ca²⁺]_i) in the presence of extracellular Ca²⁺ while in its absence the rise in [Ca²⁺]_i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca²⁺], by about 50%. Depletion of intracellular Ca²⁺ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca²⁺-free medium caused an agonist-independent increase in [Ca²⁺]_i) when extracellular Ca²⁺ was restored, which was larger than in control cells that had been incubated in Ca²⁺-free medium for the same period of time. This rise in [Ca²⁺]_i and the thapsigargin-induced increase in [Ca²⁺]_i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca²⁺ pool enhances Ca²⁺ influx through VDCC and voltage-independent Ca²⁺ channels (VICC). In conclusion, the bombesin-induced Ca²⁺ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca²⁺ transients. Intracellular Ca²⁺ is mobilized during each Ca²⁺ transient, but Ca²⁺ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca²⁺ response. Ca²⁺ influx in whole or part is activated by a capacitative Ca²⁺ entry mechanism.     

Examination of the Effects of Heterogeneous Organization of RyR Clusters,Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca²⁺]_i, regulate the contractile function of cardiac muscle cells. Measuring [Ca²⁺]_i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca²⁺]_i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca²⁺ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca²⁺]_i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca²⁺]_i transient and of the simulated fluorescence intensity signal, F/F₀, reached values similar to that found in the literature ([Ca²⁺]_i ≈1 μM; F/F₀≈5.5). However, our model predicted the variation in [Ca²⁺]_i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F₀ signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca²⁺]_i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca²⁺]_i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca²⁺]_i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.     

Nav1.7 sodium channel-induced Ca influx decreases tau phosphorylation via glycogen synthase kinase-3β in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells expressing Na_v1.7 sodium channel isoform, veratridine increased Ser⁴⁷³-phosphorylation of Akt and Ser⁹-phosphorylation of glycogen synthase kinase-3β by 217 and 195%, while decreasing Ser³⁹⁶-phosphorylation of tau by 36% in a concentration (EC₅₀ = 2.1 μM)- and time (t_1/2 = 2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca²⁺ removal. Veratridine (10 μM for 5 min) increased translocation of Ca²⁺-dependent conventional protein kinase C-α from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca²⁺ removal, or Gö6976 (an inhibitor of protein kinase C-α), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Gö6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Gö6976 alone attenuated veratridine-induced glycogen synthase kinase-3β phosphorylation by 65 or 42%; however, LY294002 plus Gö6976 completely blocked it. Veratridine (10 μM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Gö6976, but completely blocked by LY294002 plus Gö6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na⁺ influx via Na_v1.7 sodium channel and the subsequent Ca²⁺ influx via voltage-dependent calcium channel activated (1) Ca²⁺/protein kinase C-α pathway, as well as (2) Ca²⁺/phosphatidylinositol 3-kinase/Akt and (3) Ca²⁺/phosphatidylinositol 3-kinase/protein kinase C-α pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3β, decreasing tau phosphorylation.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.     

Intrinsic Islet Heterogeneity and Gap Junction Coupling Determine Spatiotemporal Ca2+ Wave Dynamics

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca²⁺]_i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca²⁺]_i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca²⁺]_i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca²⁺]_i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet.     

Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics

The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca²⁺ uptake, these include activation of countertransporters (Na⁺/Ca²⁺ exchanger and Na⁺/H⁺ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca²⁺ binding to matrix buffers. Inorganic phosphate (P_i) is known to affect both the Ca²⁺ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca²⁺ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca²⁺ and mitochondrial membrane potential (ΔΨ_m) to examine the effects of anion transport on mitochondrial Ca²⁺ flux and buffering in P_i-depleted guinea pig cardiac mitochondria. Mitochondrial Ca²⁺ uptake proceeded slowly in the absence of P_i but matrix free Ca²⁺ ([Ca²⁺]_mito) still rose to ∼50 μm. P_i (0.001–1 mm) accelerated Ca²⁺ uptake but decreased [Ca²⁺]_mito by almost 50% while restoring ΔΨ_m. P_i-dependent effects on Ca²⁺ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca²⁺ uptake rate was also increased by vanadate (V_i), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca²⁺ buffering and ΔΨ_m recovery. Interestingly, ATP or AMP-PNP prevented the effects of P_i on Ca²⁺ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca²⁺ uptake and modifies the [Ca²⁺]_mito response in a complex manner.     

Neuregulin-1β Prevents Ca<Superscript>2+</Superscript> Overloading and Apoptosis Through PI3K/Akt Activation in Cultured Dorsal Root Ganglion Neurons with Excitotoxicity Induced by Glutamate

Neuregulin (NRG) plays an important role on the genesis and differentiation of neurons in the dorsal root ganglion (DRG). Whether NRG-1β regulates Ca²⁺ homeostasis and apoptosis of cultured DRG neurons with excitotoxicity induced by Glu remains unknown. In this study, primary cultured DRG neurons were used to determine the effects of NRG-1β on Ca²⁺ overload and apoptosis of DRG sensory neurons with excitotoxicity induced by Glu. The primary cultured DRG neurons at 48 h of culture age were then exposed to Glu (0.2 mmol/l), Glu (0.2 mmol/l) plus NRG-1β (20 nmol/l), or Glu (0.2 mmol/l) plus NRG-1β (20 nmol/l) and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10 μmol/l) for additional 12 h. After that, intracellular Ca²⁺ concentration ([Ca²⁺]_i) in isolated DRG neurons using the fluorescent Ca²⁺ indicator fluo-3 was measured by confocal laser scanning microscope. Apoptotic neurons were monitored by Hoechst 33342 staining. Expression of caspase-3, procaspase-3, and pAkt was detected by Western blot assay. Administration of 0.2 mmol/l Glu evoked an increase in [Ca²⁺]_i, confirming the excitatory effect of Glu. Compared with the control group, apoptotic (condensed and fragmented nuclei) neurons were observed in Glu-treated cells after Hoechst 33342 staining. The increase caspase-3 of and decrease of procaspase-3 expression levels after administration of 0.2 mmol/l Glu suggested the apoptotic effects of Glu. These effects could be inhibited by the presence of NRG-1β. The effects of NRG-1β could be blocked by PI3K inhibitor LY294002. These results implicated that NRG-1β could prevents Ca²⁺ overload and apoptosis by activating PI3K/Akt pathway of primary cultured DRG neurons with excitotoxicity induced by Glu.     

In situ visualization of the intracellular Ca2+ dynamics at the border of the acute myocardial infarct

Ischemic insult to the heart produces myocyte Ca²⁺ ([Ca²⁺]_i) overload. However, little is known about spatiotemporal changes in [Ca²⁺]_i within the ischemic heart in situ at the cellular level. Using real-time confocal microscopy, we successfully visualized [Ca²⁺]_i dynamics at the border zone on the subepicardial myocardium of the heart 2 h after coronary ligations followed by loading with fluo 3/AM. Three distinct regions were identified in the acute infarcted heart. In intact regions, the myocytes showed spatially uniform Ca²⁺ transients synchronously to QRS complex in the electrocardiogram. The myocytes at the infarcted regions showed no fluorescence intensity (FI). At the border zones between the intact and infarcted regions, Ca²⁺ waves emerged sporadically and randomly, instead of Ca²⁺ transients, at a mean frequency of 11.5 ± 8.5 min/cell with a propagation velocity of 151.0 ± 35.7 m/sec along the longitudinal axis of the individual myocytes. In addition, some myocytes within the border zone exhibited homogeneously high static FI, indicating severe Ca²⁺ overload. In summary, we provided the first direct evidence of abnormal [Ca²⁺]_i dynamics in acute infarcted hearts at the cellular level. The observed diversity in spatiotemporal [Ca²⁺]_i dynamics at the border zone may contribute to the arrhythmias or contractile failure in acute myocardial infarction.     

Leucine Facilitates Insulin Signaling through a Gαi Protein-dependent Signaling Pathway in Hepatocytes

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt⁴⁷³ and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002), Gα_i protein (pertussis toxin or siRNA against Gα_i1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gα_i1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gα_i protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.     

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca²⁺]_i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca²⁺]_i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca²⁺]_i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca²⁺]_i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca²⁺]_i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Electronic supplementary material

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