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Ca2+ Responses (ca2+ + response)
Selected AbstractsImpairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2010Shinji Matsumura Abstract Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N -methyl- d -aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C, at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain. [source] Acute action of rotenone on nigral dopaminergic neurons , involvement of reactive oxygen species and disruption of Ca2+ homeostasisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2009Peter S. Freestone Abstract Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05,1 ,m) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca2+] ([Ca2+]i) (73.8 ± 7.7 nm) and intracellular [Na+] (3.1 ± 0.6 mm) (all with 1 ,m). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca2+]i rise was abolished by removing extracellular Ca2+, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N -(p -amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca2+]i rise resulted in a large (46.6 ± 25.3 nm) Ca2+ response when baseline [Ca2+]i was increased by a ,priming' protocol that activated voltage-gated Ca2+ channels. There was also a positive correlation between ,naturally' occurring variations in baseline [Ca2+]i and the rotenone-induced [Ca2+]i rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K+ channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca2+]i rise by a small increase in baseline [Ca2+]i. [source] Long-term potentiation of mGluR1 activity by depolarization-induced Homer1a in mouse cerebellar Purkinje neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2003Itsunari Minami Abstract Metabotropic glutamate receptor 1 (mGluR1) plays a crucial role in synaptic plasticity and motor learning in the cerebellum. We have studied activity-dependent changes in mGluR1 function in mouse cultured Purkinje neurons. Depolarizing stimulation potentiated Ca2+ and current responses to an mGluR1 agonist for several hours in the cultured Purkinje neurons. It also blocked internalization of mGluR1 and increased the number of mGluR1s on the cell membrane. We found that depolarization simultaneously increased transcription of Homer1a in Purkinje neurons. Homer1a inhibited internalization and increased cell-surface expression of mGluR1 when coexpressed in human embryonic kidney (HEK)-293 cells. Depolarization-induced Homer1a expression in Purkinje neurons was blocked by a mitogen-activated protein kinase (MAPK) inhibitor. Changes in internalization and mGluR1-mediated Ca2+ response were also blocked by inhibition of MAPK activity, suggesting that localization and activity of mGluR1 were regulated in the same signalling pathway as Homer1a expression. It is thus suggested that depolarization of the Purkinje neuron leads to the increment in mGluR1 responsiveness through MAPK activity and induction of Homer1a expression, which increases active mGluR1 on the cell surface by blocking internalization of mGluR1. [source] Reversible protein kinase C activation in PC12 cells: effect of NGF treatmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2000Jean-Luc Dupont Abstract Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura-2-AM and Fim-1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose-dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl-induced PKC responses was larger indicating an increase in the activated PKC-pool in these cells. This difference between control and NGF-treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+ -dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized. [source] Histamine-induced Ca2+ entry in human astrocytoma U373 MG cells: Evidence for involvement of store-operated channelsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2008Margarita Barajas Abstract Glial and glia-derived cells express a variety of receptors for neurotransmitters and hormones, the majority of which evoke both Ca2+ release from intracellular stores and Ca2+ entry across the plasma membrane. We investigated the links between histamine H1 receptor activation, Ca2+ release from intracellular stores and Ca2+ influx in human astrocytoma U373 MG cells. Histamine, through a H1 receptor-mediated effect, evoked an increase in cytoplasmic free calcium concentration ([Ca2+]i) that occurred in two phases: an initial, transient, increase owing to Ca2+ mobilization from intracellular pools, and a second, sustained increase dependent on both Ca2+ influx and continuous receptor occupancy. The characteristics of histamine-induced increases in [Ca2+]i were similar to the capacitative entry evoked by emptying of the Ca2+ stores with thapsigargine, and different from that observed when Ca2+ influx was activated with OAG (1-oleoyl-2-acetyl- sn -glycerol), a diacylglycerol (DAG) analog. OAG application or increased endogenous DAG, resulting from DAG kinase inhibition, reduced the histamine-induced response. Furthermore, activation of the DAG target, protein kinase C (PKC), by TPA (12-O-tetradecanoyl 4,-phorbol 13,-acetate) resulted in inhibition of the histamine-induced Ca2+ response, an action prevented by PKC inhibitors. By using reverse transcriptase,polymerase chain reaction analysis, mRNAs for transient receptor potential channels (TRPCs) 1, 4, and 6 as well as for STIM1 (stromal-interacting molecule) and Orai1 were found to be expressed in the U373 MG cells, and confocal microscopy using specific antibodies revealed the presence of the corresponding proteins. Therefore, TRPCs may be candidate proteins forming store-operated channels in the U373 MG cell line. Further, our results confirm the involvement of PKC in the regulation of H1 receptor-induced responses and point out to the existence of a feedback mechanism acting via PKC to limit the increase in [Ca2+]i. © 2008 Wiley-Liss, Inc. [source] P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca2+ rise and change in mitochondrial potentialJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2007Stefan Kahlert Abstract With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca2+ level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y1/2/4 receptor agonist in rat, caused a large cytosolic Ca2+ increase in astrocytes and a considerably smaller Ca2+ response in neighboring neurons. The P2Y1 receptor antagonist MRS2179 completely blocked the ATP-induced Ca2+ response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca2+ level. Stimulation with UDP, a P2Y6 receptor agonist; UTP, a P2Y2/4 receptor agonist; 2MeSATP, a P2Y1 receptor agonist; or 2MeSADP, a P2Y1/12/13 receptor agonist, evoked significant Ca2+ responses in astrocytes but small Ca2+ responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca2+ peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca2+ level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain. © 2007 Wiley-Liss, Inc. [source] Glutamate-mediated influx of extracellular Ca2+ is coupled with reactive oxygen species generation in cultured hippocampal neurons but not in astrocytesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Stefan Kahlert Abstract Generation of reactive oxygen species (ROS) in brain tissue leads to neurodegeneration. The major source of ROS is the mitochondrial respiratory chain. We studied regulation of Ca2+ level, mitochondrial potential, and ROS generation in defined mixed hippocampal cell cultures exposed to glutamate (100 ,M). Recordings were made from individually identified astrocytes and neurons to compare the physiologic responses in both cell types. Neurons identified by synaptotagmin immunoreactivity were characterized functionally by the fast Ca2+ increase with K+ (50 mM) stimulation, and the astrocytes identified by glial fibrillary acidic protein (GFAP) staining had the functional characteristic of a transient Ca2+ peak in response to ATP (10 ,M) stimulation. We found that the glutamate-mediated Ca2+ response in neurons is due largely to influx of extracellular Ca2+. This is consistent with our finding that in cultured hippocampal neurons, stores depending on the activity of the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pump had a low Ca2+ content, regardless of whether the neurons were challenged or not with K+ before applying the SERCA inhibitor cyclopiazonic acid (CPA). Astrocytes displayed a large CPA-mediated Ca2+ response, indicating a high level of Ca2+ load in the stores in astrocytes. Importantly, the rise in ROS generation due to glutamate application was cell-type specific. In neurons, glutamate induced a marked rise in generation of ROS, but not in astrocytes. In both astrocytes and neurons, the mitochondrial potential was increased in response to glutamate challenge. We conclude that in neurons, Ca2+ influx accounts for the increased ROS generation in response to glutamate. This might explain the high vulnerability of neurons to glutamate challenge compared to the vulnerability of astrocytes. The high resistance of astrocytes is accompanied by an efficient downregulation of cytosolic Ca2+, which is not found in neurons. © 2004 Wiley-Liss, Inc. [source] The effect of short-term low-temperature treatments on gene expression in Arabidopsis correlates with changes in intracellular Ca2+ levelsPLANT CELL & ENVIRONMENT, Issue 4 2003K. NORDIN HENRIKSSON ABSTRACT The role of changes in intracellular calcium ion concentration ([Ca2+]i) in low-temperature signal transduction in plants has lately been supported by several studies. An analysis to determine whether the low-temperature-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt) could be correlated with a downstream response such as gene expression was carried out. The induction of the low-temperature-regulated gene LTI78 was used as an end point marker of the signal transduction pathway. It was found that this gene is induced by very brief low-temperature exposures and that the induction does not depend on a continuous exposure to low temperature. By altering the cooling rate, different patterns of the Ca2+ response were obtained which could be correlated with different patterns of LTI78 induction. Furthermore, reducing the Ca2+ transients by pre-treatment with the Ca2+ channel blocker La3+ also led to a reduced level of gene induction. The results show that brief exposures to low temperature results in the onset of a signalling pathway that leads to the induction of gene expression. This indicates the involvement of changes in [Ca2+]cyt in low-temperature signalling leading to LTI78 expression but the presence of multiple signalling pathways is suggested. [source] Glutamate receptors on myelinated spinal cord axons: I. GluR6 kainate receptors,ANNALS OF NEUROLOGY, Issue 2 2009Mohamed Ouardouz PhD Objective The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Although overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptor,dependent axonal Ca2+ deregulation. Methods Dorsal column axons were loaded with a Ca2+ indicator and imaged in vitro using confocal laser-scanning microscopy. Results Activation of glutamate receptor 6 (GluR6) kainate receptors promoted a substantial increase in axonal [Ca2+]. This Ca2+ accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine-dependent intracellular stores, which, in turn, depended on activation of L-type Ca2+ channels: ryanodine, nimodipine, or nifedipine blocked the agonist-induced Ca2+ increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca2+ response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal N,-nitro-L-arginine methyl ester, blocked the Ca2+ increase. Loading axons with a peptide that mimics the C-terminal PDZ binding sequence of GluR6, thus interfering with the coupling of GluR6 to downstream effectors, greatly reduced the agonist-induced axonal Ca2+ increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Cav1.2. Interpretation Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal "nanocomplexes" to release toxic amounts of Ca2+ may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur. Ann Neurol 2009 [source] Nociceptin/orphanin FQ inhibits capsaicin-induced guinea-pig airway contraction through an inward-rectifier potassium channelBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2002Yanlin Jia Nociceptin/orphanin FQ (N/OFQ), an endogenous opioid-like orphan receptor (NOP receptor, previously termed ORL1 receptor) agonist, has been found to inhibit capsaicin-induced bronchoconstriction in isolated guinea-pig lungs and in vivo. The underlying mechanisms are not clear. In the present studies, we tested the effect of N/OFQ on VR1 channel function in isolated guinea-pig nodose ganglia cells. Capsaicin increased intracellular Ca2+ concentration in these cells through activation of vanilloid receptors. Capsaicin-induced Ca2+ responses were attenuated by pretreatment of nodose neurons with N/OFQ (1 ,M). N/OFQ inhibitory effect on the Ca2+ response in nodose ganglia cells was antagonized by tertiapin (0.5 ,M), an inhibitor of inward-rectifier K+ channels, but not by verapamil, a voltage gated Ca2+ channel blocker, indicating that an inward-rectifier K+ channel is involved in N/OFQ inhibitory effect. In isolated guinea-pig bronchus, N/OFQ (1 ,M) inhibited capsaicin-induced airway contraction. Tertiapin (0.5 ,M) abolished the N/OFQ inhibition of capsaicin-induced bronchial contraction. Capsaicin (10 ,g) increased pulmonary inflation pressure in the isolated perfused guinea-pig lungs. This response was significantly attenuated by pretreatment with N/OFQ (1 ,M). Tertiapin also abolished the N/OFQ inhibitory effect on capsaicin-induced bronchoconstriction in perfused lungs. Capsaicin increased the release of substance P and neurokinin A from isolated lungs. N/OFQ (1 ,M) blocked the capsaicin-induced tachykinin release. These results indicate that N/OFQ-induced hyperpolarization of tachykinin containing airway sensory nerves, through an inward-rectifier K+ channel activation, accounts for the inhibition of capsaicin-evoked broncoconstriction. British Journal of Pharmacology (2002) 135, 764,770; doi:10.1038/sj.bjp.0704515 [source] Determination of the physical environment within the Chlamydia trachomatis inclusion using ion-selective ratiometric probesCELLULAR MICROBIOLOGY, Issue 5 2002Scott Grieshaber Summary Chlamydia trachomatis is an obligate intracellular bacterium with a biphasic life cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial inclusion is non-fusogenic with endosomal or lysosomal compartments but intersects a pathway involved in transport of sphingomyelin from the Golgi apparatus to the plasma membrane. The physical conditions within the mature chlamydial inclusion are unknown. We used ratiometric imaging with membrane-permeant, ion-selective fluorescent dyes for microanalyis of the physical environment within the inclusion. Determination of H+, Na+, K+ and Ca2+ concentrations using CFDA (carboxy fluorescein diacetate) or BCECF-AM (2,,7, -bis (2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, SBFI-AM, PBFI-AM and fura-PE3-acetomethoxyester (Fura-PE3-AM), respectively, indicated that all ions assayed within the lumenal space of the inclusion approximated the concentrations within the cytoplasm. Stimulation of purinergic receptors by addition of extracellular ATP triggered a dynamic Ca2+ response that occurred simultaneously within the cytoplasm and interior of the inclusion. The chlamydial inclusion thus appears to be freely permeable to cytoplasmic ions. These results have implications for nutrient acquisition by chlamydiae and may contribute to the non-fusogenicity of the inclusion with endocytic compartments. [source] Neurone-to-astrocyte communication by endogenous ATP in mixed culture of rat hippocampal neurones and astrocytesDRUG DEVELOPMENT RESEARCH, Issue 1 2003Schuichi Koizumi ATP is recognized as an important intercellular signaling molecule in the peripheral and CNS. Glutamate is reported to be an important neurone-to-glia mediator being released from neurones and astrocytes that activates astrocytic and neuronal Ca2+ responses, respectively. We demonstrate here that endogenous ATP could be an extracellular molecule for neurone-to-astrocyte communication in cocultured rat hippocampal neurones and astrocytes. Hippocampal neurones reveal synchronized Ca2+ oscillation, which was due to glutamatergic synaptic transmission. When analyzed in a fura-2 method, a slight and very slow increase in intracellular Ca2+ concentration ([Ca2+]i) elevation was observed in some population of astrocytes. Such astrocytic [Ca2+]i elevation was dramatically inhibited by apyrase, though apyrase itself had no effect on neuronal Ca2+ oscillation. For a detail analysis, we investigated changes in [Ca2+]i in cells using a confocal microscopy. When cocultured hippocampal neurones and astrocytes were depolarized electronically in the presence of glutamate-receptor antagonists, a transient elevation in [Ca2+]i was observed in neurones, which was followed by a slowly initiated and small rise in [Ca2+]i in astrocytes. Apyrase or P2 receptor antagonists almost abolished the [Ca2+]i rises in astrocytes, suggesting that depolarization-evoked ATP release from neurones should produce astrocytic [Ca2+]i elevation via P2 receptors. Using a luciferin,luciferase bioluminescence assay, we found that neurones could release ATP in an activity-dependent manner. These findings suggest that endogenous ATP should be an important intercellular mediator between neurones and astrocytes and that functions of these cells should be fine-tuned by endogenously released ATP in situ. Drug Dev. Res. 59:88,94, 2003. © 2003 Wiley-Liss, Inc. [source] Pharmacological characterization of the rat brain P2Y1 receptor expressed in HEK293 cells: Ca2+ signaling and receptor regulationDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001Christian Vöhringer Abstract The increasing number of ATP- and UTP-sensitive membrane receptors identified by cloning represent either ligand-activated ion channels (P2X) or G-protein-coupled receptors (P2Y). Adenosine, ATP, and UTP have potential application in the management of pain, cancer, and some cardiovascular and pulmonary diseases and are also involved in inflammatory processes in the brain. Therefore, P2Y receptors seem to be promising therapeutic targets. Multiple P2Y receptor subtypes, classified pharmacologically, are mainly linked to activation of phospholipase C (PLC). The present study further characterizes the rat brain P2Y1 wild-type receptor (rP2Y1 -wt) and the eGFP-tagged receptor (rP2Y1 -eGFP) stably expressed in HEK293 cells, thus shedding light on receptor regulation. Both receptors were analyzed by measuring Ca2+ responses in single cells. The rP2Y1 -eGFP receptor was coupled to Ca2+ release like the rP2Y1 -wt receptor. Experiments using the PLC inhibitor U73122 confirm the functional activation of PLC, through rP2Y1 -eGFP activation. The P2Y1 -selective agonists 2-MeSADP and 2-MeSATP were most potent at the heterologously expressed receptors. We found a ligand selectivity typical for P2Y1 receptors (2-MeSADP = 2-MeSATP > ADP > ATP,S, ATP >> UTP). Fluorescence microscopy and Ca2+ measurements confirm that the rP2Y1-eGFP receptor during homologous desensitization is subjected to processes leading to agonist-induced internalization. The kinetics of receptor resensitization were also examined. Therefore, rP2Y1 -eGFP expressing cells are suitable to determine the physiological P2Y1 receptor signaling pathway and can be a helpful tool to identify drugs acting at P2Y1 receptors as possible therapeutic targets. Drug Dev. Res. 53:172,179, 2001. © 2001 Wiley-Liss, Inc. [source] Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca2+ signaling in CD8+ T cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2004Marco Averbeck Abstract The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca2+ influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8+ T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca2+ influx in T cells to long-lasting interactions and sustained Ca2+ influx of 30,min and more. Follow-up of DC/T cell interactions after 2,h using confocal microscopy revealed that long-lasting Ca2+ responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-Kb at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca2+ stimulators 7,h after initiation of maturation. Instead, the enhanced capacity of 7,h-matured DC to induce sustained Ca2+ responses in CD8+ T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8+ T cell stimulation. [source] Development of glutamate receptors in auditory neurons from long-term organotypic cultures of the embryonic chick hindbrainEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2009Carmen Diaz Abstract We used long-range organotypic cultures of auditory nuclei in the chick hindbrain to test the development of glutamate receptor activity in auditory neurons growing in a tissue environment that includes early deprivation of peripheral glutamatergic input, subsequent to removal of the otocyst. Cultures started at embryonic day (E)5, and lasted from 6 h to 15 days. Neuronal migration, clustering and axonal extension from the nucleus magnocellularis (NM) to the nucleus laminaris (NL) partially resembled events in vivo. However, the distinctive laminar organization of the NL was not observed. Glutamate receptor (GluR) activity was tested with optical recordings of intracellular Ca2+ in the NM. ,-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)/kainate receptors had Ca2+ responses with a time course similar to that in control slices. Peak amplitude, however, was significantly lower. N -methyl- d -aspartate (NMDA)-mediated Ca2+ responses were higher in 2-day cultures (E5 + 2d) than in E7 explant controls, returning later to control values. Metabotropic GluRs did not elicit Ca2+ responses at standard agonist doses. Blocking NMDA or AMPA/kainate receptors with specific antagonists for 10 days in culture did not limit neuronal survival. Blocking metabotropic GluRs resulted in complete neuronal loss. Thus, ionotropic GluRs are not required for NM neuronal survival. However, their activity during development is affected when neurons grow in an in vitro environment that includes prevention of arrival of peripheral glutamatergic input. [source] Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reportersEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005Kelly L. Rogers Abstract Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca2+ -sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi-functional Ca2+ reporter gene, GFP-aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260,7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable ,real-time' measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal-to-noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell,cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex-vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non-invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies. [source] Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003Jacqueline J. W. Van Dalen Abstract RC3 is a neuronal calmodulin-binding protein and protein kinase C substrate that is thought to play an important regulatory role in synaptic transmission and neuronal plasticity. Two molecules known to regulate synaptic transmission and neuronal plasticity are Ca2+ and calmodulin, and proposed mechanisms of RC3 action involve both molecules. However, physiological evidence for a role of RC3 in neuronal Ca2+ dynamics is limited. In the current study we utilized cultured cortical neurons obtained from RC3 knockout (RC3,/,) and wildtype mice (RC3+/+) and fura-2-based microscopic Ca2+ imaging to investigate a role for RC3 in neuronal Ca2+ dynamics. Immunocytochemical characterization showed that the RC3,/, cultures lack RC3 immunoreactivity, whereas cultures prepared from wildtype mice showed RC3 immunoreactivity at all ages studied. RC3+/+ and RC3,/, cultures were indistinguishable with respect to neuron density, neuronal morphology, the formation of extensive neuritic networks and the presence of glial fibrillary acidic protein (GFAP)-positive astrocytes and ,-aminobutyric acid (GABA)ergic neurons. However, the absence of RC3 in the RC3,/, neurons was found to alter neuronal Ca2+ dynamics including baseline Ca2+ levels measured under normal physiological conditions or after blockade of synaptic transmission, spontaneous intracellular Ca2+ oscillations generated by network synaptic activity, and Ca2+ responses elicited by exogenous application of N-methyl- d -aspartate (NMDA) or class I metabotropic glutamate receptor agonists. Thus, significant changes in Ca2+ dynamics occur in cortical neurons when RC3 is absent and these changes do not involve changes in gross neuronal morphology or neuronal maturation. These data provide direct physiological evidence for a regulatory role of RC3 in neuronal Ca2+ dynamics. [source] Intracellular Ca2+ responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell lineEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 3 2010Marit H. Aure Aure MH, Røed A, Kanli Galtung H. Intracellular Ca2+responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell line. Eur J Oral Sci 2010; 118: 237,244. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci The water channel aquaporin 5 (AQP5) seems to play a key role in salivary fluid secretion and appears to be critical in the cell volume regulation of acinar cells. Recently, the cation channel transient potential vanilloid receptor 4 (TRPV4) was shown to be functionally connected to AQP5 and also to cell volume regulation in salivary glands. We used the Simian virus 40 (SV40) immortalized cell line SMG C10 from the rat submandibular salivary gland to investigate the effect of ATP and the neurotransmitter analogue carbachol on Ca2+ signalling and cell volume regulation, as well as the involvement of TRPV4 in the responses. We used fura-2-AM imaging, cell volume measurements, and western blotting. Both carbachol and ATP increased the concentration of intracellular Ca2+, but no volume changes could be measured. Inhibition of TRPV4 with ruthenium red impaired both ATP- and carbachol-stimulated Ca2+ signals. Peak Ca2+ signalling during hyposmotic exposure was significantly decreased following inhibition of TRPV4, while the cells' ability to volume regulate appeared to be unaffected. These results show that in the SMG C10 cells, simulation of nervous stimulation did not induce cell swelling, although the cells had intact volume regulatory mechanisms. Furthermore, even though Ca2+ signals were not needed for this volume regulation, TRPV4 seems to play a role during ATP and carbachol stimulation. [source] Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytesGLIA, Issue 8 2008Erik B. Malarkey Abstract Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store-operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store-operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist-evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+ -dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells. © 2008 Wiley-Liss, Inc. [source] Effects of prolactin on intracellular calcium concentration and cell proliferation in human glioma cellsGLIA, Issue 3 2002Thomas Ducret Abstract Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription,polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca2+ concentration ([Ca2+]i) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca2+]i was measured by microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca2+ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4,4 nM) of PRL-stimulated Ca2+ entry and intracellular Ca2+ mobilization via a tyrosine kinase,dependent mechanism. The two types of Ca2+ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca2+]i. The amplitude of PRL-induced Ca2+ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [3H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells. GLIA 38:200,214, 2002. © 2002 Wiley-Liss, Inc. [source] Spatiotemporal analysis of NO production upon NMDA and tetanic stimulation of the hippocampusHIPPOCAMPUS, Issue 4 2005Norio Takata Abstract Nitric oxide (NO) is a gaseous neuromessenger. Although increasing evidence reveals significant physiological effects of NO in the hippocampal synaptic plasticity, the spatial distribution of NO production has remained largely uncharacterized due to the poor development of techniques for real-time NO imaging. In this work, using a NO-reactive fluorescent dye, diaminorhodamine-4M (DAR-4M), time-dependent heterogeneous NO production is demonstrated in hippocampal slices upon N-methyl- D -aspartate (NMDA) stimulation or tetanic stimulation. NMDA-induced DAR fluorescence increase in the CA1 was found to be twice that in the CA3 and the dentate gyrus (DG). Intracellular Ca2+ concentration was also investigated. NMDA induced similar Ca2+ responses both in the CA1 and DG, which were approx. 13% greater than that in the CA3. Subsequently, spatial distribution of NO production in the CA1 upon a tetanic stimulation of Schaffer collateral was investigated, because there are contradictory reports on the effect of NO on long-term potentiation (LTP), and that NO is known to exert various physiological effects depending on its concentration. In the stratum radiatum (sr), DAR fluorescence increase upon tetanus was largest at the vicinity of a stimulating electrode and decreased as a function of increasing distance from the stimulating electrode, suggesting the possibility that the effect of NO in LTP is dependent on the distance between stimulating and recording electrodes. The tetanus-induced Ca2+ response observed in the sr showed the same but weak distant dependence from the stimulating electrode. Taken together, the observed heterogeneity in the distribution of NO production is suggestive of region-specific effects of NO in the hippocampus. © 2005 Wiley-Liss, Inc. [source] The role of calcium in apoptosis induced by 7,-hydroxycholesterol and cholesterol-5,,6,-epoxideJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2009Sinéad Lordan Abstract Oxysterols, such as 7,-hydroxy-cholesterol (7,-OH) and cholesterol-5,,6,-epoxide (,-epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca2+) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca2+ changes on apoptosis induced by 7,-OH and ,-epoxide. Ca2+ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura-2. Over 15-min exposure of differentiated U937 cells to 30 ,M of 7,-OH induced a slow but significant rise in fura-2 ratio. The Ca2+ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca2+. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY-FLX-DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca2+ occurred through L-type channels. However, following long-term incubation with 7,-OH, elevated levels of cytoplasmic Ca2+ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca2+ may be an initial trigger of 7,-OH,induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca2+ on apoptotic cell death appears to be less significant. In contrast, Ca2+ did not appear to be involved in ,-epoxide,induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324,332, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20295 [source] Mitochondria and Ca2+ signalingJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2000Emil C. Toescu Abstract Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca2+ signaling. Mitochondria are capable to sense the levels of cytosolic Ca2+ and generate mitochondrial Ca2+ responses. Specific mechanisms for both Ca2+ uptake and Ca2+ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca2+ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca2+ transport systems and some of the functional consequences of mitochondrial Ca2+ uptake [source] Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2010Pei-Chun Wu J. Neurochem. (2010) 112, 1210,1222. Abstract In the present study, we characterized the Ca2+ responses and secretions induced by various secretagogues in mouse chromaffin cells. Activation of the acetylcholine receptor (AChR) by carbachol induced a transient intracellular Ca2+ concentration ([Ca2+]i) increase followed by two phases of [Ca2+]i decay and a burst of exocytic events. The contribution of the subtypes of AChRs to carbachol-induced responses was examined. Based on the results obtained by stimulating the cells with the nicotinic receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, high K+ and the effects of thapsigargin, it appears that activation of nAChRs induces an extracellular Ca2+ influx, which in turn activate Ca2+ -induced Ca2+ release via the ryanodine receptors. Muscarine, a muscarinic receptor (mAChRs) agonist, was found to induce [Ca2+]i oscillation and sustained catecholamine release, possibly by activation of both the receptor- and store-operated Ca2+ entry pathways. The RT-PCR results showed that mouse chromaffin cells are equipped with messages for multiple subtypes of AChRs, ryanodine receptors and all known components of the receptor- and store-operated Ca2+ entry. Furthermore, results obtained by directly monitoring endoplasmic reticulum (ER) and mitochondrial Ca2+ concentration and by disabling mitochondrial Ca2+ uptake suggest that the ER acts as a Ca2+ source, while the mitochondria acts as a Ca2+ sink. Our results show that both nAChRs and mAChRs contribute to the initial carbachol-induced [Ca2+]i increase which is further enhanced by the Ca2+ released from the ER mediated by Ca2+ -induced Ca2+ release and mAChR activation. This information on the Ca2+ signaling pathways should lay a good foundation for future studies using mouse chromaffin cells as a model system. [source] Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2008Kenji Nakao Abstract Activated astrocytes show various patterns of Ca2+ mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca2+ dynamics through investigation of thrombin-induced unique Ca2+ entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03,3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca2+ concentration ([Ca2+]i) showed that thrombin induced heterogeneous Ca2+ responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca2+ release from intracellular stores, followed by refilling of Ca2+ from the extracellular region without a direct [Ca2+]i increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca2+ mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca2+ entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca2+ responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca2+ influx. Moreover, TRPC3-mediated Ca2+ dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca2+ dynamics, including Ca2+ entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca2+ channel and might modulate astrocytic activation in response to brain hemorrhaging. © 2008 Wiley-Liss, Inc. [source] P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca2+ rise and change in mitochondrial potentialJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2007Stefan Kahlert Abstract With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca2+ level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y1/2/4 receptor agonist in rat, caused a large cytosolic Ca2+ increase in astrocytes and a considerably smaller Ca2+ response in neighboring neurons. The P2Y1 receptor antagonist MRS2179 completely blocked the ATP-induced Ca2+ response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca2+ level. Stimulation with UDP, a P2Y6 receptor agonist; UTP, a P2Y2/4 receptor agonist; 2MeSATP, a P2Y1 receptor agonist; or 2MeSADP, a P2Y1/12/13 receptor agonist, evoked significant Ca2+ responses in astrocytes but small Ca2+ responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca2+ peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca2+ level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain. © 2007 Wiley-Liss, Inc. [source] Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cellsTHE JOURNAL OF PHYSIOLOGY, Issue 20 2008Hidetada Matsuoka GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The ,1, ,3, ,2/3, ,2 and , subunits of GABAA receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl, currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only ,2-, but also ,-containing GABAA receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABAA receptors. Increases in Ca2+ signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca2+ responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only ,2-, but also ,-GABAA receptors. [source] Short-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in opposing agonist-induced Ca2+ responses in endothelial cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2008M Campos-Toimil Background and purpose: We previously reported that agonist-induced rises in cytoplasmic Ca2+ concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC) were inhibited after a short-term (2 min) pre-treatment with cAMP-elevating agents. The aim of this work was to study the effects of longer term (8 h) pre-treatment with dibutyryl-cAMP (db-cAMP) or rolipram, a specific inhibitor of phosphodiesterase-4 (PDE4), on [Ca2+]i, cAMP levels and PDE activity and expression in HUVEC. Experimental approach: [Ca2+]i changes were measured in isolated HUVEC by Fura-2 imaging. Intracellular cAMP levels and PDE4 activity were assessed by enzyme-immunoassay and radio-enzymatic assay, respectively. PDE expression was measured by northern and western blot analysis. Key results: Long-term pre-treatment of HUVEC with rolipram or db-cAMP significantly increased ATP-, histamine- and thrombin-induced [Ca2+]i rises. Short-term pre-treatment with rolipram was associated with an increase in cAMP, whereas long-term pre-treatment was associated with a decrease in cAMP. Long-term pre-treatment with rolipram or db-cAMP induced a significant increase in PDE4 activity and the expression of 74 kDa-PDE4A and 73 kDa-PDE4B was specifically enhanced. All these effects were suppressed by cycloheximide. Conclusions and implications: Our data suggest that sustained inhibition of PDE4 by rolipram induced an increase in PDE4 activity, possibly as a compensatory mechanism to accelerate cAMP degradation and that PDE4A and PDE4B were implicated in the regulation of [Ca2+]i. Thus, isozyme-specific PDE4 inhibitors might be useful as therapeutic agents in diseases where [Ca2+]i handling is altered, such as atherosclerosis, hypertension and tolerance to ,-adrenoceptor agonists. [source] Nociceptin/orphanin FQ inhibits capsaicin-induced guinea-pig airway contraction through an inward-rectifier potassium channelBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2002Yanlin Jia Nociceptin/orphanin FQ (N/OFQ), an endogenous opioid-like orphan receptor (NOP receptor, previously termed ORL1 receptor) agonist, has been found to inhibit capsaicin-induced bronchoconstriction in isolated guinea-pig lungs and in vivo. The underlying mechanisms are not clear. In the present studies, we tested the effect of N/OFQ on VR1 channel function in isolated guinea-pig nodose ganglia cells. Capsaicin increased intracellular Ca2+ concentration in these cells through activation of vanilloid receptors. Capsaicin-induced Ca2+ responses were attenuated by pretreatment of nodose neurons with N/OFQ (1 ,M). N/OFQ inhibitory effect on the Ca2+ response in nodose ganglia cells was antagonized by tertiapin (0.5 ,M), an inhibitor of inward-rectifier K+ channels, but not by verapamil, a voltage gated Ca2+ channel blocker, indicating that an inward-rectifier K+ channel is involved in N/OFQ inhibitory effect. In isolated guinea-pig bronchus, N/OFQ (1 ,M) inhibited capsaicin-induced airway contraction. Tertiapin (0.5 ,M) abolished the N/OFQ inhibition of capsaicin-induced bronchial contraction. Capsaicin (10 ,g) increased pulmonary inflation pressure in the isolated perfused guinea-pig lungs. This response was significantly attenuated by pretreatment with N/OFQ (1 ,M). Tertiapin also abolished the N/OFQ inhibitory effect on capsaicin-induced bronchoconstriction in perfused lungs. Capsaicin increased the release of substance P and neurokinin A from isolated lungs. N/OFQ (1 ,M) blocked the capsaicin-induced tachykinin release. These results indicate that N/OFQ-induced hyperpolarization of tachykinin containing airway sensory nerves, through an inward-rectifier K+ channel activation, accounts for the inhibition of capsaicin-evoked broncoconstriction. British Journal of Pharmacology (2002) 135, 764,770; doi:10.1038/sj.bjp.0704515 [source] | |||||||||||||