Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 . Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons.Ca PM) and endoplasmatic reticulum (TRPV1ER) membranes 2+ to the cytosol. These pools are inducible with: i) exo-, or endovanilloid ligands via intracellular signaling by protein kinases Recently, we and several other groups noted that TRPV1, the vanilloid ligand gated member of the TRP  super family, localizes both in the plasma membrane  channels is elicited by calmodulin binding to the C-terminus. Further elevation of [Ca2+]i and saturation of calmodulin with Ca2+ inactivate the pore opening mechanism The Ca2+-feedback theory, anti- calmodulin agents, such as calmidazolium are expected to suspend the feedback on Ca2+-entry, and thus potentiate elevation of [Ca2+]i to toxic levels 2+ increase. To test this hypothesis and better understand channel function of TRPV1, we carried out experiments with tricyclic anti-psychotic calmodulin antagonist drugs and other selective inhibitors of calmodulin. Our new data, in lieu of expectations, suggest that bona fide calmodulin antagonists blocks and do not promote Ca2+-transport via TRPV1.Consistent with this Ca2+-channel. In fact, sequence comparisons of various TRP channels suggest that a short acidic amino acid stretch at the pore loop of TRPV1 resembles to “EF-hand” and “Excalibur” motifs, both identified previously in calmodulin and calmodulin-like proteins, respectively + nociceptor/pain neurons.To address paradoxical effects of anti- calmodulin agents, we hypothesized that these compounds might not enter the cell, but inhibit the pore opening of pain/TRPV1 channel, most likely directly at the cation filter site, located at the extra-cellular orifice of the Ca2+-calmodulin feedback theory, a set of anti-calmodulin agents acutely inhibited the vanilloid/capsaicin–induced Ca2+-uptake in TRPV1 expressing cells and neurons. Extracellularly added camstatin peptide, an antagonist of calmodulin, also blocked capsaicin–induced Ca2+-uptake in intact cells. Studies carried out at the cellular levels and in animal pain models were in concert with previously noted analgesic actions of calmodulin-antagonists determined in vivoConsistent with our hypothesis, but contrary to the Ca2+- calmodulin inhibitors on nociception, activity of various phenothiazines were studied at the molecular levels in TRPV1-NIH 3T3 cells, in which the pain signal was mimicked with capsaicin-induced 45Ca2+-uptake (−5 M extracellular Ca2+ in 96 well plate formats at room temperature (22°C) for 10 min with robotic liquid handling. Briefly, cells were co-incubated with capsaicin for vanilloid-induced opening of TRPV1. Various Ca2+- calmodulin inhibitors were co-incubated in progressively increasing concentration in the 2 µM capsaicin supplemented incubation medium. Increased Ca2+-uptake was expected, due to a severe damage of shut off valve mechanism caused by the presence of calmodulin antagonists. Contrary to this theory, detailed referring to other TRP channels in the introduction, we noted that the tested calmodulin inhibitors inhibited the capsaicin-induced Ca2+-uptake in TRPV1-NIH3T3 cells with varying potency: calmidazolium (IC50 = 7 µM)>trifluoperazine (IC50 = 9 µM)>chlorpromazine (IC50 = 70 µM)>W-7 (IC50∼200 µM)>fluphenazine (IC50∼250 µM), and W-13 (IC50>300 µM), and [Ca2+]i did not accumulate in these cells at even higher concentrations of calmodulin inhibitors. The same agents were also tested in the absence of capsaicin, where none of them induced Ca2+ influx, i.e., they did not activate the vanilloid receptor. For the sake of clarity these curves were omitted from 2+-uptake in the presence of 2 mM capsaicin at low concentrations. Similar cooperative effect was noted previously between [3H] resiniferatoxin and different antipsychotic and antidepressant drugs 2+]i signal induced by inflammatory pain agonists either exo-, or endogenous.To better understand potential effect of Ca+-uptake . Assays 2+-channels 2+-transport carried out in TRPV1-NIH 3T3 cells eliminated N-benzylimidazole and clotrimazole compounds form further studies, since both groups showed either only partial inhibition or less favorable IC50 than calmidazolium and other miconazoles (i.e. M1/M2 = 8 µM>M3∼75 µM) homologues (To better define the quantitative structure-activity relationship (qSAR), other miconazole compounds, analogs of the commercially available calmidazolium, were functionally assayed in cells expressing TRPV1. A small dedicated library of miconazoles, clotrimazole and N-benzylimidazole compounds, referred to as the M-set (see M1–M7 in mologues .max) of capsaicin-induced Ca2+-uptake, but not the ED50 of capsaicin (0.3 mM of capsaicin) was determined. With progressively increasing concentrations of calmidazolium the Vmax gradually decreased, which also showed a characteristic maxima instead of a plateau due to a potential interaction with capsaicin on TRPV1. Above a calmidazolium concentration of = 2.5 µM, Ca2+-transport was almost completely abolished    that may2+-uptake medium. Likewise conventional calmodulin inhibitors, camstatin inhibited capsaicin -induced Ca2+-transport (IC50 = 11 µM) within 10 min of the assay in TRPV1-NIH3T3 cells. Prompt inhibition of inducible Ca2+-uptake suggested a rapid interaction with a potential extracellular docking site(s) of TRPV1 (2+-uptake (data not shown). Experiments with peptides suggested that camstatin and other anti-calmodulin agents indeed recognize an extracellular domain of TRPV1.From these experiments we hypothesized that maybe a calmodulin-like motif of TRPV1 is recognized extracellularly, therefore, a calmodulin antagonist with poor membrane permeability can exert inhibition without entering the cell. To address extracellular targeting of TRPV1, camstatin, a recently identified, selective anti- calmodulin peptide was coincubated with capsaicin in the Caof TRPV1 . As a co2+-uptake experiments. Among these substances amitriptyline, a structure analogue of phenothiazines, was determined the best inhibitor (IC50∼60 µM), however, its effect was significantly less prominent than that of trifluoperazine (IC50<10 µM). Carbamazepine, with shorter side chain than that in amitriptyline was completely inactive in this assay, while gabapentine showed partial inhibition  rat DRGs. Along with calmidazolium other polycyclic compounds were included in the vanilloid-induced 45Ca2+-transport assays. SB 290157, a selective, high affinity, competitive antagonist of the anaphylatoxin C3a receptor, CGP-37157, a cell-permeable benzodiazepine inhibitor of mitochondrial Na+/Ca2+ exchanger, and flunarizine, a piperazine derivative blocker of T-type cardiac Ca2+ channels were included in assays in DRG cultures. All of them were far less potent than calmidazolium. Moreover, calmidazolium inhibited capsaicin-induced Ca2+-uptake in sensory neuron cultures with a higher potency than that was determined in TRPV1-NIH3T3 cells  (To test selectivity and efficacy in an ctively) . Althougctively) .in vivo efficacy of calmidazolium, experiments were carried out in a total of 22 nociceptive specific (NS) or wide dynamic range (WDR) spinal dorsal horn neurons in 16 lightly anesthetized rats. Effects of calmidazolium were studied on the heat evoked responses of dorsal horn neurons using noxious peripheral heat stimuli delivered in every 3 min in the cutaneous receptive field of the hind paw. After establishing stable baseline control responses, calmidazolium was iontophoresed (i.e. with 100 nA) from one of the barrels of the compound electrode. Heat-evoked responses of dorsal horn neurons were significantly reduced the responses to peripheral noxious heat to a mean of 34±23%  of pre- calmidazolium baseline control  has clinically been tried in adjuvant therapy, and noted effective in post-herpetic neuralgia and painful diabetic neuropathy 50 = 60 µM) not ranked among the most potent inhibitors of TRPV1 channel in our cell-based assay.Of important finding of these studies is that classical phenothiazines/antipsychotic drugs, all of them calmodulin inhibitors, also can serve as blocker of current in silico screening of drugs available now  may even more promising in this respect. Even when a patient is not clinically depressed, antipsychotic drugs and tricyclic antidepressants could be administered to fight with various forms of neuropathic pain. Posttraumatic sympathetic dystrophy, postmastectomy pain, postherpetic neuralgia, some forms of cancer pain, and other variants of neuropathic pain syndromes maybe addressed with novel adjuvant treatment protocols, planned on our cell-based TRPV1 assays, which may help to select among a number of empirically employed drugs (30+) and rationalize adjuvants' selection. Additional lead optimization may be enhanced with able now  and 11 oable now  and 11.st place. We showed in laminectomised rat models that calmidazolium inhibits pain response to noxious heat, moreover firing evoked by NMDA and kainic acid. In the spinal dorsal horn calmidazolium inhibits other channels than the heat and inflammatory pain channel and it is active on NMDA and kainic acid receptors that usually prolong the duration of pain state. Calmidazolium, a not yet registered drug candidate, has previously been administered intrathecally in rat pain models and noted indeed analgesic According to our results obtained in our TRPV1-NIH3T3/HaCaT and DRG cell-based functional assays, as well as, in laminectomised rat models, among clinically used pain adjuvant medications, either trifluoperazine (i.e. Terfluzine®) or chlorpromazine (i.e. Thorazine®) would be a better choice than amitriptyline. Amitriptyline and fluphenazine showed almost an order of magnitude less blocking efficacy among the registered drugs on TRPV1. Terfluzine® would be an adjuvant of choice in therapeutic protocols to potentiate either efficacy of opioids or NSAIDs, although, some other factors, for example inadvertent side effects, specificity to calmodulin and ADMETox characteristics can modify optimal selection. Calmidazolium, a miconazole type calmodulin antagonist was ranked to the 1in vivo observations also contradict to the Ca2+- calmodulin feedback theory of action of these agents. Animal studies, adjuvant therapy in the clinical practice and studies in this paper suggest that antipsychotic calmodulin antagonist drugs rather elicit analgesia instead of hyperalgesia and block Ca2+-entry in peripheral nociceptive neurons. calmodulin inhibitors showed non-competitive inhibitor kinetics with increasing dose of capsaicin. Our screening technology employs vanilloid/capsaicin-induced Ca2+-uptake assays in TRPV1 expressing cell lines, in the first round, then hits are further tested and validated in primary neuron cultures from rat embryonic DRG, enriched in TRPV1+ nociceptors.Calmodulin kinase II (CaMKII) mediated intracellular signaling has recently been recognized to contribute to inflammation, pain and hyperalgesia 2+-transport experiments were carried out in “nominally zero Ca2+” buffer (2×10−5 M extracellular Ca2+ ([Ca2+]o) that reveals agonist-induced Ca2+-curents originating from either outside sources or the ER, then further amplified by Ca2+ induced Ca2+ release mechanism, has recently noted to occur in TRPV1 immunopositive nociceptor neurons. By Ca2+ induced Ca2+ release both TRPV1PM and TRPV1ER pools are actively involved in pain signaling 2+ induced Ca2+ release is usually followed by a currently recognized mechanism, so called store operated Ca2+ entry, which replenish the ER store emptied by different Ca2+-channels, including the Ca2+/vanilloid-induced TRPV1ER. The usual function of store operated Ca2+ entry is to maintain Ca2+-homeostasis, around 1 mM in the ER, and [Ca2+]i = 10−7 M, both essential for pain signaling in primary afferents sensory neurons. Coupling between Ca2+ induced Ca2+ release and store operated Ca2+ entry is now a well accepted hypothesis, therefore, [Ca2+]o is an ideal test-bead for searching specific inhibitors affecting either one or both the TRPV1PM and TRPV1ER pools. Contrary to that noted with other TRP channels, calmodulin antagonists do not eliminate Ca2+-feedback. Even more, an initial observation with the camstatin peptide prompted us to hypothesise that antipsychotic inhibitors of calmodulin eliminate vanilloid-induced Ca2+-uptake due to binding to an extracellular domain of TRPV1. Likewise, calmidazolium may block noxious heat-, NMDA- and kainic acid -induced pain signaling by a mechanism that does not need internalization of the compound in laminectomised rat. Of importance, calmidazolium has explicit positive charge, and electrostatic interactions may be required for specific docking at the pore loop of TRPV1 .A number of the Caof TRPV1  and 11. +/Ca2+ channel. The 646DLEFTENYD acidic tetrad of TRPV1 is unique and more extended stretch than that occurs in other TRPs in the TRPV1 (DXDXDGXXDXXE) and “Excalibur” (DXDXDXXXXE), both of them Ca2+ binding consensus domains, identified previously in calmodulin and bacterial calmodulin-like proteins, respectively 4W2 peptide and ruthenium red. Both are well characterized inhibitors of capsaicin-induced Ca2+-current via TRPV1 channel. Our theory is that a short segment of the pore loop may mimic some feature of Ca2+-binding proteins where anti-calmodulin drugs dock and inhibit down-stream signaling  of human erythrocytes, which indicates that these compounds can distinguish among different cation channels 2+-channel, an alternative target not used by opiates and NSAIDs. Our data in this paper consistent with specific action of calmidazolium and other anti-calmodulin analogues on a short acidic domain of TRPV1, at the cation filter region not found in other TRP channels and Ca2+-binding polypeptides mimicking this region in silico screening might be exploited to design new generations of painkillers, thereby the novel but irreversible inflammatory pain neuron ablation technology via TRPV1.From selectivity point of view, calmidazolium, trifluoperazine, and W-7 have been determined inactive on intermediate CaCGP-37157, 7-Chloro - 5 - (2 - chlorophenyl) - 1,5 - dihydro - 4,1 – benzothiazepin - 2(3H) - one; KA, Kainate, ]-2-Carboxy-4--3-pyrrolidineacetic acid; M1, Miconazole 1; M2, Miconazole 2; M3, Miconazole 3; M4, Clotrimazole 4, M5, Clotrimazole 5; M6, Clotrimazole 6; M7, N-Benzyl Imidazole; SB 290157, N2-[acetyl]-L-arginine; W-7, N- 6 – aminohexyl – 5 – chloro - 1 naphtalenesulfonamide; W-13, N - (4 - Aminobutyl) – 5 - chloro -2- naphthalenesulfonamide.TRPV1-NIH3T3 mouse fibroblast line, ectopically expressing the rat TRPV1 was established previously. For validation of some data in human cells, a permanent TRPV1-HaCaT keratinocyte line was prepared by drug selection, similar to that described previously 2+-uptake studies primary afferents neurons cultured in 96 well plates (3×103 cells/well), coated with poly-D lysine/collagen, which yielded both axons and dendrites bearing neurons due to neuronal growth factor supplementation in the culture medium 45Ca-uptake assays were carried out in “nominally zero Ca2+” (i.e. 2×10−5 M extracellular Ca2+ ([Ca2+]o) in 25 mM Tris-HCl buffered Hanks' balanced salt solution (pH 7.5), supplemented with 10 µM CaCl2 and 0.8 mM MgCl2medium (HCM) Primary DRG cultures were prepared from E16 embryonic rats as described previously then plated in 15 cm Petri dishes for 4 h to remove the most adherent fibroblasts. For Ca4 cells/well) and primary cultures prepared from embryonic DRG, seeded in poly-D lysine/collagen coated 96 well plates. The ED100 for capsaicin-induction was determined to be ∼1 µM in TRPV1-NIH3T3 cells at pH 7.5. Therefore antagonist-screening assays were carried out with 2 µM capsaicin, an excess amount of agonist, which fully activates TRPV1 either in DRG neurons or in permanent cell lines ectopically expressing the vanilloid receptor, but does not cause Ca2+-cytotoxicity during the 10 min incubation period. To probe the TRPV1 specificity of capsaicin-induced 45Ca2+-uptake, experiments were carried out in the parental NIH 3T3 and HACAT cell lines and yielded negative results (data not shown). The previously established cell lines and standardized DRG cultures were extensively characterized for functional cell-based TRPV1 studies 45Ca2+-uptake was performed for 10 min at 20°C in HCM using 0.1 µCi 45Ca2+ as radioactive tracer in a 100 µl final volume. To stop 45Ca2+-uptake, cells were rapidly changed back into 0.2 ml HCM, washed 4 additional times with 0.2 ml HCM, and then lysed in 80 µl/well RIPA buffer  for 30 min Vanilloid agonist inducible calcium transport was assayed in adherent cell lines ectopically expressing the C-terminally ε-tagged rat TRPV1  male Wistar rats weighing between 350–450 g. The head of the animal was mounted in a stereotaxic frame. The lumbar enlargement (L3–L5 segments) was exposed by a laminectomy and the spinal cord was covered with a pool of warmed mineral oil to prevent drying. Rectal temperature as well as the temperature of the mineral oil were monitored with temperature probes and kept at 37°C by a warm water-heated blanket beneath the rat and an infrared heat lamp from above. The level of anesthesia was maintained so that the rats showed no sign of discomfort but the tail flick reflex could be evoked by application of noxious heat (43–45°C) or mechanical stimulus (squeeze) to the tail. Recordings were only commenced at least 1 h after surgery. All animal experiments were performed in accordance with institutional animal welfare guidelines.Extracellular single-neuron recordings were made in chloral hydrate-anesthetized  The recording barrel contained a low impedance (1 MΩ) 7-µm carbon fiber and drugs were delivered from the surrounding barrels. Single unit extracellular recordings were made from selected dorsal horn neurons characterized by their responses to both innocuous  and noxious  stimuli applied to the excitatory receptive fields of the hind paw. Mechanical stimuli of increasing strength were delivered by a small brush and three serrated clamps with a graduated force. The experimenter released clamps so that force was standard for each mechanical stimulus delivered over a period of 3 s, and about 1 min were allowed to pass between trials. Neurons were characterized as low threshold (LT), nociceptive specific (NS) or wide dynamic range (WDR) by their responses to mechanical stimuli of increasing strength. Extracellular signals from dorsal horn neurons were amplified and filtered (300 Hz to 8 KHz) using an ExAmp-20K equipment and preamplifier (Kation Scientific). Neuronal activity was displayed on an oscilloscope and through an audio analyzer and detected with a WD-2 window discriminator . The number of action potentials per second was counted by the computer and the resulting peristimulus time histograms were displayed. Iontophoretic drug delivery and collection of experimental data were performed by a multifunction data acquisition board  placed in a computer and programmed in LabVIEW 7 . Delivery of drugs by microiontophoresis was performed using Union-36 constant current source units (Kation Scientific). Drug barrels of the combined electrodes contained one of the following freshly made solutions: 100 mM N-methyl-D-aspartate Na (NMDA) in 100 mM NaCl (pH 8.0), 20 mM kainic acid in 100 mM NaCl (pH 8.0), 5 mM calmidazolium chloride in 100 mM NaCl (pH 7.2) was prepared using a 100 mM calmidazolium in ethanol stock solution. NMDA and kainic acid were ejected with negative iontophoretic currents ranging from 10 to 100 nA for 5 s in every 2 min. calmidazolium was ejected with positive currents at 100 nA for 120 s. Retaining currents of opposite directions between 3–10 nA were used for all drugs.Single unit extracellular recordings were made from selected dorsal horn neurons responding to noxious heat delivered by a projector lamp focused on the blackened surface of the hind paw. Skin temperature was monitored using an infrared thermometer. Temperature ramps were generated from a holding temperature of 30°C to a peak of 50°C at a rate of about 2°C/s. The heat stimulus was turned off when skin temperature reached 50°C.n) of observations are given throughout. A P value of <0.05 was considered significant in all cases.Statistical evaluations were made using the total number of spikes minus background activity evoked during each epoch of excitation by heat stimuli or iontophoretic application of an excitatory compound. The background neuronal discharge was calculated by averaging a 15 s period of ongoing activity preceding each epoch of excitation and this value was subtracted from the total number of evoked spikes. Differences in magnitude between different response epochs of a single cell were confirmed by one-factor analysis of variance  by comparing the total number of spikes per excitation period. To make data from different experiments more comparable, analysis of pooled data was done after normalizing the baseline stimulus-evoked response to 100%. Means±SD of a number (+ channel KcsA The preliminary channel domain model was used in a fixed protein flexible ligand docking protocol using simulated annealing as it is implemented in the MOE 2004.03 program package from Chemical Computing Group. The homotetramer of the TM5-pore loop-TM6 domain of TRPV1 was built using the Swiss-PdbViewer molecular graphics and the SWISS-MODEL comparative homology modeling program package


Estrogen may protect against glutamate-induced cell death by reducing the excitotoxic Ca2+ influx associated with glutamate excitotoxicity. In this study, the inhibition of N-methyl-D-aspartate (NMDA) receptor and nitric oxide synthase (NOS) along with the effect of 17β-estradiol (17β-E2) and a more potent antioxidant Δ8, 17β-estradiol  on cell viability and intracellular Ca2+ ([Ca2+]i), following treatment of rat cortical cells with glutamate, was investigated.High concentrations of glutamate can accumulate in the brain and may be involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. This form of neurotoxicity involves changes in the regulation of cellular calcium  significantly reduced cell death. 17β-E2 was more potent than Δ8, 17β-E2. Glutamate caused a rapid 2.5 fold increase in [Ca2+]i. Treatment with 0.001 to 10 μM MK-801 reduced the initial Ca2+ influx by 14–41% and increased cell viability significantly. Pretreatment with 17β-E2 and Δ8, 17β-E2 had no effect on Ca2+ influx but protected the cortical cells against glutamate-induced cell death.Primary rat cortical cells were cultured for 7–12 days in Neurobasal medium containing B27 supplements. Addition of glutamate (200 μM) decreased cell viability to 51.3 ± 0.7% compared to control. Treatment with the noncompetitive NMDAR antagonist, MK-801, and the NOS inhibitor, L-NAME, completely prevented cell death. Pretreatment (24 hrs) with 17β-E-. Equine estrogens: 17β-E2 and Δ8, 17β-E2, significantly protected cortical cells against glutamate-induced excitotoxicity by a mechanism that appears to be independent of Ca2+ influx. To our knowledge, this is a first such observation. Whether the decrease in NOS related products such as ONOO-, is a mechanism by which estrogens protect against glutamate toxicity, remains to be investigated. Estrogen replacement therapy in healthy and young postmenopausal women may protect against neurodegenerative diseases by these mechanisms.Glutamate-induced cell death in cortical cultures can occur through NMDAR and NOS-linked mechanisms by increasing nitric oxide and ONOO Neuronal increases in Ca2+ can activate a number of enzymes, such as phospholipases, proteases, endonucleases and nitric oxide synthase (NOS). Increase in the activity of these enzymes is associated with neuronal cell death [2+]i required for neuronal NOS (nNOS) activation and nitric oxide (NO) production within the neuron and this can result in increased cell death i, following treatment of rat cortical cells with glutamate was studied. The concentrations of glutamate used in the present study are relatively lower than those used in previous studies where cell death may have occurred mainly via the oxidative pathway. It is hypothesized that both estrogens will protect cortical cells from glutamate induced excitotoxicity by modifying the initial Ca2+ influx in addition to acting as antioxidants.In this study, the effect of 17β-estradiol  ,22 on ceFreshly isolated embryonic cortical cells obtained from day 17–19 pregnant rats were cultured and maintained in serum-free neurobasal medium containing B27 supplements for 7 days as described under "Materials and Methods". Phase contrast microscopy indicated that the cells had characteristic morphology of neurons and their cellular extensions (dendrites) were clearly visible Figure . Immunoc4) cultured for 7 days in 96-well plates were treated with increasing concentrations (0.1 to1000 μM) of glutamate for 20 min and cell viability was measured after 24 hrs and compared with vehicle-treated samples. As depicted in Figure Cortical neuronal cells , 200 μM glutamate exposure resulted in a 50% decrease in cell viability. As depicted in Figure 2 or Δ8, 17β-E2 for 24 hrs resulted in a dose-dependent increase in cell viability against glutamate-induced neurotoxicity. No significant protection was observed with 0.01 to 0.05 μM 17β-E2, however, 0.1 to 5 μM 17β-E2 resulted in a 8.16 ± 1.93% to 20.94 ± 2.13% increase in protection (p < 0.05) respectively. Similarly, pretreatment with 0.01 to 0.5 μM Δ8, 17β-E2 resulted in an increase in protection from 0.63 ± 1.74% to 6.85 ± 2.09% respectively, however, statistical significance at these concentrations was not reached. In contrast, pretreatment with 1 and 5 μM Δ8, 17β-E2 resulted in a significant (p < 0.05) increase in protection of 9.24 ± 2.23% and 12.12 ± 2.11% respectively , were added to wells and changes in relative fluorescence units (RFU) were recorded. Baseline readings were taken every 15 seconds for a total of 60 seconds prior to an injection (10 μl) of glutamate into each well  tested. This initial rapid rise in [Ca2+]i occurs at 15 seconds after glutamate addition and is followed by a reduced rate of Ca2+ influx. The kinetics of glutamate-induced Ca2+ influx following the initial rise in [Ca2+]i remain relatively stable, but are elevated at glutamate concentration between 0.1 and 1 μM. However, at higher glutamate concentration (5 to 200 μM), [Ca2+]i continues to increase following the initial rise in [Ca2+]i. In addition, glutamate treatment results in a dose dependent increase in the initial rise in [Ca2+]i levels measured at 15 seconds, and reaches significance (p < 0.05) with 0.5 μM to 200 μM glutamate  injection and percent of maximum initial Ca2+ influx was calculated for each concentration of MK-801 (0.001 to 10 μM). As can be seen in Figure 2+ influx by 14.17 ± 2.56% to 40.30 ± 2.28% respectively. Thus, between 0 μM and 10 μM MK-801, the concentration of [Ca2+]i drops from an initial high level of 350 nM  at 15 seconds to 200 nM (glutamate +10 μM MK-801). This drop in Ca2+ influx was associated with a dose-dependent increase in cell viability compared to glutamate alone  of 17β-E2 and Δ8, 17β-E2. Representative traces for the effect of 5 μM 17β-E2 and Δ8, 17β-E2 on the glutamate-induced Ca2+ influx are presented in Figure 2+]i measurements were made every 15 seconds for a total of 60 seconds and measurements were continued every 15 seconds following glutamate addition  of estrogen. These results indicate that protection against glutamate-induced cell death in rat primary cortical neuronal cells by estrogen does not involve a reduction in the glutamate-induced, NMDAR associated, Ca2+ influx, and thus estrogen mediated protection appears to be mediated by a Ca2+ independent mechanism.The effect of estrogen on acute, glutamate (200 μM) induced CaM Figure  and 9B.  Figures  and 9B. In this study, we demonstrate that in primary cultures of pure rat embryonic cortical neurons, acute (20 min) treatment with glutamate results in a dose and time-dependent cell death. Over 50% of cells die at a glutamate concentration of 50 μM. Higher concentrations (> 50 μM) of glutamate did not result in any further increase in cell death. These findings are in keeping with previous observations with cultures of purified embryonic rat spinal cord motor neurons where NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the neurons . In contAlthough the primary cortical neurons used in our study are sensitive to small changes in glutamate concentrations 0.1 to 50 μM), the extent of neuronal death under our experimental conditions (50%) is lower than that reported in a number of studies where mixed cell cultures containing neuronal, glial and other cell types were used. In these mixed cultures over 85% of motor neurons died following treatment with glutamate ,30. It h to 50 μM2+ that occurs after treatment with glutamate or agonists i occurs that is associated with a dose-dependent decrease in cell viability. However, stimulation with glutamate above 10 μM did not further increase [Ca2+]i again suggesting that glutamate insensitive and sensitive cells may have been present in these primary cortical cultures.As we have shown, glutamate-induced excitotoxicity is dependent on NMDAR activity and therefore, changes in Call death . Indeed,2+ influx decreased significantly by MK-801 suggests that this to some extent occurs via NMDAR. Increasing concentrations of MK-801 resulted in a gradual lowering of the [Ca2+]i concentration within 15 seconds after glutamate treatment. Thus MK-801 (10 μM) reduced the [Ca2+]i from 350 nM to 200 nM in 15 seconds  and significant cell death of cortical/glial cell cultures have been previously observed i levels that activate NOS and increase the levels of NO and subsequently ONOO-free radicals. Equine estrogens, 17β-E2 and Δ8, 17β-E2, significantly protect against glutamate-induced excitotoxicity through a mechanism that is independent of Ca2+. This is to our knowledge a first such observation. Whether a decrease in NOS related products, such as ONOO-, may be one mechanism by which estrogen provides neuroprotection against glutamate toxicity, remain to be investigated.Glutamate-induced cell death in cortical cultures occurs through NMDAR and NOS-linked mechanisms, presumably through elevated i were recorded. The cells were then washed with modified Locke's buffer and NBC AO- was added and cells were incubated at 37°C, 5% CO2 for 24 hours, following which cell death/viability was measured. Measurement for Ca2+ and cell death/viability were performed on the same culture of cells.For determination of the effect of glutamate on the viability of rat primary cortical cells, the cells were washed twice with a modified Locke's buffer  treated samples were made.2. Estrogens were dissolved in ethanol and diluted to working concentrations with NBC AO- medium. The final concentration of ethanol was 0.2% for control and estrogen treated samples. Following incubation with estrogen and prior to glutamate exposure cells were prepared for [Ca2+]i measurements as outlined below. The cells were washed and incubated in Locke's buffer containing sufficient (200 μM) glutamate (pre-determined) to give 50% cell death. Incubations were carried out at 37°C for 20 minutes and changes in [Ca2+]i were recorded. Following glutamate treatment cells were washed with fresh Locke's buffer and NBC AO- was added. The cells were again incubated for 24 hours at 37°C and 5% CO2, following which cell viability was determined.For the determination of the effects of estrogen on glutamate induced changes, cells were washed twice with modified Locke's buffer and then fresh NBC AO- and estrogens were added to final concentrations of 0.01, 0.05, 0.1, 0.5, 1 and 10 μM and the cells were incubated for 24 hours at 37°C and 5% CO2+]i and cell viability were measured.In some experiments various concentrations of MK-801, an NMDAR antagonist, and 10 mM of L-NAME , a NOS i® AQueous Non-Radioactive cell proliferation assay (MTS method) (Promega G5430) were used to assess cell death and viability respectively. The LDH assay measures the amount of lactate dehydrogenase that is released from dead cells while the MTS method measures the formation of a formazan product in live cells. Changes in optical density (O.D.) at 490 nm were measured using the SpectraMax 340 microplate reader and SOFTmax® PRO software  and comparisons of treated to control values were made. Both of these methods provide a reliable measure of cell death/viability.The Lactate dehydrogenase (LDH) cytotoxicity assay  and the CellTiter 962+]i (nM) was calculated by multiplying the ratio of ΔF-Fmin and Fmax-ΔF by Kd. Where ΔF is the change in RFU, Fmax is the maximum observed RFU following NP40 treatment, Fmin is the minimum observed RFU following EGTA Ca2+ chelation and Kd is the dissociation constant for Fluo-3 AM, 390 nM, provided by supplier (Molecular Probes).Intracellular Calcium levels were assessed using the fluorescent probe Fluo-3 AM . Changes in relative fluorescence units (RFU) were monitored with the Fluoroskan Ascent FL fluorescent plate reader equipped with a micro-injection syringe pump . The protocol for intracellular dye loading was based on the method provided by the manufacturer (Molecular Probes and Labsystems Fluo-3 AM Application Note) with minor modifications. Briefly, cells were loaded with 5 μM Fluo-3 AM for 2 hours and then washed three times with Locke's buffer to remove excess dye. Cell plates were then placed in the Fluoroskan and incubated for an additional 15 minutes at 37°C to allow for complete de-esterification of dye. Measurements of relative fluorescence units (RFU) were made using an excitation and emission wavelength of 485 and 527 nm respectively. Change in [Ca2+]i were developed using Slide Write Plus V6 Software  using the non-linear curve fitting functions. Each estrogen experiment was carried out a minimum of six times with eight replicates per treatment condition and all values shown in figures are the mean ± standard error of the mean (SEM). All treatment conditions were compared to the respective controls and an effect was determined significant if p < 0.05. All statistical analyses were performed using Instat® Software . To establish the effect of estrogen against glutamate induced cell death and [Ca2+]i, nonparametric statistical analyses were performed using the Kruskal-Wallis nonparametric analysis of variance test with the Dunn post hoc test to compare each estrogen concentration against the glutamate alone control.The dose-response curves for effects of glutamate and estrogen on cell viability and [CaJP is a graduate student who participated in the development of the hypothesis, study design and carried out most of the experimental work and preparation of the manuscript.BB conceived the study and participated in the development of the hypothesis, the study design, and overall direction of the study and preparation of the manuscript.Both authors have read and approved the final preparation of the manuscript and its submission to BMC Neuroscience.


Both steady state and [Ca2+]i transients changed with depth of penetration. When the CASM tip was within 20 μm of the far side of the cell the [Ca2+]i transient time to peak was 4.4 ± 0.5 s, rising to 14.7 ± 0.7 s at a distance of 80 μm. We estimate that the Ca2+ transients travelled centripetally at an average speed of 6 μm2 s−1 and decreased in size by half over a distance of about 45 μm. Cyclopiazonic acid had little effect on the size and time to peak of Ca2+ transients but slowed their recovery significantly. This suggests that the endoplasmic reticulum curtails rather than reinforces the transients. Injecting the calcium buffer BAPTA made the Ca2+ transients more uniform in size and increased their times to peak and rates of recovery near the membrane. We have developed a computational model for the transients, which includes diffusion, uptake and Ca2+ extrusion. Good fits were obtained with a rather large apparent diffusion coefficient of about 90 ± 20 μm2 s−1.This may assist fast recovery by extrusion.We have used quartz Ca The spatiotemporal characteristics of this rise in calcium are defined by the interactions between Ca2+ ions and the cytoplasmic milieu. Calcium-induced calcium release (CICR) processes via either IP3 or ryanodine receptors can facilitate the regenerative spread of the calcium signal throughout the cell, whilst immobile calcium buffers will slow and attenuate the signal. If the increase in sub-membrane [Ca2+]i is to act as a signal at a distance, the speed at which the Ca2+ ions travel from the cell membrane to their target depends critically on the rate of diffusion of Ca2+ ions in the cytoplasm. Most studies of calcium homeostasis and signalling in nerve and muscle cells have been made using optical methods to measure [Ca2+]i via the binding of Ca2+ ions by fluorescent or other indicators 2+ buffering and mobility 2+ transients, but very few have produced data on intracellular Ca2+ movement from the periphery towards the centre inside real neurones in the absence of such alien additives. We now present such data, obtained with Ca2+-sensitive microelectrodes (CASMs), for the first time.Changes in free intracellular calcium  to move the CASM up and down in reproducible steps. Rotating the Z-control wheel by 360° moved the CASM up or down by 40 μm, thus for a 20 μm step we rotated it manually by 180°.All experiments were done on large (150–250 μm diameter) snail neurones in isolated sub-oesophageal ganglia 2.2Helix aspersa, was killed humanely by rapid removal of the circumoesophageal ring of ganglia. These ganglia were mounted dorsal side uppermost on a plastic bath insert. The thick connective tissue covering the back of the visceral and right and left pallial ganglia was then removed and the bath insert with ganglia was mounted in the experimental chamber at an angle of 50° to the vertical. Once covered with snail Ringer  the inner connective tissue was torn with a fine tungsten hook to expose some of the neurones. All experiments were carried out at room temperature, 18–22 °C, starting at least an hour after the dissection.An aestivating snail, 2.32, 5 MgCl2, 20 HEPES, titrated with NaOH to pH 7.5. Nominally Ca-free Ringers had the same composition but with no CaCl2 and 12 mM MgCl2. The EGTA Ca-free solution had an additional 1 mM EGTA. The CO2 Ringer was the same as normal Ringer except that it had 20 mM NaHCO3 instead of HEPES, was bubbled with 2.5% CO2 in air and contained 0.1 mM NaH2PO4. The high K low Ca solution for BAPTA iontophoresis calibration contained 100 KCl, 5 NaCl, 0.6 MgCl2, and 10 mM HEPES. The pH was adjusted to 7.2 with KOH. Cyclopiazonic acid (CPA) was kept frozen as a 50 mM stock solution in dimethyl sulphoxide and diluted in snail Ringer on the day of use. All these chemicals were from Sigma.The normal snail Ringer contained (mM): 80 NaCl, 4 KCl, 7 CaCl2.4Conventional micropipettes were pulled from 1.2 mm filamented borosilicate glass tubing and backfilled with either 2 M CsCl (for passing current), or 2 M KCl . Tips were broken if necessary by touching a pin in the bath to give resistances between 5 and 15 MΩ. BAPTA was iontophoresed from similar micropipettes filled with 100 mM BAPTA . CASMs were made by a method refined from those described previously 2  using a 10 ml syringe connected to the back of each micropipette via a silicone rubber tube to force air out of the tips. We then sucked a column (150–250 μm) of sensor cocktail into the tip of each micropipette, taking care to avoid any air gap between cocktail and backfill. The cocktail composition was (mg/g): 12 Ca ionophore ETH 129 (Fluka 21193), 6 Na tetrakisborate, 200 2-nitrophenyloctylether, 17 high molecular weight PVC, and 765 tetrahydrofuran . Filled microelectrodes were left in air to allow the tetrahydrofuran to evaporate for at least 2 h before use, and had useful lives of 2–3 days. During the experiment we applied 80–120 kPa of air pressure to the back of the CASM, since this maximizes its response VCa  and we have not converted them either to pCa or nM free [Ca2+]i except for modelling purposes. Calibration of CASMs is difficult because, in our hands, their responses tend to change when they are withdrawn from a cell at the end of an experiment. Previous work Individual CASMs were prepared as follows on the day of the experiment. First we backfilled 2 or 3 silanized micropipettes with 1 mM CaCl2.5VCa of 1 ml of the high K, low Ca solution from −50 to −110 mV . The BAPTA was injected into the 1 ml aliquots using an oocyte microinjector. The average quantity of BAPTA needed was 75 nmol. We then made droplets (volumes 15–25 nl) of the same solution under oil, measured VCa as with a snail neurone, and injected BAPTA iontophoretically. We found that to achieve the equivalent VCa change as in the 1 ml samples we had to inject an average 0.6 μC nl−1, giving a transport number of 0.01.We first used CASMs to measure how much BAPTA was required to change the 2.6Vm microelectrode potential as reference. Figures were prepared from the CED data after loading into Microsoft EXCEL. Large spikes in the VCa records generated by electronic pickup were erased, and the clamp current records were restricted in range. For more details of most of the apparatus see n observations. The statistical significance of observed shifts in means was determined by a one-tailed Wilcoxon rank sum test. The difference between means was considered significant when the p-value was higher than 0.95. For baseline adjustment, selecting transients, time to peak and exponential fitting, a home made Matlab script with a graphical user interface was used.Potentials from the microelectrodes were led via Ag/AgCl wires to preamplifiers in the cage. Outputs from these and the clamp amplifier were filtered (8-pole Bessel) and recorded at 20 Hz on a PC via a CED micro 1401 interface and Spike 2 data collection program . The CASM potential was differentially recorded with the 2.72+-signalling in a wide variety of cell types, for example 2+-signalling in snail neurones. We used the model to validate and explain the results obtained from the experiments. Furthermore, we used the model to estimate the mobility of Ca2+ in these large neuronal cell bodies, which have not been exposed to mobile Ca2+-indicators.There is an extensive body of literature on the modelling of Ca2+ diffusion, influx, extrusion, sequestration and buffering. We assume that influx and extrusion mechanisms at the plasma membrane are uniformly distributed over the cell's surface. The CASM and the axon attached to the soma only represent a small fraction of the cell's volume (<1%) and surface area (<5%), furthermore transients were measured near the far side of the cell, opposite to the site of penetration. Since we could not detect any major deformation of the cell looking through the microscope we assume that the Ca2+ transients measured close to the membrane can be modelled with spherical symmetry. Using these assumptions we can reduce the three-dimensional diffusion problem to a one-dimensional radial diffusion equation in a sphere. Similar computational models for Ca2+ diffusion in a spherical cell can be found elsewhere The model includes the following dynamic processes: Ca2+ after a brief influx is relatively slow, with time constants ranging from 5 to 15 s, while Ca2+ buffering occurs on the micro- and millisecond time scale. We therefore can separate these different processes kinetically and remove the explicit buffering terms from the transport equation 2+ diffusion coefficient DCa (μm2 s−1) with an apparent diffusion coefficient Dapp:The removal of Caκe andκb denote the endogenous and exogenous Ca2+ binding ratio, respectively; the associated diffusion coefficients are De and Db. The diffusion coefficient of free cytoplasmic Ca2+ is denoted by DCa and was assumed to be 220 μm2 s−12+ binding ratio κtot = 1 + κe + κb.Here we used the notation from 2+ binding ratio κtot depends on the Ca2+ concentration; however, when the concentration is much lower than the dissociation constant it may be approximated to be constant. Moreover, although some data are available on buffering in snail neurones The total CaThe general transport equation that describes the calcium dynamics in the cell, taking into account the different aforementioned considerations then becomes:jin,app and jout,app describe the apparent influx of calcium through channels and apparent efflux of calcium by the pump, respectively, Jseq,app denotes the apparent calcium flux associated with sequestration to stores and mitochondria in the cell's cytoplasm and finally jbasal,app represents the basal influx of Ca2+ at rest. The surface-to-volume ratio rsv (m−1) is necessary to convert the flux density j (mol m−2 s−1) into a concentration flux J (mM s−1). The concentration [Ca2+]i and all fluxes are functions of position r (m) and time t (s); however, for readability we do not write this explicitly.The first term on the right hand side represents the radial diffusional process that is driven by the concentration gradient in the cell. The transmembrane fluxes 2+-current denoted by ICa,app (A) relates to the real current as follows: ICa,app = ICa/κtot. S denotes the surface area (m2) of the cell and F is Faraday's constant. The extrusion of Ca2+ by the pump is modelled by the following linear equation  and relates to the real permeability as follows: Pout,app = Pout/κtot. The flux associated with sequestration was also assumed to be linear:kseq,app denotes the apparent sequestration rate constant (s−1) and relates to the real rate constant as follows: kseq,out = kseq/κtot·[Ca2+]s is the steady state Ca2+ concentration. This approximate model is also known as a pool model jbasal,app was directly calculated from the steady state Ca2+ concentration and the extrusion parameter: jbasal,app = Pout,app [Ca2+]s.There are few precise quantitative data available about the different transport processes involved. To prevent the introduction of many unknown parameters we therefore kept the mechanistic complexity to a bare minimum. Consequently we modelled the influx as a square pulse with the same duration as the depolarisation:2.82+ concentration after short depolarisations. If we assume equal activity coefficients in the Ringer and cytoplasm, the steady state cytoplasmic Ca2+ concentration can be directly estimated from the Nernst equation applied to the CASM potential:ECa (V), R is the gas constant and the temperature T was 293 K. The backfill Ca2+ concentration [Ca2+]CASM in the microelectrode was 1.0 mM. The reference potential E0 was manually adjusted prior to the experiment such that the measured potential was 0 mV in Ringer solution; the Ringer solution contained 7 mM Ca2+.In this study we used CASMs to measure the CaVCa to follow the Nernst equation over the whole range it could be used directly to estimate the cytoplasmic Ca2+ concentration. VCa is indeed close to the Nernstian value at [Ca2+]i over 100 nM VCa:τCASM (s). We assume that it is constant and depends only weakly on the microelectrode potential and [Ca2+]i.Were the resistance and capacitance of the CASMs to be infinitely small and 2.9Assuming a linear transport equation a rather complex analytical solution consisting of an infinite series of exponentials with different time constants can be obtained using standard methods An and the time constants τn are:The amplitudes αn are the positive roots of the following equation:τD = d2/(4D) and the extrusion time constant τp = d/(2P).The constants d, the extrusion rate constant and the diffusion coefficient in a complex way. However, when diffusion is much faster than extrusion (extrusion limited) the time constant is simply τ0 = d/(6Pout) and vica versa when diffusion is much slower than extrusion (diffusion limited) the time constant is simply τ0 = d2/(4πDapp).The slowest time constant determines the overall recovery time. When there is no sequestration this time constant depends on the cell diameter rsv in Eq. S and the volume of the first compartment at the membrane. Both solutions were calculated using a Matlab 6.5 mex-routine written in C++.To compare the model with the results obtained from the experiments, we solved the transport equation simultaneously with the differential equation for the microelectrode potential. Although the analytical solution is very useful for theoretical analysis and testing, it requires many terms to obtain an accurate solution and does not allow for non-linearity. Therefore, we used a numerical approach for solving both equations, allowing more flexibility. Time integration was done using the forward Euler scheme and diffusion was calculated using a finite volume scheme  of the transient measured closest to the membrane was not known exactly. We therefore allowed the depth as an adjustable parameter within the limits of ±10 μm. The transients measured closer to the middle were therefore more reliable. Furthermore, the response time of the electrode was estimated to be about 1.5–2.5 s and was allowed to vary within these limits. The remaining four unknown parameters in the transport equation, together with the response time, were obtained from an optimal fit. A simplex search algorithm was used to find this least square fit. To increase reliability of the parameter estimates, several transients measured at different depths were fitted simultaneously. Also, any slow baseline drift was removed and the first two seconds of the transients, that show an electrical artefact, were not used during the fitting process.In a typical experiment several Ca33.1Vm) and the change in VCa . In this representative example the CASM was moved down in four steps before its potential changed from zero to about −50 mV. During a further six steps downward the CASM potential gradually became more negative, eventually more negative than −150 mV. By this time the electrode tip was 200 μm from the top of the cell, which had a diameter of about 240 μm. We then inserted the membrane potential microelectrode  and clamp current microelectrode, using manually-operated micromanipulators. To test that the CASM and Vm electrodes were recording the same change in Vm, we then hyperpolarised the cell by 30 mV for 20 s. There were only small transient changes in VCa, generated by electrical pickup and imperfect subtraction of rapid changes. We then  As the CASM tip was moved down the transient increases in VCa after each depolarisation became larger, and the steady-state VCa after each recovery became more negative. After the sixth step VCa had fallen to −127 mV, although after the next two it had risen slightly to −126 mV. After the last step down VCa rose to −118 mV before any depolarisation, suggesting the CASM had gone too far; although there was no change in the clamp current. We therefore then withdrew the CASM in several steps—two each of 10 and 20 μm. After the first 20 μm step up, the VCa paradoxically decreased, as if the upward movement somehow allowed a better seal. Such paradoxical improvements on withdrawal were often seen in the early part of an experiment.As shown in detail in VCa responses to the first nine depolarisations of 2+ entered the cytoplasm only at the cell membrane. Once Vm is returned to −50 mV, entry ceased and the Ca2+ both diffused into the cell interior and was pumped out by the plasma membrane Ca ATPase, or PMCA. In some experiments we were able to make several different sequences of downward and upward movements in the same cell to a depth at which, as shown in 2+]i rose rapidly. This experiment was done on a cell of diameter about 140 μm, and the depths at which the apparent [Ca2+]i began to increase were, respectively, 140, 150 and 140 μm. In other words, it appears that when the CASM tip reached the far side of the cell it suffered a loss of sensitivity or caused a local leak. We could see no change in the clamp current associated with the sudden increases in VCa. In many other cells, once the CASM created a sudden rise in [Ca2+]i it took many minutes to recover, if at all.2+]i as the CASM was advanced deeper and deeper into a cell is that the electrode insertion induced a leak at the site of penetration. As the CASM was pushed in, its tip moved further away from the leak, which itself may be reduced by better sealing. During long experiments, as shown below, CASM movement had less dramatic effects on steady-state levels, suggesting that the leak declined with time.The simplest explanation of the decreases in apparent i. We have tested the effect of a possible leak by exposing cells to either nominally Ca-free solutions or the same with added EGTA. Measured with a CASM, the [Ca2+] of the nominal solution was about 20 μM, that of the EGTA Ca-free Ringer less than 10 nM. If calcium ions, driven by the large electrochemical gradient, normally leak into the top of the cell around the tips of the microelectrodes, reducing external calcium should reduce the apparent i, the Ca2+ transients were little changed (not shown). By 20 min after changing the superfusate to CO2 the Na-dependent chloride/bicarbonate exchanger i to normal, so we then again moved the CASM down in steps. This time the VCa baseline did not change, and the transient times to peak were very similar to those recorded in HEPES. In a total of four similar experiments, the average time to peak at distances of 0–20 μm was 4.20 ± 0.45 s in HEPES, and 4.35 ± 0.68 s in CO2-bicarbonate.We have done most of our experiments in HEPES-buffered Ringer, although it is known that CO3.4VCa gradients described above, and the higher steady-state apparent [Ca2+]i than previously reported in cells which had added buffer, were partly due to mechanically-induced leakage from the endoplasmic reticulum calcium stores. Having penetrated the cell membrane the CASM must have been moved down through the endoplasmic reticulum and might have caused significant injury. We have therefore tested the effects of the SERCA pump inhibitor cyclopiazonic acid (CPA). It is well-established that CPA empties the Ca2+stores over a period of several minutes by blocking their uptake mechanism VCa or the near-membrane VCa changes with CASM movement. The times-to-peak are trending to be later in CPA treated cells, although this was not very pronounced close to the membrane and statistically not significant ). For distances of 20, 40 and 60 μm from the membrane the times to peak were significantly longer , 0.952(5) and 0.952(5)). The recovery time after the Ca2+ transients was clearly much longer in CPA treated cells , 0.984(5), 0.972(5) and 0.996(5)), as shown in 2+]i) in CPA treated cells. Although this percentage was statistically not significant , 0.845(5), 0.79(5) and 0.726(5)), it may suggest that a small fraction of the increase in [Ca2+]i is caused by release of Ca2+.It seems possible that the 3.52+ signalling in a variety of cells; for example in nerve terminals 2+ signalling. We have therefore examined the effects of BAPTA, injected iontophoretically, on both the steady-state VCa and the Ca2+ transients induced by depolarisations. VCa transients at several different CASM depths. At the closest approach to the membrane the time to peak was 2.9 s and VCa reached a peak of −107 mV. The first two BAPTA injections loaded the cell to give an estimated BAPTA concentration of 9 μM. At the closest approach to the membrane the VCa transient then had a time to peak of 6.6 s. After two more BAPTA injections giving an estimated final concentration of 135 μm, the transient time to peak at the closest point was again slower, at 9.1 s. In three of the experiments we were able to measure times to peak and the size of the VCa transients at three different depths before and after injecting BAPTA to a final concentration of 100–150 μm. The collected data are plotted in 2+ transients more uniform throughout the cell, both in times to peak and overall sizes.Exogenous calcium buffers have been widely shown to influence CaVCa, the second two lowered VCa from −128 to −131 mV. In a total of four similar experiments large BAPTA injections (raising the BAPTA concentration by over 100 μM) also lowered the steady state VCa by an average 4 ± 1.1 mV. This effect of BAPTA is similar to that reported before in snail neurones Although the first two BAPTA injections had little effect on the steady-state 3.6ICa,app) Ca2+ enters the cell at the plasma membrane whence it diffuses into the interior with an apparent diffusion coefficient Dapp. Furthermore, Ca2+ is removed at the plasma membrane by a Ca2+ pump with extrusion permeability Pout,app; we also allow for sequestration everywhere in the cell with rate kseq,app. Further mathematical details can be found in Section In this section we present a minimal computational model that agrees fairly well with our experimental results. In the model we assume that during a 1 s current pulse  than reported for other cell types.In most of these studies the diffusion coefficient was estimated using a simple model with only a few parameters. Our model contained more parameters that we varied to obtain a good fit. The parameters for influx, extrusion and sequestration and to some extent the positions and the response time may compromise the estimate of  μm2 s−1 B. Since 2+ is bound to buffers. The properties and distribution of these Ca2+ buffers are therefore critical for Ca2+ mobility in the cytoplasm. Analysis of Ca2+ transients under differing free Ca2+ concentrations and exogenous Ca2+ buffering in snail neurones indeed showed that Ca2+ is quite well buffered in snail neurones 2+ buffers must be mobile. When we injected the cells with BAPTA, it clearly changed the VCa transients considerably, promoting spatially uniform global signals as described in Xenopus oocytes 2+]i, which may itself have influenced Ca2+ movement. Our 2+ mobility is not changed, reduction of the clearance rate slows the recovery time but also increases the time-to-peak. After normalisation of the times to peak we could only detect a slight reduction in the latency (<0.5 s) between 0 and 150 μM BAPTA, suggesting that the mobility of BAPTA is comparable to that of the endogenous Ca2+ buffers.A large fraction of the cytoplasmic Ca2+ binding ratio for each transient. The model gives a reasonable fit with an endogenous buffer diffusion coefficient of De = 100 μm2 s−1 and Ca2+ binding ratio of κe = 65. This suggests that the endogenous Ca2+-buffers have a comparable Ca2+ binding ratio to that in Aplysia axons 2+ at the different BAPTA concentrations are: Dapp = 102 μm2 s−1 (0 μM BAPTA), Dapp = 12 μm2 s−1 (50 μM BAPTA) and Dapp = 151 μm2 s−1 (150 μM BAPTA). The estimated diffusion coefficient of BAPTA was Db = 183 μm2 s−1, which slightly increases the mobility of Ca2+ at higher BAPTA concentrations. Similar values for the BAPTA diffusion coefficient were reported by others We fitted the three transients simultaneously to the model allowing a different exogenous Ca42+ transients at different depths in large snail neurones. The decrease in calcium levels as the CASM was pushed deeper was probably due to increasing the distance from a site of leakage around the CASM and other microelectrode insertions at the top of the cell. We found that the sizes and kinetics of the Ca2+transients measured after brief (1 s) depolarisations to zero or −10 mV varied systematically and repeatably with distance from the plasma membrane at the bottom of the cell. Blocking the endoplasmic reticulum SERCA pump with CPA had little effect on the transient sizes and times to peak, but significantly slowed subsequent recovery times. Injecting BAPTA both reduced and slowed the transients close to the membrane while magnifying them deep inside the cell. By fitting our results to a mathematical model we calculate that the apparent Ca2+ diffusion coefficient is about 90 μm2 s−1.We have shown that with CASMs it is possible to record steady-state calcium levels and depolarisation-induced Ca4.1+ and Ca2+. Thus, Vaughan-Jones +]i in crab muscle with a Na+-sensitive microelectrode (NASM) inserted radially (perpendicular to the long axis) and found that low external Na caused a surprisingly rapid fall in [Na+]i. He considered that this might have been caused by the low external Na reducing a leak. This was later confirmed by experiments in which it was shown that NASMs inserted along the long axis into a cannulated fibre recorded only small changes in [Na+]i when external Na was reduced, but when inserted radially the recorded [Na+]i was at first high but fell as the NASM was pushed deeper Ion-sensitive and other non-patch microelectrodes inevitably make a hole in the cell membrane, which allows leakage in or out of the cell. Given the gradients, the ions whose leakage is most likely to distort the measured intracellular level are Nai very well buffered no one has considered that pH-sensitive microelectrode measurements have been contaminated by leakage. On the other hand, [Ca2+]i is very low and only moderately buffered. In our hands CASMs unfortunately do not perform well unless their tips are at least 1 μm in diameter 2+]i which increased on depolarization. The initially high levels were recorded even in cells loaded with Fura-2 whose fluorescence indicated a normal low [Ca2+]i. Experiments in which CASMs were calibrated while inside neurones by injecting buffers led to the conclusion that the true [Ca2+]i in quiescent voltage clamped snail neurones with injected buffer was 40 nM  and that CASMs usually cause a leak at the point of insertion Perhaps because the gradient is small and pHVCa was obtained. The current results were obtained with a motor-driven and remote-controlled micromanipulator. Typically, the CASM was moved down in several 20 μm steps before its potential changed at all. This shows that the cell membrane is very extensible, since the cell as a whole did not visibly move as the CASM was inserted. For example in VCa appeared to stabilize some distance from the apparent far side of the cell, as shown in the control part of Fig 6A, and was not reduced further as the tip approached the far side of the cell. Presumably the leakage was by then too small to have a significant effect.The early experiments with CASMs were all done with a manually-operated micromanipulator, so that systematic movements were not attempted once a stable 4.2VCa transient diffuses away from the membrane at a speed of about 6 μm s−1 confirms the long-held view that Ca2+ signalling which relies purely on diffusion will inevitably have a slow response time. The lack of effect of CPA, and the decline of the signal size with distance, rather unexpectedly both rule out significant regeneration of the signal as it spreads inwards. This is surprising, since snail neurones respond well to caffeine and certainly have the capacity for Ca-induced Ca2+ release (CICR) 2+ transients Our finding that the peak of the 2+ load every 2 min, but this may have in some way have inhibited store release. On the other hand in other experiments we have avoided depolarising the cell for long periods, and not found that the first subsequent response was different. The distance over which the VCa transients fell to half their extrapolated initial size was about 45 μm, similar to the distance between the cell membrane and the nucleus. In normal-sized neurones of course the nucleus would be much closer.Either the signal is large enough without any store contribution, or something about our experimental conditions has inhibited CICR. We depolarised the cell for 1 s to ensure a large Ca2+]i is transmembrane Ca2+ influx through voltage-gated Ca2+ channels opened by depolarisation and not massive release triggered by a small Ca2+ influx as in vertebrate cardiac muscle. The only previous direct measurements of Ca2+ movement in nerve cells without added buffers were described by Gorman et al. 2+]i only within about 40 μm of the membrane in Aplysia neurones after a 5 s depolarisation. Their CASMs were made with a different sensor (ETH 1001) to that which we used, and appear to have been relatively insensitive at low values of [Ca2+]i.We conclude that the main mechanism in our experiments that leads to an increase in [Ca4.32+ influx is presumably cleared by extrusion across the cell membrane and sequestration to cytoplasmic organelles. The PMCA or Ca2+:H+ ATPase in snail neurones appears to exchange two extracellular protons for one intracellular Ca2+ at the expense of ATP 2+]i does not fully recover from an influx but ends in a plateau. This suggests that the PMCA is the essential removal mechanism, since the endoplasmic-reticulum and mitochondrial uptake mechanisms are not inhibited by cytoplasmic o-vanadate The depolarisation-induced Ca2+ clearance when Ca2+ concentrations are high 2+ concentrations Nevertheless, for many different cell types it has been shown that mitochondria participate in Ca2+ signals, and thereby generating a fast travelling Ca2+ wave into the cell, the stores clearly play a role in the recovery of the Ca2+ transients. CPA significantly increased the Ca2+ clearance time from ∼13 to ∼18 s; these were measured in the centre of the cell and were corrected for the CASM response time. From this we estimate that the stores contribute about 25% to the clearance. This is lower but comparable to the result found in Purkinje cell somata from rat cerebellar slices, where blocking of the endoplasmic Ca2+ pump also significantly slowed the recovery time after influx Instead of reinforcing the Ca4.4VCa levels were probably elevated by continuous Ca2+ entry, the recorded transients and the way they changed with depth were, we believe, physiologically significant. It seems unlikely that the kinetics of the diffusion of Ca2+ from the far side of the cell towards the centre would be changed by a constant or slowly-declining leak. Our average value for the speed at which a depolarisation-induced Ca2+ transient flows from the periphery to the centre of a nerve cell with no added mobile buffer was ∼6 μm s−1. We can find no previous measurements of this parameter in excitable cells, although several groups have modelled the process; . Hence, with diffusion coefficients <20 μm2 s−1 the cell can still be cleared readily by the pump.If recovery from a brief Ca4.62+release mechanisms for signalling and rely on extrusion at the membrane for reasonably fast recovery, require a high Ca2+ mobility to function. A high mobility will also ensure fast signal transmission to the nucleus. This is quite different from large cells that use release mechanisms to trigger rapid on/off responses such as muscle, oocytes and chromaffin cells. These cells typically have low Ca2+ mobilities and do not rely as much on extrusion, but recycle internally released Ca2+. Small neurones and axonal regions with high surface to volume ratios represent another class of geometries, which do not require high Ca2+ mobility for fast recovery.It is possible that large cells, that do not use Ca2+ is well buffered with an estimated binding ratio of 65, the mobility of the endogenous mobile buffers must be high to explain the high apparent mobility of Ca2+. Possibly the ratio of mobile buffers to immobile buffers is higher in large snail neurones, or the diffusion coefficient of the mobile buffer fraction is much higher than in other cells. This may be related to less cytoplasmic crowding, for example by reduced cytoskeleton, causing the movement of proteins to be less constricted Because Ca2+ signalling have been done with indicators such as Fura 2, which can be used on all sizes and types of cell. But the interaction of the Ca2+ signals with the indicator may have subtle effects, which are hard to predict. It is to be regretted that CASMs so far have such large tips and slow responses that they can be used only on exceptionally large cells.The great majority of studies on intracellular Ca


We investigated the effects of E2 versus several XEs representing organochlorine pesticides , plastics manufacturing by-products/detergents , a phytoestrogen (coumestrol), and a synthetic estrogen (diethylstilbestrol) on the pituitary tumor cell subline GH3/B6/F10, previously selected for expression of high levels of membrane estrogen receptor-α. Picomolar to nanomolar concentrations of both E2 and XEs caused intracellular Ca2+ changes within 30 sec of administration. Each XE produced a unique temporal pattern of Ca2+ elevation. Removing Ca2+ from the extracellular solution abolished both spontaneous and XE-induced intracellular Ca2+ changes, as did 10 μM nifedipine. This suggests that XEs mediate their actions via voltage-dependent L-type Ca2+ channels in the plasma membrane. None of the Ca2+ fluxes came from intracellular Ca2+ stores. E2 and each XE also caused unique time- and concentration-dependent patterns of prolactin (PRL) secretion that were largely complete within 3 min of administration. PRL secretion was also blocked by nifedipine, demonstrating a correlation between Ca2+ influx and PRL secretion. These data indicate that at very low concentrations, XEs mediate membrane-initiated intracellular Ca2+ increases resulting in PRL secretion via a mechanism similar to that for E2, but with distinct patterns and potencies that could explain their abilities to disrupt endocrine functions.Xenoestrogens (XEs) are widespread in our environment and are known to have deleterious effects in animal (and perhaps human) populations. Acting as inappropriate estrogens, XEs are thought to interfere with endogenous estrogens such as estradiol (E Environmental chemicals with estrogenic activities [xenoestrogens (XEs)] have been implicated in harmful endocrine effects on animals and humans such as the feminization of male animal populations , reproduo′,p′-dichlorodiphenylethylene (DDE) and its metabolites can disorder prostate maturation ] through exocytosis  to elicit cellular Ca2+ influx via a membrane version of ER-α to induce rapid intracellular Ca2+ changes leading to PRL secretion in mER-α–enriched or depleted sublines of GH3/B6 cells  from Mediatech ; horse serum from Gibco BRL ; defined supplemented calf sera and fetal bovine sera from Hyclone ; endosulfan and DDE from Ultra Scientific ; and all other XEs from Sigma . Paraformaldehyde and glutaraldehyde were purchased from Fisher Scientific . We purchased nifedipine and thapsigargin from Calbiochem  and Fura-2/AM from Molecular Probes . All other materials were purchased from Sigma.Clonal rat prolactinoma cell lines were selected for high (GH3/B6/F10) and low (GH3/B6/D9) expression of mER-α . For thed-lysine–coated coverslips in wells of a six-well plate (105 cells/well). After serum deprivation in DMEM/ITS and then DMEM, the cells were washed in Ringer’s solution , loaded with 2 μM Fura-2/AM diluted in Ringer’s, wrapped in aluminum foil, and incubated at room temperature (RT) for 1 hr. The cells were washed twice and left to equilibrate in Ringer’s for 20 min at RT before imaging. E2 and XEs were administered using a perfusion pump system at a rate of 2 mL/min. Although responses to E2 continue during a 5-min hormonal treatment, these effects are reversible, taking about 5 min to wash out , making sure that only single cells were used as the region of interest. Data were recorded every second. Signals were obtained in dual excitation mode (340/380 nm), and the intracellular Ca2+ was calculated as a ratio (R340/380) of emission data collected at 510 nm after background subtraction. Intracellular Ca2+ was quantified by calculating the change in fluorescence ratio (R – R0) during a 5-min treatment period, normalized to the basal fluorescence value (R0) for each individual cell. These calculations for individual cells were then averaged to calculate the means and SEs for the population. Test and calibration solutions included Ca2+-free solution (Ringer’s without CaCl2 and with 2 mM EGTA), Ringer’s–20 mM KCl (Ringer’s with NaCl decreased to 105 mM and KCl increased to 20 mM), and maximum Ca2+ solution (Ringer’s with NaCl decreased to 112 mM and CaCl2 increased to 10 mM). KCl treatments were used at the end of each experiment to establish cell viability. Cells that did not respond transiently to KCl depolarization at the end of the experiment were eliminated from the composite calculations.GH3/B6 cell sublines were plated on poly-wash out . Imaging6) were plated in poly-d-lysine–coated six-well plates. After serum deprivation in DMEM/ITS, this medium was removed and new DMEM/0.1% BSA with or without the appropriate reagent or vehicle control (ethanol) was added. The cells were incubated for 1, 3, 6, 10, or 15 min and centrifuged at 4°C, 350 × g, for 5 min. The supernatant was then collected and stored at −20°C until radioimmunoassay (RIA). Concentrations of PRL were determined using components of the rat PRL RIA kit from the National Institute of Diabetes and Digestive and Kidney Disease and the National Hormone and Pituitary Program . Briefly, RIA buffer , 100 μL cold standard (rat PRL-RP-3) or unknown sample, rPRL-s-9 antiserum , and [125I]-rat-PRL  were combined and incubated with shaking, overnight at 4°C. Anti-rabbit IgG  was added to a final dilution of 1:9, and the samples were incubated with shaking at RT for 2 hr. One milliliter of polyethylene glycol solution  was then added, and the samples were incubated with shaking at RT for 15 min. The samples were then centrifuged at 4,000 × g for 10 min at 4°C, the supernatant was decanted, and the pellet was counted in a Wizard 1470 Gamma Counter . The PRL concentration was then calculated and normalized to the crystal violet values representing cell number.Cells  per well. Sample plates were then rocked at RT for 30 min. The plates were then washed three times with deionized water and allowed to dry overnight. Crystal violet solution  was added to each well, and the plates were incubated with rocking at RT for 30 min. The plates were then washed and dried, and the dye was extracted with 1 mL per well of a 10% acetic acid solution (in water). After a 30 min incubation at RT, the absorbance at 590 nm was read in a model 1420 Wallac microplate reader .We calculated the statistical significance of differences using Sigma Stat  and one-way analysis of variance.2 can trigger a rapid and reversible (within 5 min) intracellular Ca2+ change  in our mER-α–enriched (F10) rat prolactinoma cell subline, whereas the mER-α–depleted (D9) subline showed no Ca2+ response  within 30 sec of administration in mER-α–enriched F10 cells (−9–10−8 M), although all XEs caused a response, generally they did so less robustly or potently when compared with E2. E2 showed a significant intracellular Ca2+ change at concentrations as low as 10−12 M, and increased with concentration. DES gave a similar response, although somewhat less robustly. Coumestrol was also effective at all tested concentrations; however, its maximal response (10−8 M) was half that caused by E2. Nonylphenol elicited concentration-dependent increases in Ca2+ influxes with similar characteristics to E2, but with the most robust response at 10−8 M being slightly lower than that for E2. BPA displayed a maximal response at 10−9 M that declined at a higher concentration. DDE produced the smallest Ca2+ elevations. Dieldrin elicited a Ca2+ change at all concentrations. Endosulfan caused no intracellular Ca2+ changes at the lowest concentrations, yet 10−9 M and 10−8 M produced quite robust influx. When we examined individual cells, we found that they were heterogeneous in their responsiveness to XEs, as we have observed previously in responses to E2 .We previously showed that Eresponse . In the 10 cells  and 2. U10 cells . Each coes to E2 . Overallurements , and theD9 cells ; for the2+  were involved in the XE-induced response, we administered thapsigargin, a cell-permeable inhibitor that releases Ca2+ from intracellular stores by specifically and irreversibly inhibiting endoplasmic reticular Ca2+ ATPase  and the Ringer’s–20 mM KCl (by displaying a large and transient Ca2+ influx due to cell depolarization), thus demonstrating cell viability.To confirm that the Ca2 causes an intracellular Ca2+ change via the L-type Ca2+ channel . After washout of the estrogens, the addition of 10 μM nifedipine caused cessation of Ca2+ influx. Subsequent addition of 10−8 M E2 or XE, in the presence of nifedipine, could not elicit a Ca2+ influx.We have recently shown that E channel . XEs mimle cells  and thenle cells  to quant−8 M E2 or XE, PRL was secreted rapidly (by 1 min), a response largely complete by 1–3 min . However, the organochlorine pesticides produced either delayed or no PRL secretion; DDE did not cause PRL secretion at any time point, and dieldrin and endosulfan stimulated PRL secretion incrementally over time with maximal secretion at 15 min, which was significantly different from their secretion level at 1 min.At 10 1–3 min  for most2 or XE concentration dependence of PRL secretion at 3 min shows differences among compounds . Nonylphenol and BPA, both detergents, show a bimodal response curve with a wide gap (interruption) at the middle concentrations; PRL secretion was only elicited at the highest and lowest concentrations. The pesticides DDE and endosulfan show similar response curves, with maximal secretion at 10−10 M. Dieldrin induced PRL secretion at all concentrations from 10−12 M to 10−8 M, but with no apparent dose-dependent changes across these concentrations.The Eompounds . The dos2+ channel mechanism, we administered nifedipine to block the influx of Ca2+  were tested at a 10−8 M concentration with and without nifedipine. Nifedipine, which blocked the entry of Ca2+ from the extracellular solution, also abolished the E2- and XE-induced PRL secretion at 3 min.To test whether PRL secretion occurs via the L-type Ca of Ca2+ . E2 and 2 by extracellular signal-regulated kinase (ERK) phosphorylation (2 induces a rapid Ca2+ influx (within 30 sec) via the L-type Ca2+ channel in these cells, which is necessary for rapidly induced PRL secretion  and rapid (nongenomic or membrane-initiated) responses . We estarylation  and PRL rylation . We alsoecretion . However2 in pituitary tumor cells  and XEs stimulate a rapid Ca2+ influx (within 30 sec) from the extracellular media (because elimination of extracellular Ca2+ abolished intracellular Ca2+ changes) that is independent of the release of endoplasmic reticulum Ca2+ stores . Blocking the L-type Ca2+ channels with nifedipine also abrogated XE-induced Ca2+ influx. Therefore, the XEs used in our study were able to cause E2-like changes in Ca2+ levels via similar mechanisms.Because we found that some XEs induced rapid mitogen-activated protein kinase (MAPK) activation via the same membrane-initiated signaling pathway used by Eor cells , we hypo like E2 , XEs pot2+ levels often lead to the release of many different kinds of stored hormones and other proteins from secretory vesicles , resulting in changes in the phosphorylation status of a variety of cellular proteins leading to rapid functional consequences. These signaling cascades are now known to be rapidly stimulated by steroid hormones , or ERK activation (2-induced PRL release was blocked in cells inhibited with the ER antagonist ICI 182,870 (Some researchers have speculated that the ER that mediates XE effects is a unique receptor . Howevertivation . This is 182,870  or treat 182,870 . The mER 182,870 . Of cour2+ influxes and PRL secretion, further study is needed to fully understand all of these differentially activated signaling cascades and their relationships to the myriad outcomes of XE exposure.XEs pose a potential environmental threat to human health because experimental animal exposures have demonstrated endocrine developmental anomalies at levels similar to those sometimes seen in environmental contamination. However, most previous studies have emphasized the genomic mechanisms of XE action, which require very high concentrations. Our research has instead focused on the rapid, nongenomic or membrane-initiated effects of these environmental contaminants. Via these alternate signaling pathways, estrogen mimetics such as XEs could interfere with endogenous estrogen actions via multiple mechanisms. By eliciting, enhancing, or inhibiting estrogenic signaling, they may interfere with physiological estrogenic signals, affecting many downstream functions. Each XE however, shows unique temporal and dose-responsive patterns, possibly due to the differential involvement of companion signaling pathways. Although we have linked XEs to rapid cellular events that trigger intracellular Ca


In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca2+-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca2+]c changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca2+ in the cytosol ([Ca2+]c) and the pattern of these changes (Ca2+ signature) is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca2+]c changes in a reproducible and dose dependant manner.Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca2+]c of the recombinant fungus in the presence of toxicants representing heavy metals – Cr6+ and Zn2+ and a phenolic polar narcotic -3,5-DCP. The fungus responds to toxicants by a decrease in the amplitude of [Ca2+]c response to 5 mM external CaCl2 and an increase in Ca2+ final resting levels and recovery time.Toxicity bioassay has been developed to monitor changes [Ca2+]c has been identified, e.g. Amplitude, Length of Transient, Final Resting Level and Recovery Time. These parameters can be used to determine the toxicity of a range of chemicals to eukaryotic cells in a 96-well microtitre plate method.A novel toxicity bioassay utilizing eukaryotic cells has been developed based on filamentous fungi transformed with the recombinant aequorin gene. A range of parameters characterising changes in [Ca V. fischeri is a gram negative free-living luminescent bacterium c have been done describing the role of calcium in animal and plant cells subjected to different treatments. However, very little work in this area has been done on filamentous fungi due to the lack of both routine and reliable methods for monitoring intracellular free Ca2+ in living fungal cells.Calcium is a well-known second messenger involved in the transduction of different external stimuli and hormonal signals in eukaryotic cells. A great number of studies describing changes in c). Using aequorin transformed fungi it has been shown that different physico-chemical stimuli result in a transient [Ca2+]c increases with a unique Ca2+ signature c at the end of the experiment) and recovery time  the phenolic inhibited the amplitude of the [Ca2+]c response at both 5 and 30 min preincubation  also increased LT50 and rise time  caused a small increase in the amplitude of the c response   Zn2+ caused an increase in amplitude of the [Ca2+]c response as well as an increase in the final Ca2+ resting level and recovery time.Chromium did not affect (P < 0.5) the final resting levels and recovery time of Cae Figure  &1d. Zinc Figure  &1f. It A. awamori to basal Ca2+ levels and light dissipation was fairly rapid in systems challenged with Cr6+ for 5 and 30 min. This was not the case for A. awamori treated with Zn2+ and 3,5-DCP. In these cases there was protracted recovery in Ca2+ content at higher toxicant challenges with both 5 and 30 min incubation.Recovery of treated 50 is one standard parameter calculated in toxicity studies and was chosen in this study to determine differences in values obtained by the fungal and bacterial luminescence bioassays. In order to calculate IC50 values for all toxicants it was decided to utilise the amplitude of [Ca2+]c changes in the fungal cell after challenge with different concentrations of toxicant. Examples of dose response curves are presented in Figures 50 values for the 5 and 30 min toxicant pre-incubations with A. awamori are presented in Table 50 for DCP, Cr6+ and Zn2+ are 36.7, 167.8 and 549.7 mg l-1 respectively. These can be compared to values for the V. fischeri test of results of 3.6, 13.95 and 0.44 mg l-1 for the same toxicants tested over a 30 min incubation period.ICude of Ca+c changeude of Ca+c changeA. awamori to different toxicant concentrations by examining changes in Relative Light Units (RLU) and [Ca2+]c in the presence of an external CaCl2 concentration of 5 mM. This study differs from that of Torrecilla et al. c was examined. The [Ca2+]c response was not significantly different from that obtained with the control solution (data not shown). A second approach studied the effect of preincubation of the fungus with toxicants on [Ca2+]c response to external CaCl2.In this paper data have been presented on the response of a et al.  which exA. awamori, using the protocol developed, responds to toxic chemical challenge in a reproducible way. The aequorin system has more parameters which can be assessed than other bioassays i.e. final [Ca2+]c resting level, recovery time and amplitude of [Ca2+]c/RLU following chemical challenge for 5 or 30 min.There is no doubt that aequorin transformed A. awamori has been shown to respond to organic and inorganic compounds by a decrease in the amplitude of [Ca2+]c response to external CaCl2 with increasing toxicant concentration. Zn2+ and DCP also affected the final Ca2+ resting levels and recovery times. In the case of 30 min preincubation with either 112 mg l-1 DCP or 700 mg l-1 Zn2+, the final [Ca2+]c resting levels remained significantly higher than after 5 min preincubation with these toxicants indicating a greater toxicity to the fungus on longer contact with the toxicant. This is not an unusual response even in the V. fischeri bioassay where it is generally observed that toxicity increases  with longer incubation times. IC50 values calculated using amplitude changes of [Ca2+]c showed decreased toxicity to Zn2+ at longer preincubation times. This needs further analytical consideration to determine toxicity values of relevance.Vibrio (e.g. V. fischeri) and related Photobacterium species. Acute toxicity tests utilising such luminescent bacteria can underestimate the toxicity of chemicals and Backhaus et al. c, toxic chemicals often exert an increase in [Ca2+]c thus affecting IC50 calculations. The calculation of IC50 for this fungal bioassay may not be appropriate. Other parameters such as LT50, rise time, final Ca2+ resting levels and recovery time would provide ideal candidate parameters, but more tests and comparisons with other bioassays are needed. The aequorin bioassay can also generate up to 15 parameters to assess the effect of toxicants due to the complex pattern of [Ca2+]c changes c response to 5 mM CaCl2 and an increase in the [Ca2+]c resting levels . Preliminary IC50 values obtained with the fungal aequorin system, and based on the inhibition of the [Ca2+]c amplitude in response to external CaCl2 treatment, indicate that it is less sensitive for detecting 3,5-DCP, Zn2+ and Cr6+ than the V. fischeri luciferase system. However, it is obvious that the changes in amplitude is not the most suitable parameter for calculating IC50 and more research are needed to determine the best possible set of parameters to be used together with the aequorin bioassay.The current paper presents the proof-of-concept study of the novel toxicity bioassay based on using filamentous fungi transformed with the recombinant aequorin gene. The research conducted has shown that the recombinant aequorin method can be used as a novel eukaryotic toxicant biosensor. It offers more parameters which can be readily analysed than the traditionally used bacterial biosensors. The fungal aequorin biosensor responded to toxic substances  Ltd.  or Sigma .A. awamori 66A, which has been transformed with the recombinant aequorin gene c concentrations the following empirically derived equation was used:The luminometer measures light emission in relative lights units (RLU). To convert RLU into c concentration was made using a Macro developed in Excel 7.0 by Mark Knight (University of Oxford).Total luminescence is measured as an integral of all luminescence up to complete aequorin discharge. Aequorin was discharged by adding 2M CaCl4 and K2Cr2O7. All toxicants were added 5 min and 30 min before treatment with 5 mM external CaCl2 in a total volume of 25 μl VS medium. 4-6 replicates were performed for each treatment. All the experiments were performed in triplicate. IC50 values were calculated only for the amplitude of the response.Three toxicants were tested: 3,5-DCP, ZnSOVibrio fischeri bioassays utilised cultures of the organism grown in Photobacterium Broth (PB) at 37°C until optimum light output. A 100 μl volume of V. fischeri suspension was added to each of the 96 wells in a microtitre plate. During the experiment a 100 μl volume of NaCl solution  containing the test substance in the appropriate concentration was added to the wells. For each measurement a control consisting of 6 wells with V. fischeri to which the NaCl solution was added. Chemicals were tested in five different concentrations and each concentration was tested in three replicates. Plates were measured in an Anthos Lucy 1 luminometer. Measurements were made over a period of 30 min. IC50 values were calculated for 5 and 30 min incubations. Recording of data started immediately after addition of the test solutions to the wells containing the culture. The standard proprietary Microtox bioassay was also carried out [ried out .OK conceived the study, designed and developed aequorin bioassay, carried out the tests and has been involved in the writing of the article.MZ has designed the microbial part of the testing and participated in carrying out the fungal tests.NC helped design the study and participated in writing of the article.All authors read and approved the final manuscript.


Electrophysiological, imaging, and behavioral studies in hippocampal or cortical neurons of rodents and rabbits have revealed aging-related increases in the slow afterhyperpolarization, Ca2+ spikes and currents, Ca2+ transients, and L-type voltage-gated Ca2+ channel (L-VGCC) activity. Several of these changes have been associated with age-related deficits in learning or memory. Consequently, one version of the Ca2+ hypothesis has been that increased L-VGCC activity drives many of the other Ca2+-related biomarkers of hippocampal aging. In addition, other studies have reported aging- or AD model-related alterations in Ca2+ release from ryanodine receptors (RyR) on intracellular stores. The Ca2+-sensitive RyR channels amplify plasmalemmal Ca2+ influx by the mechanism of Ca2+-induced Ca2+ release (CICR). Considerable evidence indicates that a preferred functional link is present between L-VGCCs and RyRs which operate in series in heart and some brain cells. Here, we review studies implicating RyRs in altered Ca2+ regulation in cell toxicity, aging, and AD. A recent study from our laboratory showed that increased CICR plays a necessary role in the emergence of Ca2+-related biomarkers of aging. Consequently, we propose an expanded L-VGCC/Ca2+ hypothesis, in which aging/pathological changes occur in both L-type Ca2+ channels and RyRs, and interact to abnormally amplify Ca2+ transients. In turn, the increased transients result in dysregulation of multiple Ca2+-dependent processes and, through somewhat different pathways, in accelerated functional decline during aging and AD.Evidence accumulated over more than two decades has implicated Ca Therefore, in this review, we summarize several lines of evidence implicating altered release from intracellular stores in aging and AD, and attempt to integrate this evidence with the role of Ca2+ influx in aging-related Ca2+ dysregulation.In addition to the accumulating evidence of increased Cang or AD . In part2+ sequestration and release by the ER in both peripheral cells  and the ryanodine receptor (RyR), each having multiple isoforms in different tissues. These receptor channels function to amplify or trigger Ca2+ rises initiated by either plasmalemmal Ca2+ influx or ligand binding, thereby inducing Ca2+ signaling cascades. Amplification is achieved through either the actions of Ca2+-induced Ca2+ release (CICR), provided by RyR, or actions of IP3-induced Ca2+ release (IICR) through IP3Rs.Several comprehensive reviews have recently considered mechanisms associated with Caal cells  and in nal cells . Accordi2+ source for excitation-contraction coupling , through interactions with the scaffold protein Homer 1a . Sarcoplasmic/endoplasmic reticulum Ca2+-ATPases maintain the relatively high levels of Ca2+ in the ER (hundreds of µm) that serve CICR, and IICR, and, in the process, contribute to the control and reduction of cytosolic Ca2+  in cell culture (excitotoxicity) is a common model used to mimic a wide range of neurological insults, including anoxia/ischemia, head and spinal cord trauma, and even chronic neurodegenerative diseases such as AD. Dysregulated Ca2+ homeostasis and altered Ca2+ influx through NMDA receptors were identified as primary contributors to neuronal cell death early in the study of excitotoxicity  release) (2+ depletion prior to an insult appears protective against necrotic (excitotoxic) cell death, long-term depletion of ER Ca2+ induces apoptosis, as indicated by elevations of apoptotic markers, stress responses and disturbance in protein synthesis, and/or massive cell death  mouse models of AD, numerous studies testing the view that altered Capatients  found ev2+ efflux from the stores in response to caffeine (an agonist at the RyR) , in combination with amyloid precursor protein (APP) mutations, increases Aβ production , nicotinic acid adenine dinucleotide phosphate (NAADP) or IP3   were able to show that the resting [Ca2+]L in sensory neurons is in the range of 200–300 µm, and high concentrations of IP3 or caffeine result in approximately a 40% decrease in luminal Ca2+  or IP3 , and also the ER . In addi2+ concentration and release, or on RyR expression. Studies focusing on measures of ER Ca2+ content have generally relied on the use of single wavelength indicators to measure changes in [Ca2+]i transients activated by caffeine, and have found varying results, depending on the experimental approach or preparation. In an early study, no net change in ER Ca2+ release with aging was reported in synaptosomes from the whole brain  if a common mechanism of Ca2+ dysregulation underlies many aspects of brain aging, then multiple Ca2+-dependent biomarkers of aging in the hippocampus should emerge at approximately the same age in adulthood; and (ii) if Ca2+ dysregulation is a major factor in cognitive decline then Ca2+ biomarkers should precede or coincide with the earliest age of cognitive impairment, which in some studies of rats has been as early as 12-months old (approximately mid-life). To test these predictions and the involvement of CICR on the emergence of Ca2+-related biomarkers, we recently conducted an extensive age course study combining electrophysiological and Ca2+ imaging techniques in hippocampal slices from male rats. Animals at five age points were used to identify the age of onset for three Ca2+-mediated markers of aging, the sAHP, spike accommodation, and the synaptically activated Ca2+ transient. A subset of hippocampal slices received a high dose of ryanodine to block the contribution of CICR to the overall Ca2+ response. In this study, we also employed the least invasive procedures available  to minimize interactions of preparation trauma and age.More broadly, in fact, several other important tenets of the overall Ca2+ response (i.e. CICR) during a 20-s train of synaptic spikes appears to be minimal in young neurons compared to aged neurons and, notably, CICR contributed most to the [Ca2+]i elevation during the first few seconds of the train , this can result in elevated intracellular Ca2+ release and greater hyperpolarization of cortical neurons from transgenic mice of all ages. Surprisingly, however, in the triple transgenic AD model, the aging-related increase in spike train-induced AHP did not differ from the aging change in the AHP seen in wild-type mice.Some types of AD mutations (e.g. presenilins), but not all, appear to alter RyR expression. Under some conditions,  indicate that elevated CICR, beginning at about 12 months of age, may be an important underlying factor in the emergence of multiple Ca2+-related biomarkers of brain aging in rats.The observed contributions of altered CICR to Ca2+ homeostasis during aging, rather than being contradictory, may instead suggest an expanded model of the Ca2+ dysregulation pathway in brain aging and, perhaps in AD (as shown in 2+ transients.The apparent strong evidence linking both L-VGCCs and RyRs to dysregulated hippocampal Ca