Intracellular Ca Release Research Articles

Vinblastine-induced apoptosis of melanoma cells is mediated by Ras homologous A protein (Rho A) via mitochondrial and non-mitochondrial-dependent mechanisms

Despite the availability of melanoma treatment at the primary site, the recurrence of local melanoma can metastasize to any distant organ. Currently, the available therapies for the treatment of metastatic melanoma are of limited benefit. Thus, the functional analysis of conventional therapies may help to improve their efficiency in the treatment of metastatic melanoma. In the present study, the exposure of melanoma cells to vinblastine was found to trigger apoptosis as evidenced by the loss of mitochondrial membrane potential, the release of both cytochrome c and apoptosis inducing factor, activation of caspase-9 and 3, and cleavage of Poly (ADP-ribose)-Polymerase. Also, vinblastine enhances the phosphorylation of Ras homologous protein A, the accumulation of reactive oxygen species, the release of intracellular Ca(2+), as well as the activation of apoptosis signal-regulating kinase 1, c-jun-N-terminal kinase, p38, inhibitor of kappaBα (IκBα) kinase, and inositol requiring enzyme 1α. In addition, vinblastine induces the DNA-binding activities of the transcription factor NF-κB, HSF1, AP-1, and ATF-2, together with the expression of HSP70 and Bax proteins. Moreover, inhibitory experiments addressed a central role for Rho A in the regulation of vinblastine-induced apoptosis of melanoma cells via mitochondrial and non-mitochondrial-dependent mechanisms. In conclusion, the present study addresses for the first time a central role for Rho A in the modulation of vinblastine-induced apoptosis of melanoma cells and thereby provides an insight into the molecular action of vinblastine in melanoma treatment.

Acute effects of angiotensin II on TRPC6 channels in the podocytes of the freshly isolated glomeruli

There has been proposed a key role of the podocytes in the pathogenesis of nephrotic diseases since the injury of these cells leads to heavy proteinuria. It was also shown that angiotensin II (Ang II) caused release of intracellular Ca2+ in podocytes; members of the TRPC‐family channels emerged as prime candidates for this effect. Mutations in the gene encoding the TRPC6 channel result in development of focal segmental glomerulosclerosis. Here, we examined the effects of Ang II (0.1 to 10 μM) on Ca2+ levels in intact podocytes of freshly isolated Sprague Dawley rat glomeruli. Changes in intracellular Ca2+ stimulated by Ang II in the presence or absence of thapsigargin or TRPC inhibitor 2‐APB were measured in podocytes of the glomeruli loaded with Fura‐2AM. Depletion of internal Ca2+ stores did not affect the calcium flux activated by Ang II, whereas 2‐APB completely blocked it. Patch‐clamp analysis demonstrated that 100 nM Ang II acutely activates native TRPC‐like channels in the podocytes of freshly isolated glomeruli as well as TRPC6 channels transiently overexpressed in CHO cells, and this effect is mediated by changes in the channel open probability. Thus, we provide direct evidence of Ang II‐dependent activation of TRPC6 channels in podocytes, which might play a significant role in the development of kidney diseases. This research was supported by the NIH (HL108880) and Zeiss‐OPTEC LLC grant’2012.

Dentin Phosphophoryn Activates Smad Protein Signaling through Ca2+-Calmodulin-dependent Protein Kinase II in Undifferentiated Mesenchymal Cells

Dentin phosphophoryn (DPP) is a major noncollagenous protein in the dentin matrix. In this study, we demonstrate that pluripotent stem cells such as C3H10T1/2 and human bone marrow cells can be committed to the osteogenic lineage by DPP. Treatment with DPP can stimulate the release of intracellular Ca(2+). This calcium flux triggered the activation of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII induced the phosphorylation of Smad1 and promoted nuclear translocation of p-Smad1. Inhibition of store Ca(2+) depletion by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or down-regulation of CaMKII by KN-62, a selective cell-permeable pharmacological inhibitor or a dominant negative plasmid of CaMKII, blocked DPP-mediated Smad1 phosphorylation. Activation of Smad1 resulted in the expression of osteogenic markers such as Runx2, Osterix, DMP1, Bone sialoprotein, Osteocalcin, NFATc1, and Schnurri-2, which have been implicated in osteoblast differentiation. These findings suggest that DPP is capable of triggering commitment of pluripotent stem cells to the osteogenic lineage.

Extraction of Sub-microscopic Ca Fluxes from Blurred and Noisy Fluorescent Indicator Images with a Detailed Model Fitting Approach

The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This ‘forward method’ allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

RETRACTED ARTICLE: Cardiomyocyte-specific deletion of endothelin receptor A rescues aging-associated cardiac hypertrophy and contractile dysfunction: role of autophagy

Cardiac aging is manifested as cardiac remodeling and contractile dysfunction although precise mechanisms remain elusive. This study was designed to examine the role of endothelin-1 (ET-1) in aging-associated myocardial morphological and contractile defects. Echocardiographic and cardiomyocyte contractile properties were evaluated in young (5-6months) and old (26-28months) C57BL/6 wild-type and cardiomyocyte-specific ET(A) receptor knockout (ETAKO) mice. Cardiac ROS production and histology were examined. Our data revealed that ETAKO mice displayed an improved survival. Aging increased plasma levels of ET-1 and Ang II, compromised cardiac function (fractional shortening, cardiomyocyte peak shortening, maximal velocity of shortening/relengthening and prolonged relengthening) and intracellular Ca(2+) handling (reduced intracellular Ca(2+) release and decay), the effects of which with the exception of ET-1 and Ang II levels was improved by ETAKO. Histological examination displayed cardiomyocyte hypertrophy and interstitial fibrosis associated with cardiac remodeling in aged C57 mice, which were alleviated in ETAKO mice. Aging promoted ROS generation, protein damage, ER stress, upregulated GATA4, ANP, NFATc3 and the autophagosome cargo protein p62, downregulated intracellular Ca(2+) regulatory proteins SERCA2a and phospholamban as well as the autophagic markers Beclin-1, Atg7, Atg5 and LC3BII, which were ablated by ETAKO. ET-1 triggered a decrease in autophagy and increased hypertrophic markers in vitro, the effect of which were reversed by the ET(A) receptor antagonist BQ123 and the autophagy inducer rapamycin. Antagonism of ET(A), but not ET(B) receptor, rescued cardiac aging, which was negated by autophagy inhibition. Taken together, our data suggest that cardiac ET(A) receptor ablation protects against aging-associated myocardial remodeling and contractile dysfunction possibly through autophagy regulation.

Group I mGluRs Evoke K-ATP Current by Intracellular Ca2+Mobilization in Rat Subthalamus Neurons

We reported previously that Ca(2+) influx through N-methly-d-aspartate-gated channels evokes ATP-sensitive K(+) (K-ATP) currents in rat subthalamic nucleus (STN) neurons. By using whole-cell patch clamp recordings in brain slices, we investigated the ability of (RS)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR) agonist, to evoke K-ATP currents. DHPG (20 µM) evoked outward current at -70 mV and was associated with a positive slope conductance of 2.7 nS. The sulfonylurea agent tolbutamide (100 µM) converted the positive slope to negative slope conductance, indicating mediation by K-ATP channels (ATP-sensitive K+ channels). Currents evoked by DHPG were significantly reduced by a combination of mGluR1 and mGluR5 negative allosteric modulators. DHPG-evoked outward current was blocked by cyclopiazonic acid and thapsigargin and mimicked by caffeine, suggesting mediation by release of intracellular Ca(2+). DHPG outward current was also blocked by ryanodine and 2-aminoethoxydiphenylborane, suggesting mediation by ryanodine- and inositol 1,4,5-triphosphate-sensitive Ca(2+) release. The nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester and inhibitors of protein kinase G activity also suppressed DHPG-induced outward current. Voltage recordings showed that tolbutamide prolonged depolarizing plateau potentials and increased the spontaneous firing rate of STN neurons recorded in the presence of DHPG. These results show that group I mGluR stimulation generates K-ATP current by a nitric oxide- and protein kinase G-dependent process that is mediated by release of Ca(2+) from intracellular stores. Because burst firing is linked to symptoms of Parkinson's disease, we suggest that K-ATP channels might provide a physiologically important inhibitory influence on STN neuronal activity.

Mutation of a NCKX Eliminates Glial Microdomain Calcium Oscillations and Enhances Seizure Susceptibility

Glia exhibit spontaneous and activity-dependent fluctuations in intracellular Ca(2+), yet it is unclear whether glial Ca(2+) oscillations are required during neuronal signaling. Somatic glial Ca(2+) waves are primarily mediated by the release of intracellular Ca(2+) stores, and their relative importance in normal brain physiology has been disputed. Recently, near-membrane microdomain Ca(2+) transients were identified in fine astrocytic processes and found to arise via an intracellular store-independent process. Here, we describe the identification of rapid, near-membrane Ca(2+) oscillations in Drosophila cortex glia of the CNS. In a screen for temperature-sensitive conditional seizure mutants, we identified a glial-specific Na(+)/Ca(2+), K(+) exchanger (zydeco) that is required for microdomain Ca(2+) oscillatory activity. We found that zydeco mutant animals exhibit increased susceptibility to seizures in response to a variety of environmental stimuli, and that zydeco is required acutely in cortex glia to regulate seizure susceptibility. We also found that glial expression of calmodulin is required for stress-induced seizures in zydeco mutants, suggesting a Ca(2+)/calmodulin-dependent glial signaling pathway underlies glial-neuronal communication. These studies demonstrate that microdomain glial Ca(2+) oscillations require NCKX-mediated plasma membrane Ca(2+) flux, and that acute dysregulation of glial Ca(2+) signaling triggers seizures.

Intracellular two-phase Ca2+ release and apoptosis controlled by TRP-ML1 channel activity in coronary arterial myocytes

Activation of the death receptor Fas has been reported to produce a two-phase intracellular Ca(2+) release response in coronary arterial myocytes (CAMs), which consists of local Ca(2+) bursts via lysosomal transient potential receptor-mucolipin 1 (TRP-ML1) channels and consequent Ca(2+) release from the sarcoplasmic reticulum (SR). The present study was designed to explore the molecular mechanism by which lysosomal Ca(2+) bursts are coupled with SR Ca(2+) release in mouse CAMs and to determine the functional relevance of this lysosome-associated two-phase Ca(2+) release to apoptosis, a common action of Fas activation with Fas ligand (FasL). By confocal microscopy, we found that transfection of CAMs with TRP-ML1 small interfering (si)RNA substantially inhibited FasL (10 ng/ml)-induced lysosome Ca(2+) bursts and consequent SR Ca(2+) release. In contrast, transfection of CAMs with plasmids containing a full-length TRP-ML1 gene enhanced FasL-induced two-phase Ca(2+) release. We further demonstrated that FasL significantly increased the colocalization of the lysosomal marker Lamp1 with ryanodine receptor 3 and enhanced a dynamic trafficking of lysosomes to the SR. When CAMs were treated with TRP-ML1 siRNA, FasL-induced interactions between the lysosomes and SR were substantially blocked. Functionally, FasL-induced apoptosis and activation of calpain and calcineurin, the Ca(2+) sensitive proteins that mediate apoptosis, were significantly attenuated by silencing TRP-ML1 gene but enhanced by overexpression of TRP-ML1 gene. These results suggest that TRP-ML1 channel-mediated lysosomal Ca(2+) bursts upon FasL stimulation promote lysosome trafficking and interactions with the SR, leading to apoptosis of CAMs via a Ca(2+)-dependent mechanism.

HEF-19-induced relaxation of colonic smooth muscles and the underlying mechanisms

To investigate the relaxant effect of chromane HEF-19 on colonic smooth muscles isolated from rabbits, and the underlying mechanisms. The relaxant effect and action mechanisms of HEF-19 were investigated using descending colon smooth muscle of the rabbits. Preparations 1 cm long were mounted in 15-mL tissue baths containing Tyrode's solution, maintained at 37 ± 0.5 °C and aerated with a mixture of 5% CO2 in oxygen (Carbogen). The tension and amplitude of the smooth muscle strips were recorded after adding HEF-19 (10(-6), 10(-5) and 10(-4) mol/L). After cumulative administration of four antispasmodic agents, including acetylcholine chloride (Ach) (10(-4) mol/L), histamine (10(-4) mol/L), high-K(+) (60 mmol/L) and BaCl2 (8.2 mmol/L), HEF-19 (3 × 10(-7)-3 × 10(-4) mol/L) was added to investigate the relaxant effect of HEF-19. CaCl2 (10(-4)-2.5 × 10(-3) mol/L) was added cumulatively to the smooth muscle preparations pretreated with and without HEF-19 (1 × 10(-6) or 3 × 10(-6) mol/L) and verapamil (1 × 10(-7) mol/L) to study the mechanisms involved. Finally, phasic contraction was induced with ACh (15 × 10(-6) mol/L), and CaCl2 (4 × 10(-3) mol/L) was added to the smooth muscle preparations pretreated with and without HEF-19 (3 × 10(-6) mol/L or 1 × 10(-5) mol/L) and verapamil (1 × 10(-7) mol/L) in calcium-free medium to further study the underlying mechanisms. HEF-19 (1 × 10(-6), 1 × 10(-5) and 1 × 10(-4) mol/L) suppressed spontaneous contraction of rabbit colonic smooth muscles. HEF-19 (3 × 10(-7)-3 × 10(-4) mol/L) relaxed in a concentration-dependent manner colonic smooth muscle preparations pre-contracted with BaCl2, high-K(+) solution, Ach or histamine with respective EC50 values of 5.15 ± 0.05, 5.12 ± 0.08, 5.58 ± 0.16 and 5.25 ± 0.24, thus showing a spasmolytic activity. HEF-19 (1 × 10(-6) mol/L and 3 × 10(-6) mol/L) shifted the concentration-response curves of CaCl2 to the right and depressed the maximum response to CaCl2. The two components contracted by Ach were attenuated with HEF-19 (3 × 10(-6) mol/L or 10(-5) mol/L) in calcium-free medium. HEF-19 inhibited rabbit colonic smooth muscle contraction, probably through inhibiting opening of voltage-dependent Ca(2+) channels. HEF-19 reduced inflow and intracellular release of Ca(2+) ions.

Advanced Glycation End Products Acutely Impair Ca2+ Signaling in Bovine Aortic Endothelial Cells

Post-translational modification of proteins in diabetes, including formation of advanced glycation end products (AGEs) are believed to contribute to vascular dysfunction and disease. Impaired function of the endothelium is an early indicator of vascular dysfunction in diabetes and as many endothelial cell processes are dependent upon intracellular [Ca2+] and Ca2+ signaling, the aim of this study was to examine the acute effects of AGEs on Ca2+ signaling in bovine aortic endothelial cells (BAEC). Ca2+ signaling was studied using the fluorescent indicator dye Fura-2-AM. AGEs were generated by incubating bovine serum albumin with 0–250 mM glucose or glucose-6-phosphate for 0–120 days at 37°C. Under all conditions, the main AGE species generated was carboxymethyl lysine (CML) as assayed using both gas-liquid chromatograph-mass spectroscopy and high-performance liquid chromatography. In Ca2+-replete solution, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated the increase in intracellular [Ca2+] caused by ATP (100 μM). In the absence of extracellular Ca2+, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated subsequent intracellular Ca2+ release caused by ATP, thapsigargin (0.1 μM), and ionomycin (3 μM), but AGEs did not affect extracellular Ca2+ entry induced by the re-addition of Ca2+ to the bathing solution in the presence of any of these agents. The anti-oxidant α-lipoic acid (2 μM) and NAD(P)H oxidase inhibitors apocynin (500 μM) and diphenyleneiodonium (1 μM) abolished these effects of AGEs on BAECs, as did the IP3 receptor antagonist xestospongin C (1 μM). In summary, AGEs caused an acute depletion of Ca2+ from the intracellular store in BAECs, such that the Ca2+ signal stimulated by the subsequent application other agents acting upon this store is reduced. The mechanism may involve generation of reactive oxygen species from NAD(P)H oxidase and possible activation of the IP3 receptor.

Periodic Calcium Waves in Cardiac Myocytes Enhance Susceptibility to Arrhythmia Triggers

Intracellular calcium (Ca) waves in cardiac myocytes can cause delayed afterdepolarizations (DADs), which are known triggers of cardiac arrhythmias. How these Ca waves are modulated by diffusive Ca-mediated coupling among Ca release units (CRUs) and promote DADs is not fully understood. Here, we hypothesized that myocytes are most susceptible to DADs due to periodic Ca wave activity at intermediate levels of Ca overload. To test this hypothesis, we strengthened CRU coupling by progressively raising intracellular free [Ca] and studied the transition from Ca sparks to waves using both confocal Ca imaging experiments in permeabilized mouse ventricular myocytes and computer simulations of a homogeneous 3D array (100x20x10) of diffusively-coupled CRUs. As free Ca was increased in experiments from 100 nM to 1,500 nM, intracellular Ca release activity evolved through four stages: Stage 1- random sparks and macrosparks arising from multiple sites; Stage 2- irregular aborted Ca waves arising from multiple sites; Stage 3- periodic full Ca waves arising from a small number of sites mostly near cell borders; Stage 4- high frequency “fibrillatory” Ca waves exhibiting mixed focal and reentrant features. Raising virtual intracellular Ca in computer simulations reproduced Stages 1-3 but not Stage 4, which may require spatial heterogeneities to occur. In both experiments and simulations, Stage 3 produced the largest whole-cell Ca transients and most synchronous Ca release, making this intermediate stage of Ca overload the most likely to generate DADs of sufficient amplitude to trigger arrhythmias. High frequency “fibrillatory” waves under severe Ca overload in Stage 4 diminished Ca transient amplitudes and reduced Ca release synchrony. In conclusion, our findings suggest that ventricular myocytes are most susceptible to DADs when intracellular Ca overload is intermediate rather than mild or severe.

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol-induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol-induced increase in intracellular calcium [Ca(2+)](i) causes the death of CGN. Alcohol induced a dose-dependent (200-800 mg/dL) neuronal death within 24 h. Ratiometric Ca(2+) imaging with Fura-2 revealed that alcohol causes a rapid (1-2 min), dose-dependent increase in [Ca(2+)](i), which persisted for the duration of the experiment (5 or 7 min). The alcohol-induced increase in [Ca(2+)](i) was observed in Ca(2+) -free media, suggesting intracellular Ca(2+) release. Pre-treatment of CGN cultures with an inhibitor (2-APB) of the inositol-triphosphate receptor (IP(3) R), which regulates Ca(2+) release from the endoplasmic reticulum (ER), blocked both the alcohol-induced rise in [Ca(2+)](i) and the neuronal death caused by alcohol. Similarly, pre-treatment with BAPTA/AM, a Ca(2+) -chelator, also inhibited the alcohol-induced surge in [Ca(2+) ](i) and prevented neuronal death. In conclusion, alcohol disrupts [Ca(2+)](i) homeostasis in CGN by releasing Ca(2+) from intracellular stores, resulting in a sustained increase in [Ca(2+)](i). This sustained increase in [Ca(2+)](i) may be a key determinant in the mechanism underlying alcohol-induced neuronal death.

Highlights from the Literature

Highlights from the Literature

MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism

Ca2+ flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca2+ signals and activation of cell death pathways1–11. Mitochondrial Ca2+ uptake occurs at regions of close apposition with intracellular Ca2+ release sites 12–14, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca2+ selective ion channel (MiCa15) called the uniporter16–18 whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore19, 20. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca2+ uptake in HeLa cells 21. Here we identified CCDC90A, hereafter referred to as MCUR1 (Mitochondrial Calcium Uniporter Regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca2+ uptake. MCUR1 binds to MCU and regulates ruthenium red-sensitive MCU-dependent Ca2+ uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca2+ uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP, and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca2+ uptake and maintenance of normal cellular bioenergetics.

Effect of Allyl Sulfides from Garlic Essential Oil on Intracellular Ca2+ Levels in Renal Tubular Cells

Diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3), which are major organosulfur compounds of garlic (Allium sativum), are recognized as a group of potential chemopreventive compounds. In this study, the early signaling effects of 3 were examined on Madin-Darby canine kidney (MDCK) cells loaded with the Ca(2+)-sensitive dye fura-2. It was found that 3 caused an immediate and sustained increase of [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 40 μM). Compound 3 also induced a [Ca(2+)](i) elevation when extracellular Ca(2+) was removed, but the magnitude was reduced by 45%. In Ca(2+)-free medium, the 3-induced [Ca(2+)](i) level was abolished by depleting stored Ca(2+) with 1 μM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). Elevation of [Ca(2+)](i) caused by 3 in the Ca(2+)-containing medium was not affected by modulation of protein kinase C activity. The 3-induced Ca(2+) influx was inhibited by nifedipine and nicardipine (1 μM). U73122, an inhibitor of phospholipase C, abolished ATP (but not the 3-induced [Ca(2+)](i) level). These findings suggest that 3 induced a significant [Ca(2+)](i) elevation in MDCK renal tubular cells by stimulating both extracellular Ca(2+) influx and thapsigargin-sensitive intracellular Ca(2+) release via as yet unidentified mechanisms. Furthermore, the order of the allyl sulfide-induced [Ca(2+)](i) elevation and cell viability was 1 < 2 < 3. The differential effect of allyl sulfides on Ca(2+) signaling and cell death appears to correlate with the number of sulfur atoms in the structure of these allyl sulfides.

SKF83959, an Agonist of Phosphatidylinositol-Linked D1-Like Receptors, Promotes ERK1/2 Activation and Cell Migration in Cultured Rat Astrocytes

Extracellular signal-regulated kinase 1/2 (ERK1/2) is a member of the mitogen-activated protein kinase family. It can mediate cell migration. Classical dopamine receptor-mediated ERK1/2 phosphorylation is widely studied in neurons. Here, we report that ERK1/2 phosphorylation is also modulated by putative phosphatidylinositol-linked D1-like receptors in cultured rat astrocytes. 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959), an agonist of the putative phosphatidylinositol-linked D1-like receptors, was found to enhance ERK1/2 phosphorylation, which then promoted the migration of cultured astrocytes. The SKF83959-induced ERK1/2 phosphorylation was found to be Ca2+-independent based on the following observations: i. chelating intracellular Ca2+ did not inhibit ERK1/2 phosphorylation and astrocyte migration; ii. blockage of the release of intracellular Ca2+ from the endoplasmic reticulum by an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor did not attenuate ERK1/2 phosphorylation. However, inhibition of phospholipase C (PLC), the upstream molecule of internal Ca2+ release, disabled SKF83959’s ability to elevate the level of ERK1/2 phosphorylation. Both non-selective protein kinase C (PKC) inhibitor and PKCδ selective inhibitor prevented ERK1/2 phosphorylation increase and astrocyte migration, but PKCα inhibitor did not. This suggests that Ca2+-independent and diacylglycerol-dependent PKCδ acts downstream of putative phosphatidylinositol-linked D1-like receptor activation and mediates SKF83959-induced elevation of ERK1/2 phosphorylation in order to modulate astrocyte migration. In conclusion, our results demonstrate that SKF83959-induced increases in ERK1/2 phosphorylation and astrocyte migration are dependent on PLC-PKCδ signals. This might help us to further understand the functions of the putative phosphatidylinositol-linked D1-like receptors in the nervous system.

Understanding the physiology of heart failure through cellular and in vivo models‐towards targeting of complex mechanisms

Heart failure (HF) is a complex disease syndrome, which affects physiology at all levels, from the molecule to the whole organism. Following a causative insult, a maladaptive response occurs, which sustains cardiac remodelling and leads to a final common pathway of debilitating HF symptoms. In terms of mechanisms, distinct defects of excitation-contraction coupling compartments and organelles have been identified in cardiac samples of patients and animal models, which include changes in Ca(2+) transport proteins and T-tubules. From a physiological standpoint, the source of regulatory intracellular Ca(2+) is defined by ∼20,000 Ca(2+) release units per cardiac myocyte, which jointly modulate contractile force production. We and others have characterized key changes in protein and membrane components of Ca(2+) release units during HF in patient samples and transgenic models to gain insight into complex disease mechanisms. While earlier HF studies identified intracellular Ca(2+) release as a major cause of contractile dysfunction, electrical dysfunction has gained attention as an important mechanism of HF mortality. In parallel, high-resolution imaging techniques have become instrumental to understand HF mechanisms in the intact cell and tissue environment, supporting translation of novel diagnostic strategies. Indeed, the increased spatial and temporal resolution of different experimental imaging techniques addresses the vastly different scales of HF pathophysiology, to correlate experimental with clinical surrogate markers, and to extend mechanisms to early, often subtle changes in HF. This last goal, in particular, will be essential to translate novel pathophysiological insight back to the growing number of asymptomatic individuals at increased risk for HF development, who may benefit most from early therapeutic interventions.

Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre-Bötzinger complex: a computational modelling study.

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre-Bötzinger complex (pre-BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre-BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na(+) current (I(NaP)), and the other involving the voltage-gated Ca(2+) current (I(Ca)) and the Ca(2+) -activated nonspecific cation current (I(CAN)), activated by intracellular Ca(2+) accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na(+)-dependent and Ca(2+)-dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I(NaP) and I(Ca) randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca(2+) release, and the Na(+)/K(+) pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I(NaP) and/or I(CAN), or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre-BötC and other brainstem/spinal cord circuits under different experimental conditions.

Polarization of Calcium Signaling and Fluid Secretion in Salivary Gland Cells

The secretion of fluid, electrolytes, and protein by exocrine gland acinar cells is a vectorial process that requires the coordinated regulation of multiple channel and transporter proteins, signaling components, as well as mechanisms involved in vesicular fusion and water transport. Most critical in this is the regulation of cytosolic free [Ca(2+)] ([Ca(2+)](i)) in response to neurotransmitter stimulation. Control of [Ca(2+)](i) increase in specific regions of the cell is the main determinant of fluid and electrolyte secretion in salivary gland acinar cells as it regulates several major ion flux mechanisms as well as the water channel that are required for this process. Polarized [Ca(2+)](i) signals are also essential for protein secretion in pancreatic acinar cells. Thus, the mechanisms that generate and modulate these compartmentalized [Ca(2+)](i) signals are central to the regulation of exocrine secretion. These mechanisms include membrane receptors for neurotransmitters, intracellular Ca(2+) release channels, Ca(2+) entry channels, as well Ca(2+) as pumps and mitochondria. The spatial arrangement of proteins involved in Ca(2+) signaling is of primary significance in the generation of specific compartmentalized [Ca(2+)](i) signals. Within these domains, both local and global [Ca(2+)](i) changes are tightly controlled. Control of secretion is also dependent on the targeting of ion channels and transporters to specific domains in the cell where their regulation by [Ca(2+)](i) signals is facilitated. Together, the polarized localization of Ca(2+) signaling and secretory components drive vectorial secretion of fluid, electrolytes, and proteins in the exocrine salivary glands and pancreas. This review will discuss recent findings which have led to resolution of the molecular components underlying the spatio-temporal control of [Ca(2+)](i) signals in exocrine gland cells and their role in secretion.

Coordination of Intercellular Ca2+ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries

To test the hypothesis that Ca(2+) responses to GPCR activation are coordinated between neighboring ECs of resistance arteries. EC tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca(2+) responses to ACh were imaged with confocal microscopy. Cell-to-cell transfer of propidium iodide confirmed functional GJCs. A 1 μm ACh stimulus evoked Ca(2+) responses (9.8 ± 0.8/min, F/F(0) = 3.11 ± 0.2) which pseudo-line-scan analysis revealed as composed of Ca(2+) waves and spatially restricted Ca(2+) release events. A 100 nm ACh stimulus induced Ca(2+) responses of lower frequency (4.5 ± 0.7/min) and amplitude (F/F(0) = 1.95 ± 0.11) composed primarily of spatially restricted events. The time interval between Ca(2+) waves in adjacent cells (0.79 ± 0.12 s) was shorter (p < 0.05) than that between nonadjacent cells (1.56 ± 0.25 s). Spatially restricted Ca(2+) release events had similar frequencies and latencies between adjacent and nonadjacent cells. Inhibiting intracellular Ca(2+) release with 2-APB, Xestospongin C or thapsigargin eliminated Ca(2+) responses. With moderate GPCR stimulation, localized Ca(2+) release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca(2+) waves via the ER. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.