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Intracellular Ca2+ Concentration (intracellular + ca2+_concentration)
Selected AbstractsMultiple P2 Receptors Contribute to a Transient Increase in Intracellular Ca2+ Concentration in Atp-Stimulated Rat Brown AdipocytesEXPERIMENTAL PHYSIOLOGY, Issue 6 2002Mariko Omatsu-Kanbe Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca2+ concentration ([Ca2+]i), which arises primarily from a release of Ca2+ from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca2+]i elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2,,4,-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 ,M) almost completely depressed the thapsigargin (100 nM)-evoked [Ca2+]i elevation mediated through store-operated Ca2+ entry. The inhibitory effect of ATP was antagonized by PPADS with IC50 of 0.7 ,M. In the presence of PPADS at concentrations of more than 5 ,M, the ATP-induced [Ca2+]i elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca2+]i elevation was reduced in a concentration-dependent manner by PPADS with an IC50 of 200 ,M. In contrast, the ATP-induced [Ca2+]i elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca2+]i responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca2+ stores, which is probably followed by the activation of store-operated Ca2+ entry. Extracellular ATP, however, would inhibit this Ca2+ entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca2+]i elevation in response to extracellular ATP. [source] Enhanced Calcium Influx in Hippocampal CA3 Neurons of Spontaneously Epileptic RatsEPILEPSIA, Issue 3 2001Hiroko Amano Summary: ,Purpose: The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous electrophysiologic studies have demonstrated that SER has abnormal excitability of hippocampal CA3 neurons, which shows a long-lasting depolarization shift by a single stimulation of mossy fibers, probably resulting from the Ca2+ channel abnormalities. The present study was performed to determine whether Ca2+ influx is actually enhanced in the CA3 area of SER. Methods: Hippocampal slices were prepared from normal Wistar rats and SER aged 11,16 weeks old, when the epileptic seizures had been observed, and loaded with fura-2AM. Intracellular Ca2+ concentration ([Ca2+]i) was monitored as the ratio of fluorescence intensities excited at wavelengths of 340 and 380 nm (RF340/F380) with photometric devices. Results: High K+ (10,60 mM) applied to the bath for 2 min increased [Ca2+]i in hippocampal CA1, CA3, and dentate gyrus (DG) areas of both the normal rats and SER in a concentration-dependent manner. However, the high K+,induced increase in [Ca2+]i was significantly more pronounced in the CA3 area of the SER than in that of the normal animals, whereas there were no significant differences in high K+,induced increases of [Ca2+]i in CA1 or DG between the SER and controls. The high K+,induced increases in [Ca2+]i of CA1, CA3, and DG were inhibited by nifedipine (1,10 nM), a Ca2+ channel antagonist in both SER and controls. However, the inhibition of the high K+,induced increase in [Ca2+]i by nifedipine (1 nM) was significantly greater in the CA3 area of SER than that of controls. Conclusions: These findings suggest that Ca2+ influx through the L-type Ca2+ channels is much greater in the CA3 area of SER than in that of normal animals and is involved in the epileptic seizures of the SER. [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] Sperm binding to the human zona pellucida and calcium influx in response to GnRH and progesteroneANDROLOGIA, Issue 5 2002P. Morales Summary. In this study the effect of the sequential exposure of spermatozoa to progesterone and gonadotrophin-releasing hormone (GnRH) upon zona binding and the intracellular free Ca2+ concentration was evaluated. Sperm aliquots were treated as follows: (a) 0.7 ,mol 1,1 progesterone or 0.1% DMSO (progesterone solvent) followed by 50 nmol 1,1 of GnRH; (b) 50 nmol 1,1 of GnRH or distilled water (GnRH solvent) followed by 0.7 ,mol 1,1 of progesterone. Additional aliquots were incubated with DMSO or distilled water (controls) and with 0.7 ,mol 1,1 of progesterone or 50 nmol 1,1 of GnRH. All treatments were for 5 min. Motile spermatozoa were incubated in modified Tyrode's medium, at 37 °, 5% CO2, 10times106 spermatozoa ml,1, for 4.5 h. Intracellular Ca2+ concentration and sperm-zona binding was evaluated using fura 2 and the hemizona assay, respectively. GnRH and progesterone increased sperm-zona binding and the Ca2+ concentration. Regarding zona binding, the effect of GnRH was significantly greater when the spermatozoa had been previously treated with progesterone (progesterone , GnRH = 185 ± 116 zona-bound spermatozoa versus DMSO , GnRH = 99 ± 15, P <0.001). On the other hand, previous treatment with GnRH did not modify their subsequent response to progesterone (GnRH , progesterone = 114 ±19 zona-bound spermatozoa versus distilled water , progesterone = 108 ± 22, NS). The results regarding intracellular Ca2+ showed a similar pattern. These findings suggest a priming effect of progesterone upon a GnRH-induced increase in sperm-zona binding and intracellular Ca2+. [source] The Cl, channel blocker niflumic acid releases Ca2+ from an intracellular store in rat pulmonary artery smooth muscle cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2003Stuart F Cruickshank The effect of the Cl, channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4,-diisothiocyanatostilbene-2,2,-disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine-induced increase in [Ca2+]i. These Cl, channel blockers also increased the half-time (t1/2) to peak for the caffeine-induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca2+ in studies involving these Cl, channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl, channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR. British Journal of Pharmacology (2003) 140, 1442,1450. doi:10.1038/sj.bjp.0705571 [source] Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of ,7 nicotinic receptors and internalization of NMDA receptorsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2010H Shen BACKGROUND AND PURPOSE Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil. EXPERIMENTAL APPROACH Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca2+ concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD. KEY RESULTS Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate-mediated Ca2+ entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of ,7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca2+ entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca2+ entry induced by donepezil. CONCLUSIONS AND IMPLICATIONS Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of ,7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system. [source] Roles of P2X receptors and Ca2+ sensitization in extracellular adenosine triphosphate-induced hyperresponsiveness in airway smooth muscleCLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2007T. Oguma Summary Background The release of adenosine triphosphate (ATP) from the airway epithelial cells during the inflammatory process is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. Objective This study was designed to determine whether extracellular ATP is involved in the bronchial hyperresponsiveness as an interaction between epithelium and smooth muscle in the airways. Methods We examined the contractile response to methacholine (MCh) before and after exposure to low concentrations (10 ,m) of ATP in isolated, epithelium-denuded guinea-pig tracheal smooth muscle by measuring isometric tension. Intracellular Ca2+ concentrations ([Ca2+]i) were assessed by fluorescent intensities of fura-2. Results MCh-induced contractile force was increased with no change in [Ca2+]i after exposure to 10 ,m ATP for 15 min. The ability of ATP to enhance the MCh-induced contraction was markedly attenuated by suramin, a non-selective P2 receptor inhibitor. Pre-incubation with ATP,S, a non-hydrolysable analogue of ATP and ,,,-meATP, a P2X agonist, also enhanced the MCh-induced contraction. In contrast, uracil triphosphate, a P2Y agonist, did not affect the MCh-induced contraction. Y-27632, a Rho-kinase inhibitor, suppressed the ability of ATP to enhance the MCh-induced contraction. Moreover, PP1 and PP2, Src tyrosin kinase inhibitors, suppressed the enhancement of MCh-induced contraction by ATP. Conclusion Pre-treatment with ATP induces hyperresponsiveness to MCh mediated by Ca2+ sensitization via the P2X receptor in airway smooth muscle. The present findings suggest the possible involvement of both the Rho-kinase and Src pathways in the intracellular mechanism of this phenomenon. [source] Developmental characteristics of AMPA receptors in chick lumbar motoneuronsDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2007Xianglian Ni Abstract Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes, including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits. In this study, we tested the possibility that changes in GluR2 expression regulate the Ca2+ permeability of AMPA receptors during a critical period of neuronal development in chick lumbar motoneurons. GluR2 expression is absent between embryonic day (E) 5 and E7, but increases significantly by E8 in the chick ventral spinal cord. Increased GluR2 protein expression is correlated with parallel changes in GluR2 mRNA in the motoneuron pool. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2+ -permeability of AMPA receptors between E6 and E11. Kainate-evoked currents were sensitive to the AMPA receptor blocker GYKI 52466. Application of AMPA or kainate generates a significant increase in the intracellular Ca2+ concentration in E6 spinal motoneurons, but generates a small response in older neurons. Changes in the Ca2+ -permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. These findings raise the possibility that Ca2+ influx through Ca2+ -permeable AMPA receptors plays an important role during early embryonic development in chick spinal motoneurons. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [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] Intracellular Calcium Increase in Epileptiform Activity: Modulation by Levetiracetam and LamotrigineEPILEPSIA, Issue 7 2004Antonio Pisani Summary:,Purpose: Alterations in neuronal calcium (Ca2+) homeostasis are believed to play an essential role in the generation and propagation of epileptiform events. Levetiracetam (LEV) and lamotrigine (LTG), novel antiepileptic drugs (AEDs), were tested on epileptiform events and the corresponding elevations in intracellular Ca2+ concentration ([Ca2+]i) recorded from rat neocortical slices. Methods: Electrophysiological recordings were performed from single pyramidal neurons from a slice preparation. Spontaneous epileptiform events consisting of long-lasting, repetitive paroxysmal depolarization shifts (PDSs) and interictal spike activity were induced by reducing the magnesium concentration from the solution and by adding bicuculline and 4-aminopyridine. Simultaneously, microfluorimetric measurements of [Ca2+]i were performed. Optical imaging with Ca2+ indicators revealed a close correlation between Ca2+ transients and epileptiform events. Results: Both LEV and LTG were able to reduce both amplitude and duration of PDSs, as well as the concomitant elevation in [Ca2+]i, in a dose-dependent fashion. Whole-cell patch-clamp recordings from isolated neocortical neurons revealed that LEV significantly reduced N-, and partially P/Q-type high-voltage-activated (HVA) Ca2+ currents, whereas sodium currents were unaffected. Interestingly, the inhibitory effects of LEV were mimicked and occluded by LTG or by a combination of ,-conotoxin GVIA and ,-agatoxin IVA, selective blockers of N- and P/Q-type HVA channels, respectively, suggesting a common site of action for these AEDs. Conclusions: These results demonstrate that large, transient elevations in neuronal [Ca2+]i correlate to epileptiform discharges. The antagonistic effects of LEV and LTG on [Ca2+]i overload might represent the basis for their anticonvulsant efficacy and could preserve neuronal viability. [source] Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell linesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2006Antonella Meini Abstract To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 µm) determined a gradual, moderate elevation in [Ca2+]i (46.8 ± 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 ± 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 ± 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50,300 µm) of DETA/NO negatively regulated cell proliferation via a Ca2+ -independent mechanism. [source] No evidence for calcium electrogenic exchanger in frog semicircular canal hair cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2002M. Martini Abstract We investigated the possibility that, in hair cells mechanically isolated from frog semicircular canals, Ca2+ extrusion occurs via a Na+ : Ca2+ (cardiac type) or a Na+ : Ca2+,K+ (retinal type) exchanger. Cells concurrently imaged during whole-cell patch-clamp recordings using the Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (100 µm) showed no voltage dependence of Ca2+ clearance dynamics following a Ca2+ load through voltage-gated Ca2+ channels. Reverse exchange was probed in hair cells dialyzed with a Ca2+ - and K+ -free solution, containing a Na+ concentration that saturates the exchanger, after zeroing the contribution to the whole-cell current from Ca2+ and K+ conductances. In these conditions, no reverse exchange current was detected upon switching from a Ca2+ -free external solution to a solution containing concentrations of Ca2+ alone, or Ca2+ + K+ that saturated the exchanger. By contrast, the same experimental protocol elicited peak exchange currents exceeding 100 pA in gecko rod photoreceptors, used as positive controls. In both cell types, we also probed the forward mode of the exchanger by rapidly increasing the intracellular Ca2+ concentration using flash photolysis of two novel caged Ca2+ complexes, calcium 2,2,-{[1-(2-nitrophenyl)ethane-1,2-diyl]bis(oxy)}bis(acetate) and calcium 2,2,-{[1-(4,5-dimethoxy-2-nitrophenyl)ethane-1,2-diyl]bis(oxy)} bis(acetate), in the presence of internal K+ and external Na+. No currents were evoked by UV-triggered Ca2+ jumps in hair cells, whereas exchanger conformational currents up to 400 pA, followed by saturating forward exchange currents up to 40 pA, were recorded in rod photoreceptors subjected to the same experimental conditions. We conclude that no functional electrogenic exchanger is present in this hair cell population, which leaves the abundant plasma membrane Ca2+ -ATPases as the primary contributors to Ca2+ extrusion. [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] Cell shrinkage evoked by Ca2+ -free solution in rat alveolar type II cells: Ca2+ regulation of Na+,H+ exchangeEXPERIMENTAL PHYSIOLOGY, Issue 2 2005Hitoshi Murao The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT-II cells) were examined. Perfusion with a Ca2+ -free solution induced shrinkage of the AT-II cell volume in the absence or presence of amiloride (1 ,m, an inhibitor of Na+ channels); however, it did not in the presence of 5-(N -methyl- N -isobutyl)-amiloride (MIA, an inhibitor of Na+,H+ exchange). MIA decreased the volume of AT-II cells. Inhibitors of Cl,,HCO3, exchange, 4,4,-diisothiocyanostilbene-2,2,-disulfonic acid (DIDS) and 4-acetamido-4,-isothiocyanatostilbene-2,2,-disulfonic acid (SITS) also decreased the volume of AT-II cells. This indicates that the cell shrinkage induced by a Ca2+ -free solution is caused by a decrease in NaCl influx via Na+,H+ exchange and Cl,,HCO3, exchange. Addition of ionomycin (1 ,m), in contrast, induced cell swelling when AT-II cells were pretreated with quinine and amiloride. This swelling of the AT-II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+ -free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA-treated AT-II cells. Ionomycin increased [Ca2+]i in fura-2-loaded AT-II cells. In conclusion, the Na+,H+ exchange activities of AT-II cells, which maintain the volume and pHi, are regulated by [Ca2+]i. [source] Calcium-sensing receptor mediates phenylalanine-induced cholecystokinin secretion in enteroendocrine STC-1 cellsFEBS JOURNAL, Issue 18 2008Tohru Hira Intraluminal l -phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK-producing enteroendocrine cells sense Phe is unknown. The calcium-sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK-producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK-producing enteroendocrine cell line STC-1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose-dependent CCK secretion and intracellular Ca2+ mobilization in STC-1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe-induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT-PCR in STC-1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC-1 cells. [source] Nucleotide-induced Ca2+ signaling in sustentacular supporting cells of the olfactory epitheliumGLIA, Issue 15 2008Thomas Hassenklöver Abstract Extracellular purines and pyrimidines are important signaling molecules acting via purinergic cell-surface receptors in neurons, glia, and glia-like cells such as sustentacular supporting cells (SCs) of the olfactory epithelium (OE). Here, we thoroughly characterize ATP-induced responses in SCs of the OE using functional Ca2+ imaging. The initial ATP-induced increase of the intracellular Ca2+ concentration [Ca2+]i always occurred in the apical part of SCs and subsequently propagated toward the basal lamina, indicating the occurrence of purinergic receptors in the apical part of SCs. The mean propagation velocity of the Ca2+ signal within SCs was 17.10 ± 1.02 ,m/s. ATP evoked increases in [Ca2+]i in both the presence and absence of extracellular Ca2+. Depletion of the intracellular Ca2+ stores abolished the responses. This shows that the ATP-induced [Ca2+]i increases were in large part, if not entirely, due to the activation of G protein-coupled receptors followed by Ca2+ mobilization from intracellular stores, suggesting an involvement of P2Y receptors. The order of potency of the applied purinergic agonists was UTP > ATP > ATP,S (with all others being only weakly active or inactive). The ATP-induced [Ca2+]i increases could be reduced by the purinergic antagonists PPADS and RB2, but not by suramin. Our findings suggest that extracellular nucleotides in the OE activate SCs via P2Y2/P2Y4 -like receptors and initiate a characteristic intraepithelial Ca2+ wave. © 2008 Wiley-Liss, Inc. [source] Alpha-fetoprotein-specific transfer factors downregulate alpha-fetoprotein expression and specifically induce apoptosis in Bel7402 alpha-fetoprotein-positive hepatocarcinoma cellsHEPATOLOGY RESEARCH, Issue 7 2007Hui Zhang Aim:, To investigate the mechanisms of AFP-specific transfer factors (AFP-TF) in induced Bel7402 cells apoptosis. Further, we investigate the interaction between AFP-TF and AFP in the apoptosis. Methods:, Bel7402 and HepG2 AFP-positive hepatocarcinoma cell lines, SK-Hep-1 AFP-negative hepatocarcinoma cell line and Changliver normal liver cell line are used. Cell viability is evaluated by MTT assay and apoptosis is measured by Hoechst33342 staining and TUNEL assay. FACS is used to analyze the cell cycle. AFP expression is examined by RT-PCR, Western blotting and immunocytochemistry. The interaction between AFP-TF and AFP in the apoptosis is investigated by addition of AFP in cultures or AFP transfection in Bel7402 cells prior to AFP-TF treatment. Mitochondrial membrane potential (,,m) and intracellular Ca2+ concentration are respectively measured by Rhodamine123 and Fluo-3 AM Ester. Western blotting detects the involvement of several apoptosis-related proteins. Finally, caspase-3 and Caspase-9 activity are respectively examined. Results:, AFP-TF can induce apoptosis in Bel7402 and HepG2 AFP-positive hepatocarcinoma cells, but not SK-Hep-1 and Changliver cells. AFP-mRNA level changes little in apoptotic Bel7402 cells; while AFP expression is downregulated and uniformly dispersed throughout the whole cell. Addition of exogenous AFP or overexpression of intracellular AFP can reduce such apoptotic effect. Besides, apoptotic Bel7402 cells show a disruption of ,,m, an immediate elevation of Ca2+ concentration, a prominently decreased ratio of bcl-2 to bax, a release of cytochrome c from mitochondria to cytosol, and ultimately an activation of caspase-9 and caspase-3. Conclusion:, AFP-TF induced Bel7402 cells apoptosis is mitochondrial-dependent and is mediated by the interaction of AFP-TF with intracellular AFP. [source] Sevoflurane and propofol depolarize mitochondria in rat and human cerebrocortical synaptosomes by different mechanismsACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 10 2009R. BAINS Background and objectives: The mitochondrial membrane potential drives the main functions of the mitochondria. Sevoflurane depolarizes neural mitochondria. There is still, however, limited information concerning the effect of anaesthetics on neural mitochondria in humans. The effect of sevoflurane and propofol on the intracellular Ca2+ concentration [Ca2+]i and the mitochondrial membrane potential (,,m) was therefore compared in rat and human synaptosomes, and the changes were related to interventions in the electron transport chain. Methods: Synaptosomes from rat and human cerebral cortex were loaded with the fluorescent probes fura-2 ([Ca2+]i) and JC-1 (,,m) before exposure to sevoflurane 1 and 2 minimum alveolar concentration (MAC), and propofol 30 and 100 ,M. The effect on the electron transport chain was investigated by blocking complex V. Results: Sevoflurane and propofol decreased ,,m in rat synaptosomes in a dose-dependent manner, and to the same extent by equipotent doses. Inhibition of complex V enhanced the depolarizing effect of sevoflurane 2 MAC, but not of propofol 100 ,M. Neither sevoflurane nor propofol affected [Ca2+]i significantly. Sevoflurane and propofol decreased ,,m in human synaptosomes to the same extent as in the rat experiments. Conclusions: Sevoflurane and propofol at equipotent doses depolarize the mitochondria in rat and human nerve terminals to the same extent. The depolarizing effect of propofol on ,m was more rapid in onset than that of sevoflurane. Whereas sevoflurane inhibits the respiratory chain sufficiently to cause ATP synthase reversal, the depolarizing effect of propofol seems to be related to inhibition of the respiratory chain from complex I to V. [source] Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Response to Acidic pH Through OGR1 in a Human Osteoblastic Cell Line,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2008Hideaki Tomura Abstract Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase-2 (COX-2) induction and prostaglandin E2 (PGE2) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX-2 induction and PGE2 production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca2+ concentration ([Ca2+]i), PGE2 production, and COX-2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification-induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca2+]i and inositol phosphate production in the cells. Acidification also induced COX-2 induction, resulting in PGE2 production. These proton-induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for Gq/11 protein, phospholipase C, and protein kinase C. We conclude that the OGR1/Gq/11/phospholipase C/protein kinase C pathway regulates osteoblastic COX-2 induction and subsequent PGE2 production in response to acidic circumstances. [source] The classic receptor for 1,,25-dihydroxy vitamin D3 is required for non-genomic actions of 1,,25-dihydroxy vitamin D3 in osteosarcoma cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2006Soraya Bravo Abstract 1,,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1,,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1,,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1,,25-dihydroxy vitamin D3. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1,,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1,,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca2+ concentration, a hallmark of the non-genomic actions of 1,,25-dihydroxy vitamin D3 in these cells. J. Cell. Biochem. 99: 995,1000, 2006. © 2006 Wiley-Liss, Inc. [source] Prolactin secretion and intracellular Ca2+ change in rat lactotroph subpopulations stimulated by thyrotropin-releasing hormone,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2002Chih-Yang Huang Abstract Thyrotropin-releasing hormone (TRH) may stimulate lactotrophs to increase intracellular Ca2+ and to secrete prolactin (PRL). In this study, PRL contents in lactotrophs were determined by the sequential cell immunoblot assay (SCIBA) and their changes in intracellular Ca2+ was analyzed by confocal microscopy. Significant correlations were found in the corresponding parameters between TRH treatments with a recovery interval of 2 h. Measuring the PRL contents after the first TRH treatment and then determining the intracellular Ca2+ changes after the second TRH treatment revealed four lactotroph subpopulations. Type I cells (51%) showed significant responses of both PRL secretion and intracellular Ca2+ concentration. Type II cells (22%) increased in PRL secretion, but without changes in intracellular Ca2+. Type III cells (17%) have increased in intracellular Ca2+, but without changes in PRL secretion. Type IV cells (10%) did not show changes in PRL secretion and intracellular Ca2+. J. Cell. Biochem. 87: 126,132, 2002. © 2002 Wiley-Liss, Inc. [source] Infiltration anesthetic lidocaine inhibits cancer cell invasion by modulating ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF)JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2002Tadanori Mammoto Although the mechanism is unknown, infiltration anesthetics are believed to have membrane-stabilizing action. We report here that such a most commonly used anesthetic, lidocaine, effectively inhibited the invasive ability of human cancer (HT1080, HOS, and RPMI-7951) cells at concentrations used in surgical operations (5,20 mM). Ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) from the cell surface plays an important role in invasion by HT1080 cells. Lidocaine reduced the invasion ability of these cells by partly inhibiting the shedding of HB-EGF from the cell surface and modulation of intracellular Ca2+ concentration contributed to this action. The anesthetic action of lidocaine (sodium channel blocking ability) did not contribute to this anti-invasive action. In addition, lidocaine (5,30 mM), infiltrated around the inoculation site, inhibited pulmonary metastases of murine osteosarcoma (LM 8) cells in vivo. These data point to previously unrecognized beneficial actions of lidocaine and suggest that lidocaine might be an ideal infiltration anesthetic for surgical cancer operations. © 2002 Wiley-Liss, Inc. [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] Mechanism of the persistent sodium current activator veratridine-evoked Ca2+ elevation: implication for epilepsyJOURNAL OF NEUROCHEMISTRY, Issue 3 2009Ádám Fekete Abstract Although the role of Na+ in several aspects of Ca2+ regulation has already been shown, the exact mechanism of intracellular Ca2+ concentration ([Ca2+]i) increase resulting from an enhancement in the persistent, non-inactivating Na+ current (INa,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na+ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na+ concentration ([Na+]i) and biphasic [Ca2+]i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca2+ response was tetrodotoxin- and extracellular Ca2+ -dependent and ionotropic glutamate receptor-independent. The first phase of [Ca2+]i rise was the net result of Ca2+ influx through voltage-gated Ca2+ channels and mitochondrial Ca2+ sequestration. The robust second phase in addition involved reverse operation of the Na+,Ca2+ exchanger and mitochondrial Ca2+ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non-inactivating Na+ current and [Ca2+]i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with INa,P. Describing the magnitude, temporal pattern and sources of Ca2+ increase induced by INa,P may provide novel targets for antiepileptic drug therapy. [source] A family of octapamine receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogasterJOURNAL OF NEUROCHEMISTRY, Issue 2 2005Sabine Balfanz Abstract In invertebrates, the biogenic-amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, ,fight or flight' responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP-binding protein (G protein)-coupled receptor types, which induce either cAMP production or Ca2+ release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca2+ concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC50 of ,3 × 10,8 m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at ,,100-fold higher concentrations, whereas dopamine and serotonin are non-effective. Developmental expression of Dmoa genes was assessed by RT,PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors. [source] 2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand in the nervous tissueJOURNAL OF NEUROCHEMISTRY, Issue 2003K. Waku 2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol identified as an endogenous cannabinoid receptor ligand by us and Mechoulam and co-workers (1). We found that 2-AG possesses binding activity toward the cannabinoid receptor in rat brain. We also found that 2-AG induces transient elevation of the intracellular Ca2+ concentration in NG108-15 cells. The response induced by 2-AG was blocked by pretreatment of cells with a cannabinoid CB1 receptor-specific antagonist SR141716A, indicating that CB1 receptor is involved in the response. Based on the results of structure,activity relationship experiments, we concluded that the cannabinoid CB1 receptor in the nervous system is originally and primary a 2-AG receptor. 2-AG was produced and released from nervous tissues following various types of stimulation through enhanced breakdown of arachidonic acid-containing phospholipids such as inositol phospholipids. Physiological and pathophysiological roles of 2-AG in the nervous system will be discussed. [source] Transient rise in intracellular calcium produces a long-lasting increase in plasma membrane calcium pump activity in rat sensory neuronsJOURNAL OF NEUROCHEMISTRY, Issue 4 2002William J. Pottorf II Abstract The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Calmodulin stimulates PMCA activity and for some isoforms this activation persists following clearance of Ca2+ owing to the slow dissociation of calmodulin. We tested the hypothesis that PMCA-mediated Ca2+ efflux from rat dorsal root ganglion (DRG) neurons in culture would remain stimulated following increases in intracellular Ca2+ concentration ([Ca2+]i). PMCA-mediated Ca2+ extrusion was recorded following brief trains of action potentials using indo-1-based photometry in the presence of cyclopiazonic acid. A priming stimulus that increased [Ca2+]i to 506 ± 28 nm (>15 min) increased the rate constant for [Ca2+]i recovery by 47 ± 3%. Ca2+ clearance from subsequent test stimuli remained accelerated for up to an hour despite removal of the priming stimulus and a return to basal [Ca2+]i. The acceleration depended on the magnitude and duration of the priming [Ca2+]i increase, but was independent of the source of Ca2+. Increases in [Ca2+]i evoked by prolonged depolarization, sustained trains of action potentials or activation of vanilloid receptors all accelerated Ca2+ efflux. We conclude that PMCA-mediated Ca2+ efflux in DRG neurons is a dynamic process in which intense stimuli prime the pump for the next Ca2+ challenge. [source] Cyclic ADP-ribose as a potential second messenger for neuronal Ca2+ signalingJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Haruhiro Higashida Cyclic ADP-ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization-induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+ -dependent inward currents. cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP-ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP-dependent phosphorylation. Stimulation of muscarinic and ,-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed. [source] Lysophosphatidic Acid Inhibits Ca2+ Signaling in Response to Epidermal Growth Factor Receptor Stimulation in Human Astrocytoma Cells by a Mechanism Involving Phospholipase C, and a G,i ProteinJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Marita Hernández Abstract: The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca2+ concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca2+ transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase C, (PLC,)-1, and G,i protein. LPA was found to induce coupling of PLC,-1 to the EGFR by a mechanism involving a G,i protein, in the absence of tyrosine phosphorylation of both PLC, and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G,i -protein-mediated translocation of PLC,-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins. [source] Bradykinin and Angiotensin II-Induced [Ca2+]i Rise in Cultured Rat Pituitary Folliculo-Stellate CellsJOURNAL OF NEUROENDOCRINOLOGY, Issue 11 2001T. Sudo Abstract Folliculo-stellate cells of the anterior pituitary are thought to modulate pituitary hormone secretion through a paracrine mechanism. Angiotensin II and pituitary adenylate cyclase-activating polypeptide (PACAP) have previously been shown to increase the intracellular Ca2+ concentration ([Ca2+]i) of these cells. In the present study, we examined the effects of various peptides such as bradykinin, angiotensin II, endothelin-1, PACAP, galanin and neurotensin by Ca2+ -imaging of folliculo-stellate cells in primary culture. Bradykinin and angiotensin II increased [Ca2+]i in folliculo-stellate cells. Both responses were completely suppressed by thapsigargin and were significantly suppressed by the phospholipase C inhibitor, U-73122. Ryanodine did not significantly modify the responses. A B2 antagonist and angiotensin II receptor antagonist inhibited the response induced by bradykinin and angiotensin II, respectively. Endothelin-1 and PACAP increased [Ca2+]i in fewer than 50% of folliculo-stellate cells but galanin and neurotensin did not influence [Ca2+]i in any of the folliculo-stellate cells tested. These results indicate that bradykinin and angiotensin II increase [Ca2+]i in folliculo-stellate cells by activating phospholipase C through B2 receptor and AT1 receptor, respectively, and that endothelin-1 and PACAP also increase [Ca2+]i in some folliculo-stellate cells. [source] | ||||||||||||||||||||||||||||