Absence Of Extracellular Ca2 Research Articles

Relationship between calcium mobilization and platelet α- and δ-granule secretion. A role for TRPC6 in thrombin-evoked δ-granule exocytosis

Changes in cytosolic Ca2+ concentration ([Ca2+]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca2+ from distinct intracellular stores, which, in turn, activates store-operated Ca2+ entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca2+ mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca2+, and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca2+ mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573–586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca2+ mobilization.

α- and γ-mangostin cause shape changes, inhibit aggregation and induce cytolysis of rat platelets

α- and γ-mangostin are natural xanthones isolated from mangosteen (Garcinia mangostana) and the major constituents responsible for the plant's diverse biological activities. In this study, the effects of α- and γ-mangostin on platelets were investigated based on their possible antiplatelet activity. Treatment of isolated platelets with α-mangostin resulted in attenuation of platelet aggregatory response to collagen, thrombin or ADP. Such antiaggregatory effects were concentration-dependent in ranges of 1–10 μM. Interestingly, α-mangostin alone induced shape changes in platelets at the same concentration, and higher levels, 25 and 50 μM caused platelet lysis. Similarly, γ-mangostin induced shape changes and inhibited aggregation at 2.5–25 μM, while 50 and 100 μM γ-mangostin exhibited cytotoxicity. Platelet shape change induced by α- and γ-mangostin was accompanied by increases in myosin light chain (MLC) phosphorylation. MLC phosphorylation and subsequent shape changes were prevented by pretreatment with Rho kinase (ROCK) inhibitor Y-27632, but not by the intracellular Ca2+ chelating with BAPTA-AM and extracellular Ca2+ removal. Cytolysis by both α- and γ-mangostin was abolished in the absence of extracellular Ca2+. Taken together, α- and γ-mangostin have differential effects on platelets depending on their concentration, which includes inducing shape change, inhibiting aggregation and causing cytolysis. Platelet shape change is attributed to stimulation of the Rho/ROCK signaling pathway, while platelet lysis is presumably mediated by extracellular Ca2+ influx. These results suggest that mangosteen consumption may have potential platelet effects, although the in vivo or clinical consequences have yet to be assessed.

Long-lasting endothelium-dependent relaxation of isolated arteries caused by an extract from the bark of Combretum leprosum.

Objective To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals.Methods Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations.Results In all rings tested, Combretumleprosum extract (1.5μg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances.Conclusions Combretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors.

Role of intracellular signaling systems in regulation of erythrocyte microrheology

Erythrocyte deformability and thus the efficiency of the oxygen delivery to tissues depends on three main factors: elasticity of cell membrane, viscosity of cytoplasm, and the erythrocyte surface/volume ratio. The membrane elasticity and stability most considerably contribute to the whole cell deformability. There are experimental data suggesting that erythrocyte deformability is regulated by intracellular signaling pathways. The aim of this work was to study the role of the adenylyl cyclase–cAMP systems and intracellular calcium signaling mechanism in the regulation of erythrocyte deformability. We found that stimulator of adenylyl cyclase (AC) forskolin (10–5 M) and cell-penetrating cAMP analog dB-cAMP (5 × 10–5 M) increased erythrocyte deformability. Inhibitors of the phosphodiesterase (PDE) activity, such as isobutyl methylxanthine, vinpocetine, and cilostazol (10–5 M), also increased the erythrocyte deformability, which provides another line of evidence of the cAMP involvement in the regulation of erythrocyte deformability. On the other hand, stimulation of Ca2+ entry into erythrocytes induced by Ca2+ ionophore A23187 (3 × 10–6 M), sodium fluoride, or sodium vanadate considerably decreased the cell deformability, while blockade of the Ca2+ entry into erythrocytes by verapamil (10–5 M) increased their deformability. Mechanical stress of the erythrocyte membrane in the absence of extracellular Ca2+ did not change the deformability, while the mechanical stress applied in the presence of Ca2+ (50–200 µM) decreased deformability by 4–18%. On the whole, the obtained results suggest that for the increase in the erythrocyte deformability the AC–cAMP–PKA cascade has to be activated, while the pathway triggered by the Ca2+ entry into the cells is required to lower the erythrocyte deformability (elasticity) and hence, to increase the whole cell membrane stability. It is possible that these pathways are coordinated at the PDE level.

Na+–Ca2+ exchanger mediates ChR2-induced [Ca2+]i elevation in astrocytes

Intracellular Ca2+ elevation resulting from different Ca2+ pathways may play different roles in astrocyte functions. Channelrhodopsin-2 (ChR2), a light-gated cation channel, has been used to selectively stimulate astrocytes by inducing intracellular Ca2+ ([Ca2+]i) elevation, but the exact underlying mechanism is still unclear. We found that in the absence of extracellular Ca2+, light stimulation failed to induce [Ca2+]i elevation in astrocytes expressed ChR2. Pharmacological experiments excluded the involvement of Ca2+-induced Ca2+ release from intracellular stores. Further experiments demonstrated that the ChR2-induced [Ca2+]i elevation was mainly mediated by reversal of the Na+–Ca2+ exchanger following Na+ influx through ChR2 channels. Since intracellular Na+ homeostasis plays important roles in astrocytes, including the modulation of [Ca2+]i, neurotransmitter uptake and cell metabolism, our results indicate that ChR2 is a good candidate which could be used for mimicking the intracellular Na+ disturbance in astrocytes that occurs in various physiological and pathological processes including the uptake of neurotransmitters and ischemia, as well as the activities of various cation channels, ion exchangers, and pumps.

Functional expression of bradykinin B1 and B2 receptors in neonatal rat trigeminal ganglion neurons.

Bradykinin (BK) and its receptors (B1 and B2 receptors) play important roles in inflammatory nociception. However, the patterns of expression and physiological/pathological functions of B1 and B2 receptors in trigeminal ganglion (TG) neurons remain to be fully elucidated. We investigated the functional expression of BK receptors in rat TG neurons. We observed intense immunoreactivity of B2 receptors in TG neurons, while B1 receptors showed weak immunoreactivity. Expression of the B2 receptor colocalized with immunoreactivities against the pan-neuronal marker, neurofilament H, substance P, isolectin B4, and tropomyosin receptor kinase A antibodies. Both in the presence and absence of extracellular Ca2+ ([Ca2+]o), BK application increased the concentration of intracellular free Ca2+ ([Ca2+]i). The amplitudes of BK-induced [Ca2+]i increase in the absence of [Ca2+]o were significantly smaller than those in the presence of Ca2+. In the absence of [Ca2+]o, BK-induced [Ca2+]i increases were sensitive to B2 receptor antagonists, but not to a B1 receptor antagonist. However, B1 receptor agonist, Lys-[Des-Arg9]BK, transiently increased [Ca2+]i in primary cultured TG neurons, and these increases were sensitive to a B1 receptor antagonist in the presence of [Ca2+]o. These results indicated that B2 receptors were constitutively expressed and their activation induced the mobilization of [Ca2+]i from intracellular stores with partial Ca2+ influx by BK. Although constitutive B1 receptor expression could not be clearly observed immunohistochemically in the TG cryosection, cultured TG neurons functionally expressed B1 receptors, suggesting that both B1 and B2 receptors involve pathological and physiological nociceptive functions.

CaMKII regulates intracellular Ca2+ dynamics in native endothelial cells

Localized endothelial Ca2+ signalling, such as Ca2+ pulsars, can modulate the contractile state of the underlying vascular smooth muscle cell through specific endothelial targets. In addition to KCa3.1 as a target, Ca2+ pulsars, an IP3R-dependent pulsatile Ca2+ release from the endoplasmic reticulum (ER) could activate a frequency-sensitive Ca2+-dependent kinase such as CaMKII. In the absence of extracellular Ca2+, acetylcholine increased endothelial CaMKII phosphorylation and activation, thereby suggesting CaMKII activation independently of Ca2+ influx. Herein, a reciprocal relation where CaMKII controls endothelial Ca2+ dynamics has been investigated in mesenteric arteries. Both CaMKIIα and β isoforms have been identified in endothelial cells and close proximity (<40nm) suggests their association in heteromultimers. Intracellular Ca2+ monitoring with high speed confocal microscopy then showed that inhibition of CaMKII with KN-93 significantly increased the population of Ca2+ pulsars active sites (+89%), suggesting CaMKII as a major regulator of Ca2+ pulsars in native endothelium. Mechanistic insights were then sought through the elucidation of the impact of CaMKII on ER Ca2+ store. ER Ca2+ emptying was accelerated by CaMKII inhibition and ER Ca2+ content was assessed using ionomycin. Exposure to KN-93 strongly diminished ER Ca2+ content (−61%) by relieving CaMKII-dependent inhibition of IP3 receptors (IP3R). Moreover, in situ proximity ligation assay suggested CaMKII-IP3R promiscuity, essential condition for a protein–protein interaction. Interestingly, segregation of IP3R within myoendothelial projection (MEP) appears to be isoform-specific. Hence, only IP3R type 1 and type 2 are detected within fenestrations of the internal elastic lamina, sites of MEP, whilst type 3 is absent from these structures. In summary, CaMKII seems to act as a Ca2+-sensitive switch of a negative feedback loop regulating endothelial Ca2+ homeostasis, including Ca2+ pulsars.

Effect of Antidepressant Doxepin on Ca^(2+) Homeostasis and Viability in PC3 Human Prostate Cancer Cells

The effect of the antidepressant doxepin on cytosolic Ca²⁺ concentrations ([Ca²⁺](i)) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺](i). Doxepin at concentrations of 500-1000 μM induced a [Ca²⁺](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Doxepin-evoked Ca²⁺ entry was suppressed by Ca²⁺ entry blockers (nifedipine, econazole, SK&F96365), and protein kinase C (PKC) modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibit doxepin-induced [Ca²⁺](i) rise. Incubation with doxepin nearly inhibited thapsigargin or BHQ-induced [Ca²⁺](i) rise. Inhibition of phospholipase C (PLC) with U73122 failed to alter doxepin-induced [Ca²⁺](i) rise. At concentrations of 200-250 μM, doxepin killed cells in a concentration-dependent manner. This cytotoxic effect was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/PI staining data implied that doxepin (200 and 250 μM) did not induce apoptosis. Collectively, in PC3 cells, doxepin induced a [Ca²⁺](i) rise by evoking PLC-independent Ca²⁺ release from stores including the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels. Doxepin caused cell death that was independent of [Ca²⁺](i) rises.

Expression and function of purinergic P2Y12 receptors in rat trigeminal ganglion neurons

Purinergic receptors play key signaling roles in neuropathic pain in the orofacial region, which is innervated by trigeminal ganglion (TG) neurons. The neuropathology of purinergic P2Y12 receptors is well characterized in glia; however, their physiological role in TG neurons remains to be fully elucidated. The present study investigated the expression and function of P2Y12 receptors in rat TG neurons. P2Y12 receptor immunoreactivity was intense in the soma, dendrites, and axons, and colocalized with a pan-neuronal marker, neurofilament H, isolectin B4, and substance P. In the presence of extracellular Ca2+, 2-methylthio-ADP (an agonist of P2Y1, 12, 13 receptors) transiently increased intracellular free Ca2+ concentrations ([Ca2+]i), an effect that was abolished by P2Y12 receptor antagonists. In the absence of extracellular Ca2+, ryanodine receptor/channel inhibitors diminished the 2-methylthio-ADP-induced increases in [Ca2+]i. A sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor gradually increased [Ca2+]i, and after a plateau, application of 2-MeS-ADP induced a rapid and transient, but additive increase in [Ca2+]i. An adenylate cyclase inhibitor transiently increased [Ca2+]i, while a phosphodiesterase inhibitor prevented the 2-methylthio-ADP-induced increase in [Ca2+]i. Our study shows that P2Y12 receptors are expressed in TG neurons, and act via a cAMP-dependent pathway to release intracellular Ca2+ from ryanodine-sensitive Ca2+ stores.

An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells

Calcium ions (Ca2+) are a ubiquitous transducer of cellular signals controlling key processes such as proliferation, differentiation, secretion and metabolism. In the context of T cells, stimulation through the T cell receptor has been shown to induce the release of Ca2+ from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca2+ that in turn activates key molecules such as calcineurin. This cascade ultimately results in gene transcription and changes in the cellular state. Traditional methods for measuring Ca2+ include spectrophotometry, conventional flow cytometry (CFC) and live cell imaging techniques. While each method has strengths and weaknesses, none can offer a detailed picture of Ca2+ mobilisation in response to various agonists. Here we report an Imaging Flow Cytometry (IFC)-based method that combines the throughput and statistical rigour of CFC with the spatial information of a microscope. By co-staining cells with Ca2+ indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca2+ flux in Jurkat cells after stimulation with anti-CD3. The multispectral, high-throughput nature of IFC means that the organelle co-staining functions to direct the measurement of Ca2+ indicator fluorescence to either the endoplasmic reticulum (ER) or the mitochondrial compartments without the need to treat cells with detergents such as digitonin to eliminate cytoplasmic background. We have used this system to look at the cellular localisation of Ca2+ after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca2+. Our data suggest that there is a dynamic interplay between the ER and mitochondrial compartments and that mitochondria act as a sink for both intracellular and extracellular derived Ca2+. Moreover, by generating an NFAT–GFP expressing Jurkat line, we were able to combine mitochondrial Ca2+ measurements with nuclear translocation. In conclusion, this method enables the high throughput study of spatiotemporal patterns of Ca2+ signals in T cells responding to different stimuli.

Redox Signal-mediated Enhancement of the Temperature Sensitivity of Transient Receptor Potential Melastatin 2 (TRPM2) Elevates Glucose-induced Insulin Secretion from Pancreatic Islets

Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive Ca(2+)-permeable cation channel expressed by pancreatic β cells where channel function is constantly affected by body temperature. We focused on the physiological functions of redox signal-mediated TRPM2 activity at body temperature. H2O2, an important molecule in redox signaling, reduced the temperature threshold for TRPM2 activation in pancreatic β cells of WT mice but not in TRPM2KO cells. TRPM2-mediated [Ca(2+)]i increases were likely caused by Ca(2+) influx through the plasma membrane because the responses were abolished in the absence of extracellular Ca(2+). In addition, TRPM2 activation downstream from the redox signal plus glucose stimulation enhanced glucose-induced insulin secretion. H2O2 application at 37 °C induced [Ca(2+)]i increases not only in WT but also in TRPM2KO β cells. This was likely due to the effect of H2O2 on KATP channel activity. However, the N-acetylcysteine-sensitive fraction of insulin secretion by WT islets was increased by temperature elevation, and this temperature-dependent enhancement was diminished significantly in TRPM2KO islets. These data suggest that endogenous redox signals in pancreatic β cells elevate insulin secretion via TRPM2 sensitization and activity at body temperature. The results in this study could provide new therapeutic approaches for the regulation of diabetic conditions by focusing on the physiological function of TRPM2 and redox signals.

Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons

Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of −60 mV. Removal of extracellular Ca2+ markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca2+ was fully inhibited by removal of extracellular Na+, indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration–inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca2+/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca2+ from intracellular Ca2+ stores and calmodulin.The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization

Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca2+-dependent PKC, and in the absence of extracellular Ca2+. Biochemical evidence indicates that either + or − extracellular Ca2+, PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.

A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca2 + microdomain in the caveolar compartment

Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1–Aequorin fusion protein (Cav1–Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca2+]cav). In HeLa cells, Cav1–Aeq reported different [Ca2+] as compared to the plasma membrane [Ca2+] in general (reported by SNAP25–Aeq) or as compared to the cytosolic [Ca2+] (reported by cyt-Aeq). The Ca2+ signals detected by Cav1–Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca2+ signaling. HeLa cells overexpressing SK1 showed increased [Ca2+]cav during histamine-induced Ca2+ mobilization in the absence of extracellular Ca2+ as well as during receptor-operated Ca2+ entry (ROCE). The SK1-induced increase in [Ca2+]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca2+]cav during ROCE. S1P treatment stimulated the [Ca2+]cav upon ROCE. The Ca2+ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca2+ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Characterization of Intracellular Ca2+ Handling in Vascular Smooth Muscle Cells Isolated from Amputated Human Limbs

High Ca2+ scores assessed by computed tomography in the lower limbs of patients with peripheral artery disease are associated with a greater risk of amputation and all‐cause mortality. Direct assessment of intracellular Ca2+ handling in peripheral vascular smooth muscle cells (VSM) of humans likely to undergo an amputation is unknown due to the invasiveness of such measures. We directly characterized intracellular VSM Ca2+ handling in arteries from amputated human limbs stratified by the number of diagnosed cardiovascular (CV) risk factors (&lt;2 vs &gt;6 CV risk factors). VSM were enzymatically isolated from peripheral arteries and were digitally imaged using the fluorescent intracellular Ca2+ indicator, fura‐2. Cells were exposed to 80 mM K+ in physiologic Ca2+ to assess Ca2+ entry through voltage‐gated Ca2+ channels (VGCC). Caffeine was used in the presence and absence of extracellular Ca2+ to release intracellular Ca2+ from the sarcoplasmic reticulum (SR). VSM from patients with &gt;6 CV risk factors had elevated basal cytosolic Ca2+ (0.76±0.01 vs 0.72±0.01 F360/F380) and greater Ca2+ entry through VGCC (0.08±0.01 vs 0.05±0.00 ΔF360/F380) vs patients with &lt;2 CV risk factors. In the absence of extracellular Ca2+, the SR Ca2+ store release was decreased in VSM in patients with &gt;6 CV risk factors (0.16±0.02 vs 0.22±0.01 F360/F380). However, this decrease was absent in the presence of extracellular Ca2+, suggesting elevated Ca2+‐activated Ca2+ influx with more CV risk factors. Taken together, these data suggest that changes in VGCC, SR Ca2+ stores, and Ca2+‐activated Ca2+ influx act to elevate cytosolic Ca2+ in VSM of amputees with more CV risk factors. NIH T32 HL079995, Health‐IUSM Strategic Research Initiative

Lysosomal Calcium: Impact on Serotonin‐Induced Contractions of Ovine Pulmonary Arteries

Mobilisation of intracellular Ca2+ from lysosomes is involved in smooth muscle contraction in response to several agonists but serotonin (5-HT) has not been examined. Given the proposed role of 5-HT in pulmonary arterial hypertension, this study aimed to investigate the role of lysosomal Ca2+ stores in pulmonary artery (PA) smooth muscle 5-HT responses. Responses of endothelium-denuded ovine PAs were measured using myography. 5-HT cumulative concentration response curves (CCRCs) were investigated in Ca2+-free medium and with nimodipine (a lysosomal Ca2+ channel blocker). Contractions are normalised to 80mM KCl responses and expressed as mean ± S.E.M. PAs showed dose-dependent contractions to 5-HT. Fourth order PAs were more responsive than third order; maximal contractions were significantly higher (P<0.05). Responses to 5-HT were reduced in the absence of extracellular Ca2+ (statistically significant at [5-HT] of 30μM and 100μM (P<0.05)). Maximal contraction: Ca2+-free medium 174±20%KCl (n=5); Ca2+-containing medium 282±17%KCl (n=5). PAs in Ca2+-free medium containing nimodipine displayed reduced contractions to 5-HT compared to control (statistically significant (P<0.05) at [5-HT] of 10μM, 30μM and 100μM). Nimodipine reduced maximal contraction at 30μM 5-HT by 56±4%: nimodipine-exposed PAs 73±7%KCl (n=5); control 174±20%KCl (n=5). Anatomical order in ovine PAs is an important factor in 5-HT responsiveness. Results also suggest both extracellular Ca2+ and mobilisation of Ca2+ from intracellular lysosomal stores play a significant role in 5-HT-mediated contraction of ovine PAs. Research supported by Institute for Health & Wellbeing Research, Robert Gordon University.

CD38 mediates angiotensin II-induced intracellular Ca(2+) release in rat pulmonary arterial smooth muscle cells.

CD38 is a multifunctional enzyme that catalyzes the formation of the endogenous Ca(2+)-mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenosine dinucleotide phosphate (NAADP) for the activation of ryanodine receptors (RyRs) of sarcoplasmic reticulum and NAADP-sensitive Ca(2+) release channels in endolysosomes, respectively. It plays important roles in systemic vascular functions, but there is little information on CD38 in pulmonary arterial smooth muscle cells (PASMCs). Earlier studies suggested a redox-sensing role of CD38 in hypoxic pulmonary vasoconstriction. This study sought to characterize its roles in angiotensin II (Ang II)-induced Ca(2+) release (AICR) in PASMCs. Examination of CD38 expression in various rat arteries found high levels of CD38 mRNA and protein in pulmonary arteries. The Ang II-elicited Ca(2+) response consisted of extracellular Ca(2+) influx and intracellular Ca(2+) release in PASMCs. AICR activated in the absence of extracellular Ca(2+) was reduced by pharmacological or siRNA inhibition of CD38, by the cADPR antagonist 8-bromo-cADPR or ryanodine, and by the NAADP antagonist Ned-19 or disruption of endolysosomal Ca(2+) stores with the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Suppression of AICR by the inhibitions of cADPR- and NAADP-dependent pathways were nonadditive, indicating interdependence of RyR- and NAADP-gated Ca(2+) release. Furthermore, AICR was inhibited by the protein kinase C inhibitor staurosporine, the nonspecific NADPH oxidase (NOX) inhibitors apocynin and diphenyleneiodonium, the NOX2-specific inhibitor gp91ds-tat, and the scavenger of reactive oxygen species (ROS) tempol. These results provide the first evidence that Ang II activates CD38-dependent Ca(2+) release via the NOX2-ROS pathway in PASMCs.

Contribution of pannexin 1 and connexin 43 hemichannels to extracellular calcium-dependent transport dynamics in human blood-brain barrier endothelial cells.

Dysregulation of blood-brain barrier (BBB) transport function is thought to exacerbate neuronal damage in acute ischemic stroke. The purpose of this study was to clarify the characteristics of pannexin (Px) and/or connexin (Cx) hemichannel(s)-mediated transport of organic anions and cations in human BBB endothelial cell line hCMEC/D3 and to identify inhibitors of hemichannel opening in hCMEC/D3 cells in the absence of extracellular Ca(2+), a condition mimicking acute ischemic stroke. In the absence of extracellular Ca(2+), the cells showed increased uptake and efflux transport of organic ionic fluorescent dyes. Classic hemichannel inhibitors markedly inhibited the enhanced uptake and efflux. Quantitative targeted absolute proteomics confirmed Px1 and Cx43 protein expression in plasma membrane of hCMEC/D3 cells. Knockdown of Px1 and Cx43 with the small interfering RNAs significantly inhibited the enhanced uptake and efflux of organic anionic and cationic fluorescent dyes. Clinically used cilnidipine and progesterone, which have neuroprotective effects in animal ischemia models, were identified as inhibitors of hemichannel opening. These findings suggest that altered transport dynamics at the human BBB in the absence of extracellular Ca(2+) is at least partly attributable to opening of Px1 and Cx43 hemichannels. Therefore, we speculate that Px1 and Cx43 may be potential drug targets to ameliorate BBB transport dysregulation during acute ischemia.

Leucine-rich repeat kinase 2-sensitive Na+/Ca2+ exchanger activity in dendritic cells.

Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca(2+)-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2(-/-)) and their wild-type littermates (Lrrk2(+/+)). According to Western blotting, Lrrk2 was expressed in Lrrk2(+/+) DCs but not in Lrrk2(-/-)DCs. Cytosolic Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) exchanger-induced current were significantly lower in Lrrk2(-/-) than in Lrrk2(+/+) DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na(+)/Ca(2+) exchanger transcription and activity leading to attenuation of Ca(2+)-signals in DCs.

Phospho-STIM1 is a downstream effector that mediates the signaling triggered by IGF-1 in HEK293 cells

STIM1 is a Ca2+ sensor of the endoplasmic reticulum (ER) that triggers the activation of plasma membrane Ca2+ channels upon depletion of Ca2+ levels within the ER. During thapsigargin-triggered Ca2+ store depletion, ERK1/2 phosphorylates STIM1 at Ser575, Ser608, and Ser621. This phosphorylation plays a role in the regulation of STIM1 dissociation from the microtubule plus-end binding protein EB1, an essential step for STIM1 activation by thapsigargin. However, little is known regarding the physiological role of this phosphorylation. Because IGF-1 triggers the activation of the RAF–MEK–ERK and the phosphoinositide pathways, the role of STIM1 phosphorylation in IGF-1 stimulation was studied. There was found to be phosphorylation of ERK1/2 in both the presence and the absence of extracellular Ca2+, demonstrating that Ca2+ influx is not essential for ERK1/2 activation. In parallel, IGF-1 triggered STIM1 phosphorylation at the aforementioned sites, an effect that was blocked by PD0325901, a MEK1/2 inhibitor used to block ERK1/2 activation. Also, STIM1-GFP was found in clusters upon IGF-1 stimulation, and STIM1-S575A/S608A/S621A-GFP strongly reduced this multimerization. Interestingly, phospho-STIM1 was mainly found in clusters when cells were treated with IGF-1, and IGF-1 triggered the dissociation of STIM1 from EB1, similarly to what has been observed for thapsigargin, suggesting that STIM1 mediates the IGF-1 signaling pathway. A study of IGF-1-stimulated NFAT translocation was therefore performed, finding that STIM1-S575A/S608A/S621A blocked this translocation, as did the fusion protein STIM1-EB1, confirming that both STIM1 phosphorylation and STIM1–EB1 dissociation are required for IGF-1-triggered Ca2+-dependent signaling, and demonstrating that STIM1 phosphorylation plays a role as a downstream effector of the RAF–MEK–ERK pathway and an upstream activator of Ca2+ entry.