μM Gd Research Articles

Biotin‐Conjugated Poly(Acrylic Acid)‐Grafted Ultrasmall Gadolinium Oxide Nanoparticles for Enhanced Tumor Imaging

AbstractHerein, biotin (Bio)‐conjugated poly(acrylic acid) (PAA)‐grafted ultrasmall gadolinium oxide nanoparticles (Bio‐PAA‐Gd2O3 NPs) were synthesized for enhanced tumor imaging using Bio as a tumor‐targeting ligand. The average particle diameter of Gd2O3 NPs was 2.1 nm. The Bio‐PAA‐Gd2O3 NPs exhibited excellent colloidal stability (i. e., no precipitation) and a high longitudinal water proton spin relaxivity (r1) of 23.8 s−1 mM−1 (r2/r1=1.6 and r2=transverse water proton spin relaxivity), which was ∼6 times higher than those of commercial Gd‐chelated magnetic resonance imaging (MRI) contrast agents. Cytotoxicity tests using two cell lines showed that the Bio‐PAA‐Gd2O3 NPs were almost non‐toxic up to the measured concentration of 500 μM Gd. The enhanced tumor imaging of the Bio‐PAA‐Gd2O3 NPs was demonstrated through their higher positive contrasts and longer contrast retention at the tumor after intravenous injection in T1 MR images, compared with those of the control PAA‐Gd2O3 NPs.

Chitosan Oligosaccharide Lactate-Coated Ultrasmall Gadolinium Oxide Nanoparticles: Synthesis, In Vitro Cytotoxicity, and Relaxometric Properties.

In this study, hydrophilic and biocompatible chitosan oligosaccharide lactate (COL)-coated ultra-small gadolinium oxide nanoparticles (NPs) were synthesized through a one-pot polyol method and characterized by various experimental techniques. The In Vitro cellular cytotoxicity assay indicated that the COL-coated gadolinium oxide NPs were non-toxic up to 500 μM Gd. In addition, their water proton spin relaxivities (i.e., r1 and r₂) were estimated to be 13.0 and 27.0 s-1mM-1, respectively, which are higher than those of commercial magnetic resonance imaging (MRI) contrast agents. The application potential of the solution sample as a T1 MRI contrast agent was demonstrated In Vitro by measuring map images in which dose-dependent contrast enhancements were observed.

In vivo evaluation of PEGylated-liposome encapsulating gadolinium complexes for gadolinium neutron capture therapy

Gadolinium neutron capture therapy (GdNCT) is a form of binary radiotherapy. It utilizes nuclear reactions that occur when gadolinium-157 is irradiated with thermal neutrons, producing high-energy γ-rays and Auger electrons. Herein, we evaluate the potential of GdNCT for cancer treatment using PEGylated liposome incorporated with an FDA-approved MRI contrast agent. The clinical gadolinium complex (Gadovist®) was successfully encapsulated inside the aqueous core of PEGylated liposomes by repeated freeze and thaw cycling. At a concentration of 152 μM Gd, the Gd-liposome showed high cytotoxicity upon thermal-neutron irradiation. In animal experiments, when a CT26 tumor model was administered with Gd-liposomes (19 mg 157Gd per kg) followed by 20-min irradiation of thermal neutron at a flux of 1.94 × 104 cm−2 s−1, tumor growth was suppressed by 43%, compared to that in the control group, on the 23rd day of post-irradiation. After two-cycle GdNCT treatment at a 10-day interval, tumor growth was more efficiently retarded. On the 31st day after irradiation, the weight of the excised tumor in the GdNCT group (38 mg 157Gd per kg per injection) was only 30% of that of the control group. These results demonstrate the potential of GdNCT using PEGylated liposomes containing MRI contrast agents in cancer treatment.

Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women.

Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca(2+) influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, are known to cause membrane depolarization in many tissue types. Previously, a Gd(3+)-sensitive, Na(+)-dependent leak current was identified in the rat myometrium, but the presence of such a current in human MSMCs and the specific ion channel conducting this current was unknown. Here, we report the presence of a Na(+)-dependent leak current in human myometrium and demonstrate that the Na(+)-leak channel, NALCN, contributes to this current. We performed whole-cell voltage-clamp on fresh and cultured MSMCs from uterine biopsies of term, non-laboring women and isolated the leak currents by using Ca(2+) and K(+) channel blockers in the bath solution. Ohmic leak currents were identified in freshly isolated and cultured MSMCs with normalized conductances of 14.6 pS/pF and 10.0 pS/pF, respectively. The myometrial leak current was significantly reduced (P < 0.01) by treating cells with 10 μM Gd(3+) or by superfusing the cells with a Na(+)-free extracellular solution. Reverse transcriptase PCR and immunoblot analysis of uterine biopsies from term, non-laboring women revealed NALCN messenger RNA and protein expression in the myometrium. Notably, ∼90% knockdown of NALCN protein expression with lentivirus-delivered shRNA reduced the Gd(3+)-sensitive leak current density by 42% (P < 0.05). Our results reveal that NALCN, in part, generates the leak current in MSMCs and provide the basis for future research assessing NALCN as a potential molecular target for modulating uterine excitability.

(Invited) Development of 4 Inch and 6 Inch GaN-on-Si for High-Voltage Operation

4 inch and 6 inch GaN-on-silicon (GOS) epitaxial wafers have been developed to enable large-volume, low-cost manufacturing technology for GaN power electronics. In an effort to reduce the buffer leakage and improve the buffer blocking voltage as well as the device off-state breakdown voltage, carbon-doped buffer epilayers and total nitride epitaxy thickness were investigated. Low normalized buffer leakage of around 3 × 10-9 A/mm and low normalized three-terminal subthreshold leakage of around 2 × 10-7 A/mm with on/off ratio of ~106 were obtained from both 4 and 6 inch GOS HEMT wafers. A three-terminal off-state breakdown voltage of 1.2 kV was achieved on a GOS HEMT with 10 μm GD spacing to confirm a strong potential for high power applications.

NALCN Ion Channels Have Alternative Selectivity Filters Resembling Calcium Channels or Sodium Channels

NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE) or sodium channels (EKEE or EEKE). NALCN channels with alternative calcium, (EEEE) and sodium, (EKEE or EEKE) -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K)-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG+ impermeant and blockable with 10 µM Gd3+ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2):371-83.

Cationic Gd-DTPA Liposomes for Highly Efficient Labeling of Mesenchymal Stem Cells and Cell Tracking with MRI

In the current study cell labeling was performed with water-soluble gadolinium (Gd)-DTPA containing liposomes, to allow for cell tracking by MRI. Liposomes were used to assure a highly concentrated intracellular build up of Gd, aiming to overcome the relatively low MRI sensitivity of Gd (compared to T2 contrast agents). Liposomes were positively charged (cationic) to facilitate uptake by binding to anionic charges in the cell membrane of bone marrow-derived mesenchymal stem cells (MSCs). We determined the cellular Gd load by variations in labeling time (1, 4, and 24 h) and liposome concentration (125, 250, 500, 1000 μM lipid), closely monitoring effects on cell viability, proliferation rate, and differentiation ability. Labeling was both time and dose dependent. Labeling for 4 h was most efficient regarding the combination of processing time and final cellular Gd uptake. Labeling for 4 h with low-dose concentration (125 μM lipid, corresponding to 52 ± 3 μM Gd) yielded an intracellular load of 30 ± 2.5 pg Gd cell(-1), without any effects on cell viability, proliferation, and cell differentiation. Gd liposomes, colabeled with fluorescent dyes, exhibited a prolonged cellular retention, with an endosomal distribution pattern. In vitro assay over 20 days demonstrated a drop in the average Gd load per cell, as a result of mitosis. However, there was no significant change in the sum of the Gd load in all daughter cells at endpoint (20 days), indicating an excellent cellular retention of Gd. MSCs labeled with Gd liposomes were imaged with MRI at both 1.5T and 3.0T, resulting in excellent visualization both in vitro and in vivo. Prolonged in vivo imaging of 500,000 Gd-labeled cells was possible for at least 2 weeks (3.0T). In conclusion, Gd-loaded cationic liposomes (125 μM lipid) are an excellent candidate to label cells, without detrimental effects on cell viability, proliferation, and differentiation, and can be visualized by MRI.

Gadolinium increases the vascular reactivity of rat aortic rings

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 µM Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 µM L-NAME, 10 µM losartan, or 10 µM enalaprilat. Gd significantly increased the maximum response (control: 72.3 ± 3.5; Gd: 101.3 ± 6.4%) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8%) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 ± 4.1; Gd: -47.4 ± 4.1%). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1%). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 ± 28.8; L-NAME + Gd: 67.9 ± 19% AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Relaxivity of gadolinium complexes detected by atomic magnetometry

Laser atomic magnetometry is a portable and low-cost yet highly sensitive method for low magnetic field detection. In this work, the atomic magnetometer was used in a remote-detection geometry to measure the relaxivity of aqueous gadolinium-diethylenetriamine pentaacetic acid Gd(DTPA) at the Earth's magnetic field (40 μT). The measured relaxivity of 9.7±2.0 s(-1) mM(-1) is consistent with field-cycling experiments measured at slightly higher magnetic fields, but no cryogens or strong and homogeneous magnetic field were required for this experiment. The field-independent sensitivity of 80 fT Hz(-1/2) allowed an in vitro detection limit of ∼10 μM Gd(DTPA) to be measured in aqueous buffer solution. The low detection limit and enhanced relaxivity of Gd-containing complexes at Earth's field motivate continued development of atomic magnetometry toward medical applications.

Optical imaging in tissue with X-ray excited luminescent sensors

We report a high-spatial resolution imaging technique to measure optical absorption and detect chemical and physical changes on surfaces embedded in thick tissue. Developing sensors to measure chemical concentrations on implanted surfaces through tissue is an important challenge for analytical chemistry and biomedical imaging. Tissue scattering dramatically reduces the resolution of optical imaging. In contrast, X-rays provide high spatial resolution imaging through tissue but do not measure chemical concentrations. We describe a hybrid technique which uses a scanning X-ray beam to irradiate Gd(2)O(2)S scintillators and detect the resulting visible luminescence through the tissue. The amount of light collected is modulated by optical absorption in close proximity to the luminescence source. By scanning the X-ray beam, and measuring total amount of light collected, one can measure the local absorption near scintillators at a resolution limited by the width of luminescence source (i.e. the width of the X-ray excitation beam). For proof of principle, a rectangular 1.7 mm scanning X-ray beam was used to excite a single layer of 8 μm Gd(2)O(2)S particles, and detect the absorption of 5 nm thick silver island film through 10 mm of pork. Lifetime and spectroscopic measurements, as well changing the refractive index of the surroundings indicate that the silver reduces the optical signal through attenuated total internal reflection. The technique was used to image the dissolution of regions of the silver island film which were exposed to 1 mM of H(2)O(2) through 1 cm of pork tissue.

Cellular uptake and imaging studies of gadolinium‐loaded single‐walled carbon nanotubes as MRI contrast agents

We quantify here, for the first time, the intracellular uptake (J774A.1 murine macrophage cells) of gadolinium-loaded ultra-short single-walled carbon nanotubes (gadonanotubes or GNTs) in a 3 T MRI scanner using R(2) and R(2)* mapping in vitro. GNT-labeled cells exhibited high and linear changes in net transverse relaxations (ΔR(2) and ΔR 2*) with increasing cell concentration. The measured ΔR(2)* were about three to four times greater than the respective ΔR(2) for each cell concentration. The intracellular uptake of GNTs was validated with inductively coupled plasma optical emission spectrometry (ICP-OES), indicating an average cellular uptake of 0.44 ± 0.09 pg Gd per cell or 1.69 × 10(9) Gd(3+) ions per cell. Cell proliferation MTS assays demonstrated that the cells were effectively labeled, without cytotoxicity, for GNTs concentrations ≤28 µM Gd. In vivo relaxometry of a subcutaneously-injected GNT-labeled cell pellet in a mouse was also demonstrated at 3 T. Finally, the pronounced R(2)* effect of GNT-labeled cells enabled successful in vitro visualization of labeled cells at 9.4 T.

Recovery of cowpea seedling roots from exposure to toxic concentrations of trace metals

Rhizotoxic effects of many trace metals are known, but there is little information on recovery after exposure. Roots of 3-d-old cowpea (Vigna unguiculata (L.) Walp. cv. Caloona) seedlings were grown for 4 or 12 h in solutions of 960 μM Ca and 5 μM B at two concentrations (which reduce growth by 50 or 85%) of nine trace metals that rupture the outer layers of roots. Measured concentrations were 34 or 160 μM Al, 0.6 or 1.6 μM Cu, 2.2 or 8.5 μM Ga, 2.3 or 12 μM Gd, 0.8 or 1.9 μM Hg, 1.0 or 26 μM In, 2.4 or 7.3 μM La, 1.8 or 3.8 μM Ru, and 1.3 or 8.6 μM Sc. Roots were rinsed, transferred to solutions free of trace metals, and regrowth monitored for up to 48 h. Recovery from exposure to Hg occurred within 4 h, but regrowth was delayed for ≥ 12 h with Al, Ga, or Ru. There was poor regrowth after 4 or 12 h exposure to Cu, Gd, In, La, or Sc. Roots recovered after being grown for 12 to 48 h in 170 μM Al, 5.1 μM Ga, 2.0 μM Hg, or 1.4 μM Ru, but the extent of recovery was reduced with longer exposure time. Microscopy showed marked differences in symptoms on roots recovering from exposure to the various trace metals. Differences in (i) concentrations that are toxic, (ii) ability of roots to recover, (iii) time for recovery to occur, and (iv) symptoms that develop, suggest that each trace metal has a unique combination of rhizotoxic effects.

Gadolinium Ions Block Mechanosensitive Channels by Altering the Packing and Lateral Pressure of Anionic Lipids

Effects of polyvalent ions on the lateral packing of phospholipids have been known for decades, but the physiological consequences have not been systematically studied. Gd 3+ is a relatively nonspecific agent that blocks mechano-gated channels with a variable affinity. In this study, we show that the large mechanosensitive channel MscL of Escherichia coli is effectively blocked by Gd 3+ only when reconstituted with negatively charged phospholipids (e.g., PS). Taking this lead, we studied effects of Gd 3+ on monolayers and unilamellar vesicles made of natural brain PS, DMPS, and its mixtures with DMPC. In monolayer experiments, we found that μM Gd 3+ present in the subphase leads to ∼8% lateral compaction of brain PS (at 35 mN/m). Gd 3+ more strongly shrinks and rigidifies DMPS films causing a spontaneous liquid expanded-to-compact transition to the limiting 40 Å 2/mol. Pressure-area isotherms of uncharged DMPC were unaffected by Gd 3+, and neutralization of DMPS surface by low pH did not produce strong compaction. Upshifts of surface potential isotherms of DMPS monolayers reflected changes in the diffuse double layer due to neutralization of headgroup charges by Gd 3+, whereas the increased packing density produced up to a 200 mV change in the interfacial dipole potential. The slopes of surface potential versus reciprocal area predicted that Gd 3+ induced a modest (∼18%) increase in the magnitude of the individual lipid dipoles in DMPS. Isothermal titration calorimetry indicated that binding of Gd 3+ to DMPS liposomes in the gel state is endothermic, whereas binding to liquid crystalline liposomes produces heat consistent with the isothermal liquid-to-gel phase transition induced by the ion. Both titration curves suggested a K b of ∼10 6 M −1. We conclude that anionic phospholipids serve as high-affinity receptors for Gd 3+ ions, and the ion-induced compaction generates a lateral pressure increase estimated as tens of mN/m. This pressure can “squeeze” the channel and shift the equilibrium toward the closed state.

Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE(-/-) mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.

A Gd-based gaseous electron multiplier detector for neutron scattering applications

The optimum configuration of a Gaseous Electron Multiplier (GEM) neutron detector using a CsI–Gd–Kapton–Gd–CsI neutron converter is investigated using Monte-Carlo simulations. Neutrons absorbed in the converter produce secondary electrons that are emitted from the CsI layers. The detector can be assembled from multiple modules where each module consists of the neutron converter, several cascaded GEMs and anode pickup plates on both sides. Position sensitive anodes and localization electronics are then used to detect, timestamp and record the resulting signal. For a single GEM module, the performance can be assessed by estimating the secondary electron (SE) leakage from the converter sandwich. The simulations show that the optimum for a single module, double-sided detector would have a neutron converter composed of 0.1 μm CsI on top of 3 μm Gd plated on each side of a 7.5 μm thick Kapton foil. This detector would have a SE yield of approximately 0.6 SE/neutron and neutron absorption of 60%. Significant enhancements of the SE yield can be obtained for detectors composed of multiple modules with thinner Gd converters. The multi-module design allows for enhanced SE leakage from each converter while maintaining the high neutron absorption efficiency of thicker converters and dividing the detector count rate among multiple sets of decoding electronics.

Influence of cholesterol depletion in plasma membrane of rat brain synaptosomes on calcium-dependent and calcium-independent exocytosis

It is well established that calcium-dependent neurotransmitter release and exocytosis can be regulated by altering the cholesterol content of the plasma membrane. We have compared the influence of cholesterol depletion of synaptosomal plasma membrane by 15 mM methyl-β-cyclodextrin (MCD) treatment on calcium-dependent release of d-[ 3H]aspartate induced by the calcium ionophore A23187 and on calcium-independent release induced by hypertonic shrinking or polyvalent cations. We found that decrease of cholesterol concentration by 9.3% inhibited calcium-dependent release of d-[ 3H]aspartate induced by calcium ionophore A23187 by four times while release induced by 300 μM Gd 3+, 150 mM and 500 mM sucrose remained unchanged. Further we have investigated the influence of MCD on exocytosis monitored by the fluorescent dye, acridine orange. Cholesterol depletion inhibited calcium-dependent exocytosis induced by calcium ionophore A23187 but had virtually no influence on calcium-independent exocytosis induced by hypertonic shrinking or Gd 3+. In summary, we found that the cholesterol content in synaptosomal plasma membrane is important for calcium-dependent exocytosis.

High-performance low-temperature solid oxide fuel cell with novel BSCF cathode

An anode-supported solid oxide fuel cell (SOFC), consisting of a dense 10 μm Gd 0.1Ce 0.9O 1.95 (GDC) electrolyte, a porous Ba 0.5Sr 0.5Co 0.8Fe 0.2O 3− δ (BSCF) cathode and a porous Ni–GDC cermet anode, is successfully assembled and electrochemically characterized. With humidified (3% water vapour) hydrogen as the fuel and air as the oxidant, the cell exhibits open-circuit voltages of 0.903 and 0.984 V when operating at 600 and 500 °C, respectively. The cell produces peak power densities of 1329, 863, 454, 208 and 83 mW cm −2 at 600, 550, 500, 450 and 400 °C, respectively. These results are impressive and demonstrate the potential of BSCF for use as the cathode material in new-generation SOFCs with GDC as the electrolyte. In addition, the sustained performance at temperatures below 600 °C warrants commercial exploitation of this SOFC in stationary and mobile applications.

Biophysical Properties of the Apoptosis-Inducing Plasma Membrane Voltage-Dependent Anion Channel

Ion channels in the plasma membrane play critical roles in apoptosis. In a recent study we found that a voltage-dependent anion channel in the plasma membrane (VDACpl) of neuronal hippocampal cell line (HT22) cells was activated during apoptosis and that channel block prevented apoptosis. Whether or not VDACpl is identical to the mitochondrial VDACmt has been debated. Here, we biophysically characterize the apoptosis-inducing VDACpl and compare it with other reports of VDACpls and VDACmt. Excised membrane patches of apoptotic HT22 cells were studied with the patch-clamp technique. VDACpl has a large main-conductance state (400 pS) and occasionally subconductance states of ∼28 pS and 220 pS. The small subconductance state is associated with long-lived inactivated states, and the large subconductance state is associated with excision of the membrane patch and subsequent activation of the channel. The open-probability curve is bell shaped with its peak around 0 mV and is blocked by 30 μM Gd 3+. The gating can be described by a symmetrical seven-state model with one open state and six closed or inactivated states. These channel properties are similar to those of VDACmt and other VDACpls and are discussed in relation to apoptosis.

Mitochondria play a critical role in shaping the exocytotic response of rat pancreatic acinar cells

We have previously demonstrated [M. Campos-Toimil, T. Bagrij, J.M. Edwardson, P. Thomas, Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca 2+ entry, Curr. Biol. 12 (2002) 211–215] that in rat pancreatic acinar cells, Gd 3+-sensitive Ca 2+ entry is instrumental in governing which second messenger pathways control secretory activity. However, in those studies, we were unable to demonstrate a significant increase in cytoplasmic [Ca 2+] during agonist application as a result of this entry pathway. In the present study, we combined pharmacology with ratiometric imaging of fura-2 fluorescence to resolve this issue. We found that 2 μM Gd 3+ significantly inhibits store-mediated Ca 2+ entry. Furthermore, both the protonophore, CCCP (5 μM) and the mitochondrial Ca 2+-uptake blocker, RU360 (10 μM), led to an enhancement of the plateau phase of the biphasic Ca 2+ response induced by acetylcholine (1 μM). This enhancement was completely abolished by Gd 3+; and as has been previously shown for Gd 3+, RU360 led to a switch to a wortmannin-sensitive form of exocytosis. Using MitoTracker Red staining we found a close association of mitochondria with the lateral plasma membrane. We propose that in rat pancreatic acinar cells, capacitative Ca 2+ entry is targeted directly to mitochondria; and that as a result of Ca 2+ uptake, these mitochondria release “third” messengers which both enhance exocytosis and suppress phosphatidylinositol 3-kinase-dependent secretion.

Arachidonic acid in astrocytes blocks Ca 2+ oscillations by inhibiting store-operated Ca 2+ entry, and causes delayed Ca 2+ influx

ATP-elicited oscillations of the concentration of free intracellular Ca 2+ ([Ca 2+] i) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca 2+ store refilling. Short-term application of AA, but not ETYA, blocked Ca 2+ influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca 2+] i oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca 2+] i increase. This AA-induced [Ca 2+] i rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 μM Gd 3+, indicative for the influx of extracellular Ca 2+. Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca 2+] i, firstly, a rapid reduction of capacitative Ca 2+ entry thereby suppressing [Ca 2+] i oscillations, and secondly inducing a delayed activation of Ca 2+ entry, also sensitive to low Gd 3+ concentration.