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Extracellular Ca2+ (extracellular + ca2+)
Terms modified by Extracellular Ca2+ Selected AbstractsRegulation of E-cadherin and ,-catenin by Ca2+ in colon carcinoma is dependent on calcium-sensing receptor expression and functionINTERNATIONAL JOURNAL OF CANCER, Issue 7 2007Narasimharao Bhagavathula Abstract An siRNA directed against the extracellular calcium-sensing receptor (CaSR) was used to down-regulate this protein in CBS colon carcinoma cells. In additional studies, we utilized a variant of the parental CBS line that demonstrates CaSR expression but does not upregulate this protein in response to extracellular Ca2+. In neither the siRNA-transfected cells nor the Ca2+ -nonresponsive variant cells did inclusion of Ca2+ in the culture medium inhibit proliferation or induce morphological alterations. Extracellular Ca2+ also failed to induce E-cadherin production or a shift in ,-catenin from the cytoplasm to the cell membrane. In mock-transfected cells and in a Ca2+ -responsive variant line derived from the same parental CBS cells, Ca2+ treatment resulted in growth-reduction. This was accompanied by increased E-cadherin production and a shift in ,-catenin distribution from the cytoplasm to the cell membrane. Additionally, down-regulation of c-myc and cyclin D1 expression was observed in mock-transfected cells and in the Ca2+ -responsive variant line (along with reduced T cell factor transcriptional activation). Neither c-myc nor cyclin D1 was significantly down-regulated in the siRNA-transfected cells or in the Ca2+ -nonresponsive variant cells upon Ca2+ stimulation. In histological sections of human colon carcinoma CaSR was significantly reduced as compared to the level in normal colonic crypt epithelial cells. Where CaSR expression was high, strong surface staining for E-cadherin and ,-catenin was observed. Where CaSR expression was reduced, ,-catenin surface expression was likewise reduced. © 2007 Wiley-Liss, Inc. [source] Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspectsACTA PHYSIOLOGICA, Issue 1 2009N. Fedirko Abstract Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca2+ store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3. We have also investigated the possible consequences of nuclear calcium signals: the role of Ca2+ in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca2+ -mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca2+: the IEG expression level was lowest in Ca2+ -free solution, increased at the physiological level of 1 mm [Ca2+]o and was maximal at 10 mm [Ca2+]o, i.e.: 102 ± 22% and 163 ± 15% for c-fos; c-myc ,73 ± 13% and 106 ± 24%; c-jun ,49 ± 8% and 59 ± 9% at 1 and 10 mm of extracellular Ca2+ respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca2+ together with nuclear Ca2+ signals induced by CCK play important roles in the induction of IEG expression. [source] Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+ACTA PHYSIOLOGICA, Issue 4 2006L. Lacinová Abstract Aim:, We have investigated the influence of Ca2+ ions on the basic biophysical properties of T-type calcium channels. Methods:, The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 ,m Ca2+ into the non-buffered intracellular solution. Various combinations of extra- and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels. Results:, The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ,100 mV for the Ca2+ current, but did not reach steady state even at ,150 mV for the Na+ current. Conclusions:, Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor. [source] Reduced calcium tolerance in rat cardiomyocytes after myocardial infarctionACTA PHYSIOLOGICA, Issue 4 2002I. Sjaastad ABSTRACT During ischaemia and reperfusion the intracellular Na+ concentration is elevated in the cardiomyocytes and the cells are depolarized, both favouring reverse mode Na,Ca-exchange loading of the cell with Ca2+. We examined whether cardiomyocytes from rats with congestive heart failure (CHF) and younger rats (HINCX) which both have a high expression of the Na,Ca-exchanger protein (NCX) showed reduced tolerance to extracellular Ca2+. The CHF was induced in Isofluran anaesthetized rats by left coronary artery ligation. Isolated cardiomyocytes were loaded with Fura-2AM and 140 mm Na+ and exposed to 0.05 mm Ca2+. Expression of the Na,Ca-exchanger protein was analysed. Fura-2 340/380 ratio rose more rapidly in HINCX and CHF than in SHAM, and the rise was abolished by Ni2+. Hypercontracture developed more frequently in HINCX and CHF than in SHAM cells. The amount of NCX was 54% higher in HINCX and 76% higher in CHF compared with SHAM. Na+ -loaded cardiomyocytes from CHF and HINCX rats are more susceptible to Ca2+ overload than SHAM cells because of the increased capacity for Na,Ca-exchange. [source] Protein tyrosine phosphatases in Chaetopterus egg activationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Shantá D. Hinton Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTP,, -,, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with ,-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process. [source] Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survivalDEVELOPMENTAL NEUROBIOLOGY, Issue 13 2009Laura Lossi Abstract Apoptosis can be modulated by K+ and Ca2+ inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca2+ signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K+/Ca2+ homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K+]e and [Ca2+]i to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K+]e under conditions of immaturity, is independent of extracellular Ca2+ and operates via IP3 channels. The second leads to influx of extracellular Ca2+ following activation of ryanodine channels in the presence of physiological [K+]e, when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Survival of mammalian B104 cells following neurite transection at different locations depends on somal Ca2+ concentrationDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2004Soonmoon Yoo Abstract We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long-standing observations that somal survival decreases with more-proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine- or thapsigargin-induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+ -induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 137,153, 2004 [source] Econazole-induced Ca2+ fluxes and apoptosis in human oral cancer cellsDRUG DEVELOPMENT RESEARCH, Issue 4 2010Daih-Huang Kuo Abstract The effect of econazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations of >1,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole-induced Ca2+ influx was sensitive to blockade of aristolochic acid (phospholipase A2 inhibitor) and GF109203X (PKC inhibitor). In Ca2+ -free medium, after treatment with 1,µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 30,µM econazole failed to induce a [Ca2+]i rise. Inhibition of phospholipase C with 2,µM U73122 substantially suppressed econazole-induced [Ca2+]i rise. At concentrations of 5,70,µM econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50,µM econazole was enhanced by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,,N,-tetraacetic acid (BAPTA). The ERK MAPK inhibitor, PD98059 (10,µM), also enhanced 20,µM econazole-induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10,70,µM. Collectively, in OC2 cells, econazole induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2/PKC-regulated Ca2+ channels. Furthermore, econazole caused cell death appeared to be regulated by ERK MAPK. Drug Dev Res 71: 240,248, 2010. © 2010 Wiley-Liss, Inc. [source] Effect of capsaicin on Ca2+ fluxes in Madin-Darby canine renal tubular cellsDRUG DEVELOPMENT RESEARCH, Issue 2 2010Jeng-Hsien Yeh Abstract The effect of capsaicin, a transient receptor potential vanniloid-1 (TRPV1) receptor agonist, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+ -sensitive fluorescent dye. Capsaicin at concentrations between 10,100,µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 80% by removing extracellular Ca2+. Capsacin induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non-selective Ca2+ entry blocker La3+, but not by store-operated Ca2+ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca2+ -free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished capsaicin-induced Ca2+ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin-induced [Ca2+]i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca2+ entry and Ca2+ release. Collectively, in MDCK cells, capsaicin induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-regulated, La3+ -sensitive Ca2+ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Nonylphenol-induced cytosolic Ca2+ elevation and death in renal tubular cellsDRUG DEVELOPMENT RESEARCH, Issue 5 2009Jeng-Yu Tsai Abstract Nonylphenol is an environmental endocrine disrupter. The effect of nonylphenol on intracellular free Ca2+ levels ([Ca2+]i) and viability in Madin-Darby canine kidney (MDCK) cells was explored. Nonylphenol increased [Ca2+]i in a concentration-dependent manner (EC50,0.8,,M). Nonylphenol-induced Mn2+ entry demonstrated Ca2+ influx and removal of extracellular Ca2+ partly decreased the [Ca2+]i rise. The [Ca2+]i rise was inhibited by the protein kinase C activator, phorbol 13-myristate acetate (PMA) but not by L-type Ca2+ channel blockers. In Ca2+ -free medium, nonylphenol-induced [Ca2+]i rise was partly inhibited by pretreatment with 1,,M thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Conversely, nonylphenol pretreatment abolished thapsigargin-induced Ca2+ release. Nonylphenol-induced Ca2+ release was unaltered by inhibition of phospholipase C. At concentrations of 5,100,,M, nonylphenol killed cells in a concentration-dependent manner. The cytotoxic effect of 100,,M nonylphenol was not affected by preventing [Ca2+]i rises with BAPTA/AM. Collectively, this study shows that nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other stores in a phospholipase C-independent manner. Nonylphenol also killed cells in a Ca2+ -independent fashion. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Independent signaling pathways in ATP-evoked secretion of plasminogen and cytokines from microgliaDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001*Article first published online: 28 AUG 200, Kazuhide Inoue Abstract We investigated the action of ATP on the secretion of plasminogen, TNF-,, and IL-6 from microglia. ATP (10,100 ,M) stimulated the release of plasminogen from rat cultured microglia in a concentration-dependent manner with a peak response at 5,10 min after the stimulation. The release was dependent on extracellular Ca2+ and was blocked by pretreatment with oxidized ATP, a blocker of P2X7. UTP, an agonist of P2Y2, also stimulated the release of plasminogen from a subpopulation (about 20% of total cells) of cultured microglia. The release was also dependent on extracellular Ca2+, suggesting a role of stocker-operated calcium entry (SOC). ATP potently stimulated TNF-, release from 2 h after the stimulation with TNF-, mRNA expression in primary cultures of rat brain microglia. The TNF-, release was maximally elicited by 1 mM ATP and 2,- and 3,-O-(4-benzoylbenzoyl)-adenosine 5,-triphosphate (BzATP), a P2X7 selective agonist, suggesting the involvement of P2X7. This TNF-, release was correlated with a sustained Ca2+ influx. The release was inhibited by PD98059, an inhibitor of MEK1 which activates extracellular signal-regulated protein kinase (ERK), and SB203580, an inhibitor of p38 MAP kinase. However, both ERK and p38 were rapidly activated by ATP even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF-, release in rat microglia via P2X7 in a manner dependent on the sustained Ca2+ influx and via the ERK/p38 cascade independently of Ca2+ influx. ATP caused the mRNA expression and release of IL-6 in a concentration-dependent manner in MG-5. The physiological meaning of these independent release mechanisms is also discussed. Drug Dev. Res. 53:166,171, 2001. © 2001 Wiley-Liss, Inc. [source] Cellular mechanisms of cobalt-induced hippocampal epileptiform dischargesEPILEPSIA, Issue 1 2009Jiwei He Summary Purpose:, To explore the cellular mechanisms of cobalt-induced epileptiform discharges in mouse hippocampal slices. Methods:, Hippocampal slices were prepared from adult mice and briefly exposed to a CoCl2 -containing external solution. Population and single cell activities were examined via extracellular and whole-cell patch recordings. Results:, Brief cobalt exposure induced spontaneous, ictal-like discharges originating from the CA3 area. These discharges were suppressed by anticonvulsants, gap junction blockers, or by raising extracellular Ca2+, but their generation was not associated with overall hyperexcitability or impairment in GABAergic inhibition in the CA3 circuit. Electroencephalographic ictal discharges of similar waveforms were observed in behaving rats following intrahippocampal cobalt infusion. Discussion:, Mechanisms involving activity-dependent facilitation of gap junctional communication may play a major role in cobalt-induced epileptiform discharges. [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] Mechanisms of ATP action on motor nerve terminals at the frog neuromuscular junctionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2005S. Grishin Abstract We have shown previously that ATP inhibits transmitter release at the neuromuscular junction through the action on metabotropic P2Y receptors coupled to specific second messenger cascades. In the present study we recorded K+ or Ca2+ currents in motor nerve endings or blocked K+ or Ca2+ channels in order to explore the nature of downstream presynaptic effectors. Endplate currents were presynaptically depressed by ATP. Blockers of Ca2+ -activated K+ -channels, such as iberiotoxin, apamin or tetraethylammonium, did not change the depressant action of ATP. By contrast, K+ channel blocker 4-aminopyridine (4-AP) and raised extracellular Ca2+ attenuated the effect of ATP. However, these effects of 4-AP and high Ca2+ were reversed by Mg2+, suggesting Ca2+ -dependence of the ATP action. Ba2+ promoted the depressant action of ATP as did glibenclamide, a blocker of ATP-sensitive K+ channels, or mild depolarization produced by 7.5 mm K+. None of the K+ channel blockers affected the depressant action of adenosine. Focal recording revealed that neither ATP nor adenosine affected the fast K+ currents of the motor nerve endings. However, unlike adenosine, ATP or UTP, an agonist of P2Y receptors, reversibly reduced the presynaptic Ca2+ -current. This effect was abolished by suramin, an antagonist of P2 receptors. Depressant effect of ATP on the endplate and Ca2+ -currents was mimicked by arachidonate, which precluded the action of ATP. ATP reduced acetylcholine release triggered by ionomycin or sucrose, suggesting inhibition of release machinery. Thus, the presynaptic depressant action of ATP is mediated by inhibition of Ca2+ channels and by mechanism acting downstream of Ca2+ entry. [source] Synaptic release of dopamine in the subthalamic nucleusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2004Stephanie J. Cragg Abstract The direct modulation of subthalamic nucleus (STN) neurons by dopamine (DA) neurons of the substantia nigra (SN) is controversial owing to the thick caliber and low density of DA axons in the STN. The abnormal activity of the STN in Parkinson's disease (PD), which is central to the appearance of symptoms, is therefore thought to result from the loss of DA in the striatum. We carried out three experiments in rats to explore the function of DA in the STN: (i) light and electron microscopic analysis of tyrosine hydroxylase (TH)-, dopamine ,-hydroxylase (D,H)- and DA-immunoreactive structures to determine whether DA axons form synapses; (ii) fast-scan cyclic voltammetry (FCV) to determine whether DA axons release DA; and (iii) patch clamp recording to determine whether DA, at a concentration similar to that detected by FCV, can modulate activity and synaptic transmission/integration. TH- and DA-immunoreactive axons mostly formed symmetric synapses. Because D,H-immunoreactive axons were rare and formed asymmetric synapses, they comprised the minority of TH-immunoreactive synapses. Voltammetry demonstrated that DA release was sufficient for the activation of receptors and abolished by blockade of voltage-dependent Na+ channels or removal of extracellular Ca2+. The lifetime and concentration of extracellular DA was increased by blockade of the DA transporter. Dopamine application depolarized STN neurons, increased their frequency of activity and reduced the impact of ,-aminobutyric acid (GABA)-ergic inputs. These findings suggest that SN DA neurons directly modulate the activity of STN neurons and their loss may contribute to the abnormal activity of STN neurons in PD. [source] Independent receptors for diadenosine pentaphosphate and ATP in rat midbrain single synaptic terminalsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2001M. Díaz-Hernández Abstract Diadenosine pentaphosphate (Ap5A) and adenosine 5,-triphosphate (ATP) stimulate a intrasynaptosomal calcium concentration [Ca2+]i increase via specific purinergic receptors in rat midbrain synaptosomes, although nothing is known about their distribution in presynaptic terminals. A microfluorimetric technique to measure [Ca2+]i increase using the dye FURA-2AM, has permitted study of the presence of dinucleotide and P2X receptors in independent isolated synaptic terminals. Our results demonstrate the existence of three populations of synaptosomes: one with dinucleotide receptors (12%), another with P2X receptors (20%) and a third with both (14%). It has been possible to demonstrate that the activation of these receptors occurs only in the presence of extracellular Ca2+ and that it is also coupled with voltage-dependent Ca2+ channels. Finally 54% of the synaptosomes that responded to K+ did not present any calcium increase mediated by the nucleotides used. In summary, ATP and dinucleotides exhibit specific ionotropic receptors that can coexist or not on the same synaptic terminal. [source] Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2001Dominique Chesnoy-Marchais Abstract Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive l -type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 µm, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10,50 µm), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels. [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] Calcium and magnesium competitively influence the growth of a PMR1 deficient Saccharomyces cerevisiae strainFEMS MICROBIOLOGY LETTERS, Issue 2 2005Réka Szigeti Abstract PMR1, the Ca2+/Mn2+ ATPase of the secretory pathway in Saccharomyces cerevisiae was the first member of the secretory pathway Ca2+ ATPases (SPCA) to be characterized. In the past few years, pmr1, yeast have received more attention due to the recognition that the human homologue of this protein, hSPCA1 is defective in chronic benign pemphigus or Hailey,Hailey disease (HHD). Recent publications have described pmr1, S. cerevisiae as a useful model organism for studying the molecular pathology of HHD. Some observations indicated that the high Ca2+ sensitive phenotype of PMR1 defective yeast strains may be the most relevant in this respect. Here we show that the total cellular calcium response of a pmr1, S. cerevisiae upon extracellular Ca2+ challenge is decreased compared to the wild type strain similarly as observed in keratinocytes. Additionally, the novel magnesium sensitivity of PMR1 defective yeast is revealed, which appears to be a result of competition for uptake between Ca2+ and Mg2+ at the plasma membrane level. Our findings indicate that extracellular Ca2+ and Mg2+ competitively influence the intracellular Ca2+ homeostasis of S. cerevisiae. These observations may further our understanding of HHD. [source] RESEARCH ARTICLE: Fungicidal activity of amiodarone is tightly coupled to calcium influxFEMS YEAST RESEARCH, Issue 3 2008Sabina Muend Abstract The antiarrhythmic drug amiodarone has microbicidal activity against fungi, bacteria and protozoa. In Saccharomyces cerevisiae, amiodarone triggers an immediate burst of cytosolic Ca2+, followed by cell death markers. Ca2+ transients are a common response to many forms of environmental insults and toxic compounds, including osmotic and pH shock, endoplasmic reticulum stress, and high levels of mating pheromone. Downstream signaling events involving calmodulin, calcineurin and the transcription factor Crz1 are critical in mediating cell survival in response to stress. In this study we asked whether amiodarone induced Ca2+ influx was beneficial, toxic or a bystander effect unrelated to the fungicidal effect of the drug. We show that downregulation of Ca2+ channel activity in stationary phase cells correlates with increased resistance to amiodarone. In actively growing cells, extracellular Ca2+ modulated the size and shape of the Ca2+ transient and directly influenced amiodarone toxicity. Paradoxically, protection was achieved both by removal of external Ca2+ or by adding high levels of CaCl2 (10 mM) to block the drug induced Ca2+ burst. Our results support a model in which the fungicidal activity of amiodarone is mediated by Ca2+ stress, and highlight the pathway of Ca2+ mediated cell death as a promising target for antifungal drug development. [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] The triakontatetraneuropeptide TTN increases [Ca2+]i in rat astrocytes through activation of peripheral-type benzodiazepine receptorsGLIA, Issue 2 2001Pierrick Gandolfo Abstract Astrocytes synthesize a series of regulatory peptides called endozepines, which act as endogenous ligands of benzodiazepine receptors. We have recently shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present study was to determine the mechanism of action of TTN on cultured rat astrocytes. Binding of the peripheral-type benzodiazepine receptor ligand [3H]Ro5-4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fragment (TTN[17,34], the octadecaneuropeptide ODN) did not compete for [3H]Ro5-4864 binding. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) with the fluorescent probe indo-1 showed that TTN (10,10 to 10,6 M) provokes a concentration-dependent increase in [Ca2+]i in cultured astrocytes. Simultaneous administration of TTN (10,8 M) and Ro5-4864 (10,5 M) induced an increase in [Ca2+]i similar to that obtained with Ro5-4864 alone. In contrast, the effects of TTN (10,8 M) and ODN (10,8 M) on [Ca2+]i were strictly additive. Chelation of extracellular Ca2+ by EGTA (6 mM) or blockage of Ca2+ channels with Ni2+ (2 mM) abrogated the stimulatory effect of TTN. The calcium influx evoked by TTN (10,7 M) or by Ro5-4864 (10,5 M) was not affected by the N- and T-type calcium channel blockers ,-conotoxin (10,6 M) and mibefradil (10,6 M), but was significantly reduced by the L-type calcium channel blocker nifedipine (10,7 M). Patch-clamp studies showed that, at negative potentials, TTN (10,7 M) induced a sustained depolarization. Reduction of the chloride concentration in the extracellular solution shifted the reversal potential from 0 mV to a positive potential. These data show that TTN, acting through peripheral-type benzodiazepine receptors, provokes chloride efflux, which in turn induces calcium influx via L-type calcium channels in rat astrocytes. GLIA 35:90,100, 2001. © 2001 Wiley-Liss, Inc. [source] Stimulation of NMDA and AMPA glutamate receptors elicits distinct concentration dynamics of nitric oxide in rat hippocampal slicesHIPPOCAMPUS, Issue 7 2009J.G. Frade Abstract Nitric oxide (,NO) is an intercellular messenger implicated in memory formation and neurodegeneration in the hippocampus. Owing to its physical and chemical properties, the concentration dynamics of ,NO is a critical issue in determining its bioactivity as a signaling molecule. Its production is closely related to glutamate N -methyl- D -aspartate (NMDA) receptors, following a rise in intracellular calcium levels. However, that dependent on ,-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors remains elusive and controversial, despite reports describing a role for these receptors in other brain regions, largely because of lack of quantitative and dynamic measurements of ,NO. Using a ,NO-selective microsensor inserted in the diffusional spread of ,NO in the CA1 region of rat hippocampal slices, we measured its real-time endogenous production, following activation of ionotropic glutamate receptors and under tissue physiological oxygen tension. Both NMDA and AMPA stimulation resulted in a concentration-dependent ,NO production but encompassing distinct kinetics for lag phases and slower rates of ,NO production were observed for AMPA stimulation. Robustness of the results was achieved instrumentally and pharmacologically, by means of nitric oxide synthase (NOS) inhibitors and antagonists of NMDA (D -(,)-2-amino-5-phosphonopentanoic acid, AP5) and AMPA (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, NBQX) receptors. When using glutamate as a stimulus, ,NO production was of lower magnitude in the presence of AP5 plus NBQX than with AP5 alone, suggesting that even when NMDA receptors are inhibited Ca2+ rises to levels to induce a peak of ,NO from the background. Whereas extracellular Ca2+ was required for the ,NO signals, Philanthotoxin-4,3,3 (PhTX-4,3,3) a toxin used to target Ca2+ -permeable AMPA receptors, attenuated ,NO production. These observations are interpreted on basis of a distinct coupling between the glutamate receptors and neuronal NOS. A role for Ca2+ -permeable AMPA receptors in the Ca2+ activation of neuronal NOS is suggested. © 2008 Wiley-Liss, Inc. [source] Azathioprine-induced suicidal erythrocyte deathINFLAMMATORY BOWEL DISEASES, Issue 8 2008Corinna Geiger Abstract Background: Azathioprine is widely used as an immunosuppressive drug. The side effects of azathioprine include anemia, which has been attributed to bone marrow suppression. Alternatively, anemia could result from accelerated suicidal erythrocyte death or eryptosis, which is characterized by exposure of phosphatidylserine (PS) at the erythrocyte surface and by cell shrinkage. Methods: The present experiments explored whether azathioprine influences eryptosis. According to annexin V binding, erythrocytes from patients indeed showed a significant increase of PS exposure within 1 week of treatment with azathioprine. In a second series, cytosolic Ca2+ activity (Fluo3 fluorescence), cell volume (forward scatter), and PS-exposure (annexin V binding) were determined by FACS analysis in erythrocytes from healthy volunteers. Results: Exposure to azathioprine (,2 ,g/mL) for 48 hours increased cytosolic Ca2+ activity and annexin V binding and decreased forward scatter. The effect of azathioprine on both annexin V binding and forward scatter was significantly blunted in the nominal absence of extracellular Ca2+. Conclusions: Azathioprine triggers suicidal erythrocyte death, an effect presumably contributing to azathioprine-induced anemia. (Inflamm Bowel Dis 2008) [source] Regulation of E-cadherin and ,-catenin by Ca2+ in colon carcinoma is dependent on calcium-sensing receptor expression and functionINTERNATIONAL JOURNAL OF CANCER, Issue 7 2007Narasimharao Bhagavathula Abstract An siRNA directed against the extracellular calcium-sensing receptor (CaSR) was used to down-regulate this protein in CBS colon carcinoma cells. In additional studies, we utilized a variant of the parental CBS line that demonstrates CaSR expression but does not upregulate this protein in response to extracellular Ca2+. In neither the siRNA-transfected cells nor the Ca2+ -nonresponsive variant cells did inclusion of Ca2+ in the culture medium inhibit proliferation or induce morphological alterations. Extracellular Ca2+ also failed to induce E-cadherin production or a shift in ,-catenin from the cytoplasm to the cell membrane. In mock-transfected cells and in a Ca2+ -responsive variant line derived from the same parental CBS cells, Ca2+ treatment resulted in growth-reduction. This was accompanied by increased E-cadherin production and a shift in ,-catenin distribution from the cytoplasm to the cell membrane. Additionally, down-regulation of c-myc and cyclin D1 expression was observed in mock-transfected cells and in the Ca2+ -responsive variant line (along with reduced T cell factor transcriptional activation). Neither c-myc nor cyclin D1 was significantly down-regulated in the siRNA-transfected cells or in the Ca2+ -nonresponsive variant cells upon Ca2+ stimulation. In histological sections of human colon carcinoma CaSR was significantly reduced as compared to the level in normal colonic crypt epithelial cells. Where CaSR expression was high, strong surface staining for E-cadherin and ,-catenin was observed. Where CaSR expression was reduced, ,-catenin surface expression was likewise reduced. © 2007 Wiley-Liss, Inc. [source] Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein KinaseJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Haruhiko Tokuda Abstract We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O -aminophinoxy)-ethane- N,N,N,N -tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase. [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] Catecholamine exocytosis is diminished in R6/2 Huntington's disease model miceJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Michael A. Johnson Abstract In this work, the mechanisms responsible for dopamine (DA) release impairments observed previously in Huntington's disease model R6/2 mice were evaluated. Voltammetrically measured DA release evoked in striatal brain slices from 12-week old R6/2 mice by a single electrical stimulus pulse was only 19% of wild-type (WT) control mice. Iontophoresis experiments suggest that the concentration of released DA is not diluted by a larger striatal extracellular volume arising from brain atrophy, but, rather, that striatal dopaminergic terminals have a decreased capacity for DA release. This decreased capacity was not due to an altered requirement for extracellular Ca2+, and, as in WT mice, the release in R6/2 mice required functioning vesicular transporters. Catecholamine secretion from individual vesicles was measured during exocytosis from adrenal chromaffin cells harvested from R6/2 and WT mice. While the number of exocytotic events was unchanged, the amounts released per vesicle were significantly diminished in R6/2 mice, indicating that vesicular catecholamines are present in decreased amounts. Treatment of chromaffin cells with 3-nitropropionic acid decreased the vesicular release amount from WT cells by 50%, mimicking the release observed from untreated R6/2 cells. Thus, catecholamine release from tissues isolated from R6/2 mice is diminished because of impaired vesicle loading. [source] Apolipoprotein E and ,-amyloid (1,42) regulation of glycogen synthase kinase-3,JOURNAL OF NEUROCHEMISTRY, Issue 5 2003A. Cedazo-Mínguez Abstract Glycogen synthase kinase-3, (GSK-3,) is implicated in regulating apoptosis and tau protein hyperphosphorylation in Alzheimer's disease (AD). We investigated the effects of two key AD molecules, namely apoE (E3 and E4 isoforms) and ,-amyloid (A,) 1,42 on GSK-3, and its major upstream regulators, intracellular calcium and protein kinases C and B (PKC and PKB) in human SH-SY5Y neuroblastoma cells. ApoE3 induced a mild, transient, Ca2+ -independent and early activation of GSK-3,. ApoE4 effects were biphasic, with an early strong GSK-3, activation that was partially dependent on extracellular Ca2+, followed by a GSK-3, inactivation. ApoE4 also activated PKC-, and PKB possibly giving the subsequent GSK-3, inhibition. A,(1,42) effects were also biphasic with a strong activation dependent partially on extracellular Ca2+ followed by an inactivation. A,(1,42) induced an early and potent activation of PKC-, and a late decrease of PKB activity. ApoE4 and A,(1,42) were more toxic than apoE3 as shown by MTT reduction assays and generation of activated caspase-3. ApoE4 and A,(1,42)-induced early activation of GSK-3, could lead to apoptosis and tau hyperphosphorylation. A late inhibition of GSK-3, through activation of upstream kinases likely compensates the effects of apoE4 and A,(1,42) on GSK-3,, the unbalanced regulation of which may contribute to AD pathology. [source] Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro studyJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Pier Andrea Serra Abstract In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso- N -acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l -type Ca2+ channel inhibitor nifedipine. The NO-donor (+/,)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+ -induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+ -dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated. [source] | ||||||||||||||||||||||||||||