Magnitude Of Ca2 Research Articles

Exploring the Impact of BKCa Channel Function in Cellular Membranes on Cardiac Electrical Activity.

This review paper delves into the current body of evidence, offering a thorough analysis of the impact of large-conductance Ca2+-activated K+ (BKCa or BK) channels on the electrical dynamics of the heart. Alterations in the activity of BKCa channels, responsible for the generation of the overall magnitude of Ca2+-activated K+ current at the whole-cell level, occur through allosteric mechanisms. The collaborative interplay between membrane depolarization and heightened intracellular Ca2+ ion concentrations collectively contribute to the activation of BKCa channels. Although fully developed mammalian cardiac cells do not exhibit functional expression of these ion channels, evidence suggests their presence in cardiac fibroblasts that surround and potentially establish close connections with neighboring cardiac cells. When cardiac cells form close associations with fibroblasts, the high single-ion conductance of these channels, approximately ranging from 150 to 250 pS, can result in the random depolarization of the adjacent cardiac cell membranes. While cardiac fibroblasts are typically electrically non-excitable, their prevalence within heart tissue increases, particularly in the context of aging myocardial infarction or atrial fibrillation. This augmented presence of BKCa channels' conductance holds the potential to amplify the excitability of cardiac cell membranes through effective electrical coupling between fibroblasts and cardiomyocytes. In this scenario, this heightened excitability may contribute to the onset of cardiac arrhythmias. Moreover, it is worth noting that the substances influencing the activity of these BKCa channels might influence cardiac electrical activity as well. Taken together, the BKCa channel activity residing in cardiac fibroblasts may contribute to cardiac electrical function occurring in vivo.

A note on estimating absolute cytosolic Ca2+ concentration in sensory neurons using a single wavelength Ca2+ indicator.

Ca2+ imaging is frequently used in the investigation of sensory neuronal function and nociception. In vitro imaging of acutely dissociated sensory neurons using membrane-permeant fluorescent Ca2+ indicators remains the most common approach to study Ca2+ signalling in sensory neurons. Fluo4 is a popular choice of single-wavelength indicator due to its brightness, high affinity for Ca2+ and ease of use. However, unlike ratiometric indicators, the emission intensity from single-wavelength indicators can be affected by indicator concentration, optical path length, excitation intensity and detector efficiency. As such, without careful calibration, it can be difficult to draw inferences from differences in the magnitude of Ca2+ transients recorded using Fluo4. Here, we show that a method scarcely used in sensory neurophysiology - first proposed by Maravall and colleagues (2000) - can provide reliable estimates of absolute cytosolic Ca2+ concentration ([Ca2+]cyt) in acutely dissociated sensory neurons using Fluo4. This method is straightforward to implement; is applicable to any high-affinity single-wavelength Ca2+ indicator with a large dynamic range; and provides estimates of [Ca2+]cyt in line with other methods, including ratiometric imaging. Use of this method will improve the granularity of sensory neuron Ca2+ imaging data obtained with Fluo4.

RETRACTED ARTICLE: Calcium-dependent transcriptional changes in human pancreatic islet cells reveal functional diversity in islet cell subtypes

Aims/hypothesisPancreatic islets depend on cytosolic calcium (Ca2+) to trigger the secretion of glucoregulatory hormones and trigger transcriptional regulation of genes important for islet response to stimuli. To date, there has not been an attempt to profile Ca2+-regulated gene expression in all islet cell types. Our aim was to construct a large single-cell transcriptomic dataset from human islets exposed to conditions that would acutely induce or inhibit intracellular Ca2+ signalling, while preserving biological heterogeneity.MethodsWe exposed intact human islets from three donors to the following conditions: (1) 2.8 mmol/l glucose; (2) 16 mmol/l glucose and 40 mmol/l KCl to maximally stimulate Ca2+ signalling; and (3) 16 mmol/l glucose, 40 mmol/l KCl and 5 mmol/l EGTA (Ca2+ chelator) to inhibit Ca2+ signalling, for 1 h. We sequenced 68,650 cells from all islet cell types, and further subsetted the cells to form an endocrine cell-specific dataset of 59,373 cells expressing INS, GCG, SST or PPY. We compared transcriptomes across conditions to determine the differentially expressed Ca2+-regulated genes in each endocrine cell type, and in each endocrine cell subcluster of alpha and beta cells.ResultsBased on the number of Ca2+-regulated genes, we found that each alpha and beta cell cluster had a different magnitude of Ca2+ response. We also showed that polyhormonal clusters expressing both INS and GCG, or both INS and SST, are defined by Ca2+-regulated genes specific to each cluster. Finally, we identified the gene PCDH7 from the beta cell clusters that had the highest number of Ca2+-regulated genes, and showed that cells expressing cell surface PCDH7 protein have enhanced glucose-stimulated insulin secretory function.Conclusions/interpretationHere we use our large-scale, multi-condition, single-cell dataset to show that human islets have cell-type-specific Ca2+-regulated gene expression profiles, some of them specific to subpopulations. In our dataset, we identify PCDH7 as a novel marker of beta cells having an increased number of Ca2+-regulated genes and enhanced insulin secretory function.Data availabilityA searchable and user-friendly format of the data in this study, specifically designed for rapid mining of single-cell RNA sequencing data, is available at https://lynnlab.shinyapps.io/Human_Islet_Atlas/. The raw data files are available at NCBI Gene Expression Omnibus (GSE196715).Graphical abstract

Alternating gemcitabine/nab-paclitaxel (GA) and 5-FU/leucovorin/irinotecan (FOLFIRI) as first-line treatment for de novo metastatic pancreatic cancer (MPC): Safety and effect.

4125 Background: Both gemcitabine and 5FU-based chemotherapy have demonstrated efficacy in MPC. Alternating regimens may 1) reduce toxicity 2) slow resistant cancer biology emergence and 3) provide a broader platform for addition of other therapeutic agents. Alternating GA and FOLFIRI in MPC has been previously reported as part of the SEENA -1 trial,our own institution, and elsewhere (Picozzi et.al. GI Cancer Symposium 2017, Picozzi et.al, ASCO 2018 Assenat et,al, ASCO 2018). An extension of our institutional observations are reported here. Methods: Pt eligibility required the following: 1) biopsy proven de novo MPC, 2) no prior evidence MPC on CT, 3) ECOG performance status ≤ 2, and 4) bi-dimensionally measurable disease. Treatment (Rx) entailed gemcitabine 1000mg/m2 and nab-paclitaxel 125mg/m2 1, (8), 15 alternating every 8 wks (2 cycles) with FOLFIRI using standard dosing. Patients were radiographically re-staged every 8 wks. Rx was continued up to 48 wks; Rx thereafter decided by pt/MD. Results: 108 pts met eligibility requirements from 10/2015 and 12/2020. Pt characteristics included median age 68 ( range 35-81), ECOG PS 0/≥1 54%/46%, # diseases sites 1/≥1 62%/38%, liver /non-liver 76%/24%, biliary obstruction yes/no 40%/60%, C 19.9 NL/ < 59XNL/ > 59X NL 12%/32%/56%; median Ca 19.9 4598 With median f/u of 19.7 mo, 17 pts remain on Rx < 48 wks, 35/91 (38%) completed 48 wks Rx, 56/91 (62%) pts progressed prior to 48 wks. Median # mos on Rx was 8.9. ≥ grade 3 heme toxicity included anemia 7%, neutropenia 9%, thrombocytopenia 5%. Neutrophil growth factors were not used in this pt cohort. ≥ 3 non-hem toxicity included neuropathy1%, nausea/vomiting 2%, mucositis 2%, diarrhea 1%. Disease control at 16 wks was 81% (35% PR/46% SD/16% PD, 95% CI 72-87%). Median OS was 13.7 mo (95% CI 10.9-18.7 mo). 6 /12/18/24 mo OS were 87%/55%/41%/ 20% respectively. Prognostic significance was seen with Rx > vs < 48 wks (21.1 vs 8.0 mo, p <.0001), and ECOG PS 0 vs. ≥1 ( 17.8 vs. 10.9 mo, p = 0.03) Age, # metastatic sites, liver involvement, biliary obstruction and magnitude of CA 19.9 elevation all failed to achieve prognostic significance at the p <.05 level. Conclusions: 1) Alternating GA/ FOLFIRI in MPC has a more favorable toxicity profile than standard regimens 2) Med OS appears superior to GA and competitive with FOLFIRINOX ; longer term (18/24 mo ) OS seemed particularly encouraging 3) ≥ 48 wks Rx and ECOG PS 0 were prognostically significant 4) Further investigation using this regimen including a) randomized comparisons, b) incorporation of molecular data and c) addition of additional agents seems indicated Updated survival data will be presented at the meeting

Modulating ALDH2 reveals a differential dependence on ROS for hypertrophy and SR Ca2+ release in aldosterone-treated cardiac myocytes

Growing evidence links high aldosterone levels with atrial fibrillation and other heart diseases. Here, we have investigated the functional consequences of culturing adult rat atrial myocytes with aldosterone, at the level of cell size, homeostasis of Ca2+, reactive oxygen species (ROS), and nitrogen oxide (NO). The protein levels of NO synthase (NOS), aldehyde dehydrogenase 2 (ALDH2), NADPH oxidase (NOX), and Na+-Ca2+ exchanger (NCX) were also studied. Aldosterone did not alter the expression of these proteins, except for the NCX, which was enhanced by nearly 100%. Additionally, the hormone inhibited and stimulated, respectively, the production of NO and ROS (the effect on ROS appeared after 24 h of treatment and reached a maximum by 4-6 days, with an EC50 of 1.2 nM). These changes in reactive species generation were blunted by tetrahydrobiopterin (BH4, a NOS cofactor), suggesting the involvement of an uncoupled NOS. An activator (Alda-1) and an inhibitor (daidzin) of ALDH2 were used, to determine if this enzyme activity is related to aldosterone effects, through possible modulation of ROS. Aldosterone produced a ∼10% increase in cell size and, remarkably, this hypertrophic effect, along with the corresponding changes in ROS and NO, were all mimicked by daidzin and prevented by Alda-1. Something different happened with SR Ca2+ release. Aldosterone increased both the magnitude of Ca2+ transients and the incidence of spontaneous Ca2+ oscillations, but these actions were not reproduced by daidzin. Moreover, rather than being prevented, they were further promoted by Alda-1, which also increased the rate of SR Ca2+ reuptake. These results suggest that NOS and ALDH2 may prevent some adverse consequences of aldosteronism (in the case of ALDH2, at the expense of exacerbating SR Ca2+ release). Our data also suggest a hierarchical model in which aldosterone promotes: SR Ca2+ release, then ROS production, and finally hypertrophy.

Abstract 3577: Pulsed focused ultrasound induces cytosolic calcium transients that increase free radical production and dsDNA breaks in tumor cells

Abstract Non-ablative pulsed focused ultrasound (pFUS) has shown promise as an immunomodulatory anti-cancer therapeutic. While largely nondestructive to tissues compared ablative ultrasound, pFUS can generate anti-tumor molecular microenvironments and enhance migration of anti-tumor immune cells into B16 and 4T1 flank tumors in immune-competent mice. pFUS also increases the number of dsDNA breaks in nuclear DNA of tumor cells by TUNEL reactions. DNA damage can elicit immune responses by causing cells to express ligands for immune cells and thus may be a critical component of generating observed immune responses in flank tumors following pFUS treatment. Here we investigate potential phyisological mechanisms underlying pFUS-associated DNA damage. B16 melanoma, 4T1 or MBA-MD-231 breast tumors, or C6 glioma cells were cultured and treated with pFUS in vitro (1 MHz, 6 MPa peak negative pressure, 10% duty cycle, 100 pulses). Cells were analyzed for DNA damage by TUNEL, reactive oxygen species (ROS) production by DCFDA, and intracellular Ca2+ by fluo-4. pFUS increased TUNEL reactivity and ROS concentrations in all cell types 2-6 hr after pFUS. Increased TUNEL reactivity was not observed when cells were incubated with the ROS scavenger Trolox (need concentration) following pFUS. pFUS generated Ca2+ transients in all cell types during pFUS treatment. Ca2+ dysregulation can increase cellular ROS production and when cytosolic Ca2+ transients were blocked by BAPTA-AM, both ROS production and TUNEL reactivity was reduced in all cell types. Further investigating mechanisms of Ca2+ transients, application of verapamil (10 uM) during pFUS to inhibit voltage-gated Ca2+ channels (VGCC) also suppressed Ca2+ transients, ROS production, and TUNEL reactivity. Lastly, the magnitudes of Ca2+ transients, ROS production, and TUNEL reactivity induced by pFUS were nonetheless different for different cell types (C6 glioma exhibited the smallest changes after pFUS, while MBA-MD-231 exhibited the largest). This suggested different sensitivities for each cell type to pFUS-induced DNA damage. As Ca2+ homeostasis can be disrupted in tumor cells, we measured resting cytosolic Ca2+ concentrations in each cell type following plasma membrane disruption with digitonin (20 uM). Resting cytosolic Ca2+ concentrations inversely correlated with increased magnitudes of ROS production or TUNEL reactivity following pFUS treatment. In conclusion, pFUS causes DNA damage in tumor cells by initiating cytosolic Ca2+ transients, in part, through VGCC that increase cellular ROS production and lead to dsDNA breaks. The effectiveness of pFUS to stimulate this physiological pathway is related to resting cytosolic Ca2+ concentrations, which different tumor cells could display varying levels of disrupted homeostasis and, thus differing magnitudes of response to pFUS. Future directions will assess potential involvement of mitochondrial dynamics in Ca2+-induced ROS production and in vivo experiments to further understand these dynamics in solid tumor models. Citation Format: Robert B. Rosenblatt, Joseph A. Frank, Scott R. Burks. Pulsed focused ultrasound induces cytosolic calcium transients that increase free radical production and dsDNA breaks in tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3577.

Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion.

TMEM16A is a Ca2+-activated Cl- channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl- currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl- channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Double Regulation of Cardiac Excitation-Contraction Coupling and Oxidant Stress by Pirfenidone

Pirfenidone (PFD) is well-known for improving cardiac performance in models of disease. Thus, here we have investigated its functional effects on cardiomyocytes. Cells were cultured under control culture conditions or the presence of PFD (1 mM, 1-2d). Subsequently, they were subjected to electrical stimulation to examine the levels of intracellular Ca2+ and contractility. Remarkably, PFD improved both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were also enhanced. Excitation-contraction coupling (ECC) was also investigated, under whole-cell patch-clamp. In keeping with a previous report, PFD elevated twofold the activity of CaV1.2. Besides, a similar increase in the magnitude of Ca2+ transients was also observed. Thus the gain of ECC was unaltered. Likewise, PFD did not alter the caffeine-sensitive Ca2+ store, indicating stimulation of Ca2+-induced Ca2+-release at constant sarcoplasmic reticulum Ca2+ load. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal during caffeine applications was 50% slower in PFD-treated cells (compared with controls), indicating that PFD downregulates the NCX activity. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidant agents (acetaldehyde and peroxide hydrogen). Conversely, the amount of nitric oxide (NO) was either, increased (in atrial myocytes) or unchanged (in ventricular myocytes). Western-blot analysis showed that endothelial nitric oxide synthase expression remains unaffected, while a dual regulation was observed for that of neuronal isoform (nNOS) which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. Thus, in atrial cardiomyocytes, an up-regulation of nNOS was enough to augment the synthesis of NO. Apart from improving our knowledge of the molecular mechanisms of PFD action, these findings may also help in explaining the corresponding cardioprotective effects.

Long-Term Regulation of Excitation-Contraction Coupling and Oxidative Stress in Cardiac Myocytes by Pirfenidone.

Pirfenidone (PFD) is used to treat human pulmonary fibrosis. Its administration to animals with distinct forms of cardiovascular disease results in striking improvement in cardiac performance. Here, its functional impact on cardiac myocytes was investigated. Cells were kept 1–2 days under either control culture conditions or the presence of PFD (1 mM). Subsequently, they were subjected to electrical stimulation to assess the levels of contractility and intracellular Ca2+. The PFD treatment promoted an increase in both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were enhanced as well. Excitation–contraction coupling (ECC) was also investigated, under whole-cell patch-clamp conditions. In keeping with a previous report, PFD increased twofold the density of Ca2+ current (ICa). Notably, a similar increase in the magnitude of Ca2+ transients was also observed. Thus, the gain of ECC was unaltered. Likewise, PFD did not alter the peak amplitude of caffeine-induced Ca2+ release, indicating stimulation of Ca2+-induced–Ca2+-release (CICR) at constant sarcoplasmic reticulum Ca2+ load. A phase-plane analysis indicated that PFD promotes myofilament Ca2+ desensitization, which is being compensated by higher levels of Ca2+ to promote contraction. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal in the presence of caffeine was 50% slower in PFD-treated cells (compared with controls), suggesting that PFD downregulates the activity of the exchanger. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidative insults (acetaldehyde and peroxide hydrogen). Conversely, the production of nitric oxide was either increased (in atrial myocytes) or remained unchanged (in ventricular myocytes). Protein levels of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) were also investigated. eNOS values did not exhibit significant changes. By contrast, a dual regulation was observed for nNOS, which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. In the latter cells, therefore, an up-regulation of nNOS was sufficient to stimulate the synthesis of NO. These findings improve our knowledge of molecular mechanisms of PFD action and may also help in explaining the corresponding cardioprotective effects.

Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

Immune therapy of cancer is among the most promising recent advances in medicine. Whether the immune system can keep cancer in check depends on, among other factors, the efficiency of immune cells to recognize and eliminate cancer cells. We describe a time-resolved single-cell assay that reports the quality, quantity, and kinetics of target cell death induced by single primary human natural killer (NK) cells. The assay reveals that single NK cells induce cancer cell death by apoptosis and necrosis but also by mixed forms. Inhibition of either one of the two major cytotoxic pathways, perforin/granzyme release or FasL/FasR interaction, unmasked the parallel activity of the other one. Ca2+ influx through Orai channels is important for tuning killer cell function. We found that the apoptosis/necrosis ratio of cancer cell death by NK cells is controlled by the magnitude of Ca2+ entry and furthermore by the relative concentrations of perforin and granzyme B. The possibility to change the apoptosis/necrosis ratio employed by NK cells offers an intriguing possibility to modulate the immunogenicity of the tumor microenvironment.

The calcium transient characteristics induced by fluid shear stress affect the osteoblast proliferation

Ca2+ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca2+ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca2+ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca2+ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca2+ uptake which were influenced by the amplitude of Ca2+ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy.

Desensitization of idiopathic pulmonary fibrosis fibroblasts to Alternaria alternata extract-mediated necrotic cell death.

Alternaria alternata is an allergenic fungus and known to cause an upper respiratory tract infection and asthma in humans with compromised immunity. Although A. alternata's effect on airway epithelial cells has previously been examined, the potential role of A. alternata on lung fibroblast viability is not understood. Since lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) display a distinct phenotype that is resistant to stress and cell death inducing conditions, the investigation of the role of Alternaria on pathological IPF fibroblasts provides a better understanding of the fibrotic process induced by an allergenic fungus. Therefore, we examined cell viability of control and IPF fibroblasts (n = 8 each) in response to A. alternata extract. Control fibroblast cell death was increased while IPF fibroblasts were resistant when exposed to 50–100 μg/mL of A. alternata extract. However, there was no significant difference in kinetics or magnitude of Ca2+ responses from control lung and IPF fibroblasts. In contrast, unlike control fibroblasts, intracellular reactive oxygen species (ROS) levels remained low when IPF cells were treated with A. alternata extracts as a function of time. Caspase 3/7 and TUNEL assay revealed that enhanced cell death caused by A. alternata extract was likely due to necrosis, and 7‐AAD assay and the use of sodium pyruvate for ATP generation further supported our findings that IPF fibroblasts become resistant to A. alternata extract‐induced necrotic cell death. Our results suggest that exposure to A. alternata potentially worsens the fibrotic process by promoting normal lung fibroblast cell death in patients with IPF.

Revealing calcium fluxes by analyzing inhibition dynamics in action potential clamp

In cardiac excitation-contraction coupling (ECC), calcium enters the cytosol via L-type Ca2+ channels (LTCC) and reverse Na+/Ca2+-exchange (NCXrev), or is released from the sarcoplasmic reticulum (SR) by Ca2+-induced Ca2+-release (CICR). The magnitude of Ca2+ influx via the different pathways varies with the state of the cell and is difficult to assess quantitatively, because changes in Ca2+ influx through one pathway affect the others. In rainbow trout ventricular myocytes, the role of the SR has been uncertain for decades. The aim of this work was therefore two-fold: 1) to develop a method to quantify the Ca2+ influx pathways, and 2) to determine the role of CICR from the SR in trout ventricular myocytes. The novelty of our developed method lies in the mathematical analysis of measured transsarcolemmal Ca2+ currents and their impact on the corresponding Ca2+ transient during gradual inhibition of the currents in action potential (AP) clamp. We tested the developed method using an excitation-contraction model and showed that the method was able to recover calcium fluxes from noisy synthetic data. We applied the approach to trout ventricular myocytes and quantified the relative contributions of different Ca2+ influx pathways in ECC and determined the kinetics of these fluxes. Under baseline conditions, NCXrev is the main transmembrane Ca2+ influx pathway contributing 29 ± 6% (of the Ca2+ influx), LTCC 18 ± 7%, and CICR 53 ± 10% to overall Ca2+ transient. Thus, NCXrev is an important regulator of contractility and probably plays a role in the negative force-frequency relationship of trout ventricular preparations. These results demonstrate that trout and neonatal mammalian cardiomyocytes resemble each other not only in terms of morphology and energetics but ECC as well. In summary, the developed method resolves the major problem how to separate highly interconnected fluxes in AP clamp and allows to study Ca2+ fluxes in cardiomyocytes under conditions close to in vivo.

Naïve and memory B cells exhibit distinct biochemical responses following BCR engagement.

Immunological memory is characterized by the rapid reactivation of memory B cells that produce large quantities of high-affinity antigen-specific antibodies. This contrasts the response of naïve B cells, and the primary immune response, which is much slower and of lower affinity. Memory responses are critical for protection against infectious diseases and form the basis of most currently available vaccines. Although we have known about the phenomenon of long-lived memory for centuries, the biochemical differences underlying these diverse responses of naïve and memory B cells is incompletely resolved. Here we investigated the nature of B-cell receptor (BCR) signaling in human splenic naïve, IgM(+) memory and isotype-switched memory B cells following multivalent BCR crosslinking. We observed comparable rapid and transient phosphorylation kinetics for proximal (phosphotyrosine and spleen tyrosine kinase) and propagation (B-cell linker, phospholipase Cγ2) signaling components in these different B-cell subsets. However, the magnitude of activation of downstream components of the BCR signaling pathway were greater in memory compared with naïve cells. Although no differences were observed in the magnitude of Ca(2+) mobilization between subsets, IgM(+) memory B cells exhibited a more rapid Ca(2+) mobilization and a greater depletion of the Ca(2+) endoplasmic reticulum stores, while IgG(+) memory B cells had a prolonged Ca(2+) uptake. Collectively, our findings show that intrinsic signaling features of B-cell subsets contribute to the robust response of human memory B cells over naïve B cells. This has implications for our understanding of memory B-cell responses and provides a framework to modulate these responses in the setting of vaccination and immunopathologies, such as immunodeficiency and autoimmunity.

Elemental Contents of Spinach and Lettuce from Irrigated Gardens in Kano, Nigeria

One way analysis (ANOVA) was used to analyze a large dataset of elemental levels of two vegetables – spinach (<em>Amaranthus cruentus</em>) and lettuce (<em>Lactuca sativa</em>) grown around River Jakara in Kano, Nigeria using data generated during 12 months of monitoring Ca, K, Mg, Na, (essential bulk elements) Cu, Zn, Cd, Ni, Cr, Co, Pb and Fe (trace/heavy elements) concentrations collected at three designated sites. The concentrations of the elements showed insignificant differences between sites but significant differences between some months. The soil was implicated as the major source of the elements. The concentrations of the trace/heavy metals exceeded those of the international permissible limits which pointed to the contamination of the vegetables. The mean concentrations of the elements occurred in the magnitude of Ca > Mg > K > Na > Fe > Zn > Pb > Co > Cr > Cu > Ni > Cd and Ca > Na > K >Mg > Fe > Zn > Pb > Cr > Co > Cu > Ni > Cd in the spinach and lettuce respectively. The continued consumption of these vegetables by the inhabitants of Kano and its environs present a public health risk with regards to their concentrations with heavy metals. It is therefore recommended that the relevant organ of government should find an alternative farmland for the farmers within the catchment area of River Jakara where unpolluted soil can be utilized for the production of the vegetables.

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca2+ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca2+ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca2+ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca2+ regulation. Basolateral Ca2+ transport across distal tubules of Drosophila varied throughout the life cycle; Ca2+ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca2+ uptake and Ca2+ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca2+ transport is bidirectional. Ca2+ uptake across the distal segment of tubules of young adults and Ca2+ release across the distal segment of tubules of older adults was also suggestive of reversible Ca2+ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca2+ sequestration during periods of high dietary calcium intake and passive Ca2+ release during periods of calcium deficiency.

Calmodulin and PI(3,4,5)P3 cooperatively bind to the Itk Pleckstrin homology domain to promote efficient calcium signaling and IL-17A production in T cells (LYM5P.710)

Abstract Precise regulation of the kinetics and magnitude of Ca2+ signaling enables this signal to mediate diverse responses, such as cell migration, differentiation, vesicular trafficking, and cell death. Here, we showed that the Ca2+-binding protein calmodulin (CaM) acted in a positive feedback loop to potentiate Ca2+ signaling downstream of the Tec kinase family member Itk. Using NMR (nuclear magnetic resonance), we mapped CaM binding to two loops adjacent to the lipid-binding pocket within the Itk pleckstrin homology (PH) domain. The Itk PH domain bound synergistically to Ca2+/CaM and the lipid phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3], such that binding to Ca2+/CaM enhanced the binding to PI(3,4,5)P3 and vice versa. Disruption of CaM binding attenuated Itk recruitment to the membrane and diminished release of Ca2+ from the endoplasmic reticulum. Moreover, disruption of this feedback loop abrogated Itk-dependent production of the proinflammatory cytokine IL-17A (interleukin-17A) by CD4+ T cells. Additionally, we found that CaM associated with PH domains from other proteins, indicating that CaM may regulate other PH domain-containing proteins.

Constitutive PKA activity is essential for maintaining the excitability and contractility in guinea pig urinary bladder smooth muscle: role of the BK channel.

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Ca(2+)-Transport through Plasma Membrane as a Test of Auxin Sensitivity.

Auxin is one of the crucial regulators of plant growth and development. The discovered auxin cytosolic receptor (TIR1) is not involved in the perception of the hormone signal at the plasma membrane. Instead, another receptor, related to the ABP1, auxin binding protein1, is supposed to be responsible for the perception at the plasma membrane. One of the fast and sensitive auxin-induced reactions is an increase of Ca2+ cytosolic concentration, which is suggested to be dependent on the activation of Ca2+ influx through the plasma membrane. This investigation was carried out with a plasmalemma enriched vesicle fraction, obtained from etiolated maize coleoptiles. The magnitude of Ca2+ efflux through the membrane vesicles was estimated according to the shift of potential dependent fluorescent dye diS-C3-(5). The obtained results showed that during coleoptiles ageing (3rd, 4th and 5th days of seedling etiolated growth) the magnitude of Ca2+ efflux from inside-out vesicles was decreased. Addition of ABP1 led to a recovery of Ca2+ efflux to the level of the youngest and most sensitive cells. Moreover, the efflux was more sensitive, responding from 10−8 to 10−6 M 1-NAA, in vesicles containing ABP1, whereas native vesicles showed the highest efflux at 10−6 M 1-NAA. We suggest that auxin increases plasma membrane permeability to Ca2+ and that ABP1 is involved in modulation of this reaction.