Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale

In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).

Objective

To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.

Methods

Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca²⁺ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca²⁺ release, F>F₅₀ within 20 ms) or their absence (delayed areas). Spontaneous Ca²⁺ release events during diastole, Ca²⁺ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.

Results

In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na⁺/Ca²⁺ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca²⁺ influx via Ca²⁺ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca²⁺ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca²⁺ removal by NCX at the membrane was significantly lower in MI.

Conclusion

TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca²⁺ loss and raise SR Ca²⁺ content, but may promote Ca²⁺ waves.     

Differential regulation and recovery of intracellular Ca2+ in cerebral and small mesenteric arterial smooth muscle cells of simulated microgravity rat

Background

The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca²⁺ determined by the alterations in the functions of plasma membrane Ca_L channels and ryanodine-sensitive Ca²⁺ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.

Methodology/Principal Findings

Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of Ca_L channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca²⁺ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.

Conclusions

The differential regulation of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca²⁺, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.     

The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling

Background

Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca²⁺. Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP₃) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.

Methodology/Principal Findings

In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca²⁺ and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca²⁺ wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca²⁺ signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.

Conclusions/Significance

Our results suggest that PIP2 plays comprehensive roles in shaping Ca²⁺ waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

Transmission Efficacy and Plasticity in Glutamatergic Synapses Formed by Excitatory Interneurons of the Substantia Gelatinosa in the Rat Spinal Cord

Background

Substantia gelatinosa (SG, lamina II) is a spinal cord region where most unmyelinated primary afferents terminate and the central nociceptive processing begins. The glutamatergic excitatory interneurons (EINs) form the majority of the SG neuron population, but little is known about the mechanisms of signal processing in their synapses.

Methodology

To describe the functional organization and properties of excitatory synapses formed by SG EINs, we did non-invasive recordings from 183 pairs of monosynaptically connected neurons. An intact presynaptic SG EIN was specifically stimulated through the cell-attached pipette while the evoked EPSCs/EPSPs were recorded through perforated-patch from a postsynaptic neuron (laminae I-III).

Principal Findings

We found that the axon of an SG EIN forms multiple functional synapses on the dendrites of a postsynaptic neuron. In many cases, EPSPs evoked by stimulating an SG EIN were sufficient to elicit spikes in a postsynaptic neuron. EPSCs were carried through both Ca²⁺-permeable (CP) and Ca²⁺-impermeable (CI) AMPA receptors (AMPARs) and showed diverse forms of functional plasticity. The synaptic efficacy could be enhanced through both activation of silent synapses and strengthening of already active synapses. We have also found that a high input resistance (R_IN, >0.5 GΩ) of the postsynaptic neuron is necessary for resolving distal dendritic EPSCs/EPSPs and correct estimation of their efficacy.

Conclusions/Significance

We conclude that the multiple synapses formed by an SG EIN on a postsynaptic neuron increase synaptic excitation and provide basis for diverse forms of plasticity. This functional organization can be important for sensory, i.e. nociceptive, processing in the spinal cord.     

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish

Background

Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood.

Methodology/Principal Findings

In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp ⁺ cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli ⁺ etsrp ⁺ hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp ⁺ cell numbers at the LPM.

Conclusions/Significance

This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.     

Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and S-glutathionylation of nitric oxide synthetase

Background

PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood.

Methodology/Principal Findings

We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca²⁺ release and causing auto-regulation of eNOS through S-glutathionylation.

Conclusions/Significance

The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca²⁺ flux and calmodulin (CaM) activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNOS activation.     

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP?/? Mice

Background

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.

Principal Findings

We used intact olfactory epithelium obtained from WT and OMP^−/− mice to monitor the Ca²⁺ dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca²⁺ channels, or Ca²⁺ stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca²⁺-homeostasis in these neurons by influencing the activity of the plasma membrane Na⁺/Ca²⁺-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca²⁺ elevation by stimulating the reverse mode of NCX in both WT and OMP^−/− mice. The resulting Ca²⁺ responses indicate that OMP facilitates NCX activity and allows rapid Ca²⁺ extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).

Conclusions

Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions.     

PI3Ks maintain the structural integrity of T-tubules in cardiac myocytes

Background

Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.

Methods and Results

Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca²⁺ channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca²⁺ transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.

Conclusions

PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca²⁺-induced Ca²⁺ release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.     

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes

Background

The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca²⁺-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/Principal Findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca²⁺-handling proteins. Detailed laser confocal Ca²⁺ imaging demonstrated spontaneous whole-cell [Ca²⁺]_i transients. These transients required Ca²⁺ influx via L-type Ca²⁺ channels, as demonstrated by their elimination in the absence of extracellular Ca²⁺ or by administration of the L-type Ca²⁺ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca²⁺ store, contributing to [Ca²⁺]_i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca²⁺) and ryanodine (decreasing [Ca²⁺]_i). Similarly, the importance of Ca²⁺ reuptake into the SR via the SR Ca²⁺ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca²⁺]_i transients elimination. Finally, the presence of an IP3-releasable Ca²⁺ pool in hiPSC-CMs and its contribution to whole-cell [Ca²⁺]_i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusions/Significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca²⁺ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca²⁺]_i transients in hiPSC-CMs on both sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and intracellular store Ca²⁺ release.     

Subcellular Min Oscillations as a Single-Cell Reporter of the Action of Polycations,Protamine, and Gentamicin on Escherichia coli

Background

In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli.

Methodology/Principal Findings

We exposed bacteria to the extracellular cations Ca⁺⁺, Mg⁺⁺, the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca⁺⁺ and Mg⁺⁺ the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca⁺⁺, and reversible freezing of the Min oscillation at high cationic concentrations.

Conclusions/Significance

Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.     

Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level

Background

Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination.

Methodology/Principal Findings

We applied BRET to develop an autoluminescent Ca²⁺ indicator, BRAC, which is composed of Ca²⁺-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein''s chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca²⁺ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca²⁺-independent signal drifts due to change in cell shape, focus shift, etc.

Conclusions/Significance

The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca²⁺ imaging not only in single live cells but also in small living subjects.     

Causes of Abnormal Ca2+ Transients in Guinea Pig Pathophysiological Ventricular Muscle Revealed by Ca2+ and Action Potential Imaging at Cellular Level

Background

Abnormal Ca²⁺ transients are often observed in heart muscles under a variety of pathophysiological conditions including ventricular tachycardia. To clarify whether these abnormal Ca²⁺ transients can be attributed to abnormal action potential generation or abnormal Ca²⁺ handling/excitation-contraction (EC) coupling, we developed a procedure to determine Ca²⁺ and action potential signals at the cellular level in isolated heart tissues.

Methodology/Principal Findings

After loading ventricular papillary muscle with rhod-2 and di-4-ANEPPS, mono-wavelength fluorescence images from rhod-2 and ratiometric images of two wavelengths of emission from di-4-ANEPPS were sequentially obtained. To mimic the ventricular tachycardia, the ventricular muscles were field-stimulated in non-flowing Krebs solution which elicited abnormal Ca²⁺ transients. For the failed and alternating Ca²⁺ transient generation, there were two types of causes, i.e., failed or abnormal action potential generation and abnormal EC coupling. In cells showing delayed initiation of Ca²⁺ transients with field stimulation, action potential onset was delayed and the rate of rise was slower than in healthy cells. Similar delayed onset was also observed in the presence of heptanol, an inhibitor of gap junction channels but having a non-specific channel blocking effect. A Na⁺ channel blocker, on the other hand, reduced the rate of rise of the action potentials but did not result in desynchronization of the action potentials. The delayed onset of action potentials can be explained primarily by impaired gap junctions and partly by Na⁺ channel inactivation.

Conclusions/Significance

Our results indicate that there are multiple patterns for the causes of abnormal Ca²⁺ signals and that our methods are useful for investigating the physiology and pathophysiology of heart muscle.     

Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery

Background

The rate-limiting step that determines the dominant time constant (τ_D) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca²⁺-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τ_D is modulated by background light in mouse rods.

Methodology/Principal Findings

We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca²⁺ shortens the dominant time constant (τ_D) of saturated photoresponse recovery, i.e., when extracellular free Ca²⁺ is decreased from 1 mM to ∼25 nM, the τ_D is reversibly increased ∼1.5–2-fold.

Conclusions

We conclude that the increase in τ_D during low Ca²⁺ treatment is not due to increased [cGMP], increased [Na⁺] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca²⁺-dependent mechanism controls the life time of active PDE.     

Cardiomyocyte Contractile Dysfunction in the APPswe/PS1dE9 Mouse Model of Alzheimer's Disease

Objectives

Ample clinical and experimental evidence indicated that patients with Alzheimer''s disease display a high incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening, intracellular Ca²⁺ homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER) stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for Alzheimer''s disease.

Methods

Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR), maximal velocity of shortening and relengthening (±dL/dt), intracellular Ca²⁺ transient rise and decay.

Results

Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or PS1 single mutation mice exhibited depressed PS, reduced±dL/dt, normal TPS and TR compared with WT mice_. Rise in intracellular Ca²⁺ was lower accompanied by unchanged resting/peak intracellular Ca²⁺ levels and intracellular Ca²⁺ decay in APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness to adrenergic agonists was dampened although β₁-adrenergic receptor expression was unchanged in APP/PS1 hearts. Expression of the Ca²⁺ regulatory protein phospholamban and protein carbonyl formation were downregulated and elevated, respectively, associated with unchanged SERCA2a, Na⁺-Ca²⁺ exchanger and ER stress markers in APP/PS1 hearts. Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice.

Conclusions

Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer''s disease model, possibly due to oxidative stress.     

Fibro-vascular coupling in the control of cochlear blood flow

Background

Transduction of sound in the cochlea is metabolically demanding. The lateral wall and hair cells are critically vulnerable to hypoxia, especially at high sound levels, and tight control over cochlear blood flow (CBF) is a physiological necessity. Yet despite the importance of CBF for hearing, consensus on what mechanisms are involved has not been obtained.

Methodology/Principal Findings

We report on a local control mechanism for regulating inner ear blood flow involving fibrocyte signaling. Fibrocytes in the super-strial region are spatially distributed near pre-capillaries of the spiral ligament of the albino guinea pig cochlear lateral wall, as demonstrably shown in transmission electron microscope and confocal images. Immunohistochemical techniques reveal the inter-connected fibrocytes to be positive for Na+/K+ ATPase β1 and S100. The connected fibrocytes display more Ca²⁺ signaling than other cells in the cochlear lateral wall as indicated by fluorescence of a Ca²⁺ sensor, fluo-4. Elevation of Ca²⁺ in fibrocytes, induced by photolytic uncaging of the divalent ion chelator o-nitrophenyl EGTA, results in propagation of a Ca²⁺ signal to neighboring vascular cells and vasodilation in capillaries. Of more physiological significance, fibrocyte to vascular cell coupled signaling was found to mediate the sound stimulated increase in cochlear blood flow (CBF). Cyclooxygenase-1 (COX-1) was required for capillary dilation.

Conclusions/Significance

The findings provide the first evidence that signaling between fibrocytes and vascular cells modulates CBF and is a key mechanism for meeting the cellular metabolic demand of increased sound activity.     

Carbenoxolone blocks the light-evoked rise in intracellular calcium in isolated melanopsin ganglion cell photoreceptors

Background

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca²⁺ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca²⁺ signals in ipRGCs independent of gap junction blockade.

Methodology/Principal Findings

To test the possibility that carbenoxolone directly inhibits light-evoked Ca²⁺ responses in ipRGCs, the light-evoked rise in intracellular Ca²⁺ ([Ca²⁺]_i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca²⁺]_i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.

Conclusions/Significance

We demonstrate that the light-evoked rise in [Ca²⁺]_i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca²⁺]_i in isolated ipRGCs is almost entirely due to Ca²⁺ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca²⁺]_i in ipRGCs by blocking L-type voltage-gated Ca²⁺ channels. The ability of carbenoxolone to block evoked Ca²⁺ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca²⁺]_i is the output being measured.     

Hypoxia Activates a Ca2+-Permeable Cation Conductance Sensitive to Carbon Monoxide and to GsMTx-4 in Human and Mouse Sickle Erythrocytes

Background

Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca²⁺] ([Ca²⁺]_i) and subsequent activation of K_Ca 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.

Methodology/Principal Findings

We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca²⁺- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca²⁺]_i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca²⁺]_i in mouse sickle erythrocytes did not require KCa3.1 activity.

Conclusions/Significance

The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca²⁺-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca²⁺ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.     

Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin

Background

Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood.

Methodology/Principal Findings

Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca²⁺/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca²⁺/CaM formed a 1∶1 complex with an equilibrium association constant around 10⁵ M⁻¹, whereas no binding reaction of K-RasB-(DESGPC) with Ca²⁺/CaM is detected. Furthermore, the interaction of K-RasB with Ca²⁺/CaM is found to be enhanced by the farnesylation of K-RasB.

Conclusions/Significance

We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca²⁺/CaM, but also defines its isoform specific interaction with Ca²⁺/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca²⁺/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.     

Involvement of AMPK in Alcohol Dehydrogenase Accentuated Myocardial Dysfunction Following Acute Ethanol Challenge in Mice

Objectives

Binge alcohol drinking often triggers myocardial contractile dysfunction although the underlying mechanism is not fully clear. This study was designed to examine the impact of cardiac-specific overexpression of alcohol dehydrogenase (ADH) on ethanol-induced change in cardiac contractile function, intracellular Ca²⁺ homeostasis, insulin and AMP-dependent kinase (AMPK) signaling.

Methods

ADH transgenic and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Oral glucose tolerance test, cardiac AMP/ATP levels, cardiac contractile function, intracellular Ca²⁺ handling and AMPK signaling (including ACC and LKB1) were examined.

Results

Ethanol exposure led to glucose intolerance, elevated plasma insulin, compromised cardiac contractile and intracellular Ca²⁺ properties, downregulated protein phosphatase PP2A subunit and PPAR-γ, as well as phosphorylation of AMPK, ACC and LKB1, all of which except plasma insulin were overtly accentuated by ADH transgene. Interestingly, myocardium from ethanol-treated FVB mice displayed enhanced expression of PP2Cα and PGC-1α, decreased insulin receptor expression as well as unchanged expression of Glut4, the response of which was unaffected by ADH. Cardiac AMP-to-ATP ratio was significantly enhanced by ethanol exposure with a more pronounced increase in ADH mice. In addition, the AMPK inhibitor compound C (10 µM) abrogated acute ethanol exposure-elicited cardiomyocyte mechanical dysfunction.

Conclusions

In summary, these data suggest that the ADH transgene exacerbated acute ethanol toxicity-induced myocardial contractile dysfunction, intracellular Ca²⁺ mishandling and glucose intolerance, indicating a role of ADH in acute ethanol toxicity-induced cardiac dysfunction possibly related to altered cellular fuel AMPK signaling cascade.