Extracellular Ca2 Research Articles

Extracellular H+ Ions are a Novel Signal for Tyrosine Hydroxylase Activation in Catecholaminergic Cells.

Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthetic pathway. Short-term TH activity is proposed to be regulated by the phosphorylation/dephosphorylation of regulatory domains Ser 40, 31, and/or 19 in response to membrane depolarization coupled increase in intracellular Ca2+. Here, we present in situ evidence to support that extracellular H+ ions ([H+]o) are an intracellular or extracellular Ca2+-independent novel signal for TH activation in catecholaminergic MN9D and PC12 cells. [H+]o-mediated TH activation is a short-term process coupled with a Na+-independent Cl-/HCO3- exchanger-mediated increase of intracellular hydrogen ions ([H+]i). While extracellular Ca2+ is not required for [H+]o-mediated TH activation, [H+]o does not increase the cytosolic Ca2+ levels in neuronal or non-neuronal cells in the presence or absence of extracellular Ca2+. Although [H+]o-mediated TH activation is associated with a significant increase in Ser 40 phosphorylation, major protein kinases proposed to be responsible for this process appear to be not involved. However, we have not been able to identify the protein kinase(s) involved in [H+]o-mediated phosphorylation of TH at present. Studies with a pan-phosphatase inhibitor, okadaic acid (OA), appear to indicate that the inhibition of phosphatase activities may not play a significant role in H+-mediated activation of TH. The relevance of these findings to the physiological TH activation mechanism and hypoxia, ischemia, and trauma-induced selective dopaminergic neural death is being discussed in this paper.

Piezo1 and TRPV4 complementary contribute to [Ca2+]i homeostasis in collecting duct cells

Mechanical stress arising from elevated fluid flow is an important regulator of the water-electrolyte transport in the collecting duct (CD). It is well established that mechano-sensitive TRPV4 Ca2+-permeable channel mediates intracellular Ca2+ ([Ca2+]i) responses to high flow in these cells. Piezo1, a Ca2+-permeable channel directly activated by the plasma membrane stretch, is a recent arrival at this scene. Piezo1 expression has been reported in the CD. However, its functional significance is yet to be determined. Here, we applied a combination of Ca2+ imaging and confocal microscopy in freshly isolated split-opened CDs to investigate a potential interplay between TRPV4 and Piezo1 in mediating [Ca2+]i signaling in the CD. Immunofluorescent analysis of split-opened CDs showed presence of Piezo1 in both AQP2-positive (principal cells, PC) and -negative (intercalated cells, IC) with higher intensity in the former. Importantly, Piezo1 was apparent on both apical and basolateral membrane of CD cells. Piezo1 agonists, Yoda-1 (20μM) and Jedi2 (500μM) acutely increase [Ca2+]i in both principal and intercalated cells. The responses were abolished when extracellular Ca2+ was buffered with EGTA suggesting a direct Ca2+ influx via Piezo1. Interestingly, we detected two disparate calcium responses to Piezo1 activation with the majority (70%) cells showing a fast transient response with a sustained plateau, whereas the remaining (30%) population had much slower gradual calcium increase to the same plateau level. Subsequent immunofluorescent analysis revealed that fast Ca2+ responses were attributed to PC, whereas responses with slow kinetics corresponded to IC. Consistently, PCs have ~3 fold higher Piezo-1 signal when compared to neighbor ICs. We next tested whether Piezo1 activity depends on TRPV4 status. Genetic deletion and pharmacological inhibition of TRPV4 with GSK2193874 did not affect Piezo1-dependent elevations in [Ca2+]i. However, high K+ diet, which is known to elevate the flow in the CD and augment both TRPV4 activity and expression, led to diminished Piezo1 responses to Yoda-1. In summary, our results are consistent with the view that Piezo1 and TRPV4 are functional in both principal and intercalated cells of the collecting duct and operate independently to modulate mechanosensitive [Ca2+]i signaling. We hypothesize that augmented TRPV4-dependent [Ca2+]i is compensated by the diminished activity of Piezo1 to account for elevated flow rate upon increased K+ intake. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-19CDA34660148 (to V. N. Tomilin). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of GPER in the expression and secretion of pro-inflammatory cytokines and calcium signaling in bone marrow-derived macrophages

Macrophages play an important role in mediating inflammation in numerous disease states by secreting pro-inflammatory cytokines including IL-1β and IL-18. Macrophages are driven into the M1-proinflammatory phenotype upon detection of pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). As macrophages are polarized into the M1 phenotype, they upregulate the expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. An additional activating stimulus is required to induce the assembly of the NLRP3 inflammasome, which facilitates the activation of pro-caspase 1 and the subsequent conversion of the pro-forms of both IL-18 and IL-1β into their mature forms for their secretion through gasdermin D membrane pores. A canonical activator of the NLRP3 inflammasome is extracellular ATP. ATP activation of the P2X7 receptor induces potassium efflux and calcium influx, both of which are necessary for NLRP3 inflammasome activation. Previous studies suggest that the G protein-coupled receptor (GPER) agonist G-1 reduced inflammation in animal disease models. However, the GPER-dependent effect of G-1 on pro-inflammatory cytokine expression and NLRP3 inflammasome activation in macrophages is unknown. In the current study, we utilized bone marrow-derived macrophages (BMDMs) from either WT or GPER-/- mice to test the hypothesis that G-1 reduces inflammatory cytokine production and NLRP3 inflammasome expression in a GPER-dependent manner. We found that the treatment of WT BMDMs with 100 nM G-1 for 24 hrs inhibited LPS-induced NLRP3, pro-IL-1β, IL-6, CCL5 and expression, and IL-1β secretion. Importantly, G-1 had no effect on GPER-/- BMDMs. To begin to determine if GPER modulates NLRP3 inflammasome activation, we characterized ATP-induced Ca2+ signals in both WT and GPER BMDMs with and without LPS pretreatment. No differences were observed between unprimed WT and GPER-/- BMDMs with respect to basal Ca2+ level, ATP-induced intracellular Ca2+ release, and subsequent bulk Ca2+ signal upon reestablishment of extracellular Ca2+. However, priming with LPS significantly increased ATP-induced intracellular Ca2+ release in both WT and GPER-/- BMDMs. Compared to WT cells, LPS-primed GPER-/- BMDMs exhibit significantly higher basal Ca2+ yet significantly lower ATP-induced Ca2+ release. Consistently, 15-minute treatment with 100 nM G-1 agonist strongly reduces ATP-induced Ca2+ release in WT BMDMs but had no effect in GPER-/- BMDMs. These results suggest a role for GPER in mediating Ca2+ uptake into IP3-sensitive intracellular compartments in BMDMs during LPS priming. In keeping with the known role for mobilization of intracellular Ca2+ in ATP-induced secretion of pro-inflammatory cytokines, preliminary data suggests that short-term G-1 pretreatment reduced IL-1β secretion induced by ATP in LPS-primed BMDMs. Overall, these data indicate a role for GPER in the priming and activation of the NLRP3 inflammasome. This research was supported by Des Moines University IOER Research & Grant Award #03-22-10. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Two distinct calcium signaling behaviors are present in interstitial cells of Cajal of the gastroesophageal junction

Background/Objectives: The lower esophageal sphincter (LES) is located within the gastroesophageal junction (GEJ) and generates tone to prevent gastric reflux into the esophagus. Previously, we demonstrated that the generation and maintenance of tone in the LES clasp muscle involves intramuscular interstitial cells of Cajal (ICC-IM) (Drumm et al., 2022; PMID: 35229888). The aim of the current study was to further characterize Ca2+ signaling in ICC-IM within the GEJ. We hypothesize that Ca2+ release from the endoplasmic reticulum (ER) activates Ca2+-activated Cl- channels (ANO1) in ICC leading to depolarization that is conducted to smooth muscle cells (SMCs) via gap junctions; this in turn leads to Ca2+ influx via L-type Ca2+ channels (CavL) and the development of tone. Methods: Confocal spinning disc microscopy was used to image Ca2+ transients in the LES and distal esophagus of mice expressing GCaMP6f in ICC (Kit-GCaMP6f). Muscles of the GEJ imaged with the submucosal surface upwards at 20x. Ani9 (3mM), nifedipine (1mM) and CPA (10mM) were used to inhibit ANO1, CavL and SERCA, respectively. Pinacidil (10mM) was used to activate KATP channels. Results: ICC-IM within the GEJ displayed two types of Ca2+ signaling behaviors. Ca2+ transients in ICC-IM of the LES clasp muscle were stochastic, arising from multiple firing sites within the same cell. These Ca2+ transients were not synchronized within the same cell or between adjacent cells. In contrast, two Ca2+ signaling patterns were observed in ICC-IM of the distal esophagus where smooth muscle transitions to skeletal muscle. One ICC-IM population (Type I) shared the Ca2+ signaling behaviors of ICC-IM in the LES clasp. Another ICC population (Type II) exhibited rhythmic whole-cell Ca2+ transients that were synchronized between adjacent Type II ICC. Whole cell Ca2+ transients in Type II ICC were abolished by Ani9, nifedipine or pinacidil, revealing underlying Ca2+ transients similar to those observed in Type I ICC. In contrast, Ca2+ transients in Type I ICC were resistant to Ani9, nifedipine and pinacidil. Ca2+ transients were abolished in both types of ICC by removal of extracellular Ca2+, however the activity in Type I ICC persisted longer than that of Type II ICC. Discussion/Conclusions: This study demonstrates two distinct Ca2+ signaling behaviors in ICC-IM of the GEJ with differing pharmacology. This is similar to what has been previously described within the internal anal sphincter (IAS) (Hannigan et al., 2020; PMID: 32587396). In the IAS, it has been suggested that Type II ICC-IM generate slow waves however their role in the GEJ remains unclear as slow waves have not been described within this region of the mouse. Thus, the functional role of ICC-IM in the GEJ, particularly Type II ICC, warrants further investigation. NIH DK129528 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pressure induced changes in endothelial cell morphology and Ca2+ signaling in ex vivo pressurized resistance arteries revealed using 4D diSPIM myography and AI deep-learning

The vascular endothelium is critical in promoting vasodilation of resistance-size arteries through Ca 2+ dependent pathways. Vascular endothelial cells (ECs) are exposed to mechanical stimulation through changing intravascular pressure and flow, yet it is unclear how these forces impact EC morphology and Ca 2+ signaling in pressurized arteries. Visualizing how the endothelium of an intact resistance-size vessel responds to changing physical forces requires isometric, sub-micron, resolution. Conventional confocal fluorescent microscope systems are limited by poor axial resolution. 4D dual inverted selective plane of illumination microscopy (diSPIM; 3i) uniquely provides isometric resolution at ~330 nm with high temporal resolution and minimal photobleaching or phototoxicity. We designed custom pressure myography chambers to allow simultaneous 4D diSPIM fluorescent imaging and vessel diameter tracking measurements via transmitted IR light (diSPIM Myography). We captured fluorescent images of ex vivo pressurized resistance-size mouse mesenteric arteries at 80, 60, 40, and 20 mm Hg before and after the generation of myogenic tone and in the absence of extracellular Ca 2+ . Representative images were annotated for ECs, nuclei, myoendothelial projections (MEPS), and Ca 2+ signals. Annotations were used as ground truth for CNN AI model generation and validation using Apeer (Zeiss). Arivis Vision4D (Zeiss) was used to run the AI model and quantify data. As static intravascular pressure is increased in arteries without tone (with or without Ca 2+ ), ECs increase in width as the vessel dilates. Myogenic tone minimizes pressure dilation and changes in EC width, however, ECs compress by up to 1 micron as pressure increases from 20 to 80 mm Hg. Additionally, increasing intravascular pressure in arteries with tone decreases local EC Ca 2+ signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca 2+ signaling in intact resistance arteries under conditions of physiologic temperature, pressure, pH, and tone. diSPIM myography provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function. AI image annotation reduces analysis bias and increases the breadth of quantifiable microscopy data. DMC is supported by NIH R00HL133451 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Piezo1 regulates expression of BK and TRPV4 channels in mouse cortical collecting duct (CCD)

Within the CCD, an acute increase in tubular fluid flow rate (TFFR) leads to an immediate high amplitude increase in [Ca 2+ ] i , due to release of IP3-sensitive internal Ca 2+ stores coupled to extracellular Ca 2+ entry at the basolateral membrane in both principal (PC) and intercalated (IC) cells therein (Liu et al., 2003, 2005, 2007). This is followed by a decay in [Ca 2+ ] i to a sustained plateau elevation due, at least in part, to luminal Ca 2+ entry through TRPV4 (Berrout et al., 2012). This flow-induced increase in [Ca 2+ ] i is necessary for BK channel-mediated flow induced K + secretion (FIKS) in the microperfused mammalian CCD. We generated a mouse with IC-specific targeted deletion of the mechanosensitive Ca 2+ permeable channel, Piezo1, which is expressed on the basolateral membranes of PCs and ICs in the mouse CCD (Dalghi et al., 2019). Microperfused CCDs isolated from these IC-PIEZO1-KO mice fed a high K (HK, 5% K + ) diet and subject to an increase in TFFR did not exhibit either a peak or plateau elevation in [Ca 2+ ] i in response to flow, and FIKS was eliminated. The absence of FIKS and the plateau elevation in [Ca 2+ ] during sustained flow in the CCDs of KO mice led us to examine whether BK and TRPV4 channel expression was impacted by deletion of PIEZO1.Indirect immunofluorescence (IF) staining was performed to examine expression and localization of the BK and TRPV4 channels in PCs and ICs of IC-PIEZO1-KO mice. Whole cell and apical + subapical fluorescence intensities in cryosections were analyzed using LAS X software from a total of 3 male and 3 female control mice and 3 male and 3 female IC-PIEZO1-KO mice fed HK diet x 10d.IF analysis showed reduced whole cell BKα expression in ICs of both male (p≤0.05) and female (p≤0.01) IC-PIEZO1-KO mice. We also saw reduced BKα expression in PCs from female but not male KOs. In addition, ICs in female but not male KO mice had reduced whole cell TRPV4 expression vs. controls (p≤0.01). Also, whole cell expression of TRPV4 in PCs from female KO mice was reduced vs. female controls (p≤0.01). The ratio of apical-to-whole cell expression of BKα was similar in PCs and ICs of KO and control mice. Male, but not female, KO mice had modestly reduced apical TRPV4 expression in PCs vs. controls (p≤0.01). Piezo1 plays a role in the regulation of BK and TRPV4 channel expression in CCDs. In addition, sex differences exist in whole cell expression of these channels in response to IC-selective deletion of PIEZO1. R01 DK038470 (TK, LMS); R01 DK129285 (TK, LMS); P30 DK079307 (TK) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular histone proteins are novel activators of P2XR7 channel current

Extracellular histone proteins have been identified as important damage associated molecules that contribute to vascular dysfunction. Histones are elevated in circulation after traumatic injury and activation of the innate immune response. In prior work, we demonstrated that extracellular histone proteins induced endothelial cell (EC) Ca 2+ influx, increased EC propidium iodide labeling, and decreased vasodilatory responses. These paradoxical observations could be explained by activation of a non-selective cation channel. The ionotropic purinergic receptor 7 (P2XR7) stands out as a candidate as it has been shown to form a high-conductance, poorly selective, cation channel capable of conducting large cationic dyes. Herein, we test the hypothesis that P2XR7 channels are activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells ( Xenopus oocytes) and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was much slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). GW791343, a selective P2XR7 antagonist with a known binding site distinct from the ATP binding site, inhibited ATP-evoked currents but did not inhibit histone-evoked currents. As with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca 2+ . These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents. These results suggest a new allosteric mechanism of P2XR7 activation by histone proteins that may guide the development of more effective P2XR7 pharmacology and therapeutic strategies for a wide array of pathologies that involve P2XR7 function. DMC is supported by NIH R00HL133451 and R01HL155180 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Allelic effects on cerebral and carotid artery passive mechanical properties in young, adult, and aged hAPOE3 and hAPOE4 mice

Alzheimer’s disease (AD) is the most common form of dementia and among the leading mortalities in the US. There is growing evidence supporting a connection between AD and cardiovascular disease (CVD). Variant isoforms of apolipoprotein E (APOE) are risk factors for AD and CVD. Compared to the most common APOE isoform, APOE3 (E3), APOE4 (E4) increases AD risk. In order to study APOE-mediated processes, posterior cerebral arteries (PCA) and common carotid arteries (CCA) were isolated from mice expressing human-ApoE targeted replacement of APOE3 (B6.129P2- Apoe tm2(APOE*3)Mae N8) and APOE4 (B6.129P2- Apoe tm3(APOE*4)Mae N8)(Taconic Labs). Our objective was to determine whether the E3 and E4 allele cause different mechanical properties in the absence of extracellular Ca 2+ in the PCA and CCA isolated from young (Y, 3-4mo), adult (Ad, 12-15mo), and aged (Ag, 18-22mo), homozygous E3 and E4, male and female mice. We hypothesized that E4 would alter PCA and CCA structure/function when compared to age matched E3 cohorts. 5-8mm segments of PCA and CCA were isolated, cleaned of connective tissue, cannulated, and placed into an arteriograph. Upon equilibration, all vessels were challenged with 60mM [K + ] to assess viability. Vessels not responding to 60mM [K + ] were discarded. Mechanical characteristics, including lumen diameter (LD), wall thickness (WT), passive distensibility (PD), and stress/strain (S/S) were measured at intraluminal pressures ranging from 10-140mmHg. We observed no statistical differences in LD, PD, or S/S in PCAs and CCAs of Y and Ad, E3 and E4 mice. For LD and WT, Ag E3 was greater than Ag E4 (p<0.05). In contrast, LD in PCAs and CCAs of Ag E3 mice was larger than in Ag E4 mice (p<0.05). We further observed that PD and S/S in CCAs from Ag E3 was greater than in Ag E4 mice (p<0.05), whereas there was no difference in PD and S/S in PCAs isolated from Ag E3 or E4 mice. The WT of PCA in Y E4 was significantly greater than Y E3 mice (p<0.05). In contrast, WT in Ag E3 mice was larger than Ag E4 mice (p<0.05). There was no difference between WT of Ad E3 and E4 mice. The WT of CCAs was significantly greater in Ag E3 vs Ag E4 mice (p<0.05). There is no difference in WT in CCAs from Y (E3 vs E4) or Ad (E3 vs E4) mice. This data indicates aging has minimal effect on mechanical properties in the PCA. In contrast, CCAs in Ag E4 exhibited greater arterial stiffening as indicated by a decrease in their distensibility and leftward shift in S/S, as evidenced by greater stress for given amount of strain observed in PCAs of Ag E4 mice at increased intraluminal pressures. ABRC/ADHS18-205211 (JVE, TBJ, CBJ, DME), Arizona Alzheimer's Consortium (funded by the Arizona Department of Health Services, Contract No. CTR040636) and matching funds from Midwestern University (DME), Biomedical Sciences Program (BG, LJS, DME), Biomedical Sciences Start-up Funds (DME). LJS and ASH equally contributed as first authors. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The interplay of intracellular calcium and zinc ions in response to electric field stimulation in primary rat cortical neurons in vitro.

Recent pharmacological studies demonstrate a role for zinc (Zn2+) in shaping intracellular calcium (Ca2+) dynamics and vice versa in excitable cells including neurons and cardiomyocytes. Herein, we sought to examine the dynamic of intracellular release of Ca2+ and Zn2+ upon modifying excitability of primary rat cortical neurons using electric field stimulation (EFS) in vitro. We show that exposure to EFS with an intensity of 7.69 V/cm induces transient membrane hyperpolarization together with transient elevations in the cytosolic levels of Ca2+ and Zn2+ ions. The EFS-induced hyperpolarization was inhibited by prior treatment of cells with the K+ channel opener diazoxide. Chemical hyperpolarization had no apparent effect on either Ca2+ or Zn2+. The source of EFS-induced rise in Ca2+ and Zn2+ seemed to be intracellular, and that the dynamic inferred of an interplay between Ca2+ and Zn2+ ions, whereby the removal of extracellular Ca2+ augmented the release of intracellular Ca2+ and Zn2+ and caused a stronger and more sustained hyperpolarization. We demonstrate that Zn2+ is released from intracellular vesicles located in the soma, with major co-localizations in the lysosomes and endoplasmic reticulum. These studies further support the use of EFS as a tool to interrogate the kinetics of intracellular ions in response to changing membrane potential in vitro.

Transient Receptor Potential Ankyrin 1 (TRPA1) Channel as a Sensor of Oxidative Stress in Cancer Cells.

Moderate levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), fuel tumor metastasis and invasion in a variety of cancer types. Conversely, excessive ROS levels can impair tumor growth and metastasis by triggering cancer cell death. In order to cope with the oxidative stress imposed by the tumor microenvironment, malignant cells exploit a sophisticated network of antioxidant defense mechanisms. Targeting the antioxidant capacity of cancer cells or enhancing their sensitivity to ROS-dependent cell death represent a promising strategy for alternative anticancer treatments. Transient Receptor Potential Ankyrin 1 (TRPA1) is a redox-sensitive non-selective cation channel that mediates extracellular Ca2+ entry upon an increase in intracellular ROS levels. The ensuing increase in intracellular Ca2+ concentration can in turn engage a non-canonical antioxidant defense program or induce mitochondrial Ca2+ dysfunction and apoptotic cell death depending on the cancer type. Herein, we sought to describe the opposing effects of ROS-dependent TRPA1 activation on cancer cell fate and propose the pharmacological manipulation of TRPA1 as an alternative therapeutic strategy to enhance cancer cell sensitivity to oxidative stress.

Patch repair protects cells from the small pore-forming toxin aerolysin.

Aerolysin family pore-forming toxins damage the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four proposed membrane repair mechanisms include toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which repair mechanism aerolysin triggers is unknown. Membrane repair requires Ca2+, but it is controversial if Ca2+ flux is triggered by aerolysin. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to what is seen with cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Intracellular Ca2+ chelation increased cell death, indicating that Ca2+-dependent repair pathways were triggered. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to what is seen with CDCs, expression of the patch repair protein dysferlin protected cells from aerolysin. We propose aerolysin triggers a Ca2+-dependent death mechanism that obscures repair, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

Platelet-activating factor (PAF) enhances guinea pig detrusor smooth muscle contractile activities by stimulating voltage-dependent Ca2+ channels and store-operated Ca2+ channels

We investigated the extracellular Ca2+ influx pathways involved in platelet-activating factor (PAF)-enhanced guinea pig detrusor smooth muscle (DSM) contractile activities. One micromolar PAF-enhanced DSM contractile activities were completely inhibited by extracellular Ca2+ removal and strongly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors. PAF-enhanced DSM contractile activities remaining in the presence of verapamil (10 μM) were not inhibited by LOE-908 (30 μM, an inhibitor of receptor-operated Ca2+ channels (ROCCs)), but were almost completely inhibited by SKF-96365 (30 μM, an inhibitor of store-operated Ca2+ channels (SOCCs) and ROCCs). These results suggest that VDCCs and SOCCs are responsible for PAF-enhanced DSM contractile activities.

Contractile effect of trimethylamine and trimethylamine-n-oxide on isolated human umbilical arteries.

The aim of this study is to investigate the effect of trimethylamine (TMA) and trimethylamine-n-oxide (TMAO) on the contractility of human umbilical artery and the possible mechanisms involved. Vasoactive responses to TMA and TMAO on human umbilical artery rings were measured in isolated organ baths. Cumulative dose-response curves for TMA and TMAO were obtained before and after incubation with atropine, yohimbine, prazosin, indomethacin, verapamil, and Ca+2 -free Krebs-Henselite solution. Administration of cumulative TMA and TMAO resulted in dose-dependent contraction at concentrations ranging from 10 to 100 mM on human umbilical artery rings. TMA-induced contractions were more potent than TMAO-induced contractions (TMA: -logEC50 = 1.00 ± 0.02, TMAO: -logEC50 = 0.57 ± 0.02). Contraction responses to TMA were significantly lower in the presence of verapamil and in the absence of external Ca+2 (p < 0.001, p < 0.05, respectively). Our results showed that TMA and TMAO caused vasoconstriction in isolated human umbilical artery rings. Our findings also indicated that TMA but not TMAO-induced vasoconstriction was partially dependent on extracellular Ca2+ and calcium influx through L-type Ca2+ channels. Our results suggest that TMA and TMAO may have the potential to contribute to cardiovascular diseases through their direct effect on vascular contractility in human arteries.

Ca2+-induced myelin pathology precedes axonal spheroid formation and is mediated in part by store-operated Ca2+ entry after spinal cord injury.

The formation of axonal spheroid is a common feature following spinal cord injury. To further understand the source of Ca2+ that mediates axonal spheroid formation, we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca2+. We performed two-photon excitation imaging of spinal cords isolated from Thy1YFP+ transgenic mice and applied the lipophilic dye, Nile red, to record dynamic changes in dorsal column axons and their myelin sheaths respectively. We selectively released Ca2+ from internal stores using the Ca2+ ionophore ionomycin in the presence or absence of external Ca2+. We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 mM Ca2+ artificial cerebrospinal fluid. In contrast, removal of external Ca2+ significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment. Using mice that express a neuron-specific Ca2+ indicator in spinal cord axons, we confirmed that ionomycin induced significant increases in intra-axonal Ca2+, but not in the absence of external Ca2+. Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation. Pretreatment with YM58483 (500 nM), a well-established blocker of store-operated Ca2+ entry, significantly decreased myelin injury and axonal spheroid formation. Collectively, these data reveal that ionomycin-induced depletion of internal Ca2+ stores and subsequent external Ca2+ entry through store-operated Ca2+ entry contributes to pathological changes in myelin and axonal spheroid formation, providing new targets to protect central myelinated fibers.

Ablation of TRPC3 disrupts Ca2+ signaling in salivary ductal cells and promotes sialolithiasis

Clinical studies and structural analyses of salivary stones strongly suggest a linkage between higher saliva calcium (Ca2+) and salivary stone formation, sialolithiasis; however, the process and the mechanism leading to Ca2+ overload during sialolithiasis is not well understood. Here, we show that TRPC3 null (−/−) mice presented with a reduction in Ca2+ entry and current in ductal cells with higher saliva [Ca2+] suggesting diminished transepithelial Ca2+ flux across the salivary ductal cells, leaving more Ca2+ in ductal fluid. Significantly, we found that TRPC3 was expressed in mice and human salivary ductal cells, while intraductal stones were detected in both mice (TRPC3−/−) and patient (sialolithiasis) salivary glands. To identify the mechanism, we found that TRPC3 was crucial in preventing the expression of calcification genes (BMP2/6, Runx2) in ductal cells which may be due to higher extracellular Ca2+ in SMG tissues. Similarly, inflammatory (IL6, NLRP3), fibrotic (FN1, TGFβ1) and apoptotic (Bax1/Bcl2) markers were also elevated, suggesting that the loss of TRPC3 induces genetic changes that leads to salivary gland cell death and induction of inflammatory response. Overall, ablation of TRPC3−/− leads to higher saliva [Ca2+], along with elevated detrimental gene expressions, altogether contributing to salivary gland stone formation.

The activation of mGluR4 rescues parallel fiber synaptic transmission and LTP, motor learning and social behavior in a mouse model of Fragile X Syndrome

BackgroundFragile X syndrome (FXS), the most common inherited intellectual disability, is caused by the loss of expression of the Fragile X Messenger Ribonucleoprotein (FMRP). FMRP is an RNA-binding protein that negatively regulates the expression of many postsynaptic as well as presynaptic proteins involved in action potential properties, calcium homeostasis and neurotransmitter release. FXS patients and mice lacking FMRP suffer from multiple behavioral alterations, including deficits in motor learning for which there is currently no specific treatment.MethodsWe performed electron microscopy, whole-cell patch-clamp electrophysiology and behavioral experiments to characterise the synaptic mechanisms underlying the motor learning deficits observed in Fmr1KO mice and the therapeutic potential of positive allosteric modulator of mGluR4.ResultsWe found that enhanced synaptic vesicle docking of cerebellar parallel fiber to Purkinje cell Fmr1KO synapses was associated with enhanced asynchronous release, which not only prevents further potentiation, but it also compromises presynaptic parallel fiber long-term potentiation (PF-LTP) mediated by β adrenergic receptors. A reduction in extracellular Ca2+ concentration restored the readily releasable pool (RRP) size, basal synaptic transmission, β adrenergic receptor-mediated potentiation, and PF-LTP. Interestingly, VU 0155041, a selective positive allosteric modulator of mGluR4, also restored both the RRP size and PF-LTP in mice of either sex. Moreover, when injected into Fmr1KO male mice, VU 0155041 improved motor learning in skilled reaching, classical eyeblink conditioning and vestibuloocular reflex (VOR) tests, as well as the social behavior alterations of these mice.LimitationsWe cannot rule out that the activation of mGluR4s via systemic administration of VU0155041 can also affect other brain regions. Further studies are needed to stablish the effect of a specific activation of mGluR4 in cerebellar granule cells.ConclusionsOur study shows that an increase in synaptic vesicles, SV, docking may cause the loss of PF-LTP and motor learning and social deficits of Fmr1KO mice and that the reversal of these changes by pharmacological activation of mGluR4 may offer therapeutic relief for motor learning and social deficits in FXS.

Modulation of NK cell activation by exogenous calcium from alginate dressings in vitro.

Natural Killer (NK) cells participate in the defense against infection by killing pathogens and infected cells and secreting immuno-modulatory cytokines. Defects in NK cell activity have been reported in obese, diabetic, and elderly patients that are at high risk of developing infected chronic wounds. Calcium alginate dressings are indicated for the debridement during the inflammatory phase of healing. Since calcium ions are major activators of NK cells, we hypothesized that these dressings could enhance NK functions, as investigated in vitro herein. Primary human blood NK cells were freshly-isolated from healthy volunteers and exposed to conditioned media (CM) from two alginate dressings, Algosteril® (ALG, pure Ca2+ alginate) and Biatain® Alginate (BIA, Ca2+ alginate with CMC), in comparison with an exogenous 3mM calcium solution. Our results demonstrated that exogenous calcium and ALG-CM, but not BIA-CM, induced NK cell activation and enhanced their capacity to kill their targets as a result of increased degranulation. NK cell stimulation by ALG depended on the influx of extracellular Ca2+ via the SOCE Ca2+ permeable plasma membrane channels. ALG-CM also activated NK cell cytokine production of IFN-γ and TNF-α through a partly Ca2+-independent mechanism. This work highlights the non-equivalence between alginate dressings for NK cell stimulation and shows that the pure calcium alginate dressing Algosteril® enhances NK cell cytotoxic and immuno-modulatory activities. Altogether, these results underline a specific property of this medical device in innate defense that is key for the cutaneous wound healing process.

Prolonged Ca2+ release refractoriness and T-tubule disruption as determinants of increased propensity to cardiac alternans in the hypertensive heart disease.

Cardiac alternans is a dynamical phenomenon linked to the genesis of severe arrhythmias and sudden cardiac death. It has been proposed that alternans is caused by alterations in Ca2+ handling by the sarcoplasmic reticulum (SR), in both the SR Ca2+ uptake and release processes. The hypertrophic myocardium is particularly prone to alternans, but the precise mechanisms underlying its increased vulnerability are not known. Mechanical alternans (intact hearts) and Ca2+ alternans (cardiac myocytes) were studied in spontaneously hypertensive rats (SHR) during the first year of age after the onset of hypertension and compared with age-matched normotensive rats. Subcellular Ca2+ alternans, T-tubule organization, SR Ca2+ uptake, and Ca2+ release refractoriness were measured. The increased susceptibility of SHR to high-frequency-induced mechanical and Ca2+ alternans appeared when the hypertrophy developed, associated with an adverse remodeling of the T-tubule network (6 mo). At the subcellular level, Ca2+ discordant alternans was also observed. From 6 mo of age, SHR myocytes showed a prolongation of Ca2+ release refractoriness without alterations in the capacity of SR Ca2+ removal, measured by the frequency-dependent acceleration of relaxation. Sensitizing SR Ca2+ release channels (RyR2) by a low dose of caffeine or by an increase in extracellular Ca2+ concentration, shortened refractoriness of SR Ca2+ release, and reduced alternans in SHR hearts. The tuning of SR Ca2+ release refractoriness is a crucial target to prevent cardiac alternans in a hypertrophic myocardium with an adverse T-tubule remodeling.

Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication.

Calcium (Ca2+), a ubiquitous second messenger, plays a crucial role in many cellular functions. Viruses often hijack Ca2+ signaling to facilitate viral processes such as entry, replication, assembly, and egress. Here, we report that infection by the swine arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV), induces dysregulated Ca2+ homeostasis, subsequently activating calmodulin-dependent protein kinase-II (CaMKII) mediated autophagy, and thus fueling viral replication. Mechanically, PRRSV infection induces endoplasmic reticulum (ER) stress and forms a closed ER-plasma membrane (PM) contacts, resulting the opening of store operated calcium entry (SOCE) channel and causing the ER to take up extracellular Ca2+, which is then released into the cytoplasm by inositol trisphosphate receptor (IP3R) channel. Importantly, pharmacological inhibition of ER stress or CaMKII mediated autophagy blocks PRRSV replication. Notably, we show that PRRSV protein Nsp2 plays a dominant role in the PRRSV induced ER stress and autophagy, interacting with stromal interaction molecule 1 (STIM1) and the 78 kDa glucose-regulated protein 78 (GRP78). The interplay between PRRSV and cellular calcium signaling provides a novel potential approach to develop antivirals and therapeutics for the disease outbreaks.

S100B chaperone multimers suppress the formation of oligomers during Aβ42 aggregation.

Extracellular aggregation of the amyloid-β 1-42 (Aβ42) peptide is a major hallmark of Alzheimer's disease (AD), with recent data suggesting that Aβ intermediate oligomers (AβO) are more cytotoxic than mature amyloid fibrils. Understanding how chaperones harness such amyloid oligomers is critical toward establishing the mechanisms underlying regulation of proteostasis in the diseased brain. This includes S100B, an extracellular signaling Ca2+-binding protein which is increased in AD as a response to neuronal damage and whose holdase-type chaperone activity was recently unveiled. Driven by this evidence, we here investigate how different S100B chaperone multimers influence the formation of oligomers during Aβ42 fibrillation. Resorting to kinetic analysis coupled with simulation of AβO influx distributions, we establish that supra-stoichiometric ratios of dimeric S100B-Ca2+ drastically decrease Aβ42 oligomerization rate by 95% and AβO levels by 70% due to preferential inhibition of surface-catalyzed secondary nucleation, with a concomitant redirection of aggregation toward elongation. We also determined that sub-molar ratios of tetrameric apo-S100B decrease Aβ42 oligomerization influx down to 10%, while precluding both secondary nucleation and, more discreetly, fibril elongation. Coincidently, the mechanistic predictions comply with the independent screening of AβO using a combination of the thioflavin-T and X-34 fluorophores. Altogether, our findings illustrate that different S100B multimers act as complementary suppressors of Aβ42 oligomerization and aggregation, further underpinning their potential neuroprotective role in AD.