Carbenoxolone blocks the light-evoked rise in intracellular calcium in isolated melanopsin ganglion cell photoreceptors

Background

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca²⁺ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca²⁺ signals in ipRGCs independent of gap junction blockade.

Methodology/Principal Findings

To test the possibility that carbenoxolone directly inhibits light-evoked Ca²⁺ responses in ipRGCs, the light-evoked rise in intracellular Ca²⁺ ([Ca²⁺]_i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca²⁺]_i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.

Conclusions/Significance

We demonstrate that the light-evoked rise in [Ca²⁺]_i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca²⁺]_i in isolated ipRGCs is almost entirely due to Ca²⁺ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca²⁺]_i in ipRGCs by blocking L-type voltage-gated Ca²⁺ channels. The ability of carbenoxolone to block evoked Ca²⁺ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca²⁺]_i is the output being measured.     

Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca²⁺]_i) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod).Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca²⁺]_i) were calculated. 200 μM FFA induced an increase in [Ca²⁺]_i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca²⁺]_i, which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca²⁺]_i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca²⁺]_i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6.In conclusion, FFA activates TRPC6 [Ca²⁺]_i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Hypoxia Activates a Ca2+-Permeable Cation Conductance Sensitive to Carbon Monoxide and to GsMTx-4 in Human and Mouse Sickle Erythrocytes

Background

Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca²⁺] ([Ca²⁺]_i) and subsequent activation of K_Ca 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.

Methodology/Principal Findings

We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca²⁺- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca²⁺]_i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca²⁺]_i in mouse sickle erythrocytes did not require KCa3.1 activity.

Conclusions/Significance

The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca²⁺-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca²⁺ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.     

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes

Background

The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca²⁺-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/Principal Findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca²⁺-handling proteins. Detailed laser confocal Ca²⁺ imaging demonstrated spontaneous whole-cell [Ca²⁺]_i transients. These transients required Ca²⁺ influx via L-type Ca²⁺ channels, as demonstrated by their elimination in the absence of extracellular Ca²⁺ or by administration of the L-type Ca²⁺ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca²⁺ store, contributing to [Ca²⁺]_i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca²⁺) and ryanodine (decreasing [Ca²⁺]_i). Similarly, the importance of Ca²⁺ reuptake into the SR via the SR Ca²⁺ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca²⁺]_i transients elimination. Finally, the presence of an IP3-releasable Ca²⁺ pool in hiPSC-CMs and its contribution to whole-cell [Ca²⁺]_i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusions/Significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca²⁺ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca²⁺]_i transients in hiPSC-CMs on both sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and intracellular store Ca²⁺ release.     

Growth hormone secretagogues protect mouse cardiomyocytes from in vitro ischemia/reperfusion injury through regulation of intracellular calcium

Background

Ischemic heart disease is a leading cause of mortality. To study this disease, ischemia/reperfusion (I/R) models are widely used to mimic the process of transient blockage and subsequent recovery of cardiac coronary blood supply. We aimed to determine whether the presence of the growth hormone secretagogues, ghrelin and hexarelin, would protect/improve the function of heart from I/R injury and to examine the underlying mechanisms.

Methodology/Principal Findings

Isolated hearts from adult male mice underwent 20 min global ischemia and 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nM) or hexarelin (1 nM) was introduced into the perfusion system either 10 min before or after ischemia, termed pre- and post-treatments. In freshly isolated cardiomyocytes from these hearts, single cell shortening, intracellular calcium ([Ca²⁺]_i) transients and caffeine-releasable sarcoplasmic reticulum (SR) Ca²⁺ were measured. In addition, RT-PCR and Western blots were used to examine the expression level of GHS receptor type 1a (GHS-R1a), and phosphorylated phospholamban (p-PLB), respectively. Ghrelin and hexarelin pre- or post-treatments prevented the significant reduction in the cell shortening, [Ca²⁺]_i transient amplitude and caffeine-releasable SR Ca²⁺ content after I/R through recovery of p-PLB. GHS-R1a antagonists, [D-Lys3]-GHRP-6 (200 nM) and BIM28163 (100 nM), completely blocked the effects of GHS on both cell shortening and [Ca²⁺]_i transients.

Conclusion/Significance

Through activation of GHS-R1a, ghrelin and hexarelin produced a positive inotropic effect on ischemic cardiomyocytes and protected them from I/R injury probably by protecting or recovering p-PLB (and therefore SR Ca²⁺ content) to allow the maintenance or recovery of normal cardiac contractility. These observations provide supporting evidence for the potential therapeutic application of ghrelin and hexarelin in patients with cardiac I/R injury.     

Amino Alcohol- (NPS-2143) and Quinazolinone-Derived Calcilytics (ATF936 and AXT914) Differentially Mitigate Excessive Signalling of Calcium-Sensing Receptor Mutants Causing Bartter Syndrome Type 5 and Autosomal Dominant Hypocalcemia

Introduction

Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants.

Methods

All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca²⁺]_o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca²⁺]_o in the presence or absence of NPS-2143, ATF936 or AXT914.

Results

All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca²⁺]_o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.

Conclusion

The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

Background

Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).

Methods

Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O₂ for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) and store-operated Ca²⁺ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.

Results

Hypoxia increased the basal [Ca²⁺]_i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O₂, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.

Conclusions

Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca²⁺]_i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.     

Acid-sensing ion channel 3 decreases phosphorylation of extracellular signal-regulated kinases and induces synoviocyte cell death by increasing intracellular calcium

Introduction

Acid-sensing ion channel 3 (ASIC3) is expressed in synoviocytes, activated by decreases in pH, and reduces inflammation in animal models of inflammatory arthritis. The purpose of the current study was to characterize potential mechanisms underlying the control of inflammation by ASIC3 in fibroblast-like synoviocytes (FLS).

Methods

Experiments were performed in cultured FLS from wild-type (WT) and ASIC3-/- mice, ASIC1-/- mice, and people with rheumatoid arthritis. We assessed the effects of acidic pH with and without interleukin-1β on FLS and the role of ASICs in modulating intracellular calcium [Ca²⁺]_i, mitogen activated kinase (MAP kinase) expression, and cell death. [Ca²⁺]_i was assessed by fluorescent calcium imaging, MAP kinases were measured by Western Blots; ASIC, cytokine and protease mRNA expression were measured by quantitative PCR and cell death was measured with a LIVE/DEAD assay.

Results

Acidic pH increased [Ca²⁺]_i and decreased p-ERK expression in WT FLS; these effects were significantly smaller in ASIC3-/- FLS and were prevented by blockade of [Ca²⁺]_i. Blockade of protein phosphatase 2A (PP2A) prevented the pH-induced decreases in p-ERK. In WT FLS, IL-1β increases ASIC3 mRNA, and when combined with acidic pH enhances [Ca²⁺]_i, p-ERK, IL-6 and metalloprotienase mRNA, and cell death. Inhibitors of [Ca²⁺]_i and ERK prevented cell death induced by pH 6.0 in combination with IL-1β in WT FLS.

Conclusions

Decreased pH activates ASIC3 resulting in increased [Ca²⁺]_i, and decreased p-ERK. Under inflammatory conditions, acidic pH results in enhanced [Ca²⁺]_i and phosphorylation of extracellular signal-regulated kinase that leads to cell death. Thus, activation of ASIC3 on FLS by acidic pH from an inflamed joint could limit synovial proliferation resulting in reduced accumulation of inflammatory mediators and subsequent joint damage.     

Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Background

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca²⁺] ([Ca²⁺]_o) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca²⁺]_o sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.

Methodology/Principal Findings

By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca²⁺]_o than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.

Conclusions/Significance

Our experiments identify CaSR as the first component in the [Ca²⁺]_o sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca²⁺]_o will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca²⁺]_o following its entry via voltage-activated Ca²⁺ channels. CaSR may detects such falls in [Ca²⁺]_o and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca²⁺]_o on release probability. CaSR is positioned to detect the dynamic changes of [Ca²⁺]_o and provide presynaptic feedback that will alter brain excitability.     

Fine-Tuning of the Cytoplasmic Ca2+ Concentration Is Essential for Pollen Tube Growth

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca²⁺ concentration ([Ca²⁺]) gradient and regular oscillations of the cytosolic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region. Whether this [Ca²⁺] gradient and/or [Ca²⁺]_cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca²⁺]_cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca²⁺]_cyt gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca²⁺]_cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca²⁺]_cyt in the tip region is essential for pollen tube growth, whereas a regular [Ca²⁺] oscillation is not. Next, we monitored [Ca²⁺] dynamics in the endoplasmic reticulum ([Ca²⁺]_ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca²⁺ compared with yellow cameleon 3.60. The [Ca²⁺]_ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 μm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca²⁺-ATPases, inhibited growth and decreased the [Ca²⁺]_ER. Our observations suggest that the ER serves as one of the Ca²⁺ stores in the pollen tube and cyclopiazonic acid-sensitive Ca²⁺-ATPases in the ER are required for pollen tube growth.In many flowering plants, a pollen grain that lands on the top surface of a stigma will hydrate and germinate a pollen tube. Following germination, the pollen tube enters the style and grows through the wall of transmitting tract cells on the way to the ovary, where the tube emerges to release the sperm for double fertilization. Therefore, pollen tube growth is essential for reproduction in flowering plants.Since Brewbaker and Kwack (1963) revealed that Ca²⁺ is essential for in vitro pollen tube cultures, the relationship between the Ca²⁺ concentration ([Ca²⁺]) and pollen tube growth has been further examined under in vitro germination culture conditions. Ratiometric ion imaging using fluorescent dye has revealed that the apical domain of a pollen tube grown in vitro contains a tip-focused [Ca²⁺] gradient (Pierson et al., 1994, 1996; Cheung and Wu, 2008) and that the cytoplasmic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region and the growth rate oscillate with the same periodicity (Pierson et al., 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997). Therefore, oscillation of the [Ca²⁺]_cyt has been thought to correlate with pollen tube growth. It is not clear, however, whether regular [Ca²⁺]_cyt oscillations in the tip region occur in pollen tubes growing through stigmas and styles.The [Ca²⁺]_cyt is controlled temporally and spatially by transporters in the membranes of intracellular compartments and in the plasma membrane (Sze et al., 2000). Studies using a Ca²⁺-sensitive vibrating electrode revealed Ca²⁺ influx in the tip region of the pollen tube (Pierson et al., 1994; Holdaway-Clarke et al., 1997; Franklin-Tong et al., 2002). Stretch-activated Ca²⁺ channels have been found in the plasma membrane using patch-clamp electrophysiology (Kuhtreiber and Jaffe, 1990; Dutta and Robinson, 2004). Recently, CNGC18 was identified as a Ca²⁺-permeable channel in the plasma membrane that is essential for pollen tube growth (Frietsch et al., 2007). The intracellular compartments that store Ca²⁺ in the pollen tube and the relevant Ca²⁺ transporters, however, have yet to be identified.Yellow cameleons are genetically encoded Ca²⁺ indicators that were developed to monitor the [Ca²⁺] in living cells (Miyawaki et al., 1997). These indicators are chimeric proteins consisting of enhanced cyan fluorescent protein (ECFP), calmodulin (CaM), a glycylglycine linker, the CaM-binding domain of myosin light chain kinase (M13), and enhanced yellow fluorescent protein (EYFP). When the CaM domain binds Ca²⁺, the domain associates with the M13 peptide and induces fluorescence resonance energy transfer (FRET) between ECFP and EYFP. Several types of cameleons have been developed by tuning the CaM domain binding affinity for Ca²⁺. Yellow cameleon 2.1 (YC2.1) is a high-affinity indicator that has been used to monitor the [Ca²⁺]_cyt in Arabidopsis (Arabidopsis thaliana) guard cells (Allen et al., 1999, 2000, 2001), Lilium longiflorum and Nicotiana tabacum pollen tubes (Watahiki et al., 2004), and the root hair of Medicago truncatula (Miwa et al., 2006). YC3.1 is a low-affinity indicator that has been used to monitor the [Ca²⁺]_cyt during pollen germination and in papilla cells of Arabidopsis (Iwano et al., 2004).Recently, YC3.60 was developed as a new YC variant (Nagai et al., 2004), in which the acceptor fluorophore is a circularly permuted version of Venus rather than EYFP (Nagai et al., 2002). YC3.60 has a monophasic Ca²⁺ dependency with a dissociation constant (K_d) of 0.25 μm. Compared with YC3.1, YC3.60 is equally bright with a 5- to 6-fold larger dynamic range. Thus, YC3.60 results in a markedly enhanced signal-to-noise ratio, thereby enabling Ca²⁺ imaging experiments that were not possible with conventional YCs. On the other hand, YC4.60 was developed by mutating the Ca²⁺-binding loop of CaM in YC3.60. Because YC4.60 has a significantly lower Ca²⁺ affinity with a biphasic Ca²⁺ dependency (K_d: 58 nm and 14.4 μm), it allows changes in [Ca²⁺] dynamics to be detected against a high background [Ca²⁺] (Nagai et al., 2004).To examine whether the [Ca²⁺]_cyt oscillates in pollen tubes growing through a stigma after pollination (in vivo condition), in those placed in germination medium immediately after passing through a style (semi-in vivo condition), or in those grown in germination medium (in vitro condition), we generated transgenic Arabidopsis and N. tabacum lines expressing the YC3.60 gene in their pollen grains and monitored Ca²⁺ dynamics in the pollen tube tip. We also examined how inhibitors of pollen tube growth affect Ca²⁺ dynamics in pollen tubes growing under the semi-in vivo condition. To examine Ca²⁺ dynamics in the endoplasmic reticulum (ER), we generated transgenic Arabidopsis plants expressing YC4.60 in the pollen tube ER. The results are discussed in relation to the physiological relevance of [Ca²⁺] oscillations for pollen tube growth.     

Spent Culture Medium from Virulent Borrelia burgdorferi Increases Permeability of Individually Perfused Microvessels of Rat Mesentery

Background

Lyme disease is a common vector-borne disease caused by the spirochete Borrelia burgdorferi (Bb), which manifests as systemic and targeted tissue inflammation. Both in vitro and in vivo studies have shown that Bb-induced inflammation is primarily host-mediated, via cytokine or chemokine production that promotes leukocyte adhesion/migration. Whether Bb produces mediators that can directly alter the vascular permeability in vivo has not been investigated. The objective of the present study was to investigate if Bb produces a mediator(s) that can directly activate endothelial cells resulting in increases in permeability in intact microvessels in the absence of blood cells.

Methodology/Principal Findings

The effects of cell-free, spent culture medium from virulent (B31-A3) and avirulent (B31-A) B. burgdorferi on microvessel permeability and endothelial calcium concentration, [Ca²⁺]_i, were examined in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca²⁺]_i, a necessary signal initiating hyperpermeability, was measured in Fura-2 loaded microvessels. B31-A3 spent medium caused a rapid and transient increase in Lp and endothelial [Ca²⁺]_i. Within 2–5 min, the mean peak Lp increased to 5.6±0.9 times the control, and endothelial [Ca²⁺]_i increased from 113±11 nM to a mean peak value of 324±35 nM. In contrast, neither endothelial [Ca²⁺]_i nor Lp was altered by B31-A spent medium.

Conclusions/Significance

A mediator(s) produced by virulent Bb under culture conditions directly activates endothelial cells, resulting in increases in microvessel permeability. Most importantly, the production of this mediator is associated with Bb virulence and is likely produced by one or more of the 8 plasmid(s) missing from strain B31-A.     

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺]_i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca2 channels preserved K_Ca2 expression and significantly reduced pathological increases in [Ca²⁺]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.     

Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells

Background

The present study investigates the effects of high external calcium concentration ([Ca²⁺]_o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.

Methodology/Principal Findings

A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca²⁺]_o (0.37 mM); 2) high [Ca²⁺]_o (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca²⁺]_o and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca²⁺]_o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca²⁺]_o was not effective in this cell line. In small cells, both higher [Ca²⁺]_o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca²⁺]_o and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.

Conclusions/Significance

In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.     

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Regulatory effect of connexin 43 on basal Ca2+ signaling in rat ventricular myocytes

Background

It has been found that gap junction-associated intracellular Ca²⁺ [Ca²⁺]_i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca²⁺ signaling, in particular the basal [Ca²⁺]_i activities, is unclear.

Methods and Results

Global and local Ca²⁺ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca²⁺ transients and local Ca²⁺ sparks in monolayer NRVMs and Ca²⁺ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP₃ butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca²⁺ signal and LY uptake by gap uncouplers, whereas blockade of IP₃ receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca²⁺ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca²⁺ signaling regulation in cardiomyocytes.

Conclusions

These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca²⁺ signaling in normal ventricular myocytes, in which IP₃/IP₃ receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca²⁺ activities in cardiomyocytes.     

Demand for Zn2+ in acid-secreting gastric mucosa and its requirement for intracellular Ca2+

Background and Aims

Recent work has suggested that Zn²⁺ plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn²⁺ in gastric mucosa under baseline conditions and its regulation during secretory stimulation.

Methods and Findings

Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn²⁺ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, ⁷⁰Zn²⁺, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of ⁷⁰Zn²⁺ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn²⁺ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn²⁺ {[Zn²⁺]_i} during exposure to standard extracellular concentrations of Zn²⁺ (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn²⁺ increased with exposure to secretagogues (forskolin, carbachol/histamine) and under conditions associated with increased intracellular Ca²⁺ {[Ca²⁺]_i}. Uptake of Zn²⁺ was abolished following removal of extracellular Ca²⁺ or depletion of intracellular Ca²⁺ stores, suggesting that demand for extracellular Zn²⁺ increases and depends on influx of extracellular Ca²⁺.

Conclusions

This study is the first to characterize the content and distribution of Zn²⁺ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca²⁺ integrates basolateral demand for Zn²⁺ with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues.     

Identification of novel DNA methylation inhibitors via a two-component reporter gene system

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.