Modulation Of Intracellular Ca2 Research Articles

Purinergic regulation of pulmonary vascular tone.

Purinergic signaling is a crucial determinant in the regulation of pulmonary vascular physiology and presents a promising avenue for addressing lung diseases. This intricate signaling system encompasses two primary receptor classes: P1 and P2 receptors. P1 receptors selectively bind adenosine, while P2 receptors exhibit an affinity for ATP, ADP, UTP, and UDP. Functionally, P1 receptors are associated with vasodilation, while P2 receptors mediate vasoconstriction, particularly in basally relaxed vessels, through modulation of intracellular Ca2+ levels. The P2X subtype receptors facilitate extracellular Ca2+ influx, while the P2Y subtype receptors are linked to endoplasmic reticulum Ca2+ release. Notably, the primary receptor responsible for ATP-induced vasoconstriction is P2X1, with α,β-meATP and UDP being identified as potent vasoconstrictor agonists. Interestingly, ATP has been shown to induce endothelium-dependent vasodilation in pre-constricted vessels, associated with nitric oxide (NO) release. In the context of P1 receptors, adenosine stimulation of pulmonary vessels has been unequivocally demonstrated to induce vasodilation, with a clear dependency on the A2B receptor, as evidenced in studies involving guinea pigs and rats. Importantly, evidence strongly suggests that this vasodilation occurs independently of endothelium-mediated mechanisms. Furthermore, studies have revealed variations in the expression of purinergic receptors across different vessel sizes, with reports indicating notably higher expression of P2Y1, P2Y2, and P2Y4 receptors in small pulmonary arteries. While the existing evidence in this area is still emerging, it underscores the urgent need for a comprehensive examination of the specific characteristics of purinergic signaling in the regulation of pulmonary vascular tone, particularly focusing on the disparities observed across different intrapulmonary vessel sizes. Consequently, this review aims to meticulously explore the current evidence regarding the role of purinergic signaling in pulmonary vascular tone regulation, with a specific emphasis on the variations observed in intrapulmonary vessel sizes. This endeavor is critical, as purinergic signaling holds substantial promise in the modulation of vascular tone and in the proactive prevention and treatment of pulmonary vascular diseases.

New Insights into the Reparative Angiogenesis after Myocardial Infarction.

Myocardial infarction (MI) causes massive loss of cardiac myocytes and injury to the coronary microcirculation, overwhelming the limited capacity of cardiac regeneration. Cardiac repair after MI is finely organized by complex series of procedures involving a robust angiogenic response that begins in the peri-infarcted border area of the infarcted heart, concluding with fibroblast proliferation and scar formation. Efficient neovascularization after MI limits hypertrophied myocytes and scar extent by the reduction in collagen deposition and sustains the improvement in cardiac function. Compelling evidence from animal models and classical in vitro angiogenic approaches demonstrate that a plethora of well-orchestrated signaling pathways involving Notch, Wnt, PI3K, and the modulation of intracellular Ca2+ concentration through ion channels, regulate angiogenesis from existing endothelial cells (ECs) and endothelial progenitor cells (EPCs) in the infarcted heart. Moreover, cardiac repair after MI involves cell-to-cell communication by paracrine/autocrine signals, mainly through the delivery of extracellular vesicles hosting pro-angiogenic proteins and non-coding RNAs, as microRNAs (miRNAs). This review highlights some general insights into signaling pathways activated under MI, focusing on the role of Ca2+ influx, Notch activated pathway, and miRNAs in EC activation and angiogenesis after MI.

Optical modulation of excitation-contraction coupling in human-induced pluripotent stem cell-derived cardiomyocytes.

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), and modulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology.

E as in Enigma: The Mysterious Role of the Voltage-Dependent Anion Channel Glutamate E73

The voltage-dependent anion channel (VDAC) is the main passageway for ions and metabolites over the outer mitochondrial membrane. It was associated with many physiological processes, including apoptosis and modulation of intracellular Ca2+ signaling. The protein is formed by a barrel of 19 beta-sheets with an N-terminal helix lining the inner pore. Despite its large diameter, the channel can change its selectivity for ions and metabolites based on its open state to regulate transport into and out of mitochondria. VDAC was shown to be regulated by a variety of cellular factors and molecular partners including proteins, lipids and ions. Although the physiological importance of many of these modulatory effects are well described, the binding sites for molecular partners are still largely unknown. The highly symmetrical and sleek structure of the channel makes predictions of functional moieties difficult. However, one residue repeatedly sticks out when reviewing VDAC literature. A glutamate at position 73 (E73) located on the outside of the channel facing the hydrophobic membrane environment was repeatedly proposed to be involved in channel regulation on multiple levels. Here, we review the distinct hypothesized roles of E73 and summarize the open questions around this mysterious residue.

KCNJ8/ABCC9-containing K-ATP channel modulates brain vascular smooth muscle development and neurovascular coupling

Loss- or gain-of-function mutations in ATP-sensitive potassium channel (K-ATP)-encoding genes, KCNJ8 and ABCC9, cause human central nervous system disorders with unknown pathogenesis. Here, using mice, zebrafish, and cell culture models, we investigated cellular and molecular causes of brain dysfunctions derived from altered K-ATP channel function. We show that genetic/chemical inhibition or activation of KCNJ8/ABCC9-containing K-ATP channel function leads to brain-selective suppression or promotion of arterial/arteriolar vascular smooth muscle cell (VSMC) differentiation, respectively. We further show that brain VSMCs develop from KCNJ8/ABCC9-containing K-ATP channel-expressing mural cell progenitor and that K-ATP channel cell autonomously regulates VSMC differentiation through modulation of intracellular Ca2+ oscillation via voltage-dependent calcium channels. Consistent with defective VSMC development, Kcnj8 knockout mice showed deficiency in vasoconstrictive capacity and neuronal-evoked vasodilation leading to local hyperemia. Our results demonstrate a role for KCNJ8/ABCC9-containing K-ATP channels in the differentiation of brain VSMC, which in turn is necessary for fine-tuning of cerebral blood flow.

Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer's Disease on the Pump Activity.

Since dysregulation of intracellular calcium (Ca2+) levels is a common occurrence in neurodegenerative diseases, including Alzheimer’s disease (AD), the study of proteins that can correct neuronal Ca2+ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca2+-ATPase (PMCA), a high-affinity Ca2+ pump, is functionally impaired in AD and is inhibited by amyloid-β peptide (Aβ) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca2+-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and other modulators of intracellular Ca2+ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aβ and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aβ and tau on the pump, and counteracts the neurotoxicity of Aβ and tau by interacting with them.

Sevoflurane modulates breast cancer cell survival via modulation of intracellular calcium homeostasis

BackgroundSome retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis.MethodsEstrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects.ResultsSevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell.ConclusionSevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.

Numerical Study of Voltage-Gated Ca2+ Transport Irradiated by Terahertz Electromagnetic Wave

This paper simulates Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> transmembrane transport through voltage-gated Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> channels in response to terahertz electromagnetic irradiation. The active transport of Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> ions is taken into considerations in the Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> transport. Temperature variations due to terahertz electromagnetic loss in physiological medium are simulated. The electromagnetic interaction between terahertz fields and physiological mobile ions at the cellular level is deduced from relativistic electrodynamics. It shows that effects of 0.1 ~ 3 THz electromagnetic fields on cell mobile ions are primarily due to effects of electric fields, and effects of magnetic fields at the cellular level are insignificant. In addition, numerical simulation reveals that terahertz irradiation causes vibration of membrane potential, which is able to activate voltage-gated Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> channels. Besides, bioeffects of terahertz frequency, irradiation duration and electric intensity on the increment of intracellular Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> concentration due to activation of voltage-gated Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> channels are revealed. Meanwhile, numerical results show that temperature rises are inconsequential in the case of different irradiation parameters, indicating the non-thermal bioeffects of voltage-gated Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> transmembrane transport due to terahertz irradiation. Furthermore, the results also reveal that thermal bioeffect can be significant if the irradiation duration is raised long enough for high-dose terahertz irradiation. The numerical simulations lay the basis for understanding the bioeffects of terahertz irradiation on Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> transmembrane transport and pave the way for further exploration in modulation of intracellular Ca <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2+</sup> concentration with terahertz electromagnetic wave.

TRP Channels Mediated Pathological Ca2+-Handling and Spontaneous Ectopy.

Ion channel biology offers great opportunity in identifying and learning about cardiac pathophysiology mechanisms. The discovery of transient receptor potential (TRP) channels is an add-on to the opportunity. Interacting with numerous signaling pathways, being activated multimodally, and having prescribed signatures underlining acute hemodynamic control and cardiac remodeling, TRP channels regulate cardiac pathophysiology. Impaired Ca2+-handling cause contractile abnormality. Modulation of intracellular Ca2+ concentration ([Ca2+]i) is a major part of Ca2+-handling processes in cardiac pathophysiology. TRP channels including TRPM4 regulate [Ca2+]i, Ca2+-handling and cardiac contractility. The channels modulate flux of divalent cations, such as Ca2+ during Ca2+-handling and cardiac contractility. Seminal works implicate TRPM4 and TRPC families in intracellular Ca2+ homeostasis. Defective Ca2+-homeostasis through TRP channels interaction with Ca2+-dependent regulatory proteins such as sodium calcium exchanger (NCX) results in abnormal Ca2+ handling, contractile dysfunction and in spontaneous ectopy. This review provides insight into TRP channels mediated pathological Ca2+-handling and spontaneous ectopy.

PSEN2 (presenilin 2) mutants linked to familial Alzheimer disease impair autophagy by altering Ca2+ homeostasis

ABSTRACTPSEN2 (presenilin 2) is one of the 3 proteins that, when mutated, causes early onset familial Alzheimer disease (FAD) cases. In addition to its well-known role within the γ-secretase complex (the enzyme ultimately responsible for Aβ peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca2+ homeostasis. Here, by using different FAD-PSEN2 cell models, we demonstrate that mutated PSEN2 impairs autophagy by causing a block in the degradative flux at the level of the autophagosome-lysosome fusion step. The defect does not depend on an altered lysosomal functionality but rather on a decreased recruitment of the small GTPase RAB7 to autophagosomes, a key event for normal autophagy progression. Importantly, FAD-PSEN2 action on autophagy is unrelated to its γ-secretase activity but depends on its previously reported ability to partially deplete ER Ca2+ content, thus reducing cytosolic Ca2+ response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca2+ signaling in autophagy and reveal a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for a dysfunctional quality control pathway in AD neurodegeneration.Abbreviations: Aβ: amyloid β; AD: Alzheimer disease; ACTB: actin beta; AMPK: AMP-activated protein kinase; APP: amyloid-beta precursor protein; BafA: bafilomycin A1; BAPTA-AM: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; CFP: cyan fluorescent protein; EGTA-AM: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; ER: endoplasmic reticulum; EGFP-HDQ74: enhanced GFP-huntingtin exon 1 containing 74 polyglutamine repeats; FAD: familial Alzheimer disease; FCS: fetal calf serum; FRET: fluorescence/Förster resonance energy transfer; GFP: green fluorescent protein; IP3: inositol trisphosphate; KD: knockdown; LAMP1: lysosomal associated membrane protein 1; MAP1LC3-II/LC3-II: lipidated microtubule-associated protein 1 light chain 3; MCU: mitochondrial calcium uniporter; MICU1: mitochondrial calcium uptake 1; MEFs: mouse embryonic fibroblasts; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; SQSTM1/p62: sequestosome 1; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB7: RAB7A: member RAS oncogene family; RFP: red fluorescent protein; ATP2A/SERCA: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting; siRNA: small interference RNA; V-ATPase: vacuolar-type H+-ATPase; WT: wild type

Electropharmacological effects of intracellular Ca2+ handling modulator caldaret on the heart assessed in the halothane-anesthetized dogs

We analyzed how the enhancement of net sarcoplasmic reticulum (SR) Ca2+ uptake may affect cardiac electrophysiological properties in vivo by using caldaret which can decrease SR diastolic Ca2+ leak, enhance SR Ca2+ reuptake and inhibit reverse-mode Na+/Ca2+ exchanger. Caldaret in doses of 0.5, 5 and 50 μg/kg was intravenously administered over 10 min to the halothane-anesthetized beagle dogs (n = 5), attaining pharmacologically active plasma concentration. The low and middle doses of caldaret increased the ventricular contraction, which could be explained by its on-target pharmacological activities. The high dose enhanced the sinus automaticity followed by its suppression in addition to the increase of the total peripheral resistance, which may be unfavorable for treating diastolic heart failure. The low and middle doses enhanced the atrioventricular conduction, which may have some potential for predisposing the atria to the onset of atrial fibrillation via an induction of mitral and/or tricuspid regurgitation. The middle and high doses of caldaret prolonged the ventricular effective refractory period without altering the intraventricular conduction or repolarization period, which may prevent the onset of ventricular arrhythmias. Thus, modulation of intracellular Ca2+ handling by caldaret can induce not only inotropic effect, but also various electrophysiological actions on the in situ heart.

Catalpol Inhibits Ischemia-Induced Premyelinating Oligodendrocyte Damage through Regulation of Intercellular Calcium Homeostasis via Na⁺/Ca2+ Exchanger 3.

The heightened vulnerability of premyelinating oligodendrocytes (PreOLs) in response to hypoxia–ischemia may contribute to perinatal white matter injury and subsequent neurobehavioral dysfunction. Intracellular Ca2+ overload is considered a crucial mechanism predisposing PreOLs to ischemic injury. We previously reported that catalpol, an iridoid glycoside extracted from Rehmannia root, inhibits intracellular Ca2+ overload of PreOLs in an in vitro ischemia model. However, the exact underlying mechanisms remain elusive. In the present study, we aimed to investigate the protective effects of catalpol on PreOLs and to explore the underlying mechanisms involved in the modulation of intracellular Ca2+ homeostasis. Postnatal day 2 (P2) Sprague-Dawley (SD) rats subjected to bilateral common carotid artery ligation followed by exposure to 8% oxygen for 10 min were used as a rat model of neonatal hypoxia–ischemia. We found that catalpol significantly improved behavioral functions and prevented PreOL loss and myelination deficit after hypoxia–ischemia. Our in vitro studies also confirmed the direct effects of catalpol on oxygen-glucose deprivation (OGD)-induced cell death and arrested maturation of PreOLs. Moreover, we demonstrated that catalpol significantly inhibited intracellular Ca2+ overload and promoted the expression of Na+/Ca2+ exchanger 3 (NCX3). Finally, we found that catalpol significantly reduced mitochondrial damage and subsequent extracellular signal-regulated kinase 1/2 (ERK1/2) and poly-ADP-ribose polymerase-1 (PARP-1) activation. Treatment with NCX3-preferring inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943) significantly reversed the protective effects of catalpol on PreOLs under OGD. Overall, our data suggest that catalpol protects PreOLs from ischemic injury through regulation of intercellular Ca2+ homeostasis via upregulation of NCX3 activity.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca²⁺ in Cat Esophageal Smooth Muscle Cells.

A comprehensive collection of proteins senses local changes in intracellular Ca2+ concentrations ([Ca2+]i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca2+ concentrations in cat esophageal smooth muscle cells. To measure [Ca2+]i levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca2+]i in the cells. Pretreatment with EGTA, an extracellular Ca2+ chelator, decreased the S1P-induced increase in [Ca2+]i, and an L-type Ca2+-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca2+ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP3 receptor blocker, the S1P-evoked increase in [Ca2+]i was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of Gi-protein, suppressed the increase in [Ca2+]i evoked by S1P. These results suggest that the S1P-induced increase in [Ca2+]i in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca2+ from the InsP3-sensitive Ca2+ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca2+ via the L type Ca2+ channel, which was dependent on activation of the S1P4 receptor coupled to PTX-sensitive Gi protein, via phospholipase C-mediated Ca2+ release from the InsP3-sensitive Ca2+ pool in cat esophageal smooth muscle cells.

Induction of oxidative stress and sensitization of cancer cells to paclitaxel by gold nanoparticles with different charge densities and hydrophobicities.

An elevated reactive oxygen species (ROS) level leads to cellular oxidative stress, which has long been associated with diseases, such as cancer. Thus, the understanding and appropriate manipulation of cellular oxidative stress are needed for disease treatment. It has been reported that nanoparticles induce oxidative stress in human cells through different pathways. However, how the physicochemical properties of nanoparticles perturb cellular oxidative stress remains unclear. In this paper, we explored the effects of the positive/negative charge density and hydrophobicity of gold nanoparticles (GNPs) on the induction of oxidative stress and related mechanisms. In multiple human cell lines, we found that only the positive charge density and hydrophobicity of nanoparticles were correlated with the induction of cellular oxidative stress. Hydrophobic nanoparticles generated oxidative stress mainly through NADPH oxidase activation while positively charged nanoparticles generated it through perturbations of the mitochondria and modulation of intracellular Ca2+ concentration. Furthermore, nanoparticle-induced oxidative stress sensitized paclitaxel-induced cancer cell killing by 200%. These findings provided unequivocal structural parameters for the design of future nanomedicine and biocompatible nanocarriers.

Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/β-catenin pathway.

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1. We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt/β-Catenin pathway.

TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway

ABSTRACTRationale: Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored.Objective: To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action.Methods and Results: Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+]i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca2+]i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca2+]i and velocity of shortening as well as an acceleration in [Ca2+]i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1−/− mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14–22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) (1–5 min) that correlated with increases in CM [Ca2+]i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca2+]i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L).Conclusions: These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca2+ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.

ConBr, A Lectin Purified from the Seeds of Canavalia brasiliensis, Protects Against Ischemia in Organotypic Culture of Rat Hippocampus: Potential Implication of Voltage-Gated Calcium Channels.

Lectins are proteins that bind cellular glycans and can modulate various neuronal functions. We have evaluated the neuroprotective effect of ConBr, a lectin purified from the seeds of Canavalia brasiliensis in a model of rat organotypic hippocampal cultures (OHCs) exposed to oxygen and glucose deprivation (OGD). OGD for 15min followed by 24h re-oxygenation significantly increased cell death, caused mitochondrial depolarization and increased reactive oxygen species (ROS) in CA1 region of OHCs. ConBr (0.1μg/mL) added during the re-oxygenation period counteracted cell death, mitochondrial depolarization and overproduction of ROS induced by OGD. Moreover, ConBr restored the levels of Akt and ERK1 phosphorylation that were reduced by OGD. Modulation of intracellular Ca2+ by ConBr was evaluated in isolated hippocampal neurons loaded with the fluorescent calcium dye Fluo-4/AM. ConBr (0.1 and 1µg/mL) reduced by 25-30 % the Ca2+ increment induced by 70mM K+. A sub effective concentration of ConBr (0.01µg/mL) together with a sub effective concentration of the L-type calcium channel antagonist nifedipine (0.3µM) conferred a synergic neuroprotective effect in OHCs subjected to OGD. In conclusion, ConBr provides OHCs neuroprotection against OGD. The mechanism was not fully addressed but it may involve modulation of L-type voltage-gated Ca2+ channels by ConBr.

Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase

BackgroundSERCA maintains intracellular Ca2+ homeostasis by sequestering cytosolic Ca2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:19-cis) and linoleamide (18:29,12-cis), induce an increase in intracellular Ca2+ levels, which might be caused by their inhibition of SERCA. MethodsThree major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca2+-ATPase activity of SERCA was examined. ResultsThe Ca2+-ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC50 15–53μM) and partially by oleamide (IC50 8.3–34μM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:19-trans), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca2+ and the apparent maximum velocity of Ca2+-ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45μM in linoleamide and from 1.6 to 55μM in oleamide depending on the isoform. ConclusionsLinoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca2+ affinity of the enzyme. General significanceOur findings imply a novel role of these PFAAs as modulators of intracellular Ca2+ homeostasis via regulation of SERCA activity.

Berberine alleviates the cerebrovascular contractility in streptozotocin-induced diabetic rats through modulation of intracellular Ca²⁺ handling in smooth muscle cells.

BackgroundVascular dysfunction is a distinctive phenotype in diabetes mellitus. Current treatments mostly focus on the tight glycemic control and few of these treatments have been designed to directly recover the vascular dysfunction in diabetes. As a classical natural medicine, berberine has been explored as a possible therapy for DM. In addition, it is reported that berberine has an extra-protective effect in diabetic vascular dysfunction. However, little is known whether the berberine treatment could ameliorate the smooth muscle contractility independent of a functional endothelium under hyperglycemia. Furthermore, it remains unknown whether berberine affects the arterial contractility by regulating the intracellular Ca2+ handling in vascular smooth cells (VSMCs) under hyperglycemia.MethodsSprague–Dawley rats were used to establish the diabetic model with a high-fat diet plus injections of streptozotocin (STZ). Berberine (50, 100, and 200 mg/kg/day) were intragastrically administered to control and diabetic rats for 8 weeks since the injection of STZ. The intracellular Ca2+ handling of isolated cerebral VSMCs was investigated by recording the whole-cell L-type Ca2+ channel (CaL) currents, assessing the protein expressions of CaL channel, and measuring the intracellular Ca2+ in response to caffeine. Our results showed that chronic administration of 100 mg/kg/day berberine not only reduced glucose levels, but also inhibited the augmented contractile function of cerebral artery to KCl and 5-hydroxytryptamine (5-HT) in diabetic rats. Furthermore, chronic administration of 100 mg/kg/day berberine significantly inhibited the CaL channel current densities, reduced the α1C-subunit expressions of CaL channel, decreased the resting intracellular Ca2+ ([Ca2+]i) level, and suppressed the Ca2+ releases from RyRs in cerebral VSMCs isolated from diabetic rats. Correspondingly, acute application of 10 μM berberine could directly inhibit the hyperglycemia-induced CaL currents and suppress the hyperglycemia-induced Ca2+ releases from RyRs in cerebral VSMCs isolated from normal control rats.ConclusionsOur study indicated that berberine alleviated the cerebral arterial contractility in the rat model of streptozotocin-induced diabetes via regulating the intracellular Ca2+ handling of smooth muscle cells.

Drugs That Inhibit The Sarcoplasmic Reticulum Ca2+ ATPase (SERCA) and Prevention of Breast Cancer Cell Proliferation

Breast cancer is the most common cancer in women and a leading cause of death worldwide. Pharmacological modulation of intracellular Ca2+ homeostasis has been proposed as a potential target for therapeutic intervention for breast cancer. In this regard, thapsigargin and cyclopiazonic acid inhibit the Ca2+ ATPase of sarcoplasmic reticulum (SERCA) that pumps Ca2+ into the endoplasmic reticulum (ER) intracellular Ca2+ stores and display high efficacy for killing breast cancer cells. However, these “classic” SERCA blockers are highly toxic and also kill normal cells, thereby preventing use for anticancer therapy. We have determined that new drugs in the benzothiazepine (BZT) family (K201, CGP37157), as well as agents like pimozide and sipatrigine can inhibit SERCA through a novel Ca2+ dependent mechanism. These agents would not affect Ca2+ sequestration in ER when cytosolic Ca2+ levels are high, but potently inhibit this process when cytosolic Ca2+ approaches resting cell levels, which could help prevent ER overload under pathological conditions (ischemia) and provide cellular protection in normal tissues. Therefore, we tested the effectiveness of these drugs to inhibit growth of two breast cancer cell lines, MCF‐7 and MDA‐231, in culture. Our preliminary studies indicate that K201 inhibits MCF‐7 breast cancer cell growth and synergizes with anti‐cancer drugs to increase their effectiveness in killing these cells; thus, MCF‐7 cells are not “protected” by the decreased ER Ca2+ load provided by the SERCA inhibitors; in fact these drugs make them less viable. Building upon this data, we compared the effects of a “classic” SERCA blocker (thapsigargin) to the Ca2+ dependent SERCA blockers above on MCF‐7 and MDA‐231 breast cancer cells. We determined that all SERCA blockers effectively disrupted the metabolic activity of MCF‐7 cells but were much less effective in MDA‐231. In MCF‐7 cells, SERCA blockers also increased the effects of chemotherapeutic drugs (cisplatin, doxorubicin). The results indicate that targeting SERCA has potential to interfere with cancer cell growth. However, our results also suggest that resistance to chemotherapy in MDA‐231 may relate to these cancer cells becoming less dependent on intracellular Ca2+ signaling. We have also determined some of the structural characteristics that are required for BZT to block SERCA. Finally, we also found that drugs reported to do both, protection of normal cells in ischemia and inhibition of cancer cell proliferation, can also display significant SERCA blocking ability. These agents may serve as potential templates for developing new anti‐cancer drugs that target intracellular Ca2+ homeostasisSupport or Funding InformationAPS Frontiers in Physiology Research Fellowship to MLSIUSOM Team Development Award to SAT and JAC