Removal Of Extracellular Na Research Articles

The removal of extracellular Na+ induces intracellular Ca2+ oscillations in human cerebral microvascular endothelial cells: Emerging evidence for a Na+-sensitive GPCR

The removal of extracellular Na+ induces intracellular Ca2+ oscillations in human cerebral microvascular endothelial cells: Emerging evidence for a Na+-sensitive GPCR

Enhancement of receptor-mediated calcium responses by phenytoin through the suppression of calcium excretion in human gingival fibroblasts.

Gingival overgrowth caused by phenytoin is proposed to be associated with Ca2+ signaling; however, the mechanisms that increase the intracellular Ca2+ concentration ([Ca2+ ]i ) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin-induced increase in [Ca2+ ]i in human gingival fibroblasts (HGFs). Effects of 100 μM phenytoin on [Ca2+ ]i in HGFs were examined at the single-cell level using fluorescence images of fura-2 captured by an imaging system consisting of an EM-CCD camera coupled to an inverted fluorescence microscope at room temperature. Exposure of HGFs to 100 μM phenytoin induced a transient increase in [Ca2+ ]i in the absence of extracellular Ca2+ , indicating that the phenytoin-induced increase in [Ca2+ ]i does not require an influx of extracellular Ca2+ . In addition, phenytoin increased [Ca2+ ]i in HGFs depleted of intracellular Ca2+ stores by thapsigargin, indicating that neither Ca2+ release from stores nor inhibition of Ca2+ uptake is involved. Furthermore, the phenytoin-induced [Ca2+ ]i elevation was reduced to 18.8% in the absence of extracellular Na+ , and [Ca2+ ]i elevation upon removal of extracellular Na+ was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca2+ ]i of HGFs by suppressing the Na+ /Ca2+ exchanger. Suppression of intracellular Ca2+ excretion is thought to enhance the Ca2+ responses induced by various stimuli. Analysis at the single-cell level showed that stimulation with 1μM ATP or 3μM histamine increased [Ca2+ ]i in 20-50% of cells, and [Ca2+ ]i increased in many unresponsive cells in the presence of phenytoin. Our findings demonstrate that phenytoin induced increase in [Ca2+ ]i by the inhibition of Ca2+ efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca2+ responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca2+ efflux. These findings suggest a possibility that phenytoin causes drug-induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.

The activity of the serotonergic 5-HT1A receptor is modulated by voltage and sodium levels

G protein–coupled receptors are known to play a key role in many cellular signal transduction processes, including those mediating serotonergic signaling in the nervous system. Several factors have been shown to regulate the activity of these receptors, including membrane potential and the concentration of sodium ions. Whether voltage and sodium regulate the activity of serotonergic receptors is unknown. Here, we used Xenopus oocytes as an expression system to examine the effects of voltage and of sodium ions on the potency of one subtype of serotonin (5-hydroxytryptamine [5-HT]) receptor, the 5-HT1A receptor. We found that the potency of 5-HT in activating the receptor is voltage dependent and that it is higher at resting potential than under depolarized conditions. Furthermore, we found that removal of extracellular Na+ resulted in a decrease of 5-HT potency toward the 5-HT1A receptor and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. Our results suggest that this allosteric effect of Na+ does not underlie the voltage dependence of this receptor. We propose that the characterization of modulatory factors that regulate this receptor may contribute to our future understanding of various physiological functions mediated by serotonergic transmission.

Cellular Pathophysiology of Mutant Voltage-Dependent Ca2+ Channel CACNA1H in Primary Aldosteronism

The physiological stimulation of aldosterone production in adrenocortical glomerulosa cells by angiotensin II and high plasma K+ depends on the depolarization of the cell membrane potential and the subsequent Ca2+ influx via voltage-activated Ca2+ channels. Germline mutations of the low-voltage activated T-type Ca2+ channel CACNA1H (Cav3.2) have been found in patients with primary aldosteronism. Here, we investigated the electrophysiology and Ca2+ signaling of adrenal NCI-H295R cells overexpressing CACNA1H wildtype and mutant M1549V in order to understand how mutant CACNA1H alters adrenal cell function. Whole-cell patch-clamp measurements revealed a strong activation of mutant CACNA1H at the resting membrane potential of adrenal cells. Both the expression of wildtype and mutant CACNA1H led to a depolarized membrane potential. In addition, cells expressing mutant CACNA1H developed pronounced action potential-like membrane voltage oscillations. Ca2+ measurements showed an increased basal Ca2+ activity, an altered K+ sensitivity, and abnormal oscillating Ca2+ changes in cells with mutant CACNA1H. In addition, removal of extracellular Na+ reduced CACNA1H current, voltage oscillations, and Ca2+ levels in mutant cells, suggesting a role of the partial Na+ conductance of CACNA1H in cellular pathology. In conclusion, the pathogenesis of stimulus-independent aldosterone production in patients with CACNA1H mutations involves several factors: i) a loss of normal control of the membrane potential, ii) an increased Ca2+ influx at basal conditions, and iii) alterations in sensitivity to extracellular K+ and Na+. Finally, our findings underline the importance of CACNA1H in the control of aldosterone production and support the concept of the glomerulosa cell as an electrical oscillator.

Sodium ions allosterically modulate the M2 muscarinic receptor

G protein coupled receptors (GPCRs) play a key role in the vast majority of cellular signal transduction processes. Previous experimental evidence has shown that sodium ion (Na+) allosterically modulate several class A GPCRs and theoretical studies suggested that the same also holds true for muscarinic receptors. Here we examined, using Xenopus oocytes as an expression system, the effect of Na+ on a prototypical GPCR, the M2 muscarinic receptor (M2R). We found that removal of extracellular Na+ resulted in a decrease in the potency of ACh toward the M2R and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. We further show that this allosteric effect of Na+ does not underlie the voltage-dependence of this receptor.

Treating TB human neuroectodermal cell line with retinoic acid induces the appearance of neuron-like voltage-gated ionic currents

TB is a cell line derived from the cerebrospinal fluid sample of a patient with primary leptomeningeal melanomatosis. Our previous immunological and ultrastructural analysis revealed that TB cells differentiate towards a neuronal phenotype when grown in vitro up to 7 days in presence of 10 µM all-trans retinoic acid (RA). Recently, we reported that TB cells are sensitive to the cytotoxic effects of β-amyloid peptides, activating the cytosolic phospholipase A2. To date, it is not known if RA, in addition to inducing morphological changes, also causes functional modification in TB cells, by regulating voltage-gated ionic currents. To this purpose, we performed electrophysiological characterization of undifferentiated (TB) and differentiated (RA-TB) cells by means of whole-cell patch clamp recordings. Upon depolarizing stimuli, both groups displayed voltage-gated K+ outward currents of similar amplitude. By contrast, the low amplitude voltage-gated Na+ currents recorded in undifferentiated TB cells were largely up-regulated by RA exposure. This current was strongly reduced by TTX and lidocaine and completely abolished by removal of extracellular sodium. Furthermore, treatment with RA caused the appearance of a late-onset inward current carried by Ca2+ ions in a subpopulation of TB cells. This current was not affected by removal of extracellular Na+ and was completely blocked by Cd2+, a broad-spectrum blocker of Ca2+ currents. Altogether, our results indicate that RA-differentiation of TB cells induces functional changes by augmenting the amplitude of voltage-gated sodium current and by inducing, in a subpopulation of treated cells, the appearance of a voltage-gated calcium current.

Na+ /Ca2+ exchanger 1 is a key mechanosensitive molecule of the esophageal myenteric neurons.

Our earlier studies showed that mechanical stretch activates inhibitory motor neurons of the oesophagus; however, the underlying molecular mechanisms are unclear. Here, we sought to examine if Na+ /Ca2+ exchanger 1 (NCX1) is responsible for the mechanosensitivity in the esophageal myenteric neurons (EMN) of rats and humans. The function of NCX1 in primary culture of neurons was determined using calcium imaging, and mechanosensitivity was tested using osmotic stretch and direct mechanical stretch. Axial stretch-induced relaxation of the lower esophageal sphincter (LES) was also studied in vivo in rats. The expression and co-localization of NCX1 with nNOS were identified in the EMN from both rats and humans. The extracellular Ca2+ entry caused by ATP through purinergic signalling in the rat EMN was significantly inhibited by selective NCX blockers. Removal of extracellular Na+ to activate the Ca2+ entry mode of NCX1 induced an increase in the cytoplasmic calcium ([Ca2+ ]cyt ), which was attenuated by NCX blockers. Osmotic stretch and mechanical stretch-induced [Ca2+ ]cyt signalling in the rat and human EMN were attenuated by NCX blockers as well as specific NCX1 knockdown. Osmotic stretch and mechanical stretch also induced [Ca2+ ]cyt signalling in the Chinese hamster ovary (CHO) cells with NCX1 over-expression, which was attenuated by NCX blockers. Finally, NCX blockade inhibited axial stretch-activated LES relaxation in vivo experiments in the rats. We demonstrate a novel NCX1/Ca2+ pathway in the mechanosensitive neurons of rat and human oesophagus, which may provide a potential therapeutic target for the treatment of oesophageal motility disorders.

Na+/Ca2+ Exchanger 1 in Airway Smooth Muscle of Allergic Inflammation Mouse Model.

Cytosolic free Ca2+ ([Ca2+]cyt) is essential for airway contraction, secretion and remodeling. [Ca2+]cyt homeostasis is controlled by several critical molecules, one of which is the Na+/Ca2+ exchanger 1 (NCX1) in the plasma membrane. Since little is currently known about NCX1 in the airway smooth muscle and its involvement in airway diseases, the present study was designed to investigate the expression and function of NCX1 in normal airway smooth muscle and its relevance to airway inflammation. Western blot analysis, tracheal smooth muscle contraction, and [Ca2+]cyt measurements were performed in mouse tracheal smooth muscle tissues and primary airway smooth muscle cell cultures. Additional studies were performed in a mouse model of allergic airway inflammation. Our data showed that NCX1 proteins were expressed in the human bronchial smooth muscle cells (HBSMCs), murine airway and whole lung. Carbachol raised [Ca2+]cyt in mouse tracheal smooth muscle cells and induced murine tracheal contraction, all of which were significantly attenuated by KB-R7943, a selective NCX inhibitor. Removal of extracellular Na+ increased [Ca2+]cyt in HBSMCs and mouse tracheal SMCs, which was dependent on extracellular Ca2+ and sensitive to KB-R7943. TNF-α treatment of HBSMCs significantly upregulated mRNA and protein expression of NCX1 and enhanced NCX activity. Finally, KB-R7943 abolished the airway hyperresponsiveness to methacholine in an ovalbumin-induced mouse model of allergic airway inflammation. Together, these findings indicate that NCX1 in airway smooth muscle may play an important role in the development of airway hyperresponsiveness, and downregulation or inhibition of NCX1 may serve as a potential therapeutic approach for asthma.

Modulation of Mg2+ influx and cytoplasmic free Mg2+ concentration in rat ventricular myocytes.

To examine whether TRPM7, a member of the melastatin family of transient receptor potential channels, is a physiological pathway for Mg2+ entry in mammalian cells, we studied the effect of TRPM7 regulators on cytoplasmic free Mg2+ concentration ([Mg2+]i) of rat ventricular myocytes. Acutely isolated single cells were AM-loaded with the fluorescent indicator furaptra, and [Mg2+]i was estimated at 25°C. After [Mg2+]i was lowered by soaking the cells with a high-K+ and Mg2+-Ca2+-free solution, [Mg2+]i was recovered by extracellular perfusion of Ca2+-free Tyrode's solution that contained 1mMMg2+. The initial rate of increase in [Mg2+]i was analyzed as the Mg2+ influx rate. The Mg2+ influx rate was increased by the TRPM7 activator, naltriben (2-50μM), in a concentration-dependent manner with a half maximal effective concentration (EC50) of 24μM. This EC50 value is similar to that reported for the activation of recombinant TRPM7 overexpressed in HEK293 cells. Naltriben (50μM) caused little change in basal [Mg2+]i (~ 0.9mM) in Ca2+-free Tyrode's solution, but significantly raised [Mg2+]i to 1.31 ± 0.03mM in 94min after the removal of extracellular Na+. Re-introduction of extracellular Na+ lowered [Mg2+]i back to the basal level even in the presence of naltriben. Application of 10μM NS8593, an inhibitor of TRPM7, significantly lowered [Mg2+]i to 0.72 ± 0.03mM in 50-60min independent of extracellular Na+. The results suggest that Mg2+ entry through TRPM7 significantly contributes to physiological Mg2+ homeostasis in mammalian heart cells.

TRPC6 and TRPC4 Heteromultimerization Mediates Store Depletion-Activated NCX1 Reversal in Proliferative Vascular Smooth Muscle Cells

ABSTRACTStore depletion has been shown to induce Ca2+ entry by Na+/Ca+ exchange (NCX) 1 reversal in proliferative vascular smooth muscle cells (VSMCs). The study objective was to investigate the role of transient receptor potential canonical (TRPC) channels in store depletion and NCX1 reversal in proliferative VSMCs. In cultured VSMCs, expressing TRPC1, TRPC4, and TRPC6, the removal of extracellular Na+ was followed by a significant increase of cytosolic Ca2+ concentration that was inhibited by KBR, a selective NCX1 inhibitor. TRPC1 knockdown significantly suppressed store-operated, channel-mediated Ca2+ entry, but TRPC4 knockdown and TRPC6 knockdown had no effect. Separate knockdown of TRPC1, TRPC4, or TRPC6 did not have a significant effect on thapsigargin-initiated Na+ increase in the peripheral regions with KBR treatment, but knockdown of both TRPC4 and TRPC6 did. Stromal interaction molecule (STIM)1 knockdown significantly reduced TRPC4 and TRPC6 binding. The results demonstrated that TRPC4–TRPC6 heteromultimerization linked Ca2+ store depletion and STIM1 accumulation with NCX reversal in proliferative VSMCs.

Hypocretin/Orexin Peptides Excite Rat Neuroendocrine Dopamine Neurons through Orexin 2 Receptor-Mediated Activation of a Mixed Cation Current

Hypocretin/Orexin (H/O) neurons of the lateral hypothalamus are compelling modulator candidates for the chronobiology of neuroendocrine output and, as a consequence, hormone release from the anterior pituitary. Here we investigate the effects of H/O peptides upon tuberoinfundibular dopamine (TIDA) neurons – cells which control, via inhibition, the pituitary secretion of prolactin. In whole cell recordings performed in male rat hypothalamic slices, application of H/O-A, as well as H/O-B, excited oscillating TIDA neurons, inducing a reversible depolarising switch from phasic to tonic discharge. The H/O-induced inward current underpinning this effect was post-synaptic (as it endured in the presence of tetrodotoxin), appeared to be carried by a Na+-dependent transient receptor potential-like channel (as it was blocked by 2-APB and was diminished by removal of extracellular Na+), and was a consequence of OX2R receptor activation (as it was blocked by the OX2R receptor antagonist TCS OX2 29, but not the OX1R receptor antagonist SB 334867). Application of the hormone, melatonin, failed to alter TIDA membrane potential or oscillatory activity. This first description of the electrophysiological effects of H/Os upon the TIDA network identifies cellular mechanisms that may contribute to the circadian rhythmicity of prolactin secretion.

Extracellular phosphates enhance activities of voltage-gated proton channels and production of reactive oxygen species in murine osteoclast-like cells.

Osteoclasts are highly differentiated bone-resorbing cells and play a significant role in bone remodelling. In the resorption pit, inorganic phosphate (Pi) concentrations increase because of degradation of hydroxyapatite. We studied effects of extracellular Pi on voltage-gated H+ channels in osteoclast-like cells derived from a macrophage cell line (RAW264). Extracellular Pi (1.25-20mM) increased the H+ channel currents dose dependently and reversibly. The Pi-induced increases were attenuated by removal of extracellular Na+ and by phosphonoformic acid, a blocker of Na+-dependent Pi transporters. Pi increased the maximal conductance, decreased activation time constant, increased deactivation time constant, and shifted the conductance-voltage relationship to more negative voltages. The most marked change was enhanced gating which was mainly caused by elevation of intracellular Pi levels. The Pi-induced enhanced gating was partially inhibited by protein kinase C (PKC) inhibitors, GF109203X and staurosporine, indicating that PKC-mediated phosphorylation was involved in part. The increase in the maximal conductance was mainly due to accompanying decrease in intracellular pH. These effects of Pi were not affected by intracellular Mg2+, bafilomycin A1 (V-ATPase inhibitor) and removal of intracellular ATP. Extracellular Pi also upregulated reactive oxygen species (ROS). Diphenyleneiodonium chloride, an inhibitor of NADPH oxidases, decreased ROS production and partially attenuated the enhanced gating. In the cells during later passages where osteoclastogenesis declined, H+ channel activities and ROS production were both modest. These results suggest that, in osteoclasts, ambient Pi is a common enhancer for H+ channels and ROS production and that potentiation of H+ channels may help ROS production.

Essential role of Na+/Ca2+ exchanger 1 in smoking-induced growth and migration of esophageal squamous cell carcinoma

Tobacco-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a major environmental risk factor for the pathogenesis of human esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms by which tobacco induces ESCC are not well understood. Na+/Ca2+ exchanger 1 (NCX1) is a plasma membrane transporter protein that plays an essential role in maintaining cytosolic Ca2+ ([Ca2+]cyt) homeostasis under physiological conditions and is implicated in tumorigenesis as well. In this study, we found that NCX1 expression was significantly higher in ESCC primary tissues compared to the noncancerous tissues and was overexpressed in tumor samples from the smoking patients. The expression of NCX1 proteins was also significantly higher in human ESCC cell lines compared to normal esophageal epithelial cell line. Moreover, NNK potentiated the [Ca2+]cyt signaling induced by removal of extracellular Na+, which was abolished by KB-R7943 or SN-6. NNK dose-dependently promoted proliferation and migration of human ESCC cells induced by NCX1 activation. Therefore, NCX1 expression correlates with the smoking status of ESCC patients, and NNK activates the Ca2+ entry mode of NCX1 in ESCC cells, leading to cell proliferation and migration. Our findings suggest NCX1 protein is a novel potential target for ESCC therapy.

NF-κB-Dependent Upregulation of NCX1 Induced by Angiotensin II Contributes to Calcium Influx in Rat Aortic Smooth Muscle Cells

BackgroundThe reverse mode of Na+/Ca2+ exchanger (NCX) 1 can transport Ca2+ into cells and is involved in the contractile regulation of vascular smooth muscle cells (VSMCs) and the development of hypertension. We hypothesized that upregulation of NCX1 expression induced by angiotensin II (Ang II) could be dependent on activation of nuclear factor–kappa B (NF-κB) and contributes to Ca2+ influx in VSMCs. MethodsAn osmotic minipump was implanted for administration of Ang II in Sprague-Dawley rats, and blood pressure, as well as NCX1 expression, in the aorta was measured. VSMCs were cultured to verify that Ang II–upregulated NCX1 expression is dependent on activation of NF-κB and contributes to Ca2+ influx. ResultsAng II–upregulated NCX1 expression in rat aortas (2.1-fold at day 6) and VSMCs (1.7-fold at 24 hours) and NF-κB knockdown and p38 mitogen-activated protein kinase (MAPK) inhibitor resulted in 2.1- and 2.0-fold decreases in Ang II–upregulated NCX1 expression in VSMCs. KB-R7943 (an inhibitor of NCX1 reversal) and NCX1 knockdown decreased Ang II–induced Ca2+ influx 1.4- and 1.3- fold, respectively. KB-R7943 and removal of extracellular Na+ decreased Ang II–initiated store depletion–mediated Ca2+ entry by 1.5- and 1.3-fold, respectively. Moreover, NF-κB knockdown and use of a p38 MAPK inhibitor resulted in about 1.3-fold decreases in Ang II–induced Ca2+ influx through activation of reverse-mode NCX1. ConclusionsAng II upregulates NCX1 expression through p38 MAPK and NF-κB pathways, and reverse-mode NCX1 plays an important part in Ang II-induced Ca2+ influx in VSMCs, which may be associated with Ang II–initiated store-operated channel entry.

Effects of Leptin on Na+/Ca2+ Exchanger in PC12 Cells

Background/Aims: Alzheimer's disease (AD) is known to be related to alterations in neuronal intracellular calcium activity ([Ca²⁺]_i). The present study revealed the distinct role of leptin in Na⁺/Ca²⁺-exchanger activity. Methods: [Ca²⁺]_i was determined utilizing Fura-2 fluorescence. The activity of NCX was measured by removal of extracellular Na⁺ in the presence of external Ca²⁺. Na⁺/Ca²⁺-exchanger activity was further quantified from whole cell currents following removal of extracellular Na⁺. Na⁺/Ca²⁺-exchanger isoform NCX1 transcript levels and protein abundance were quantified by RT-PCR and Western blotting, respectively. Results: Exposure of PC12 cells to 30 µM amyloid (Aβ₄₂) increased [Ca²⁺]_i, an effect significantly blunted by 6 hours incubation with leptin before Aβ₄₂ treatment. Moreover, leptin treatment significantly increased Na⁺/Ca²⁺-exchanger mediated Ca+ transport and current, NCX1 transcript level as well as NCX1 membrane protein abundance. Conclusion: We show that leptin blunts Aβ₄₂-evoked [Ca²⁺]_i increase by increasing expression and activity of Na⁺/Ca²⁺-exchanger NCX1.

Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms

Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose.

Up-Regulation of Na+/Ca2+ Exchange in Megakaryocytes Following TGFβ1 Treatment

Background: Blood platelets are activated by increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). Ca²⁺ entry is accomplished in part by store operated Ca²⁺ entry (SOCE) involving Ca²⁺ release activated Ca²⁺-channel (CRAC) moiety Orai1 and its regulator STIM1, which are stimulated by depletion of intracellular Ca²⁺ stores. An increase of [Ca²⁺]_i is terminated by Na⁺/Ca²⁺-exchange. The expression of both, Orai1 and STIM1 in megakaryocytes is up-regulated by tumor growth factor TGFβ1, a powerful regulator of megakaryocyte differentiation. The present study explored whether TGFβ1 similarly modifies megakaryocyte Na⁺/Ca²⁺-exchanger activity. Methods: [Ca²⁺]_i was determined utlizing Fura-2 fluorescence, SOCE from increase of [Ca²⁺]_i, following readdition of extracellular Ca²⁺ after store depletion, and Na⁺/Ca²⁺-exchanger activity from increase of [Ca²⁺]_i and whole cell currents following removal of extracellular Na⁺. Results: TGFβ1 treatment not only augments the increase of [Ca²⁺]_i following store depletion and SOCE, but significantly up-regulates Na⁺/Ca²⁺-exchanger activity as apparent from [Ca²⁺]_i measurements and whole cell currents. Conclusions: TGFβ1 is a powerful stimulator of both, SOCE and Na⁺/Ca²⁺-exchanger activity in megakaryocytes.

Regulation of mechanosensitive biliary epithelial transport by the epithelial Na(+) channel.

Intrahepatic biliary epithelial cells (BECs), also known as cholangiocytes, modulate the volume and composition of bile through the regulation of secretion and absorption. While mechanosensitive Cl(-) efflux has been identified as an important secretory pathway, the counterabsorptive pathways have not been identified. In other epithelial cells, the epithelial Na(+) channel (ENaC) has been identified as an important contributor to fluid absorption; however, its expression and function in BECs have not been previously studied. Our studies revealed the presence of α, β, and γ ENaC subunits in human BECs and α and γ subunits in mouse BECs. In studies of confluent mouse BEC monolayers, the ENaC contributes to the volume of surface fluid at the apical membrane during constitutive conditions. Further, functional studies using whole-cell patch clamp of single BECs demonstrated small constitutive Na(+) currents, which increased significantly in response to fluid-flow or shear. The magnitude of Na(+) currents was proportional to the shear force, displayed inward rectification and a reversal potential of +40 mV (ENa+ = +60 mV), and were abolished with removal of extracellular Na(+) (N-methyl-d-glucamine) or in the presence of amiloride. Transfection with ENaCα small interfering RNA significantly inhibited flow-stimulated Na(+) currents, while overexpression of the α subunit significantly increased currents. ENaC-mediated currents were positively regulated by proteases and negatively regulated by extracellular adenosine triphosphate. These studies represent the initial characterization of mechanosensitive Na(+) currents activated by flow in biliary epithelium; understanding the role of mechanosensitive transport pathways may provide strategies to modulate the volume and composition of bile during cholestatic conditions. (Hepatology 2016;63:538-549).

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Rises in cytosolic Ca2+ concentration ([Ca2+]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca2+]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca2+]cyt (Ca2+ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca2+ signalling, we here monitor Ca2+ flux around the platelet by measuring net Ca2+ fluxes to or from the extracellular space and the intracellular Ca2+ stores, which act as the major sources and sinks for Ca2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na+ concentration ([Na+]cyt), which influences platelet Ca2+ fluxes via Na+/Ca2+ exchange. The intracellular store Ca2+ concentration ([Ca2+]st) was monitored using Fluo-5N, the extracellular Ca2+ concentration ([Ca2+]ext) was monitored using Fluo-4 whilst [Ca2+]cyt and [Na+]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca2+]cyt in the absence of extracellular Ca2+. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca2+ release and Ca2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca2+]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na+]cyt which would be expected to reduce Ca2+ removal via the Na+/Ca2+ exchanger (NCX). Thrombin-evoked rises in [Na+]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca2+]cyt following SERCA inhibition and either removal of extracellular Na+ or inhibition of Na+/K+-ATPase activity by removal of extracellular K+ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca2+]cyt by acceleration of SERCA activity, whilst rises in [Ca2+]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na+/K+-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na+]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca2+ signalling.

Lung Beractant Increases Free Cytosolic Levels of Ca2+ in Human Lung Fibroblasts.

Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF). As intracellular Ca2+ signalling has been related to programmed cell death, we aimed to assess the effect of beractant on intracellular Ca2+ concentration ([Ca2+]i) in NHLF in vitro. Cultured NHLF were loaded with Fura-2 AM (3 μM) and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+]i with a EC50 of 0.82 μg/ml. The application of beractant, at a concentration of 500 μg/ml, which has been shown to exert an apoptotic effect in human fibroblasts, elicited different patterns of Ca2+ signals in NHLF: a) a single Ca2+ spike which could be followed by b) Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by Ca2+ oscillations. The amplitude and pattern of Ca2+ transients evoked by beractant were dependent on the resting [Ca2+]i. Pharmacological manipulation revealed that beractant activates a Ca2+ signal through Ca2+ release from intracellular stores mediated by phospholipase Cβ (PLCβ), Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP3Rs) and Ca2+ influx via a store-operated pathway. Moreover, beractant-induced Ca2+ release was abolished by preventing membrane depolarization upon removal of extracellular Na+ and Ca2+. Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α1(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.