Mediator Of Ca2 Research Articles

Insights into the mediation of Ca2+ signaling in the promoting effects of LETX-VI on the synthesis and release of dopamine.

Latroeggtoxin-VI (LETX-VI) is an active protein and was previously demonstrated to have effects on the synthesis and release of dopamine. Hererin, the involvement of Ca2+ signaling in the effects of LETX-VI on dopamine was systematically investigated, using PC12 cells as a neuron model. LETX-VI was shown to promote dopamine release from PC12 cells both in the presence and absence of extracellular Ca2+; however the presence of extracellular Ca2+ was favorable for enhancing the promoting effects of LETX-VI on dopamine, because LETX-VI facilitated the influx of extracellular Ca2+ through the L-type calcium channels in plasma membrane (PM) to increase cytosolic Ca2+ concentration. LETX-VI was able to penetrate the PM of PC12 cells to act on the Ca2+ channel proteins IP3Rs and RyRs in the endoplasm reticulum (ER) membrane, opening the Ca2+ channels and promoting the release of ER Ca2+ to elevate cytosolic Ca2+ level. With the help of intracellular Ca2+ chelator BAPTA, the elevated cytosolic Ca2+ level was proven to play crucial role for the enhanced promoting effects of LETX-VI on dopamine. Taken together, LETX-VI is able to open the Ca2+ channels in both PM and ER membrane simultaneously to facilitate extracellular Ca2+ influx and ER Ca2+ release, and thus increases the cytosolic Ca2+ concentration to enhance the promoting effects on the synthesis and release of dopamine.

A biomimetic peptide–drug supramolecular hydrogel as eyedrops enables controlled release of ophthalmic drugs

The rapid clearance of instilled drugs from the ocular surface due to tear flushing and excretion results in low drug bioavailability, necessitating the development of new drug delivery routes. Here, we generated an antibiotic hydrogel eye drop that can extend the pre-corneal retention of a drug after topical instillation to address the risk of side effects (e.g., irritation and inhibition of enzymes), resulting from frequent and high-dosage administrations of antibiotics used to obtain the desired therapeutic drug concentration. The covalent conjugation of small peptides to antibiotics (e.g., chloramphenicol) first endows the self-assembly ability of peptide-drug conjugate to generate supramolecular hydrogels. Moreover, the further addition of calcium ions, which are also widely present in endogenous tears, tunes the elasticity of supramolecular hydrogels, making them ideal for ocular drug delivery. The in vitro assay revealed that the supramolecular hydrogels exhibited potent inhibitory activities against both gram-negative (e.g., Escherichia coli) and gram-positive (e.g., Staphylococcus aureus) bacteria, whereas they were innocuous toward human corneal epithelial cells. Moreover, the in vivo experiment showed that the supramolecular hydrogels remarkably increased pre-corneal retention without ocular irritation, thereby showing appreciable therapeutic efficacy for treating bacterial keratitis. This work, as a biomimetic design of antibiotic eye drops in the ocular microenvironment, addresses the current issues of ocular drug delivery in the clinic and further provides approaches to improve the bioavailability of drugs, which may eventually open new directions to resolve the difficulty of ocular drug delivery. Statement of SignificanceHerein, we present a biomimetic design for antibiotic hydrogel eye drops mediated by calcium ions (Ca2+) in the ocular microenvironment, which can extend the pre-corneal retention of antibiotics after topical instillation. The mediation of Ca2+ which is widely present in endogenous tears, tunes the elasticity of hydrogels, making them ideal for ocular drug delivery. Since increasing the ocular retention of antibiotic eye drops enhances its action and reduces its adverse effects, this work may lead to an approach of peptide–drug-based supramolecular hydrogel for ocular drug delivery in clinics to combat ocular bacterial infections.

S100A1 expression is increased in spinal cord injury and promotes inflammation, oxidative stress and apoptosis of PC12 cells induced by LPS via ERK signaling.

Spinal cord injury (SCI) is a severe neurological disorder and the molecular mechanisms leading to its poor prognosis remain to be elucidated. S100A1, a mediator of Ca2+ handling of sarcoplasmic reticulum and mitochondrial function, operates as an endogenous danger signal (alarmin) associated with inflammatory response and tissue injury. The aim of the present study was to investigate the expression and biological effects of S100A1 in SCI. A rat model of SCI and a PC12 cell model of lipopolysaccharide (LPS)‑induced inflammation were established to examine S100A1 expression at the mRNA and protein levels. The inflammation level, which was mediated by S100A1, was determined based on inflammatory factor (IL‑1β, IL‑6 and TNF‑α) and anti‑inflammatory factor (IL‑10) expression. The effects of S100A1 on cellular oxidation and anti‑oxidation levels were observed by detecting the levels of reactive oxygen species, superoxide dismutase, catalase activities and nuclear factor erythroid 2‑related factor 2 expression. The protein levels of Bax, Bcl2 and cleaved caspase‑3 were used for the evaluation of the effects of S100A1 on apoptosis. Phosphorylated (p‑)ERK1/2 expression was used to evaluate the effects of S100A1 on ERK signaling. The results revealed that S100A1 expression was significantly upregulated invivo and invitro in the PC12 cell model of LPS‑inflammation. The silencing and overexpression of S100A1 helped alleviate and aggravate LPS‑induced inflammation, oxidative stress and apoptosis levels, respectively. S100A1 was found to regulate the ERK signaling pathway positively. An inhibitor of ERK signaling (MK‑8353) partially abolished the promoting effects of the overexpression of S100A1 on inflammation, oxidative stress damage and apoptosis. In conclusion, S100A1 expression was elevated in model of SCI and in the PC12 cell model of LPS‑induced inflammation. Furthermore, the overexpression/silencing S100A1 aggravated/mitigated the inflammation, oxidative stress damage and the apoptosis of LPS‑stimulated PC12 cells via the ERK signaling pathway. The present study revealed the mechanism of S100A1 in SCI, which provided a new theoretic reference for future research on SCI.

Role of Ca2+ channels in non-alcoholic fatty liver disease and their implications for therapeutic strategies (Review)

Non-alcoholic fatty liver disease (NAFLD) is a clinically progressive illness that can advance from simple fatty liver to non-alcoholic hepatitis and liver fibrosis. Cirrhosis and hepatocellular carcinoma are two of the most common diseases caused by NAFLD. As there are no early disease biomarkers and no US Food and Drug Administration-approved medications, treatment for NAFLD is still focused on altering lifestyle and dietary habits, which makes it difficult to treat effectively. As a result, a novel treatment is urgently needed to prevent NAFLD progression. Calcium (Ca2+) channels regulate intracellular Ca2+ homeostasis via the mediation of Ca2+ flow. Previous studies have reported that Ca2+ channel expression varies throughout the development and progression of NAFLD, which results in the dysregulation of intracellular Ca2+ homeostasis, endoplasmic reticulum stress, mitochondrial dysfunction and autophagy suppression, all of which contribute to NAFLD progression. Several types of Ca2+ channels (including two-pore segment channel 2, transient receptor potential, inositol triphosphate receptor, voltage-dependent anion channel 1, store-operated Ca2+ entry, purinergic receptor X7 and potassium Ca2+-activated channel subfamily N member 4) have been identified as potential targets for preventing NAFLD development and controlling intracellular Ca2+ homeostasis. To achieve this, these channels can be blocked or activated, which exerts anti-steatotic, anti-inflammatory, anti-fibrotic and other effects, which ultimately prevents the development of NAFLD. In the present review NAFLD therapeutics and the treatments that target Ca2+ channels that are currently being developed were examined.

Transcriptome analysis of primary podocytes reveals novel calcium regulated regulatory networks.

Podocytes are pivotal in establishing the selective permeability of the glomerular filtration barrier. Recently, we showed that an increase of the intracellular calcium ion concentration [Ca2+ ] causes a rapid and transient actin reset (CaAR) measurable through live imaging microscopy using lifeact-mCherry as an actin dye in different cell types including the podocyte. This and other studies show the critical role [Ca2+ ] and the actin cytoskeleton play in podocyte homeostasis. To further investigate the role of [Ca2+ ] and the actin cytoskeleton in podocytes, we used a double fluorescent reporter mouse model to establish a primary podocyte culture system. We treated these podocytes temporarily with a Calcium Ionophore and facultatively with Latrunculin A, an inhibitor of actin polymerization. Unbiased genome wide transcriptional analysis identified a transcriptional response in podocytes to elevated [Ca2+ ] levels, affecting mRNA levels of PDGF-BB, RICTOR, and MIR17HG as mediators of Ca2+ -signaling. Comparison of the ex vivo transcriptional response from the primary podocyte culture with glomerular transcripts across a wide spectrum of CKD disease confirmed co-regulation of transcript sets, establishing the disease relevance of the model system. Our findings demonstrate novel [Ca2+ ] regulated gene networks in podocytes deepening our understanding of podocyte biology and disease.

PbGLR3.3 Regulates Pollen Tube Growth in the Mediation of Ca2+ Influx in Pyrus bretschneideri

Glutamate receptors (GluRs) are sensors of extracellular signals; they play important roles in the regulation of multiple physiological and developmental processes in eukaryotes. However, their functional roles in fruit trees are largely unknown. Here, based on the pear genome database, which was established in this lab, we identified 34 PbGLRs in pear (Pyrus bretschneideri Rehd), and they were divided into four groups by phylogenetic analysis. In comparisons with other groups, phylogenetic analyses and structural information of the PbGLRs in group 3 suggest that these genes underlie specific characteristics. Among the ten genes in group 3, we observed that the expression of PbGLR3.3 increased gradually during pollen germination and continuous growth, indicating that this gene might play a vital role in the development of pear pollen tubes. Using a combination of antisense oligodeoxy nucleotides and Ca2+-sensitive fluorescent probe methods, we verified that PbGLR3.3 participates in DSer-elicited intracellular Ca2+ signaling and Ca2+ regulation of growth in pear pollen tubes.

Abstract 3552: Induction of store operated calcium entry (SOCE) suppresses glioblastoma growth by inhibiting the Hippo pathway transcriptional coactivators YAP/TAZ

Abstract Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here, we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca2+ entry by enhancing store-operated Ca2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca2+-induced Lats1/2 activation. Ca2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca2+ is a crucial intracellular cue that regulates the Hippo pathway, and that triggering SOCE could be a strategy to target YAP/TAZ in GBM. Citation Format: Zhijun Liu. Induction of store operated calcium entry (SOCE) suppresses glioblastoma growth by inhibiting the Hippo pathway transcriptional coactivators YAP/TAZ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3552.

Abstract 2221: Necroptosis and apoptosis in human colorectal tissue

Abstract Background: The molecular mechanisms for colorectal carcinogenesis still remain elusive. In recent years, necroptosis has emerged as a programmed cell death regulated by RIP kinases /mixed lineage kinase domain-like (MLKL) pathway, involving the release of intracellular "danger signals" which results in inflammation. On the other hand, classic apoptosis is regulated by Bax-induced caspase pathway and does not lead to inflammation. Transient receptor potential melastatin related 7 (TRPM7), a MLKL downstream target for the mediation of Ca2+ influx, was found to be involved in TNF-induced necroptosis (Nat Cell Bio 2014;16:55). Both necroptosis and apoptosis are important barriers to prevent normal cells from developing cancer. No study has examined how necroptosis biomarkers (MLKL, TRPM7) and pro-apoptosis biomarker (Bax) are related to inflammation (COX-2) and cell proliferation (Ki-67) and how MLKL relates to TRPM7 in human colorectal tissue. Methods: In our randomized trial (R01CA149633, NCT01105169) “Personalized Prevention of Colorectal Cancer Trial” conducted among colorectal polyp patients or those at high risk of colorectal cancer, colorectal biopsies from 250 participants were collected prior to intervention in the trial. Formalin-fixed paraffin-embedded tissues were serially sectioned, and three levels of serial sections spaced 50 µm apart were mounted on one slide for each tissue block. The protein expressions of COX-2, Bax, pMLKL, TRPM7, and Ki-67 were detected immunohistochemically following the EnVision+ System-HRP kit. Positive cells in the upper and bottom zones were evaluated separately by a zonal quantitative analysis procedure using BioQuant imaging software. Correlation analysis was used to compute Pearson product-moment correlation coefficient between necroptosis, apoptosis and inflammation biomarkers. Results: The expression of selected biomarkers showed stronger positive signals in the upper zone than in the bottom zone, with the exception of Ki-67 which showed stronger staining in the bottom zone of crypts. Epithelial Bax had strong positive correlation with COX-2 in the same zones (r=0.25 - 0.36, p< 0.001), and overall Bax expression showed weaker association with stromal COX-2 (r=0.13, p=0.04). pMLKL and TRPM7 were not significantly correlated with COX-2 except a weak correlation between stromal COX-2 and overall TRPM7 (r=-0.174, p=0.008). pMLKL expression was positively correlated with TRPM7 in the upper zone of crypts (r=0.19, p=0.004), and both pMLKL and TRPM7 were not significantly correlated with Bax expression. Conclusions: Our study confirmed a positive correlation of pMLKL and TRPM7, as suggested by previous cell studies, in human non-tumoral colorectal tissue. The positive correlation between Bax and COX-2 suggests that elevated inflammation may result in increased apoptosis. On the other hand, pMLKL is not related to COX-2, although stromal COX-2 is inversely associated with overall TRPM7. Citation Format: Timothy Su, Yong Li, Chang Yu, Martha J. Shrubsole, Xiangzhu Zhu, Xinqing Deng, Eugene Shubin, Wei Zheng, Harvey J. Murff, Douglas L. Seidner, Reid M. Ness, Qi Dai. Necroptosis and apoptosis in human colorectal tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2221.

The Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis via Ca2+ CaMKII–Dependent MAPK and Akt/ mTOR Pathways in a Rat Osteoarthritis Model

Background/Aims: Chondrocyte apoptosis is a central pathological feature of cartilage in osteoarthritis (OA). Accumulating evidence suggests that calcium ions (Ca²⁺) are an important regulator of apoptosis. Previously, we reported that the transient receptor potential channel vanilloid (TRPV5) is upregulated in monoiodoacetic acid (MIA)-induced OA articular cartilage. Methods: The protein levels of TRPV5, phosphorylated Ca²⁺/calmodulin-dependent kinase II (p-CaMKII), and total CaMKII were detected in vivo using western blotting techniques. Primary chondrocytes were isolated and cultured in vitro. Then, p-CAMKII was immunolocalized by immunofluorescence in chondrocytes. Fluo-4AM staining was used to assess intracellular Ca²⁺. Annexin V-fluorescein isothiocyanate / propidium iodide flow cytometric analysis was performed to determine chondrocyte apoptosis. Western blotting techniques were used to measure the expression of apoptosis-related proteins. Results: We found that ruthenium red (aTRPV5inhibitor)or(1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaze (KN-62) (an inhibitor of Ca^2+/calmodulin-dependent kinase II (CaMKII) phosphorylation) can relieve or even reverse OA in vivo. We found that TRPV5 has a specific role in mediating extracellular Ca²⁺ influx leading to chondrocyte apoptosis in vitro. The apoptotic effect in chondrocytes was inhibited by KN-62. We found that activated p-CaMKII could elicit the phosphorylation of extracellular signal-regulated protein kinase 1/2, c-Jun N-terminal kinase, and p38, three important regulators of the mitogen-activated protein kinase (MAPK) cascade. Moreover, we also showed that activated p-CaMKII could elicit the phosphorylation of protein kinase B (Akt) and two important downstream regulators of mammalian target of rapamycin (mTOR): 4E-binding protein, and S61 kinase. Conclusion: Our results demonstrate that upregulated TRPV5 may be an important initiating factor that activates CaMKII phosphorylation via the mediation of Ca²⁺ influx. In turn, activated p-CaMKII plays a critical role in chondrocyte apoptosis via MAPK and Akt/mTOR pathways.

Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis in a Rat Osteoarthritis Model Through the Mediation of Ca2+ Influx

Background/Aims: Chondrocyte apoptosis is the most common pathological feature in cartilage in osteoarthritis (OA). Transient receptor potential channel vanilloid 5 (TRPV5) is important in regulating calcium ion (Ca²⁺) influx. Accumulating evidences suggest that Ca²⁺ is a major intracellular second messenger that can trigger cell apoptosis. Therefore, we investigate the potential role of TRPV5 in mediating Ca²⁺ influx to promote chondrocyte apoptosis in OA. Methods: The monoiodoacetic acid (MIA)-induced rat OA model was assessed by macroscopic and radiographic analyses. Calmodulin protein immunolocalization was detected by immunohistochemistry. The mRNA and protein level of TRPV5, calmodulin and cleaved caspase-8 in articular cartilage were assessed by real time polymerase chain reaction and western blotting. Primary chondrocytes were isolated and cultured in vitro. TRPV5 small interfering RNA was used to silence TRPV5 in chondrocytes. Then, calmodulin and cleaved caspase-8 were immunolocalized by immunofluorescence in chondrocyte. Fluo-4AM staining was used to assess intracellular Ca²⁺ to reflect TRPV5 function of mediation Ca²⁺ influx. Annexin V-fluorescein isothiocyanatepropidium iodide flow cytometric analysis was performed to determine chondrocytes apoptosis. Western blotting techniques were used to measure the apoptosis-related proteins in chondrocyte level. Results: Here, we reported TRPV5 was up-regulated in MIA-induced OA articular cartilage. Ruthenium red (a TRPV5 inhibitor) can relieve progression of joint destruction in vivo which promoted us to demonstrate the effect of TRPV5 in OA. We found that TRPV5 had a specific role in mediating extracellular Ca²⁺ influx leading to chondrocytes apoptosis in vitro. The apoptotic effect was inhibited even reversed by silencing TRPV5. Furthermore, we found that the increase Ca²⁺ influx triggered apoptosis by up-regulating the protein of death-associated protein, FAS-associated death domain, cleaved caspase-8, cleaved caspase-3, cleaved caspase-6, and cleaved caspase-7, and the up-regulated proteins were abolished by silencing TRPV5 or 1, 2-bis-(o-Aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (a Ca²⁺ chelating agent). Conclusion: The up-regulated TRPV5 could used be as an initiating factor that induces extrinsic chondrocyte apoptosis via the mediation of Ca²⁺ influx. These findings suggested TRPV5 could be an intriguing mediator for drug target in OA.

Carbon Monoxide as a Mediator of Ca2+ Signaling for Insulin Secretion in Pancreatic Islets

Glucagon‐like peptide‐1 (GLP‐1) increases intracellular Ca2+ concentrations ([Ca2+]i), resulting in insulin secretion from pancreatic β‐cells through the sequential production of Ca2+ mobilizing second messengers: nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP‐ribose (cADPR). Ca2+ signals and gasotransmitter formation are closely related in maintaining cellular signaling function, including insulin secretion from pancreatic β‐cells. However, the interrelationship between Ca2+ signals and gas formation is poorly understood. Previously, we demonstrated that NAADP activates the neuronal type of nitric oxide (NO) synthase (nNOS) during the stimulation of insulin secretion in β‐cells. NO activates guanylyl cyclase (GC) to produce cyclic GMP (cGMP), which in turn induces cADPR formation. We investigated the influence of GLP‐1/NAADP on nNOS activity. NAADP regulates nNOS enzyme activity by increasing CaMKII phosphorylation levels, which was abolished by pre‐treatment with a CaMKII inhibitor (AIP). We then studied the effects of NO and carbon monoxide (CO) on cGMP formation and Ca2+ signals by using a NO donor (SNP) and a CO donor (CORM‐3). Exogenous NO and CO amplify cGMP formation, Ca2+ signal strength, and insulin secretion in mouse islets. However, this signal is impeded when exposed to combined treatment with NO and CO. Furthermore, CO potentiates cGMP formation in a dose‐dependent manner in the range of 10–100 μM, whereas higher doses of CORM‐3 inhibited cGMP formation, suggesting that high CO concentrations block GC activity. Furthermore, our data with regards to ZnPP, a HO inhibitor, and heme oxygenase (HO)‐2 knockdown revealed that NO‐induced cADPR formation and insulin secretion is dependent on HO. HO‐2 knockdown or ZnPP blocked NO‐induced cGMP formation and insulin secretion in pancreatic islets, whereas exogenous CO repaired the blockade. These results demonstrate that CO, a downstream regulator of NO, plays a role in bridging the gap between the Ca2+ signaling second messengers.

Transient Receptor Potential Vanilloid 5 Mediates Ca2+ Influx and Inhibits Chondrocyte Autophagy in a Rat Osteoarthritis Model

Background: Autophagy, a self-protective mechanism of chondrocytes, has become a promising target to impede the progress of osteoarthritis (OA). Autophagy is regulated by cytosolic Ca²⁺ activity and may thus be modified by the Ca²⁺ permeable transient receptor potential channel vanilloid 5 (TRPV5). Therefore, we investigated the potential role of TRPV5 in mediating Ca²⁺ influx and in inhibiting chondrocyte autophagy in a rat OA model. Methods: The rat OA model was assessed by macroscopic and histological analyses. light chain 3B (LC3B) immunolocalization was detected by immunohistochemistry. TRPV5, LC3B and calmodulin in OA articular cartilage were assessed by real time polymerase chain reaction (RT-PCR) and western blotting. TRPV5 small interfering RNA (TRPV5 siRNA) were transfected into rat primary chondrocyte then the calmodulin and LC3B was detected by immunofluorescence. The functionality of the TRPV5 was assessed by Ca²⁺ influx. Western blot was used to measure autophagy-related proteins. Results: We constructed a monosodium iodoacetate (MIA) -induced rat OA model and found that ruthenium red (TRPV5 inhibitor) slowed the progression of joint destruction. We found that the TRPV5 and calmodulin were up-regulated but LC3B was down-regulated in articular cartilage following prolonged progression of OA. Furthermore, the up-regulated TRPV5 channel caused an increase in the Ca²⁺ influx in chondrocytes. The up-regulation of TRPV5 stimulated Ca²⁺ influx, which inhibited autophagy by increasing the production of calmodulin, phosphorylation of calmodulin dependent protein kinases II (p-CAMK II), phosphorylation of Beclin1 (p-Beclin1), and protein of B-cell lymphoma-2 (Bcl-2), and attenuating ratio of LC3-II/ LC3-. Conclusion: Up-regulated TRPV5 as an initiating factor inhibited chondrocyte autophagy via the mediation of Ca²⁺ influx.

The MAPK pathway is involved in the regulation of rapid pacing-induced ionic channel remodeling in rat atrial myocytes

Alterations to the expression L‑type calcium channels (LTCCs) and Kv4.3 potassium channels form the possible basis of atrial electrical remodeling during rapid pacing. The mitogen‑activated protein kinase (MAPK) pathway is affected by increases in cytoplasmic Ca2+, and therefore represents an attractive candidate for the regulation and mediation of Ca2+‑induced ion channel remodeling. The present study aimed to investigate alterations to the ion channel‑MAPK axis, and to determine its influence on ion channel remodeling during atrial fibrillation. Rat atrial myocytes were isolated, cultured, and in vitro rapid pacing was established. Intracellular Ca2+ signals were monitored using the Fluo‑3/AM Ca2+ indicator. Verapamil, PD98058 and SB203580 were added to the culture medium of various groups at specific time‑points. The mRNA expression levels of LTCC‑α1c and Kv4.3 potassium channels were detected by reverse transcription‑polymerase chain reaction. Western blotting was performed to determine the expression levels of channel and signaling proteins. The results demonstrated that fast pacing significantly increased the intracellular Ca2+ concentration in atrial myocytes, whereas treatment with verapamil markedly inhibited this increase. In addition, verapamil significantly antagonized the rapid pacing‑induced activation of extracellular signal‑regulated kinase (ERK) and p38MAPK. These results indicated that the MAPK pathway may have an important role in the opening of LTCCs, and alterations to MAPK molecule expression could affect the expression and remodeling of ion channels.

Gap Junction-associated Na+ Influx Is Involved in the Mediation of Ca2+ Transients in Neonatal Rat Ventricular Myocytes.

Gap junction (GJ) coupling is involved in the regulation of spontaneous Ca(2+) transients in neonatal rat ventricular myocytes (NRVMs); however, the underlying mechanism(s) remains incompletely clear. In this study, we investigated Na influx mediated by Cx43-associated GJ modulating the spontaneous NRVM excitation. Intracellular Na(+) and Ca(2+) concentrations ([Na(+)](i) and [Ca(2+)](i)) were assessed using CoroNa Green and Fluo-4 fluorescent probes, respectively, by confocal microscopy. GJ function was evaluated by measuring fluorescence recovery after photobleaching. The results showed that [Na(+)](i) and spontaneous Ca(2+) oscillating were significantly decreased by the treatment of NRVMs with GJ uncouplers or adenovirus-mediated Cx43 gene knockdown using short hairpin RNA, while both of the intracellular cation levels were increased by adenovirus-mediated Cx43 overexpression. Neither Na(+) channel blocker (tetrodotoxin) nor the GJ uncoupler (Gap27) or Cx43-short hairpin RNA adenovirus could completely suppress the Ca(2+) transients, but their combination usage could. In addition, Ca(2+) transients disappeared in NRVMs incubated in Na(+)-free, but not in Ca(2+) -free medium, in which the remained transients could be further abolished by Gap27. Collectively, our findings suggest that Cx43-associated GJ function is involved in the regulation of Na(+) influx into cardiomyocytes, which contributes, at least in part, to triggering spontaneous excitation and regulation of cardiomyocyte automaticity.

STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration

STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca2+ entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca2+ emptying triggered by the inhibition of the sarco(endo)plasmic Ca2+-ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca2+, indicating that both events are upstream steps for Ca2+ entry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca2+ channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.

The impact of nitric oxide on calcium homeostasis in PE/CA-PJ15 cells

ObjectiveNitric oxide (NO) production and Ca2+ homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca2+ homeostasis in a highly complex and cell-specific manner and although Ca2+ homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca2+]c homeostasis in PE/CA-PJ15 cells. DesignCells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca2+ concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La3+ were employed to perturb intracellular calcium levels. ResultsNO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca2+]c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca2+ homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca2+]c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca2+]c was also reduced in the presence of La3+ or thapsigargin. ConclusionsNO induces a concentration-dependent [Ca2+]c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca2+ buffering in the presence of TG or La3+.Further, we show that exogenous NO deregulates Ca2+ homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.

The Role of Tyrosine Kinase in Ca^(2+)-Independent Contraction of the Ropivacaine on Rat Aortic Smooth Muscle

The tyrosine kinase signaling pathway plays an important role in the mediation of Ca²⁺ independent mechanisms of smooth muscle contraction. Several components of this pathway, including protein kinase C (PKC), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and Rho-kinase are involved in Ca²⁺ independent mechanisms. Whether the tyrosine kinase pathway mediates vasoconstriction induced by the anesthetic ropivacaine remains unclear. The present study was designed to examine the role of tyrosine kinase in ropivacaine-induced, Ca²⁺-independent contraction of rat aortic smooth muscle. The effects of tyrosine kinase inhibitor on ropivacaine-induced contractile response were observed by isometric force measurement. The protein tyrosine phosphorylation, PKC, p44/42 MAPK, and membrane translocation of Rho-kinase were examined by Western blotting. Ropivacaine induced a concentration-dependent contractile response, and showed a number of effects on protein tyrosine phosphorylation. In this study, phosphorylation levels were shown to increase at lower concentrations of ropivacaine, but the levels decline at higher concentrations in rat aortic rings attenuated by the tyrosine kinase inhibitor genistein in a concentration-dependent fashion. Ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and Rho-kinase membrane translocation were also significantly attenuated by genistein in similar decreasing manner as the PKC inhibitor bisindolylmaleimide I (Bis I) and the Rho-kinase inhibitor, Y27632, but to a lesser degree than that by the p44/42 MAPK inhibitor, PD 098059. Our results showed that the ropivacaine-induced, Ca²⁺ independent-mediated contraction of rat aortic smooth muscle is, in part, regulated by tyrosine kinase-catalyzed protein tyrosine phosphorylation.

Calcium-Mediated Fusion between Endo-Lysosomal Compartments Enhances Virus-Like Particles Release

Assembly of the human immunodeficiency virus (HIV) is governed by the structural polyprotein Gag, which is necessary and sufficient for the release of virus-like particles (VLPs) from the host cell. Although the plasma membrane has been recognized as the major site for the production of VLPs, in some cell-types Gag is targeted to late endosomes/multivesicular bodies (LE/MVBs), where assembly and budding take place. Virus release into the extracellular space then occurs after regulated exocytosis. It is well accepted that the release of VLPs requires participation of different host cell components. In particular, it was shown that induction of a transient rise in cytoplasmic Ca2+ increased the amounts of VLPs in MVBs, and resulted in a dramatic enhancement of VLPs release (Perlman M. et al., 2006). However, although cellular factors have been already proposed as mediators of Ca2+ provision (Ehrlich L. et al., 2010), how Ca2+ can promote the release of VLPs remains to be determined.With FACS analyses on live cells, we could identify variations of intracellular Ca2+ in Gag-expressing cells treated with Ca2+ fluorescent indicators. High-resolution confocal and electron microscopy have confirmed that Gag can assemble and bud into VLPs in lysosomes (Ly) and LE. Furthermore, we could show for the first time that Ca2+ released specifically from those compartments causes formation of Ly/LE hybrid organelles, which in turn fuse with the PM and release VLPs into the extracellular space. This heterotypic fusion process requires components of the SNARE complex and the Ca2+ sensor protein Synaptotagmin VII, which regulates Ly exocytosis. All these elements constitute a productive pathway for virus assembly and release. We believe that the Gag protein itself, or a cellular factor recruited by Gag, might promote the increase of Ca2+ required for this process to function.

A transiently expressed connexin is essential for anterior neural plate development in Ciona intestinalis

A forward genetic screen in the ascidian Ciona intestinalis identified a mutant line (frimousse) with a profound disruption in neural plate development. In embryos with the frimousse mutation, the anteriormost neural plate cells, which are products of an FGF induction at the blastula and gastrula stages, initially express neural plate-specific genes but fail to maintain the induced state and ultimately default to epidermis. The genetic lesion in the frimousse mutant lies within a connexin gene (cx-11) that is transiently expressed in the developing neural plate in a temporal window corresponding to the period of a-lineage neural induction. Using a genetically encoded calcium indicator we observed multiple calcium transients throughout the developing neural plate in wild-type embryos, but not in mutant embryos. A series of treatments at the gastrula and neurula stages that block the calcium transients, including gap junction inhibition and calcium depletion, were also found to disrupt the development of the anterior neural plate in a similar way to the frimousse mutation. The requirement for cx-11 for anterior neural fate points to a crucial role for intercellular communication via gap junctions, probably through mediation of Ca(2+) transients, in Ciona intestinalis neural induction.

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca(2+) to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca(2+) provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca(2+) stores. Following activation by binding of its ligand, IP3, it releases Ca(2+) from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca(2+) signaling as a potential novel cofactor in viral particle release.