Dependent Ca2 Research Articles

Voltage-gated Ca2+ channel activity modulates smooth muscle cell calcium waves in hamster cremaster arterioles.

Cremaster muscle arteriolar smooth muscle cells (SMCs) display inositol 1,4,5-trisphosphate receptor-dependent Ca2+ waves that contribute to global myoplasmic Ca2+ concentration and myogenic tone. However, the contribution made by voltage-gated Ca2+ channels (VGCCs) to arteriolar SMC Ca2+ waves is unknown. We tested the hypothesis that VGCC activity modulates SMC Ca2+ waves in pressurized (80 cmH2O/59 mmHg, 34°C) hamster cremaster muscle arterioles loaded with Fluo-4 and imaged by confocal microscopy. Removal of extracellular Ca2+ dilated arterioles (32 ± 3 to 45 ± 3 μm, n = 15, P < 0.05) and inhibited the occurrence, amplitude, and frequency of Ca2+ waves ( n = 15, P < 0.05), indicating dependence of Ca2+ waves on Ca2+ influx. Blockade of VGCCs with nifedipine (1 μM) or diltiazem (10 μM) or deactivation of VGCCs by hyperpolarization of smooth muscle with the K+ channel agonist cromakalim (10 μM) produced similar inhibition of Ca2+ waves ( P < 0.05). Conversely, depolarization of SMCs with the K+ channel blocker tetraethylammonium (1 mM) constricted arterioles from 26 ± 3 to 14 ± 2 μm ( n = 11, P < 0.05) and increased wave occurrence (9 ± 3 to 16 ± 3 waves/SMC), amplitude (1.6 ± 0.07 to 1.9 ± 0.1), and frequency (0.5 ± 0.1 to 0.9 ± 0.2 Hz, n = 10, P < 0.05), effects that were blocked by nifedipine (1 μM, P < 0.05). Similarly, the VGCC agonist Bay K8644 (5 nM) constricted arterioles from 14 ± 1 to 8 ± 1 μm and increased wave occurrence (3 ± 1 to 10 ± 1 waves/SMC) and frequency (0.2 ± 0.1 to 0.6 ± 0.1 Hz, n = 6, P < 0.05), effects that were unaltered by ryanodine (50 μM, n = 6, P > 0.05). These data support the hypothesis that Ca2+ waves in arteriolar SMCs depend, in part, on the activity of VGCCs. NEW & NOTEWORTHY Arterioles that control blood flow to and within skeletal muscle depend on Ca2+ influx through voltage-gated Ca2+ channels and release of Ca2+ from internal stores through inositol 1,4,5-trisphosphate receptors in the form of Ca2+ waves to maintain pressure-induced smooth muscle tone.

Basal Spontaneous Firing of Rabbit Sinoatrial Node Cells Is Regulated by Dual Activation of PDEs (Phosphodiesterases) 3 and 4.

Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.

The role of tripartite interaction of calcium sensors and transporters in the accumulation of calcium in finger millet grain

Finger millet (Eleusine coracana) is one of important crops, and its grains contain an exceptionally high content of calcium. In order to investigate the molecular mechanism by which it orchestrate the accumulation of Ca2+ during grain filling, some candidate genes encoding calcium transporters [calcium exchangers (CAX1, CAX3)] and sensors [calcineurin-B like (CBL4 and 10)], a CBL-interacting protein kinase (CIPK24), and calmodulin (CaM) were identified using transcriptomics and differential expression analysis in two genotypes of finger millet differing in grain calcium content. These transporters and sensors are highly expressed in leaves and developing spikes of the genotype with a high grain Ca2+ indicating their potential role in Ca2+ accumulation. Calcium transporters, mainly CAXs, pump Ca2+ inside the cell through plasmalemma and tonoplast, and their activities are regulated by CaM dependent and independent Ca2+ sensor proteins of CaM and CBL-CIPK networks. Abundance of CaM in a high grain Ca2+ genotype is suggestive that CaM might also contribute for grain calcium accumulation by interaction with Ca2+ATPase. The upregulation of CAX1 in vegetative tissues and developing spikes and CAX3 only in developing spikes provides the most plausible clue for calcium transport and accumulation regulated by tripartite interaction in finger millet.

Enhanced ZnR/GPR39 Activity in Breast Cancer, an Alternative Trigger of Signaling Leading to Cell Growth

Acquired resistance to the estrogen receptor (ER) antagonist tamoxifen, is a major obstacle in treatment of breast cancer. Changes in Zn2+ accumulation and distribution are associated with tamoxifen-resistance and breast cancer progression. The Zn2+-sensing G-protein coupled receptor, ZnR/GPR39, triggers signaling leading to cell growth, but a role for this receptor in breast cancer in unknown. Using fluorescence imaging, we found Zn2+-dependent Ca2+ release, mediated by ZnR/GPR39 activity, in TAMR tamoxifen-resistant cells derived from MCF-7 cells, but not in ER-expressing MCF-7 or T47D cells. Furthermore, ZnR/GPR39 signaling was monitored in ER negative BT20, MDA-MB-453 and JIMT-1 cells. Expression of ZnR/GPR39 was increased in grade 3 human breast cancer biopsies compared to grade 2. Consistently, analysis of two breast cancer patient cohorts, GDS4057 and TCGA, indicated that in ER-negative tumors higher ZnR/GPR39 mRNA levels are associated with more aggressive tumors. Activation of ZnR/GPR39 in TAMR cells triggered MAPK, mTOR and PI3K signaling. Importantly, enhanced cell growth and invasiveness was observed in the ER negative breast cancer cells, TAMR, MDA-MB-453 and BT20 cells but not in the ER expressing MCF-7 cells. Thus, we suggest ZnR/GPR39 as a potential therapeutic target for combination treatment in breast cancer, particularly relevant in ER negative tumors.

Norepinephrine Increases Cytosolic Ca2+ in Neurons of the Paraventricular Nucleus of the Hypothalamus

The paraventricular nucleus (PVN), an important nucleus of the hypothalamus, is involved in cardiorespiratory and central autonomic responses to a variety of stimuli or stressors. The PVN receives dense innervation from catecholaminergic neurons in the nucleus tractus solitarii, ventrolateral medulla, and locus coeruleus. Stressors, including hypoxia, activate these projections to the PVN, releasing neurotransmitters and modulators including norepinephrine (NE). Within the PVN, adrenergic receptor activation by NE increases excitatory postsynaptic currents, and induces depolarization and elevated action potential discharge. However, NE's role in modulating cytosolic Ca2+ in PVN neurons remains to be determined. In this study, we determined the effects of NE on PVN neuron cytosolic Ca2+ using fura‐2 ratiometric fluorescent Ca2+ imaging. PVN neurons were dissociated from 3–4 wk old male Sprague‐Dawley rats and used within 12 hours of isolation. Neurons were exposed to aCSF baseline (Bsl, 5 min), followed by vehicle (5 min) or NE (100 mM, 5 min). Compared to aCSF Bsl, PVN neurons showed stable basal Ca2+ during vehicle controls (n=34). In contrast, addition of 100 mM NE elevated basal Ca2+ (n=69). To examine the contribution of Ca2+ from internal sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) stores in this elevation, neurons were exposed to the SERCA blocker Thapsigargin (TG, 1 mM, 30 min). When NE was added in the presence of TG the increase in basal Ca2+ persisted, although slightly attenuated (n=11). To examine if NE elevates cytosolic Ca2+ via extracellular Ca2+ entry, cells were exposed to aCSF with zero Ca2+. In the absence of extracellular Ca2+, NE did not elevate basal Ca2+ and rather showed a significant decrease (n=63). Spontaneous Ca2+ peaks in PVN neurons were also recorded. While NE did not affect the number of spontaneous peaks or the number of cells that exhibited spontaneous peaks, NE did increase spontaneous peak amplitude (n=69). NE in presence of TG replicated NE's increase in spontaneous peak amplitude (n=11). NE in the absence of extracellular Ca2+ significantly decreased spontaneous peak amplitude (n=63). To study the potential influence of NE on use (voltage)‐dependent Ca2+ channels, cells were repeatedly depolarized with 55 mM K+ (5×, 20 sec, 5 min intervals). NE or vehicle was applied between the 3rd and 4th depolarization and the amplitude of Ca2+ peaks and basal Ca2+ was evaluated. Across the 5 peaks, there was a slight decrease in amplitude over time that was not altered by vehicle (n=34). Application of 100 mM NE induced a small, non‐significant increase in Ca2+ peak amplitude, and a significant increase in basal Ca2+ (n=34). In contrast to the NE response that occurred in neurons studied immediately after isolation, PVN neurons incubated for 2–5 days saw the effects of NE greatly diminished, if not completely abolished, and 24 hour incubation in 10 mM NE did not restore NE sensitivity. These preliminary findings demonstrate NE increases cytosolic Ca2+, likely from an influx of extracellular Ca2+, and support previous work that NE is associated with depolarization of PVN neurons.Support or Funding InformationHL 098602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alterations in Arginase-NO-synthase System of Spermatozoa in Human Subjects with Different Fertility Potential.

SummaryBackgroundInfertility is an important worldwide problem which affects 10–15% of couples globally. Altered NO production has also been implicated in the pathogenesis of the male infertility. The present study was designed to evaluate the changes in the activity of NO-synthase (NOS) and arginase in spermatozoa of patients with infertility.MethodsThe total NOS, Ca2+-dependent constitutive (cNOS) and Ca2+-independent inducible (iNOS) activity and arginase activity were assessed in sperm cells of patients with different forms of pathospermia.ResultsWe found a significant increase in iNOS activity, but significantly decreased cNOS and arginase activity in sperm cells of infertile men vs fertile, normozoospermic men (p<0.001). The arginase/NOS ratio significantly decreased compared to control group. The iNOS/cNOS ratio was drastically increased in patients with decreased fertility potential indicating predominance of iNOS. Men with leuko cytospermia were distinguished to have the most express iNOS activity.ConclusionsThese observations provide evidence for a disturbed balance between the L-arginine metabolic pathways in sperm cells of infertile men. This imbalance includes the considerable activation of the inducible isoform of NO-synthase accompanied by significant inhibition of its constitutive isoform which indicates disturbances in NO production. In patients with decreased fertility potential the arginase/NOS was shifted towards predominance of iNOS-derived NO production.

Ca2+-dependent regulation and binding of calmodulin to multiple sites of Transient Receptor Potential Melastatin 3 (TRPM3) ion channels

TRPM3 proteins assemble to Ca2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca2+. Conceivably, this effect is attributed to the Ca2+ binding protein calmodulin, which binds to TRPM3 in a Ca2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin.

The role of proteins Stim and Orai as molecular components of the store-dependent current Ca2+ in lymphocytes

In the process of evolution of eukaryotes has formatted a highly organized mechanism for maintaining and regulating intracellular calcium homeostasis, which is one of the most important components of cell signaling in all branches of the phylogenetic tree. Intracellular calcium controls numerous physiological processes in the cell. Ca2+ forms signals as their spatial-temporal distribution. The frequency and amplitude of calcium oscillations depends on the signal strength. Calcium signals causing long-term or short-term responses of cells. Mainly, calcium signals in lymphocytes mediate gene expression program initiation that leads to proliferation, differentiation and production of proinflammatory cytokines also activate formation of inflammasome. Therefore, calcium signals mediate immune, and inflammatory response, autoimmune reaction of lymphocytes. The main mechanism of calcium signaling in lymphocytes is store-dependent Ca2+ current. Mobilization of cellular Ca2+ in response to receptor stimulation commonly occurs through release of Ca2+ ions from intracellular Ca2+ stores or influx across the plasma membrane through calcium - selective channels. Calciumselective channels are assembled from two protein families: the Orai proteins which form the ion channel pore, and the stromal interaction molecule (STIM) proteins which function as endoplasmic reticulum calcium sensors and activators of the channel. Stim protein is a transmembrane monomer which is localized at the membrane of the endoplasmic reticulum. This molecule is a sensor Ca2+ in response to emptying store activates calciumselective channels the plasma membrane. These channels express proteins Orai which are tetramers forming inside the channel pore and act as a site Ca2+. Orai binds to Stim. Orai proteins are activated after receiving information from Stim about Store depletion. Thus, the relationship and coordination of Stim and Orai proteins provides store - dependent Ca2+ current and causes cellular functional responses. Increased Ca2+ levels induce the activation of transcription factors such as NFAT, JNK1, MEF2, CREB, and, in most cases, is a crucial factor in the all differentiation or death. In this review, the mechanism of the store-dependent Ca2+ current in lymphocytes is presented.

Heart automaticity in mice lacking L-type Cav1.3 and T-type Cav3.1 Ca2+ channels: Insights into the cardiac pacemaker mechanism

Sino-atrial node (SAN) pacemaker activity is generated by ion channels of the plasma membrane, such as hyperpolarization-activated “funny” f-(HCN), Ca 2+ channels and ryanodine receptor (RyR) – dependent Ca 2+ release from the sarcoplasmic reticulum (SR). It is currently disputed whether Ca 2+ release from RyRs could sustain viable pacemaker activity provided preserved SR Ca 2+ content. While working myocytes express L-type Ca v 1.2 channels to maintain SR Ca 2+ content, SAN cells express also L-type Ca v 1.3 and T-type Ca v 3.1 channels to generate pacemaking. We used mutant mice carrying concomitant ablation of Ca v 1.3 and Ca v 3.1 (Ca v 1.3 −/− /Ca v 3.1 −/− ) to study the importance of these channels in automaticity. We also investigated the role of f-HCN channels and RyR-dependent Ca 2+ release in residual pacemaker activity of mutant mice. We employed in vivo telemetric recordings of heart rate (HR) in Ca v 1.3 −/− , Ca v 3.1 −/− and Ca v 1.3 −/− /Ca v 3.1 −/− mice. We studied the consequences of pharmacologic inhibition of f-HCN and TTX-sensitive Na + channels in mutant mice using Langendorff perfused hearts or optical mapping (OM) of the pacemaker impulse in intact SAN preparations (SANs). Ca v ablation reduced HR in mice: Ca v 3.1 −/− (−7.6%, n = 11), Ca v 1.3 −/− (−24.4%, n = 8), Ca v 1.3 −/− /Ca v 3.1 −/− (−35%, n = 11). In OM experiments on SANs, concomitant inhibition of f-HCN and Na v 1.1 channels slowed pacemaking in wild-type (−48%, n = 7) and Ca v 3.1 −/− (−37%, n = 7), while arresting automaticity in 4/6 of Ca v 1.3 −/− , 3/6 of Ca v 1.3 −/− /Ca v 3.1 −/− . When present, residual pacemaking was reduced by 82%. Similar results were obtained using isolated Ca v 1.3 −/− /Ca v 3.1 −/− pacemaker cells were automaticity arrested in 5/9 cells tested, or was reduced by 80% in 4/9 cells. Heart automaticity is primarily generated by Ca v 1.3 and f-HCN channels. RyR-dependent Ca 2+ release cannot sustain automaticity following concomitant targeting of Ca v 1.3 and f-HCN channels.

Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone.

In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1 -adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3 )-dependent Ca2+ transients activate intermediate conductance Ca2+ -activated K+ (IKCa ) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel-mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel-mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+ -activated K+ (SKCa ) channels in response to increases in shear stress appeared to be the primary mediators of endothelial modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel-mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction. Constriction of isolated resistance arteries in response to α1 -adrenoceptor agonists is limited by reciprocal engagement of inhibitory endothelial mechanisms via myoendothelial feedback. In the current model of feedback, agonist stimulation of smooth muscle cells results in localized InsP3 -dependent Ca2+ transients that activate endothelial IKCa channels. The subsequent hyperpolarization of the endothelial membrane potential then feeds back to the smooth muscle to limit further reductions in vessel diameter. We hypothesized that the functional contribution of InsP3 -IKCa channel-mediated myoendothelial feedback to limiting arterial diameter may be influenced by the nature of the vasoconstrictor stimulus. To test this hypothesis, we investigated the functional role of myoendothelial feedback in modulating responses of rat mesenteric resistance arteries to the adrenoceptor agonist noradrenaline, the thromboxane A2 mimetic U46619, increases in intravascular pressure and stimulation of perivascular sympathetic nerves. In isolated arteries, responses to noradrenaline and stimulation of sympathetic nerves, but not to U46619 and increases in intravascular pressure, were modulated by IKCa channel-dependent myoendothelial feedback. In the intact mesenteric bed perfused under conditions of constant flow, responses to exogenous noradrenaline were modulated by myoendothelial feedback, but shear stress-induced release of NO and activation of endothelial SKCa channels appeared to be the primary mediators of endothelial modulation of vasoconstriction to agonists and nerve stimulation. Thus, we propose that myoendothelial feedback may contribute to local control of diameter within arterial segments, but at the level of the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.

Cooperative CRF and α1 Adrenergic Signaling in the VTA Promotes NMDA Plasticity and Drives Social Stress Enhancement of Cocaine Conditioning

SUMMARYStressful events rapidly trigger activity-dependent synaptic plasticity, driving the formation of aversive memories. However, it remains unclear how stressful experience affects plasticity mechanisms to regulate appetitive learning, such as intake of addictive drugs. Using rats, we show that corticotropin-releasing factor (CRF) and α1 adrenergic receptor (α1AR) signaling enhance the plasticity of NMDA-receptor-mediated glutamatergic transmission in ventral tegmental area (VTA) dopamine (DA) neurons through distinct effects on inositol 1,4,5-triphosphate (IP3)-dependent Ca2+ signaling. We find that CRF amplifies IP3-Ca2+ signaling induced by stimulation of α1ARs, revealing a cooperative mechanism that promotes glutamatergic plasticity. In line with this, acute social defeat stress engages similar cooperative CRF and α1AR signaling in the VTA to enhance learning of cocaine-paired cues. These data provide evidence that CRF and α1ARs act in concert to regulate IP3-Ca2+ signaling in the VTA and promote learning of drug-associated cues.

The Spatial Distribution of the Na+/Ca2+ Exchanger in Cardiac Mitochondria Enhances the Efficincy of the Mitochondrial Ca2+ Signal Generation

In cardiac muscle, mitochondria take up Ca2+ through the Ca2+ uniporter (mtCU), while Ca2+ is extruded predominantly via the 3Na+/Ca2+ exchanger (NCLX). Both transports are electrogenic, thus consume mitochondrial membrane potential (dPsim). We have reported that mtCU is strategically positioned in cardiac muscle at the mitochondria-junctional SR (Mito-jSR) association to support efficient local Ca2+ signaling. Last year we presented data suggesting that NCLX-mediated Ca2+ extrusion is largely excluded from Mito-jSR association areas. We developed fractionation-based assay comparing mitochondria in the mitochondrial fraction (8,500∗g, less Mito-jSR associations) to mitochondria in the jSR (40,000∗g, more jSR associations) fraction. WB analysis found most NCLX in the mitochondrial fraction and 45Ca2+ flux assays showed more efficient Na+-dependent Ca2+ extrusion in the mitochondrial fraction. Now, we have validated these assays using NCLX cKO mice. Mitochondrial fraction from NCLX cKO showed both, diminished NCLX levels and Na+-dependent Ca2+ extrusion, confirming that the Ca2+ extrusion is indeed mediated by NCLX. Furthermore, transgenic NCLX overexpression resulted in significant increase in Na+-dependent Ca2+ extrusion in the mitochondrial but not the jSR fraction, underscoring the efficacy of NCLX exclusion from the dyad area (overexpression cannot override it). Finally, we wanted to test the concept whether increasing NCLX in the same domain as mtCU increased the energy (dPsim) expense to attain a given [Ca2+]m signal. Thus, we performed dPsim and [Ca2+]m measurements in permebilized H9c2 cells overexpressing NCLX (transiently transfected with NCKX6-FLAG). Upon 10µM Ca2+ addition, by the time of the [Ca2+]m peak the associated dPsim drop was larger in the NCLX-overexpressing than control cells. Collectively, mitochondrial Ca2+ uptake and Na+-dependent extrusion are reciprocally polarized, likely to optimize the energy-efficiency of [Ca2+]m signals arisen mostly from Ca2+ entry at the dyadic interface.

Brain-state dependent astrocytic Ca2+ signals are coupled to both positive and negative BOLD-fMRI signals

Astrocytic Ca2+-mediated gliovascular interactions regulate the neurovascular network in situ and in vivo. However, it is difficult to measure directly both the astrocytic activity and fMRI to relate the various forms of blood-oxygen-level-dependent (BOLD) signaling to brain states under normal and pathological conditions. In this study, fMRI and GCaMP-mediated Ca2+ optical fiber recordings revealed distinct evoked astrocytic Ca2+ signals that were coupled with positive BOLD signals and intrinsic astrocytic Ca2+ signals that were coupled with negative BOLD signals. Both evoked and intrinsic astrocytic calcium signal could occur concurrently or respectively during stimulation. The intrinsic astrocytic calcium signal can be detected globally in multiple cortical sites in contrast to the evoked astrocytic calcium signal only detected at the activated cortical region. Unlike propagating Ca2+ waves in spreading depolarization/depression, the intrinsic Ca2+ spikes occurred simultaneously in both hemispheres and were initiated upon the activation of the central thalamus and midbrain reticular formation. The occurrence of the intrinsic astrocytic calcium signal is strongly coincident with an increased EEG power level of the brain resting-state fluctuation. These results demonstrate highly correlated astrocytic Ca2+ spikes with bidirectional fMRI signals based on the thalamic regulation of cortical states, depicting a brain-state dependency of both astrocytic Ca2+ and BOLD fMRI signals.

Actin polymerization contributes to enhanced pulmonary vasoconstrictor reactivity after chronic hypoxia.

Chronic hypoxia (CH) augments basal and endothelin-1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction after CH. To test this hypothesis, both end points were monitored in pressurized, endothelium-disrupted pulmonary arteries (fourth-fifth order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous actin (F-actin)-to-globular actin (G-actin) ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor small-molecule inhibitor of formin homology domain 2. Immunoblot analysis revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin but not phosphorylated cofilin-to-total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity after CH in a ROS- and ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization after CH is dependent on mDia but independent of changes in the phosphorylated cofilin-to-total cofilin ratio. NEW & NOTEWORTHY This research is the first to demonstrate a role for actin polymerization in chronic hypoxia-induced basal pulmonary arterial constriction and enhanced agonist-induced vasoconstrictor activity. These results suggest that a reactive oxygen species-Rho kinase-actin polymerization signaling pathway mediates this response and may provide a mechanistic basis for the vasoconstrictor component of pulmonary hypertension.

Voltage dependence of the Ca2+ transient in endocardial and epicardial myocytes from the left ventricle of Goto-Kakizaki type 2 diabetic rats.

Diabetes mellitus is a major global health disorder and, currently, over 450million people have diabetes with 90% suffering from type 2 diabetes. Left untreated, diabetes may lead to cardiovascular diseases which are a leading cause of death in diabetic patients. Calcium is the trigger and regulator of cardiac muscle contraction and derangement in cellular Ca2+ homeostasis, which can result in heart failure and sudden cardiac death. It is of paramount importance to investigate the regional involvement of Ca2+ in diabetes-induced cardiomyopathy. Therefore, the aim of this study was to investigate the voltage dependence of the Ca2+ transients in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of the Goto-Kakizaki (GK) rats, an experimental model of type 2 diabetes mellitus. Simultaneous measurement of L-type Ca2+ currents and Ca2+ transients was performed by whole-cell patch clamp techniques. GK rats displayed significantly increased heart weight, heart weight/body weight ratio, and non-fasting and fasting blood glucose compared to controls (CON). Although the voltage dependence of L-type Ca2+ current was unaltered, the voltage dependence of the Ca2+ transients was reduced to similar extents in EPI-GK and ENDO-GK compared to EPI-CON and ENDO-CON myocytes. TPK L-type Ca2+ current and Ca2+ transient were unaltered. THALF decay of L-type Ca2+ current was unaltered; however, THALF decay of the Ca2+ transient was shortened in ENDO and EPI myocytes from GK compared to CON rat hearts. In conclusion, the amplitude of L-type Ca2+ current was unaltered; however, the voltage dependence of the Ca2+ transient was reduced to similar extents in EPI and ENDO myocytes from GK rats compared to their respective controls, suggesting the possibility of dysfunctional sarcoplasmic reticulum Ca2+ transport in the GK diabetic rat hearts.

Multivariate statistics and hydrogeochemical modeling for source identification of major elements and heavy metals in the groundwater of Qareh-Ziaeddin plain, NW Iran

The present study aims to evaluate the possible source of major and some minor elements and heavy metals in the groundwater of Qareh-Ziaeddin plain, NW Iran with respect to chemical elements, saturation index, and multivariate statistics including correlation coefficient, cluster analysis, and factor analysis. Groundwater samples were collected in Jun 2016 and measured with respect to EC, pH, major and some minor elements and heavy metals including Fe, Mn, Zn, Cr, Pb, Cd, Al, and As. Among all the measured parameters, some of the samples exceed the World Health Organization (WHO) guideline value for EC, Na, Mg, HCO3, SO4, Cl, NO3, F, As, Zn, and Pb. The results of correlation analysis show that weathering and dissolution of minerals especially evaporites and silicates, water-rock interaction, and cation exchange are dominant occurred processes in the groundwater of the study area. Also, denitrification process is occurred in the groundwater system. Cluster analysis categorizes the samples into three distinct groups which are different based on their EC and dependent variables, e.g., Na, Ca, Cl, SO4 and pH, Pb, Cd, and As. It can be found that volcanic, evaporite, and clay formations have the least impact on the chemistry of the cluster 1 samples while clay and evaporite formations have the highest impact on the cluster 3 and also calcareous formations on cluster 2. Factor analysis shows that five factors, with total variance of 83%, are effective in the release of heavy metals and groundwater chemistry which are mostly geogenic.

Angiotensin II, a unique vasoactive agent dissociates myosin light chain phosphorylation from contraction.

Angiotensin II (100 nM) induced bi-phasic increases in cytosolic Ca2+ level ([Ca2+]i) through the activation of angiotensin II type 1 receptor. Pharmacological examinations using 10 µM verapamil, 30 µM La3+, and 1 µM thapsigargin indicated that the first phase of the [Ca2+]i-increase was mediated by Ca2+ release from sarcoplasmic reticulum (SR) and Ca2+ influx independently of voltage dependent Ca2+ channel (VDC). In contrast, the second phase of [Ca2+]i-increase was mediated by Ca2+ influx through VDC. Although both [Ca2+]i and myosin light chain (MLC)-phosphorylation at the first phase was apparently exceeded the threshold for contraction as estimated by high K+-induced responses, there was no appreciable contraction, indicating the dissociation between MLC phosphorylation and force during this phase. In contrast, the second phase of [Ca2+]i was associated with the increases in both MLC phosphorylation and force. These results suggest that angiotensin II is a unique agonist which dissociates MLC-phosphorylation from muscle force during the Ca2+ releases from SR.

AMP-activated protein kinase–mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β

The Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr144 phosphorylation by activated AMPK converts CaMKKβ into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKKβ.

The Role of Na:K:2Cl Cotransporter 1 (NKCC1/SLC12A2) in Dental Epithelium during Enamel Formation in Mice.

Na+:K+:2Cl− cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl− transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl−-dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl− ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.