Effectiveness and Utility of Flowcharts on Learning in a Classroom Setting: A Mixed-Methods Study.

OBJECTIVE: Graphical representation of information organizes and promotes meaningful learning. As an example of graphical organizers, flowcharts can simplify and summarize complex information. The evidence of classroom use of flowcharts as an instructional tool is unclear. We investigated the effectiveness of flowcharts on student learning as an in-class instructional tool in a cardiovascular therapeutic course. Student experiences with the use and application of flowcharts were explored. METHODS: An explanatory sequential mixed-methods study was conducted with pharmacy students enrolled in an acute-care cardiovascular course from 2019-2021. The quantitative phase comprised a survey to determine flowchart effectiveness and a comparison of student performance in three content areas. The qualitative phase of the study used focused group interviews to understand student perceptions of flowchart use. RESULTS: Survey results indicated that using flowcharts improved understanding (110/128, 86%), integration of material (114/128, 89%), and overall knowledge (111/128, 87%). Student performance in the 3 content areas, shock, arrhythmia, and acute coronary syndrome were statistically significant with flowcharts implementation. Emerging themes from student interviews were (1) used as a medium for retention and recall, (2) used as a study tool, and (3) used as a decision-making framework. CONCLUSION: Flowcharts provide an alternative approach to teaching complex content, which allows students to organize and summarize information that promotes meaningful learning. The ease of implementation combined with the generalized nature of flowcharts makes it an effective graphical organizer that can be used across various disciplines.

Calcium handling genes are regulated by promoter DNA methylation in colorectal cancer cells.

Calcium signaling regulates various cellular processes, including proliferation and cell death. DNA methylation of gene promoters is an epigenetic modification that facilitates transcriptional suppression. Disruption of calcium homeostasis and DNA methylation in cancer are each linked to tumor development and progression. However, the possible connection between these two processes has not been thoroughly studied. Therefore, we measured the expression of six gene families involved in calcium regulation (ATP2A, ITPR, ORAI, RyR, STIM, and TRPC) in a colorectal cancer cell model, HCT116, with either genetic (Double Knock-out/DKO) or pharmacological (5-aza-2'-deoxycytidine/DAC) inhibition of DNA methyltransferases. Fourteen of the 20 examined calcium handling genes were expressed at higher levels in DKO cells as compared to HCT116. Expression of five genes was increased in HCT116 cells treated with DAC, three matching DKO. Due to a unique expression pattern of the three ATP2A genes in our model, encoding the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) pumps, we chose to evaluate the methylation status of these genes, protein expression, and potential associated physiological effects, using the SERCA inhibitor thapsigarin (TG). We observed an expected pattern of promoter methylation coinciding with reduced expression and vice versa. This differential mRNA expression was associated with altered SERCA3 protein expression and cytosolic calcium levels with TG exposure. As a result, DKO cells displayed less TG-induced cytotoxicity, as compared to HCT116 cells. Overall, it is likely that at least several calcium regulatory genes are transcriptionally regulated by DNA methylation, and this may play a role in tumorigenesis through altering apoptosis in cancer.

The polyphenol ellagic acid exerts anti-inflammatory actions via disruption of store-operated calcium entry (SOCE) pathway activators and coupling mediators.

Ellagic acid, a naturally occurring phenol found in a variety of fruits and nuts has been shown to possess anti-inflammatory properties. However, the mechanism of action behind its anti-inflammatory action is unclear. Using human Jurkat T cells, our study examined the effects of ellagic acid (EA) on Ca2+ handling, in particular, store-operated Ca2+ entry (SOCE), a process critical to proper T cell function. We observed that the acute addition of EA-induced Ca2+ release with an EC50 of 63 µM. The Ca2+ release was significantly attenuated by Xestospongin C, a known inhibitor of the Inositol 1,4,5-trisphosphate receptor (IP3R) channel and was unaffected by the phospholipase C (PLC) inhibitor, U73122. Furthermore, chronic incubation of Jurkat T cells with EA not only decreased the ATP-induced Ca2+ release but also diminished the SOCE-mediated Ca2+ influx in a dose-dependent manner. This inhibition was confirmed by reduced Mn2+ entry rates in the EA-treated cells. The ATP-induced Ca2+ entry was also attenuated in EA-treated HEK293 cells transiently transfected with SOCE channel Orai1-myc and ER-sensor stromal interaction molecule (STIM1) (HEKSTIM/Orai). Moreover, EA treatment interfered with the Orai1 and STIM1 coupling by disrupting STIM1 puncta formation in the HEKSTIM/Orai cells. We observed that EA treatment reduced cytokine secretion and nuclear factor of activated T-cell transcriptional activity in stimulated T cells. Hence, by inhibiting SOCE mediated Ca2+ influx, EA decreased downstream activation of pro-inflammatory mediators. These results suggest a novel target for EA-mediated effects and provide insight into the mechanisms underlying EA-mediated anti-inflammatory effects.

The Na+/Ca2+ exchange inhibitor 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) also blocks ryanodine receptors type 1 (RyR1) and type 2 (RyR2) channels.

Na(+)/Ca(2+) exchanger (NCX) is a plasma membrane transporter that moves Ca(2+) in or out of the cell, depending on membrane potential and transmembrane ion gradients. NCX is the main pathway for Ca(2+) extrusion from excitable cells. NCX inhibitors can ameliorate cardiac ischemia-reperfusion injury and promote high-frequency fatigue of skeletal muscle, purportedly by inhibiting the Ca(2+) inward mode of NCX. Here we tested two known NCX inhibitors, 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)-isothiourea methanesulfonate (KB-R7943) and the structurally related 2-[[4-[(4-Nitrophenyl)methoxy]phenyl]methyl]-4-thiazoli dinecarboxylic acid ethyl ester (SN-6), for their influence on electrically or caffeine-evoked Ca(2+) transients in adult dissociated flexor digitorum brevis (FDB) skeletal muscle fibers and human embryonic kidney (HEK) 293 cells that have stable expression of type 1 ryanodine receptor (RyR1). KB-R7943 (< or = 10 microM) reversibly attenuates electrically evoked Ca(2+) transients in FDB and caffeine-induced Ca(2+) release in HEK 293, whereas the structurally related NCX inhibitor SN-6 does not, suggesting that KB-R7943 directly inhibits RyR1. In support of this interpretation, KB-R7943 inhibits high-affinity binding of [(3)H]ryanodine to RyR1 (IC(50) = 5.1 +/- 0.9 microM) and the cardiac isoform RyR2 (IC(50) = 13.4 +/- 1.8 microM). KB-R7943 interfered with the gating of reconstituted RyR1 and RyR2 channels, reducing open probability (P(o)), shortening mean open time, and prolonging mean closed time. KB-R7943 was more effective at blocking RyR1 with cytoplasmic conditions favoring high P(o) compared with those favoring low P(o). SN-6 has negligible activity toward altering [(3)H]ryanodine binding of RyR1 and RyR2. Our results identify that KB-R7943 is a reversible, activity-dependent blocker of the two most broadly expressed RyR channel isoforms and contributes to its pharmacological and therapeutic activities.

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways.

Regulation of bi-directional communication between intracellular Ca(2+) pools and surface Ca(2+) channels remains incompletely characterized. We report Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca(2+) release, Ca(2+) influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP(3)R induced Ca(2+) release, whereas RyR-mediated Ca(2+) release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP(3)R and RyR linked Ca(2+) influx even though Ca(2+) pool sensitivities were different. These findings suggest discrete Ca(2+) store/Ca(2+) channel coupling mechanisms in the IP(3)R and RyR pathways as revealed by the differential sensitivity to actin perturbation.

Enantiomeric specificity of (-)-2,2',3,3',6,6'-hexachlorobiphenyl toward ryanodine receptor types 1 and 2.

Polychlorinated biphenyls (PCBs) with unsymmetrical chlorine substitutions and multiple orthosubstitutions that restrict rotation around the biphenyl bond may exist in two stable enantiomeric forms.Stereospecific binding and functional modification of specific biological signaling targets have not been previously described for PCB atropisomers. We report that (-)-2,2',3,3',6,6'-hexachlorobiphenyl [(-)-PCB 136] enhances the binding of [3H]ryanodine to high-affinity sites on ryanodine receptors type 1(RyR1) and type 2 (RyR2) (EC50 values ~0.95 microM), whereas (+)-PCB 136 is inactive at < or =10 microM.(-)-PCB 136 induces a rapid release of Ca2+ from microsomal vesicles by selective sensitization of RyRs, an effect not antagonized by (+)-PCB 136. (-)-PCB 136 (500nM) enhances the activity of reconstituted RyR1 channels 3-fold by stabilizing the open and destabilizing the closed conformational states. The enantiomeric specificity is also demonstrated in intact HEK 293 cells expressing RyR1 where exposure to (-)-PCB 136 (100 nM; 12 h) sensitizes responses to caffeine, whereas (+)-PCB 136 does not. These data show enantiomeric specificity of (-)-PCB 136 toward a broadly expressed family of microsomal Ca2+ channels that may extend to other chiral noncoplanar PCBs and related structures.Evidence for enantioselective enrichment of PCBs in biological tissues that express RyR1 and RyR2channels may provide new mechanistic leads about their toxicological impacts on human health

The size of zinc oxide nanoparticles controls its toxicity through impairing autophagic flux in A549 lung epithelial cells.

Zinc oxide nanoparticles (ZnONPs) widely used in various products, have been concerned with its impact on human health, in particular, on the risk of pulmonary toxicity. Our previous study indicated that ZnONPs could harness autophagy and impair the autophagic flux, which was positively linked to ZnONPs-induced toxicity. The objective of this study was to investigate whether ZnONPs-induced impairment of autophagic flux and cell death in lung epithelial cells is related to the size of ZnONPs. We demonstrate that ZnONPs with the average size of 50 nm could induce toxic effects in A549 lung epithelial cells, including accumulation of autophagosomes (the elevation of LC3B-II/LC3B-I ratio), impaired autophagic flux (the increase of p62 expression), the release of intracellular zinc ions (the increase of FluoZin-3 signal and ZnT1 mRNA expression), mitochondrial damage (the decrease of TMRE signal), lysosomal dysfunction (the aberrant expression of LAMP-2), oxidative stress (the increase of DCFH-DA signal and HO-1 expression) and cell death. Interestingly, ZnONPs with the average size of 200 nm failed to induce autophagy-mediated toxicity. Taken together, our results indicate that the size of ZnONPs is closely correlated with its toxicity, which is probably mediated by induction of impaired autophagic flux. This finding provides an insight into better understating of ZnONPs-associated toxicity, and mitigating the risk to humans and allowing the safer application.

Autophagy-dependent release of zinc ions is critical for acute lung injury triggered by zinc oxide nanoparticles.

Pulmonary exposure to zinc oxide nanoparticles (ZnONPs) could cause acute lung injury (ALI), but the underlying molecular mechanism remains unclear. Herein, we established a ZnONPs-induced ALI mouse model, characterized by the histopathological changes (edema and infiltration of inflammatory cells in lung tissues), and the elevation of total protein and cytokine interleukin-6 in bronchoalveolar lavage fluid in time- and dose-dependent manners. This model also exhibited features like the disturbance of redox-state (reduced of glutathione to glutathione disulfide ratio, elevation of heme oxygenase-1 and superoxide dismutase 2), the decrease of adenosine triphosphate synthesis and the release of zinc ions in the lung tissues. Interestingly, we found that ZnONPs exposure caused the accumulation of autophagic vacuoles and the elevation of microtubule-associated proteins 1A/1B light chain (LC)3B-II and p62, indicating the impairment of autophagic flux. Our data indicated that the above process might be regulated by the activation of AMP-activated protein kinase but not the mammalian target of rapamycin pathway. The association between ZnONPs-induced ALI and autophagy was further verified by a classical autophagy inhibitor, 3-methyladenine (3-MA). 3-MA administration reduced the accumulation of autophagic vacuoles, the expression of LC3B-II and p62, followed by a significant attenuation of histopathological changes, inflammation, and oxidative stress. More importantly, 3-MA could directly decrease the release of zinc ions in lung tissues. Taken together, our study provides the evidence that ZnONPs-induced pulmonary toxicity is autophagy-dependent, suggests that limiting the release of zinc ions by inhibiting autophagy could be a feasible strategy for the prevention of ZnONPs-associated pulmonary toxicity.

Zinc oxide nanoparticles harness autophagy to induce cell death in lung epithelial cells.

Although zinc oxide nanoparticles (ZnONPs) are widely used, they have raised concerns of toxicity in humans. Previous studies have indicated that reactive oxygen species (ROS) and autophagy are involved in the cytotoxicity of ZnONPs, but the regulatory mechanisms between autophagy and ROS remain to be elucidated. Herein, we comprehensively investigated the regulatory mechanism of autophagy and the link between autophagy and ROS in ZnONPs-treated lung epithelial cells. We demonstrated that ZnONPs could induce autophagy, and this process could enhance the dissolution of ZnONPs in lysosomes to release zinc ions. Sequentially, zinc ions released from ZnONPs were able to damage not only lysosomes, leading to impaired autophagic flux, but also mitochondria. Impaired autophagic flux resulted in the accumulation of damaged mitochondria, which could generate excessive ROS to cause cell death. We further demonstrated that the inhibition of autophagy by either pharmacological inhibitors or small interfering RNA (siRNA)-mediated knockdown of Beclin-1 and AMP-activated protein kinase could ameliorate ZnONPs-induced cell death. Moreover, we found that lysosomal-associated membrane protein 1/2 (LAMP-1/2), which were the most abundant highly glycosylated protein in late endosomes/lysosomes, exhibited aberrant expression pattern upon treatment with ZnONPs. Intriguingly, LAMP-2 knockdown, but not LAMP-1 knockdown, could exacerbate the ROS generation and cell death induced by ZnONPs treatment. Meanwhile, LAMP-2 overexpression alleviated ZnONPs-induced cell death, suggesting that LAMP-2 was linked to this toxic phenotype induced by ZnONPs. Our results indicate that autophagic dysfunction could contribute to excessive ROS generation upon treatment with ZnONPs in lung epithelial cells, suggesting that modulating the autophagy process would minimize ZnONPs-associated toxicity.

2-Aminoethoxydiphenyl borate (2-APB) stimulates a conformationally coupled calcium release pathway in the NG115-401L neuronal cell line.

We report in this study a 2-aminoethoxydiphenyl borate (2-APB) activated Ca2+ pathway in NG115-401L (401L) neuronal cells bearing resemblance to hormonal and ryanodine receptor activated pathways. We observed that 2-APB, in contrast to much earlier work, did not inhibit store operated Ca2+ channel (SOC) function, but rather induced potent Ca2+ discharge responses that robustly activated SOC-mediated Ca2+ influx. Further, these studies intriguingly revealed that the 2-APB-induced Ca2+ release pathway likely couples conformationally to targets in the plasma membrane, as membrane permeabilization or actin perturbation abolished the ability of the compound to stimulate Ca2+ signals. These findings suggest that conformationally sensitive complexes form between endoplasmic reticulum and plasma membrane components that not only regulate Ca2+ influx, previously proposed as the conformational coupling hypothesis, but are also required to promote Ca2+ release from intracellular stores. These observations further characterize the 401L neuronal cell line as having unique characteristics that may prove useful in gaining insight into the nature of the coupling mechanism linking Ca2+ release to Ca2+ influx.

Activation of ryanodine receptors induces calcium influx in a neuroblastoma cell line lacking calcium influx factor activity.

We have further characterized the Ca2+ signalling properties of the NG115-401L (or 401L) neuroblastoma cell line, which has served as an important cell line for investigating SOC (store-operated channel) influx pathways. These cells possess an unusual Ca2+ signalling phenotype characterized by the absence of Ca2+ influx when Ca2+ stores are depleted by inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). Previous studies found that Ca2+-store depletion does not produce a CIF (Ca2+ influx factor) activity in 401L cells. These observations have prompted the question whether 401L cells possess the signalling machinery that permits non-voltage-gated Ca2+ influx to occur. We tested the hypothesis that ryanodine-sensitive Ca2+ pools and activation of RyRs (ryanodine receptors) constitute a signalling pathway capable of inducing Ca2+ influx in 401L cells. We found that 401L cells express mRNA for RyR1 and RyR2 and that RyR activators induced Ca2+ release. Activation of RyRs robustly couples with Ca2+ influx responses in 401L cells, in sharp contrast with absence of Ca2+ influx when cells are treated with SERCA inhibitors. Thus it is clear that 401L cells, despite lacking depletion-induced Ca2+ influx pathways, express the functional components of a Ca2+ influx pathway under the control of RyR function. These findings further support the importance of the 401L cell line as an important cell phenotype for deciphering Ca2+ influx regulation.

An Elective Course in Cardiovascular Electrophysiology for Pharmacy Learners.

Objective. To implement an integrated, comprehensive, and learner-centered elective course focused at exposing learners to the interpretation of electrocardiograms and highlighting the mechanisms underlining the abnormal electrophysiological events. Design. Learners were presented with foundational information on the mechanisms underlying electrophysiological changes associated with the development of arrhythmias. They then discussed the interpretation of electrocardiogram (ECG) recordings and diagnosis of cardiovascular events. Teaching formats included "chalk-talk" and didactic sessions, case-based exercises providing hands-on evaluation of ECG recordings, and high-fidelity simulation presenting cases of arrhythmias. The course design emphasized critical thinking, learner engagement, and development of problem-solving skills. Learners were assessed by formal assignments, examinations, and in-class quizzes. Assessment. Learner comprehension of the material was assessed using cumulative examinations, in-class quizzes, assignments, and in-class presentations. Learner evaluations showed that the case-based discussions, practice ECGs, review tables, and illustrations enhanced course performance and retention of complex material. Conclusion. The elective course provided in-depth exposure to the mechanisms underlying electrophysiological aberrations resulting in arrhythmias. It gave learners an opportunity to learn the art of ECG interpretation and to apply their knowledge in simulated scenarios. As clinical teams adopt a multidisciplinary team approach to patient care, acquiring these skills enriches learner experiences and allows them to expand their role and professional opportunities as pharmacists.

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells.

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.

Paradoxical effects of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activator gingerol on NG115-401L neuronal cells: failure to augment ER Ca(2+) uptake and protect against ER stress-induced cell death.

Perturbation of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress are thought to underlie a spectrum of defects encompassing major societal diseases such as diabetes and neurodegeneration. In this report we used the NG115-401L neuronal cell line to test the hypothesis that neuroprotection against ER stress may be conferred by pharmacological stimulation of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps. We report that the SERCA activator gingerol stimulates SR microsomal Ca(2+)-ATPase activity and restores enzymatic function in the presence of potent SERCA blockers. Yet, enzyme protection in isolated membranes does not extend to protection from ER stress in intact NG115-401L cells. Surprisingly, gingerol not only failed to protect cells from SERCA blocker-induced ER stress and cell death, the compound itself potently induced cell death. Also, we report that gingerol failed to augment ER Ca(2+) uptake, a result contradictory to what has been observed in muscle. Unexpectedly, gingerol discharged ER Ca(2+) stores and coupled robustly to Ca(2+) influx pathways. These observations suggest that gingerol is not acting as a traditional SERCA blocker as thapsigargin mediated ER Ca(2+) store depletion fails to stimulate Ca(2+) influx in the NG115-401L cell phenotype. Moreover, cell death induced by gingerol, in contrast to the classic SERCA inhibitors, is not accompanied by increases in reactive oxygen species production or enzymatic caspase activity. These results argue for a finer regulatory control on SERCA function with gingerol's actions revealing potentially novel routes of coupling altered pump regulation to the assembly of functional Ca(2+) influx units and activation of cell death pathways.

PCB 136 atropselectively alters morphometric and functional parameters of neuronal connectivity in cultured rat hippocampal neurons via ryanodine receptor-dependent mechanisms.

We recently demonstrated that polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substitutions sensitize ryanodine receptors (RyRs), and this activity promotes Ca²âº-dependent dendritic growth in cultured neurons. Many ortho-substituted congeners display axial chirality, and we previously reported that the chiral congener PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) atropselectively sensitizes RyRs. Here, we test the hypothesis that PCB 136 atropisomers differentially alter dendritic growth and other parameters of neuronal connectivity influenced by RyR activity. (-)-PCB 136, which potently sensitizes RyRs, enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. The dendrite-promoting activity of (-)-PCB 136 is observed at concentrations ranging from 0.1 to 100 nM and is blocked by pharmacologic RyR antagonism. Neither atropisomer alters axonal growth or cell viability. Quantification of PCB 136 atropisomers in hippocampal cultures indicates that atropselective effects on dendritic growth are not due to differential partitioning of atropisomers into cultured cells. Imaging of hippocampal neurons loaded with Ca²âº-sensitive dye demonstrates that (-)-PCB 136 but not (+)-PCB 136 increases the frequency of spontaneous Ca²âº oscillations. Similarly, (-)-PCB 136 but not (+)-PCB 136 increases the activity of hippocampal neurons plated on microelectrode arrays. These data support the hypothesis that atropselective effects on RyR activity translate into atropselective effects of PCB 136 atropisomers on neuronal connectivity, and suggest that the variable atropisomeric enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure.

PCB-95 promotes dendritic growth via ryanodine receptor-dependent mechanisms.

BACKGROUND: Aroclor 1254 (A1254) interferes with normal dendritic growth and plasticity in the developing rodent brain, but the mechanism(s) mediating this effect have yet to be established. Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) enhance the activity of ryanodine receptor (RyR) calcium ion (Ca(2+)) channels, which play a central role in regulating the spatiotemporal dynamics of intracellular Ca(2+) signaling. Ca(2+) signaling is a predominant factor in shaping dendritic arbors, but whether PCB potentiation of RyR activity influences dendritic growth is not known. OBJECTIVE: We determined whether RyR activity is required for PCB effects on dendritic growth. METHODS AND RESULTS: Golgi analysis of hippocampi from weanling rats confirmed that developmental exposure via the maternal diet to NDL PCB-95 (2,2',3,5'6-pentachlorobiphenyl), a potent RyR potentiator, phenocopies the dendrite-promoting effects of A1254. Dendritic growth in dissociated cultures of primary hippocampal neurons and in hippocampal slice cultures is similarly enhanced by PCB-95 but not by PCB-66 (2,3,4',4-tetrachlorobiphenyl), a congener with negligible effects on RyR activity. The dendrite-promoting effects of PCB-95 are evident at concentrations as low as 2 pM and are inhibited by either pharmacologic blockade or siRNA knockdown of RyRs. CONCLUSIONS: Our findings demonstrate that environmentally relevant levels of NDL PCBs modulate neuronal connectivity via RyR-dependent effects on dendritic arborization. In addition, these findings identify RyR channel dysregulation as a novel mechanism contributing to dysmorphic dendritogenesis associated with heritable and environmentally triggered neurodevelopmental disorders.

PCB-95 modulates the calcium-dependent signaling pathway responsible for activity-dependent dendritic growth.

BACKGROUND: Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) promote dendritic growth in hippocampal neurons via ryanodine receptor (RyR)-dependent mechanisms; however, downstream signaling events that link enhanced RyR activity to dendritic growth are unknown. Activity-dependent dendritic growth, which is a critical determinant of neuronal connectivity in the developing brain, is mediated by calcium ion (Ca(2+))-dependent activation of Ca(2+)/calmodulin kinase-I (CaMKI), which triggers cAMP response element binding protein (CREB)-dependent Wnt2 transcription. RyRs regulate the spatiotemporal dynamics of intracellular Ca(2+) signals, but whether RyRs promote dendritic growth via modulation of this signaling pathway is not known. OBJECTIVE: We tested the hypothesis that the CaMKI-CREB-Wnt2 signaling pathway couples NDL PCB-enhanced RyR activity to dendritic arborization. METHODS AND RESULTS: Ca(2+) imaging of dissociated cultures of primary rat hippocampal neurons indicated that PCB-95 (2,2',3,5'6-pentachlorobiphenyl; a potent RyR potentiator), enhanced synchronized Ca(2+) oscillations in somata and dendrites that were blocked by ryanodine. As determined by Western blotting and quantitative polymerase chain reaction, PCB-95 also activated CREB and up-regulated Wnt2. Blocking CaMKK, CaMKIα/γ, MEK/ERK, CREB, or Wnt2 prevented PCB-95-induced dendritic growth. Antagonism of γ-aminobutyric acid (GABA) receptors with bicuculline (BIC) phenocopied the dendrite-promoting effects of PCB-95, and pharmacological antagonism or siRNA knockdown of RyR blocked BIC-induced dendritic growth in dissociated and slice cultures of hippocampal neurons. CONCLUSIONS: RyR activity contributes to dynamic remodeling of dendritic architecture in response to NDL PCBs via CaMKI-CREB-Wnt2 signaling in rats. Our findings identify PCBs as candidate environmental risk factors for neurodevelopmental disorders, especially in children with heritable deficits in calcium signaling associated with autism.

Para- and ortho-substitutions are key determinants of polybrominated diphenyl ether activity toward ryanodine receptors and neurotoxicity.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants that bioaccumulate in human tissues. Their neurotoxicity involves dysregulation of calcium ion (Ca(2+))signaling; however, specific mechanisms have yet to be defined. OBJECTIVE: We aimed to define the structure-activity relationship (SAR) for PBDEs and their metabolites toward ryanodine receptors type 1 (RyR1) and type 2 (RyR2) and to determine whether it predicts neurotoxicity. METHODS: We analyzed [3H]ryanodine binding, microsomal Ca(2+) fluxes, cellular measurements of Ca(2+) homeostasis, and neurotoxicity to define mechanisms and specificity of PBDE-mediated Ca(2+) dysregulation. RESULTS: PBDEs possessing two ortho-bromine substituents and lacking at least one para-bromine substituent (e.g., BDE-49) activate RyR1 and RyR2 with greater efficacy than corresponding congeners with two para-bromine substitutions (e.g., BDE-47). Addition of a methoxy group in the free para position reduces the activity of parent PBDEs. The hydroxylated BDEs 6-OH-BDE-47 and 4´-OH-BDE-49 are biphasic RyR modulators. Pretreatment of HEK293 cells (derived from human embryonic kidney cells) expressing either RyR1 or RyR2 with BDE-49 (250 nM) sensitized Ca2+ flux triggered by RyR agonists, whereas BDE-47 (250 nM) had negligible activity. The divergent activity of BDE-49, BDE-47, and 6-OH-BDE-47 toward RyRs predicted neurotoxicity in cultures of cortical neurons. CONCLUSIONS: We found that PBDEs are potent modulators of RyR1 and RyR2. A stringent SAR at the ortho and para position determined whether a congener enhanced, inhibited, or exerted nonmonotonic actions toward RyRs. These results identify a convergent molecular target of PBDEs previously identified for noncoplanar polychlorinated biphenyls (PCBs) that predicts their cellular neurotoxicity and therefore could be a useful tool in risk assessment of PBDEs and related compounds.

Effects of 17 ß-estradiol on lipopolysacharride-induced intracellular adhesion molecule-1 mRNA expression and Ca²+ homeostasis alteration in human endothelial cells.

Recent evidence showed that 17 ß-estradiol (E2) decreased cytokine-induced expression of cell adhesion molecules (CAM). Changes in intracellular Ca²+ concentration ([Ca²+](i)) has been shown to be associated with CAM expression in endothelial cells. Here, the effects of E2 (1 µM, 24 h) on the expression of intracellular adhesion molecule-1 (ICAM-1) and [Ca²+](i) were investigated in a lipopolysaccharide (LPS) (100 ng/mL, 18 h)-stimulated human endothelial cell line, EA.hy926, using real-time PCR and spectrofluorometry, respectively. PCR analysis revealed a significant increase in ICAM-1 expression in calcium ionophore A23187 (1 nM)- or LPS-stimulated cells. Pretreatment of cells with E(2) significantly inhibited LPS-induced ICAM-1 mRNA expression. [Ca²+](i) was monitored in Fura-2AM-loaded cells in the presence and absence of extracellular Ca²+ with thapsigargin (TG, 1 µM), a sarco/endoplasmic reticulum ATPase inhibitor or ATP (100 µM). The extent of TG- or ATP-induced [Ca²+](i) increase was significantly higher in LPS-stimulated cells than in control cells. Pre-treatment of LPS-stimulated cells with E2 limited the Ca²+ response to the same level as in control cells. Furthermore, ICI 182,780, an estrogen receptor antagonist, attenuated the inhibitory actions of E2 on ICAM-1 mRNA expression and Ca²+ responses, suggesting that estrogen receptors mediate, at least in part, the effects of estrogen. These data suggest a potential underlying mechanism for the protective effect of E2 against atherosclerosis.