Role Of Intracellular Calcium Research Articles

1512-P: The Novel Roles of Intracellular Calcium and Phosphoinositides in Insulin Signaling and Metabolic Disease

Insulin resistance is closely associated with elevated intracellular Ca2+ concentration under ectopic lipid accumulation and hyperglycemia-induced intracellular stress conditions. We recently identified that obesity impairs intracellular Ca2+ homeostasis, which prevents membrane localization of PH domain-containing proteins such as AKT, PLCδ, and IRS through the formation of Ca2+-phosphoinositides (PIPs), resulting in dysregulation of glucose homeostasis and insulin resistance. It is unclear, however, whether insulin sensitivity can be improved by targeting intracellular Ca2+ overload. Therefore, we screened nine angiotensin-II-receptor blockers (ARBs), a class of antihypertensive agents, for beneficial effects on palmitic acid (PA)-induced insulin resistance in human HepG2 cells. Two of the ARBs attenuated PA-induced intracellular Ca2+ overload by inhibiting dysregulated store-operated channel (SOC)-mediated Ca2+ entry into cells, which ameliorated insulin resistance by promoting insulin-stimulated membrane localization of AKT and by increasing the phosphorylation of AKT and its downstream substrates in PA-treated HepG2 cells. Furthermore, certain ARBs ameliorated obesity-induced insulin resistance, hepatic steatosis, and tissue inflammation in mice fed a high-fat diet. Meanwhile, certain ARBs normalized intracellular Ca2+ homeostasis by regulating obesity-associated SOC-mediated Ca2+ entry, which rescued impaired insulin signaling in liver and muscle tissues by promoting postprandial membrane localization of AKT and IRS2. These findings underscore the contribution of dysregulated intracellular Ca2+ homeostasis to the pathophysiology of insulin resistance and suggest a therapeutic strategy to use specific ARBs to ameliorate insulin resistance. Disclosure J.Lee: None. Y.Jung: None. S.Im: None. D.Lee: None. H.Lee: None. I.Hong: None. O.Kim: None. B.Oh: None. Funding Korean Ministry of Health & Welfare (HI14C1135 to B-C.O.); Basic Science Research Program (2021R1I1A1A01051429 to O-H.K.); Mid-Career Researcher Program (2019R1A2C2008130, 2022R1A2C2092700 to B-C.O.); National Research Foundation of Korea (2021R1A5A203033)

The role of intracellular calcium and Rho kinase pathways in G protein-coupled receptor-mediated contractions of urinary bladder urothelium and lamina propria.

The influence of extracellular and intracellular calcium on smooth muscle contractile activity varies between organs. In response to G protein-coupled receptor (GPCR) stimulation, the urinary bladder detrusor muscle has shown a 70% dependence on extracellular calcium, whereas the urothelium and lamina propria (U&LP) has a 20%-50% dependence. However, as this only accounts for partial contractile activity, the contribution of intracellular calcium and calcium sensitization pathways remains unclear. This study assessed the role of intracellular signaling pathways on GPCR-mediated urinary bladder U&LP contraction. Porcine U&LP responses to activation of the Gq/11-coupled muscarinic, histamine, 5-hydroxytryptamine (serotonin), neurokinin, prostaglandin, and angiotensin II receptors were assessed with three selective inhibitors of store-released intracellular calcium, 2-aminoethyl diphenylborinate (2-APB), cyclopiazonic acid (CPA), and ruthenium red, and three Rho kinase inhibitors, fasudil, Y-27632, and GSK269962. There was no discernible impact on receptor agonist-induced contractions of the U&LP after blocking intracellular calcium pathways, suggesting that this tissue is more sensitive to alterations in the availability of extracellular calcium. However, an alternative mechanism of action for GPCR-mediated contraction was identified to be the activation of Rho kinase, such as when Y-27632 significantly reduced the GPCR-mediated contractile activity of the U&LP by approximately 50% (P < 0.05, n = 8). This suggests that contractile responses of the bladder U&LP do not involve a significant release of calcium from intracellular stores, but that Gq/11-coupled receptor activation causes calcium sensitization via Rho kinase. This study highlights a key role for Rho kinase in the urinary bladder, which may provide a novel target in the future pharmaceutical management of bladder contractile disorders.

Therapeutic advances in ADPKD: the future awaits.

Autosomal dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder included in ciliopathies, representing the fourth cause of end stage renal disease (ESRD), with an estimated prevalence between 1:1000 and 1:2500. It is mainly caused by mutations in the PKD1 and PKD2 genes encoding for polycystin 1 (PC1) and polycystin 2 (PC2), which regulate differentiation, proliferation, survival, apoptosis, and autophagy. The advances in the knowledge of multiple molecular pathways involved in the pathophysiology of ADPKD led to the development of several treatments which are currently under investigation. Recently, the widespread approval of tolvaptan and, in Italy, of long-acting release octreotide (octreotide-LAR), represents but the beginning of the new therapeutic management of ADPKD patients. Encouraging results are expected from ongoing randomized controlled trials (RCTs), which are investigating not only drugs acting on the calcium/cyclic adenosin monoposphate (cAMP) pathway, the most studied target so far, but also molecules targeting specific pathophysiological pathways (e.g. epidermal growth factor (EGF) receptor, AMP-activated protein kinase (AMPK) and KEAP1-Nrf2) and sphingolipids. Moreover, studies on animal models and cultured cells have also provided further promising therapeutic strategies based on the role of intracellular calcium, cell cycle regulation, MAPK pathway, epigenetic DNA, interstitial inflammation, and cell therapy. Thus, in a near future, tailored therapy could be the key to changing the natural history of ADPKD thanks to the vigorous efforts that are being made to implement clinical and preclinical studies in this field. Our review aimed to summarize the spectrum of drugs that are available in the clinical practice and the most promising molecules undergoing clinical, animal, and cultured cell studies.

Location, location, location: lessons from airway epithelial anion channels.

Location, location, location: lessons from airway epithelial anion channels.

Role of intracellular Ca2+-based mechanotransduction of human periodontal ligament fibroblasts.

Human periodontal ligament (hPDL) fibroblasts are thought to receive mechanical stress (MS) produced by orthodontic tooth movement, thereby regulating alveolar bone remodeling. However, the role of intracellular calcium ([Ca2+]i)-based mechanotransduction is not fully understood. We explored the MS-induced [Ca2+]i responses both in isolated hPDL fibroblasts and in intact hPDL tissue and investigated its possible role in alveolar bone remodeling. hPDL fibroblasts were obtained from healthy donors’ premolars that had been extracted for orthodontic reasons. The oscillatory [Ca2+]i activity induced by static compressive force was measured by a live-cell Ca2+ imaging system and evaluated by several feature extraction method. The spatial pattern of cell-cell communication was investigated by Moran’s I, an index of spatial autocorrelation and the gap junction (GJ) inhibitor. The Ca2+-transporting ionophore A23187 was used to further investigate the role of [Ca2+]i up-regulation in hPDL cell behavior. hPDL fibroblasts displayed autonomous [Ca2+]i responses. Compressive MS activated this autonomous responsive behavior with an increased percentage of responsive cells both in vitro and ex vivo. The integration, variance, maximum amplitude, waveform length, and index J in the [Ca2+]i responses were also significantly increased, whereas the mean power frequency was attenuated in response to MS. The increased Moran’s I after MS indicated that MS might affect the pattern of cell-cell communication via GJs. Similar to the findings of MS-mediated regulation, the A23187-mediated [Ca2+]i uptake resulted in the up-regulation of receptor activator of NF-κB ligand (Rankl) and Sost along with increased sclerostin immunoreactivity, suggesting that [Ca2+]i signaling networks may be involved in bone remodeling. In addition, A23187-treated hPDL fibroblasts also showed the suppression of osteogenic differentiation and mineralization. Our findings suggest that augmented MS-mediated [Ca2+]i oscillations in hPDL fibroblasts enhance the production and release of bone regulatory signals via Rankl/Osteoprotegerin and the canonical Wnt/β-catenin pathway as an early process in tooth movement–initiated alveolar bone remodeling.—Ei Hsu Hlaing, E., Ishihara, Y., Wang, Z., Odagaki, N., Kamioka, H. Role of intracellular Ca2+–based mechanotransduction of human periodontal ligament fibroblasts.

A computational study of pancreatic beta cell dynamics in the progression to diabetes

Complex interactions within the pancreatic β-cell are responsible for the range of phenomena observed in the progression to type 2 diabetes. Much is now known about the biophysics of this progression, but more remains to be elucidated through experimental and theoretical collaborations. Recent experimental results identify changes in oscillation magnitude and frequency for fluorescent measures of Ca2+ from isolated pancreatic islets over the progression of diabetes in leptin-receptor deficient mice, and in response to varying levels of glucose stimulation. This paper is a computational study of a subset of those results, using adaptations from classic whole cell models for the role of intracellular calcium and ATP/ADP levels in the regulation of membrane potential and insulin secretion for pancreatic β-cells.While the data was collected from probes within a pancreatic islet, the whole-cell models used for simulations represent a single β-cell. Within the Islets of Langerhans, these cells synchronize electrically, and disrupting cell-cell communication may cause disruptions in insulin secretion. Simulations qualitatively recover the data, observe the behavior of inter-dependent variables not reported in the data, and interpret the implications for parameter values required to obtain these results, constrained by the data. Results reproduce frequency and proportional changes in magnitude for Ca2+ traces from both non-diabetic heterozygous controls and new onset diabetic mice at a fixed age and level of glucose.

Acute cocaine exposure elicits rises in calcium in arousal-related laterodorsal tegmental neurons.

Cocaine has strong reinforcing properties, which underlie its high addiction potential. Reinforcement of use of addictive drugs is associated with rises in dopamine (DA) in mesoaccumbal circuitry. Excitatory afferent input to mesoaccumbal circuitry sources from the laterodorsal tegmental nucleus (LDT). Chronic, systemic cocaine exposure has been shown to have cellular effects on LDT cells, but acute actions of local application have never been demonstrated. Using calcium imaging, we show that acute application of cocaine to mouse brain slices induces calcium spiking in cells of the LDT. Spiking was attenuated by tetrodotoxin (TTX) and low calcium solutions, and abolished by prior exhaustion of intracellular calcium stores. Further, DA receptor antagonists reduced these transients, whereas DA induced rises with similar spiking kinetics. Amphetamine, which also results in elevated levels of synaptic DA, but via a different pharmacological action than cocaine, induced calcium spiking with similar profiles. Although large differences in spiking were not noted in an animal model associated with a heightened proclivity of acquiring addiction‐related behavior, the prenatal nicotine exposed mouse (PNE), subtle differences in cocaine's effect on calcium spiking were noted, indicative of a reduction in action of cocaine in the LDT associated with exposure to nicotine during gestation. When taken together, our data indicate that acute actions of cocaine do include effects on LDT cells. Considering the role of intracellular calcium in cellular excitability, and of the LDT in addiction circuitry, our data suggest that cocaine effects in this nucleus may contribute to the high addiction potential of this drug.

Photobiomodulation (blue and green light) encourages osteoblastic-differentiation of human adipose-derived stem cells: role of intracellular calcium and light-gated ion channels.

Human adipose-derived stem cells (hASCs) have the potential to differentiate into several different cell types including osteoblasts. Photobiomodulation (PBM) or low level laser therapy (LLLT) using red or near-infrared wavelengths has been reported to have effects on both proliferation and osteogenic differentiation of stem cells. We examined the effects of delivering four different wavelengths (420 nm, 540 nm, 660 nm, 810 nm) at the same dose (3 J/cm2) five times (every two days) on hASCs cultured in osteogenic medium over three weeks. We measured expression of the following transcription factors by RT-PCR: RUNX2, osterix, and the osteoblast protein, osteocalcin. The 420 nm and 540 nm wavelengths were more effective in stimulating osteoblast differentiation compared to 660 nm and 810 nm. Intracellular calcium was higher after 420 nm and 540 nm, and could be inhibited by capsazepine and SKF96365, which also inhibited osteogenic differentiation. We hypothesize that activation of light-gated calcium ion channels by blue and green light could explain our results.

The S100B-RAGE pathway is dysregulated in the ALS-linked neuroinflammatory process

The comprehension of the mechanisms at the basis of astrocytic dysfunction in ALS is crucial to limit neuronal injury. Most of the toxic astrocytic effects highlight the role of intracellular calcium. S100B is a Ca2+-binding protein particularly present in astrocytes, behaving as a neuroinflammatory mediator as it is secreted by astrocytes under pathological conditions and can display paracrine toxicity by binding to RAGE. During ALS progression S100B increases in patient astrocytes and, in a rat model of the disease, S100B is augmented in “aberrant astrocytes”, characterized by their neurotoxic potential. The induction of S100B in astrocytes, its release and its interaction with RAGE in motoneurons could represent a hazardous mechanism that takes place during ALS. Main objectives of this work were to investigate 1) if the expression of S100B protein and RAGE change during the course of the disease in rodent models of ALS, 2) if the expression of mutant SOD1 protein per se is sufficient to modify S100B levels in astrocytic cultures. We observed that S100B levels and localization are modulated in the spinal cord and in the brain cortex of rat and mouse models of ALS. We also demonstrated a differential expression of RAGE subunits in SOD1-G93A-derived CNS tissues. Moreover, we showed that the overexpression of mutant SOD1 in astrocytic cell line is sufficient to increase the intracellular levels and release of S100B, while it is not enough to induce a differential expression of RAGE. Thus, the expression of mutant SOD1 interferes with the physiological expression of S100B and RAGE and reveals that in astrocytes S100B modulation is an early event related to the mere expression of mutant SOD1, while the dysregulation of RAGE might be a phenomenon possibly requiring a more complex interplay between different cell types and pathways. Overall, these data suggest that S100B might be a toxic mediator released by astrocytes in the ALS-linked neuroinflammatory process.

Properties of Atrial Myocyte Calcium Handling in Canine Model of Chronic Heart Failure

Weakened atrial contractility is a common comorbidity in heart failure (HF) that increases severity of HF symptoms. The role of intracellular calcium (Ca) handling remodeling in pathogenesis of ventricular contractile failure has been well established. However, the relationship between Ca signaling and atrial dysfunction in HF is not well recognized. We studied intracellular Ca handling in atrial myocytes isolated from control dogs and dogs with tachypacing-induced chronic HF. Ca transients and cellular shortening were reduced, while sarcoplasmic reticulum (SR) Ca content was increased in field-stimulated myocytes from HF compared to controls. Both control and HF myocytes have scarce t-tubule density, and Ca transients originated predominantly at the periphery of myocytes in both groups. Diminished Ca transients in HF myocytes were associated with decreased peripheral Ca release and impaired propagation of Ca signal from sarcolemma to the central areas of the myocyte. Peak density of the L-type Ca currents and gain of excitation-contraction coupling were reduced in voltage-clamped HF myocytes compared to control myocytes. Analysis of decay of caffeine- and electrically-induced Ca transients suggests reduced Na/Ca exchanger activity and increased SR Ca ATPase (SERCA) activity in HF myocytes. SERCA inhibition with cyclopiazonic acid did not improve centripetal propagation of Ca signal in HF myocytes. In contrast, L-type Ca channel agonist BayK 8644 increased peripheral Ca release, improved centripetal Ca wave propagation and restored shortening of HF myocytes to control levels.We conclude that defective activation and impaired propagation of the SR Ca release in atrial myocytes contribute to compromised atrial contractility in chronic HF. Our results suggest L-type Ca channels as a therapeutic target for treatment of atrial contractile dysfunction in HF.

Tu1671 Calcium Signaling in Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Tu1674 Cannabinoid Agonist Interfere the Human Bone Marrow-Derived Mesenchymal Stem Cell-Induced Gastric Cancer Cell Migration

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Tu1672 Involvement of p21-Activated Kinase 4 (PAK4) in Gastrin-Induced Migration of Gastric Cancer Cells

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.

Different mechanism of LPS-induced calcium increase in human lung epithelial cell and microvascular endothelial cell: a cell culture study in a model for ARDS

Acute respiratory distress syndrome (ARDS) is a contemporary term incorporating the historic 'acute lung injury' and the colloquial term 'shock lung'. ARDS remains a serious and enigmatic human disease, causing significant mortality. The mechanisms involved at the alveolar cell/capillary endothelial interface have been explored but to date we lack clarity on the role of intracellular calcium ([Ca(2+)]i) fluxes across this interface. To explore the mechanisms of Ca(2+) induced inflammatory reaction in epithelial cells and pulmonary microvascular endothelial cells (HMVEC) located at the two sides of blood-air barrier, lung epithelial A549 and HMVEC cells were treated with LPS. Our results demonstrated that LPS evoked the increase of [Ca(2+)]i, TNF-α and IL-8 in both cells types. The [Ca(2+)]i increases involved intracellular but not extracellular Ca(2+) sources in A549, but both intracellular and extracellular Ca(2+) sources in HMVEC cells. The effects of LPS on both cells types were completely inhibited by the combination of LPS and CaSR-targeted siRNA. Furthermore, LPS-inhibited cell proliferations were significantly reversed by the combined treatment. Therefore, LPS induced different mechanisms of [Ca(2+)]i increase during the activation of CaSR in A549 and HMVEC cells, which translates into functional outputs related to ARDS.

Role of intracellular calcium and reactive oxygen species in microbubble-mediated alterations of endothelial layer permeability

Drugs will be delivered to diseased tissue more efficiently if the vascular endothelial permeability is increased. Ultrasound in combination with an ultrasound contrast agent is known to increase the permeability of the endothelial layer, but the mechanism is not known. The goal of this study was to elucidate whether intracellular calcium ions, [Ca(2+)]i, and reactive oxygen species (ROS) are part of the mechanism that leads to an increased endothelial layer permeability following ultrasound and microbubble treatment. Human umbilical vein endothelial cells (HUVECs) treated for 2 min with ultrasound-activated microbubbles (1 MHz, 210 kPa, 10 000 cycles, 20 Hz repetition rate) had an increased permeability that lasted up to 12 h. Recovery of permeability after 2 h was only found when HUVECs were preincubated with the [Ca(2+)]i chelator BAPTA-AM or the antioxidant butylated hydroxytoluene (BHT). This suggests that both [Ca(2+)]i and ROS play an important role in the mechanism of increased permeability following ultrasound in combination with microbubble treatment.

Role of Intracellular Calcium in Proteasome Inhibitor-Induced Endoplasmic Reticulum Stress, Autophagy, and Cell Death

Proteasome inhibition induces endoplasmic reticulum (ER) stress and compensatory autophagy to relieve ER stress. Disturbance of intracellular calcium homeostasis can lead to ER stress and alter the autophagy process. It has been suggested that inhibition of the proteasome disrupts intracellular calcium homeostasis. However, it is unknown if intracellular calcium affects proteasome inhibitor-induced ER stress and autophagy. Human colon cancer HCT116 Bax positive and negative cell lines were treated with MG132, a proteasome inhibitor. BAPTA-AM, a cell permeable free calcium chelator, was used to modulate intracellular calcium levels. Autophagy and cell death were determined by fluorescence microscopy and immunoblot analysis. MG132 increased intracellular calcium levels in HCT116 cells, which was suppressed by BAPTA-AM. MG132 suppressed proteasome activity independent of Bax and intracellular calcium levels in HCT116 cells. BAPTA-AM inhibited MG132-induced cellular vacuolization and ER stress, but not apoptosis. MG132 induced autophagy with normal autophagosome-lysosome fusion. BAPTA-AM seemed not to affect autophagosome-lysosome fusion in MG132-treated cells but further enhanced MG132-induced LC3-II levels and GFP-LC3 puncta formation, which was likely via impaired lysosome function. Blocking intracellular calcium by BAPTA-AM relieved MG132-induced ER stress, but it was unable to rescue MG132-induced apoptosis, which was likely due to impaired autophagic degradation.

Abstract 2184: The roles of intracellular calcium and ER stress in the synergistic apoptotic effect of the plant polyphenols curcumin and carnosic acid in acute myeloid leukemia cells.

Abstract Failure to cure acute myeloid leukemia (AML) by conventional chemotherapy still represents a challenge for hematological oncology. Small molecule plant-derived agents and their derivatives (e.g., vinblastine, paclitaxel, etoposide, topotecan) have been widely employed for the treatment of several types of malignancies. However, it is still unclear whether active phytochemicals can be used for the management of AML. We have recently reported that the combination of the plant polyphenols curcumin (CUR) and carnosic acid (CA) at low, bioavailable concentrations synergistically arrests cell growth and induces robust apoptotic response in KG-1a and HL60 human AML cell lines [Pesakhov et al. Nutr. Cancer 2010, 62:811-824], but the underlying mechanisms remain obscure. Here, employing pharmacologic inhibitors of caspases as well as dominant-negative mutants of caspase-8 and -9 and the death receptor adaptor protein FADD, we reveal the involvement of both extrinsic and intrinsic apoptotic pathways as well as their interdependence in CUR/CA-induced cytotoxicity. The activation of both pathways was preceded by upregulation of the expression of endoplasmatic reticulum (ER) stress markers (GRP78 and CHOP) and by the sustained elevation of cytosolic Ca2+ ([Ca2+]cyt). CUR/CA-induced apoptosis was only partially attenuated by the ER stress blocker salubrinal but was fully prevented by lowering [Ca2+]cyt with the membrane-permeable Ca2+ chelator BAPTA-AM. Notably, each polyphenol alone caused modest reduction in ER Ca2+ content, whereas their combination dramatically depleted ER Ca2+ stores thereby triggering cytotoxic [Ca2+]cyt rise. Preincubation of AML cells with 2-aminoethoxydiphenyl borate (2-APB), the ER inositol trisphosphate receptor antagonist, which blocks the main route of Ca2+ release from the ER, also prevented CUR/CA-induced apoptosis. Importantly, the Ca2+ store depletion as well as [Ca2+]cyt accumulation were not observed in CUR/CA-treated peripheral blood mononuclear cells obtained from healthy donors, consistent with the lack of CUR/CA-induced cytotoxicity in these cells. In summary, our findings indicate that the malignant cell-selective synergistic CUR/CA cytotoxicity is mediated by disruption of cellular Ca2+ homeostasis and, partially, through the ER stress response, leading to stimulation of apoptotic pathways. These results suggest that this combination may have potential for the treatment and/or prevention of AML and, possibly, other malignant diseases. (Supported by the American Institute for Cancer Research grant #10A049 to G.P.S. and M.D.) Citation Format: Stella Pesakhov, Nasma Aqaqe, George P. Studzinski, Daniel Fishman, Michael Danilenko. The roles of intracellular calcium and ER stress in the synergistic apoptotic effect of the plant polyphenols curcumin and carnosic acid in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2184. doi:10.1158/1538-7445.AM2013-2184

Role of Intracellular Calcium in Release from Nerve Terminals

Oxytocin (OT) and vasopressin (AVP) are released from terminals in the neurohypophysis (NH). These neuropeptides (NP) are contained in large dense core vesicles (LDCV). Ryanodine receptors (RyR) in NH terminals can induce spontaneous focal Ca2+ transients (DeCrescenzo, et al., 2004 J. Neurosci.24:1226-1235), making them ideal for studying the role of intraterminal Ca2+ in NP release.Fluorescent immunolabeling and immunogold micrographs of NH terminals show that RyR are localized specifically to LDCV. Furthermore, a large conductance non-specific cation channel, previously identified in the LDCV membrane with properties similar to that of a RyR (Lee, et al., 1992 Neuron 8:335-342), is pharmacologically affected in the same characteristic manner as a RyR.We found that individual Ca2+ release events alone are not enough to drive peptide release (McNally, et al., 2009 J. Neurosci. 29:14120-14126). However, these RyR sensitive events could potentially play a role in modulating NP release. To test this hypothesis, the association of LDCV within an area 0.45 µm of the plasma membrane was assessed using immunolabeling of Neurophysins I (OT) and II (AVP) in isolated NH terminals. We found that the amount of membrane associated NP-immunoreactivity varies significantly between terminal types and that this distribution pattern can be enhanced by agonist concentrations of ryanodine, but only in OT terminals.Importantly, Ca2+-evoked NP release from permeabilized-terminals was increased by agonist concentrations of ryanodine and decreased by antagonist concentrations of this drug. Amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Agonist concentrations of ryanodine were also able to increase the asynchronous phase of low frequency electrically-stimulated capacitance increases in NH terminals. Thus, the ryanodine-sensitive mobilization of LDCV seems to have a functional role in modulating secretion of NP from NH terminals. [Support: NS29470, NS40966, P60037094900000].

Monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc Aplysia.

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation). These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3). Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc &lt;em&gt;Aplysia&lt;/em&gt;

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.