Protective effect of fangchinoline on cyanide-induced neurotoxicity in cultured rat cerebellar granule cells

The present study was performed to examine the effect of fangchinoline, a bis- benzylisoquinoline alkaloid, which exhibits the characteristics of a Ca2+ channel blocker, on cyanide-induced neurotoxicity using cultured rat cerebellar granule neurons. NaCN produced a concentration-dependent reduction of cell viability, which was blocked by MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, verapamil, L-type Ca2+ channel blocker, and L-NAME, a nitric oxide synthase inhibitor. Pretreatment with fangchinoline over a concentration range of 0.1 to 10 microM significantly decreased the NaCN-induced neuronal cell death, glutamate release into medium, and elevation of [Ca2+]i and oxidants generation. These results suggest that fangchinoline may mitigate the harmful effects of cyanide-induced neuronal cell death by interfering with [Ca2+]i influx, due to its function as a Ca2+ channel blocker, and then by inhibiting glutamate release and oxidants generation.     

Muscarinic receptor stimulation reduces NMDA responses in CA3 hippocampal pyramidal cells via Ca2+-dependent activation of tyrosine phosphatase

N-methyl-D-aspartate (NMDA)-type glutamate receptors perform critical functions during the development of the nervous system and in the initiation of synaptic plasticity. An important mechanism in setting the gain of NMDA receptors involves the stimulation of G-protein-coupled receptors (GPCRs), which through activation of protein tyrosine kinases leads to an upregulation of NMDA receptors. In contrast, little is known about how NMDA receptors are downregulated. In the present study, we characterized a signaling pathway that mediates the depression of NMDA receptor function in response to stimulation of muscarinic acetylcholine receptors. Whole-cell patch-clamp recordings obtained from CA3 pyramidal cells in organotypic slice cultures revealed that under conditions of low intracellular calcium buffering application of muscarine-depressed NMDA receptor current. The sensitivity of this response to pirenzipine indicated that the M1 acetylcholine receptor is mediating this depression. The muscarine-induced depression of NMDA current was prevented by blocking G-protein function or after depleting intracellular Ca2+ stores with cyclopiazonic acid. Inhibitors of calmodulin prevented the depression whereas blocking calcineurin enhanced the depression of NMDA currents. Blocking tyrosine phosphatase activity with pervanandate converted the muscarine-induced depression into a potentiation of NMDA currents, whereas blocking protein kinase A (H-89), Src kinase (PP2, SU6656), or PKC (GF 109203X) failed to prevent the depression of NMDA currents. As Src tyrosine kinase is known to phosphorylate and upregulate NMDA receptors, we propose that a protein tyrosine phosphatase(s) counteracting the action of Src is the final target in the mAChR-dependent inhibitory signaling cascade. Our data are consistent with a transduction cascade comprising an M1 acetylcholine receptor-->G-protein-->Ca2+ release-->calmodulin-->tyrosine phosphatase.     

Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells

Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases. The present study was performed to evaluate the inhibitory effect of fangchinoline, a bis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline (1 and 5 microM) inhibited glutamate (1 mM), N-methyl-D-aspartate (NMDA; 1 mM) and kainate (100 microM)-induced neuronal cell death which was measured by trypan blue exclusion test. Fangchinoline (1 and 5 microM) inhibited glutamate release into medium induced by NMDA (1 mM) and kainate (100 microM), which was measured by HPLC. And fangchinoline (5 microM) inhibited glutamate (1 mM)-induced elevation of intracellular calcium concentration. These results suggest that inhibition of Ca2+ influx by fangchinoline may contribute to the beneficial effects on neurodegenerative effect of glutamate in pathophysiological conditions.     

Differential inhibitory effects of honokiol and magnolol on excitatory amino acid-evoked cation signals and NMDA-induced seizures

The effects of honokiol and magnolol, two major bioactive constituents of the bark of Magnolia officinalis, on Ca(2+) and Na(+) influx induced by various stimulants were investigated in cultured rat cerebellar granule cells by single-cell fura-2 or SBFI microfluorimetry. Honokiol and magnolol blocked the glutamate- and KCl-evoked Ca(2+) influx with similar potency and efficacy, but did not affect KCl-evoked Na(+) influx. However, honokiol was more specific for blocking NMDA-induced Ca(2+) influx, whereas magnolol influenced with both NMDA- and non-NMDA activated Ca(2+) and Na(+) influx. Moreover, the anti-convulsant effects of these two compounds on NMDA-induced seizures were also evaluated. After honokiol or magnolol (1 and 5 mg/kg, i.p.) pretreatment, the seizure thresholds of NMRI mice were determined by tail-vein infusion of NMDA (10 mg/ml). Data showed that both honokiol and magnolol significantly increased the NMDA-induced seizure thresholds, and honokiol was more potent than magnolol. These results demonstrated that magnolol and honokiol have differential effects on NMDA and non-NMDA receptors, suggesting that the distinct therapeutic applications of these two compounds for neuroprotection should be considered.     

Glycine-evoked release of [3H]acetylcholine from rat striatal slices is independent of the NMDA receptor

Summary KCl-, NMDA-, and glycine-evoked release of [³H]acetylcholine was studied in superfused rat striatal slices. KCl-evoked release of [³H]acetylcholine was inhibited by 1.2 mM MgC12 and 100 M lidocaine. Similarly, NMDA-evoked release was inhibited by MgCl₂ and lidocaine as well as 10 M CGS 19755, a competitive antagonist at NMDA receptors, and 10 nM MK-801, a noncompetitive antagonist of NMDA-induced responses. Glycine-evoked release was calcium-dependent and was inhibited by 0.1 M strychnine whereas KCl- and NMDA-evoked release were resistant to strychnine. In addition, lidocaine inhibited the glycine-induced response. Cross-tachyphylaxis was not observed between NMDA- and glycine-evoked release. These results indicate that the strychnine-sensitive, glycine-evoked release of [³H]acetylcholine is independent of the NMDA receptor.     

Glycogen synthase kinase-3 inhibitors prevent caspase-dependent apoptosis induced by ethanol in cultured rat cortical neurons

The effect of ethanol on cell viability was examined in rat cultured cortical neurons. Ethanol induced apoptosis, which was characterized by cell shrinkage, nuclear condensation or fragmentation and internucleosomal DNA fragmentation. Ethanol-induced apoptosis was prevented by N-methyl-d-aspartate (NMDA), an agonist of the NMDA receptor, which is a subtype of ionotropic glutamate receptors. Incubation with glycogen synthase kinase-3 (GSK-3) inhibitors 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) and alsteropaullone, but not a cyclin-dependent protein kinase 5 inhibitor roscovitine, completely protected the neurons from ethanol-induced apoptosis. Apoptosis was accompanied by the activation of caspase-3 and prevented by a caspase-3 inhibitor. These results suggest that ethanol induces caspase-dependent apoptosis mediated by glycogen synthase kinase-3 activation in cultured rat cortical neurons.     

Neonatal toluene exposure alters agonist and antagonist sensitivity and NR2B subunit expression of NMDA receptors in cultured cerebellar granule neurons.

Toluene has been reported to antagonize the function of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of neonatal toluene exposure on NMDA receptors in primarily cultured cerebellar granule neurons were examined. Sprague-Dawley rats were treated with toluene (0, 200, 500, and 1000 mg/kg, i.p.) from postnatal day (PN) 4 to PN 7. Under toluene-free conditions, Ca2+ signals of cultured neurons in response to glutamate and NMDA were measured for up to 14 days. The expression of NMDA receptor subunits (NR1, NR2A, and NR2B) at 5-14 days in vitro (DIV) were also determined. Neonatal toluene exposure dose-dependently reduced intracellular Ca2+ signals in response to glutamate/glycine and NMDA/glycine in cultured cerebellar granule neurons, and these effects were gradually decreased with time. Such toluene exposure did not influence the inhibition of Mg2+ or MK801 on NMDA-evoked responses, but it decreased the potency of ifenprodil (an NR2B preferring antagonist). The protein levels of NMDA receptor subunit NR2B were consistently reduced by toluene exposure at 5 DIV, but not at 14 DIV. These results demonstrate that neonatal toluene exposure induces long-term but reversible changes in the function and composition of NMDA receptors. Such changes during developmental stages may contribute to the cerebellar dysfunction observed in fetal solvent syndrome.     

Nitric oxide synthase inhibitor decreases NMDA-induced elevations of extracellular glutamate and intracellular Ca2+ levels via a cGMP-independent mechanism in cerebellar granule neurons

These studies were designed to examine the differential effect of nitric oxide (NO) and cGMP on glutamate neurotransmission. In primary cultures of rat cerebellar granule cells, the glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulates the elevation of intracellular calcium concentration ([Ca2+]i), the release of glutamate, the synthesis of NO and an increase of cGMP. Although NO has been shown to stimulate guanylyl cyclase, it is unclear yet whether NO alters the NMDA-induced glutamate release and [Ca2+]i elevation. We showed that the NO synthase inhibitor, N(G)-monomethyl-L-arginine (NMMA), partially prevented the NMDA-induced release of glutamate and elevation of [Ca2+]i and completely blocked the elevation of cGMP. These effects of NO on glutamate release and [Ca2+]i elevation were unlikely to be secondary to cGMP as the cGMP analogue, dibutyryl cGMP (dBcGMP), did not suppress the effects of NMDA. Rather, dBcGMP slightly augmented the NMDA-induced elevation of [Ca2+]i with no change in the basal level of glutamate or [Ca2+]i. The extracellular NO scavenger hydroxocobalamine prevented the NMDA-induced release of glutamate providing indirect evidence that the effect of NO may act on the NMDA receptor. These results suggest that low concentration of NO has a role in maintaining the NMDA receptor activation in a cGMP-independent manner.     

Behavioral sensitization to ethanol does not result in cross-sensitization to NMDA receptor antagonists

Rationale Fluoxetine is used to treat unipolar depression and is thought to act by increasing the concentration of serotonin (5-HT) in the synaptic cleft, leading to increased serotonin signaling. The 5-HT_2A/2C receptor subtypes are coupled to a phospholipase A₂ (PLA₂). We hypothesized that chronic fluoxetine would increase the brain activity of PLA₂ and the turnover rate of arachidonic acid (AA) in phospholipids of the unanesthetized rat.Materials and methods To test this hypothesis, rats were administered fluoxetine (10 mg/kg) or vehicle intraperitoneally daily for 21 days. In the unanesthetized rat, [1-¹⁴C]AA was infused intravenously and arterial blood plasma was sampled until the animal was killed at 5 min and its brain was subjected to chemical, radiotracer, or enzyme analysis.Results Using equations from our fatty acid model, we found that chronic fluoxetine compared with vehicle increased the turnover rate of AA within several brain phospholipids by 75–86%. The activity and protein levels of brain cytosolic PLA₂ (cPLA₂) but not of secretory or calcium-independent PLA₂ were increased in rats administered fluoxetine. In a separate group of animals that received chronic fluoxetine followed by a 3-day saline washout, the turnover of AA and activity and protein levels of cPLA₂ were not significantly different from controls. The protein levels of cyclooxygenases 1 and 2 as well as the concentration of prostaglandin E₂ in rats chronically administered fluoxetine did not differ significantly from controls.Conclusion The results support the hypothesis that fluoxetine increases the cPLA₂-mediated turnover of AA within brain phospholipids.     

Diclofenac, a nonsteroidal anti-inflammatory drug, activates the transient outward K+ current in rat cerebellar granule cells

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its direct effect on ion channels. Here, the effect of diclofenac on voltage-dependent transient outward K+ currents (I(A)) in cultured rat cerebellar granule cells was investigated using the whole-cell voltage-clamp technique. At concentrations of 10(-5)-10(-3) M, diclofenac reversibly increased the I(A) amplitude in a dose-dependent manner and significantly modulated the steady-state inactivation properties of the I(A) channels, but did not alter the steady-state activation properties. Furthermore, diclofenac treatment resulted in a slightly accelerated recovery from I(A) channel inactivation. Intracellular application of diclofenac could mimic the effects induced by extracellular application, although once the intracellular response reached a plateau, extracellular application of diclofenac could induce further increases in the current. These observations indicate that diclofenac might exert its effects on the channel protein at both the inner and outer sides of the cell membrane. Our data provide the first evidence that diclofenac is able to activate transient outward potassium channels in neurons. Although further work will be necessary to define the exact mechanism of diclofenac-induced I(A) channel activation, this study provides evidence that the nonsteroidal anti-inflammatory drug, diclofenac, may play a novel neuronal role that is worthy of future study.     

Pharmacologic and radioligand binding analysis of the actions of 1,4-dihydropyridine activators related to Bay K 8644 in smooth muscle,cardiac muscle and neuronal preparations

Summary The structure-activity relationships of a series of 1,4-dihydropyridine Ca²⁺ channel activators, including Bay K 8644, have been determined by pharmacologic and radioligand binding techniques. Pharmacologic techniques included tension responses and the measurement of pA₂ values for nifedipine antagonism of Bay K 8644 responses in guinea pig ileal, rat femoral and rat atrial and papillary muscle preparations. Radioligand binding experiments employed competition against [³H]nitrendipine binding in ileal smooth muscle and rat ventricular membranes and rat brain synaptosomal preparations. The series of compounds was employed as the racemates. Binding affinities were not significantly different between smooth muscle, cardiac muscle and brain preparations and the same rank order of pharmacologic activities is observed in smooth and cardiac muscle, where the effects of the 4-phenyl substituents, o m > p, parallel those observed for 1,4-dihydropyridine antagonists. In the ileal and femoral artery smooth muscle preparations a 1:1 correlation is observed between pharmacologic and radioligand binding affinities. However, in the cardiac muscle preparations, left atrium and papillary muscle, there is an approximately 10-fold difference between the binding affinities and the lower pharmacologic affinities. A similar difference between smooth and cardiac muscle is observed with the pA₂ values of 6.97 and 7.06 in atrial and papillary muscle respectively, which are significantly lower than the values of 8.54 and 8.72 measured in ileal and femoral artery respectively. The structure-activity expressions measured for this small series of 1,4-dihydropyridine activators parallel those observed in the larger series of 1,4-dihydropyridine antagonists. This is consistent with proposals that activators and antagonists interact at common binding sites that are components of a voltage-dependent Ca²⁺ channel. Send offprint requests to D. J. Triggle at the above address     

NMDA enhances a depolarization-activated inward current in subthalamic neurons

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor stimulation evokes Ca2+- and Na+-dependent burst firing in subthalamic nucleus (STN) neurons. Using whole-cell patch pipettes to record currents under voltage-clamp, we identified a time-dependent depolarization-activated inward current (DIC) that may underlie NMDA-induced burst firing in STN neurons in rat brain slices. Continuous superfusion with NMDA (20 microM) elicited a marked TTX-insensitive inward current when the membrane was depolarized to the level of -70 or -50 mV, from a holding potential of -100 mV. This current had a long duration, and its peak amplitude occurred at a test potential of -60 mV. DIC could not be evoked using the non-NMDA receptor agonist D,L-alpha-amino-3-hydroxy-5-methylisoxalone-4-propionic acid (AMPA). DIC was blocked by either intracellular BAPTA or by removal of extracellular Ca2+, but selective blockers of T-type (mibefradil), L-type (nifedipine) and N-type (omega-conotoxin GVIA) Ca2+ channels did not. Perfusing slices with a low extracellular concentration of sodium abolished the NMDA-induced DIC, implying that both Ca2+ and Na+ are necessary for the expression of DIC. Transient receptor potential (TRP) channel blockers flufenamic acid and SKF96365 severely reduced DIC amplitude, whereas NMDA-gated currents were either increased or were unchanged. These results suggest that the activation of NMDA receptors enhances a Ca2+-activated non-selective cation current that may be mediated by a member of the TRP channel family in STN neurons.     

Acute toxic effect of the algal yessotoxin on Purkinje cells from the cerebellum of Swiss CD1 mice.

Swiss CD1 mice died less than 2 h after intraperitoneal injection of 420 microg/kg of algal yessotoxin (YTX). The morphological, histochemical and immunocytochemical studies performed on the cerebellar cortex revealed damage to the Purkinje cells. The main cytological alterations were observed in the cytoplasm, while less sufferance was detected in the nucleus. The immunocytochemical experiments showed an increased positivity to S100 protein while there was a decreased response to calbindin D-28K, beta-tubulin and neurofilaments. These changes in intracellular Ca(2+)-binding proteins and the modifications in the cytoskeletal components of Purkinje cells suggest that YTX may be involved in neurological disorders.     

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced, partly, by removing extracellular Ca²⁺. Bisphenol A induced Mn²⁺ influx, leading to quenching of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca²⁺ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca²⁺-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca²⁺ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca²⁺]_i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca²⁺-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca²⁺]_i rises by causing phospholipase C–dependent Ca²⁺ release from the endoplasmic reticulum and mitochondria and Ca²⁺ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca²⁺ channels.     

Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons

Context: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities.

Objective: The effects of fucoidan on Ca²⁺ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined.

Materials and methods: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca²⁺ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5?mg/mL or 1.5?mg/mL was applied for 3?min to determine its effects on Ca²⁺ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5?mg/mL for 3?h.

Results: The Ca²⁺ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca²⁺ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca²⁺ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca²⁺ channels, PR1/PR2.

Discussion and conclusions: Our data indicate that fucoidan suppresses the intracellular Ca²⁺ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca²⁺ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.     

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim:

To investigate the reverse mode function of Na⁺/Ca²⁺ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.

Methods:

CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca²⁺ level ([Ca²⁺]_i) was measured using Ca²⁺ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na⁺-free medium. Ca²⁺ paradox was induced by Ca²⁺-free EBSS medium, followed by Ca²⁺-containing solution (1.8 or 3.8 mmol/L CaCl₂).

Results:

The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca²⁺]_i, which was followed by a Ca²⁺ level plateau at higher external Ca²⁺ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca²⁺]_i at higher external Ca²⁺ concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).

Conclusion:

Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca²⁺]_i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.     

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Background and Purpose

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca²⁺]_i) in beta cells, in the absence of any co-stimulating factor.

Experimental Approach

Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca²⁺]_i were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ channel currents were recorded with the whole-cell patch-clamp technique.

Key Results

Quercetin concentration-dependently increased insulin secretion and elevated [Ca²⁺]_i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L⁻¹), but were nearly abolished by the L-type Ca²⁺ channel antagonist nifedipine (1 μmol·L⁻¹). Similar to the L-type Ca²⁺ channel agonist Bay K 8644, quercetin enhanced the L-type Ca²⁺ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca²⁺]_i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L⁻¹), with the two drugs having cumulative effects on [Ca²⁺]_i.

Conclusions and Implications

Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca²⁺ influx through an interaction with L-type Ca²⁺ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin''s mechanism of action on insulin secretion.     

Effect of the antidepressant maprotiline on calcium movement and the viability of renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of maprotiline, an antidepressant, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura-2. Maprotiline (>2.5 µM) caused a rapid rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ 200 µM). Maprotiline-induced [Ca²⁺]_i rise was reduced by removal of extracellular Ca²⁺ or by addition of La³⁺, but was not altered by voltage-gated Ca²⁺-channel blockers. Maprotiline-induced Mn²⁺ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca²⁺ influx. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, caused a monophasic [Ca²⁺]_i rise, after which the increasing effect of maprotiline on [Ca²⁺]_i was nearly abolished; also, pretreatment with maprotiline reduced a portion of thapsigargin-induced [Ca²⁺]_i rise. U73122, an inhibitor of phospholipase C, abolished [Ca²⁺]_i rise induced by ATP (but not by maprotiline). Overnight incubation with 1–10 µM maprotiline enhanced cell viability, but 20–50 µM maprotiline decreased it. These findings suggest that maprotiline rapidly increases [Ca²⁺]_i in renal tubular cells by stimulating both extracellular Ca²⁺ influx and intracellular Ca²⁺ release, and may modulate cell proliferation in a concentration-dependent manner.     

Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study

Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 ± 199, 345 ± 47, and 243 ± 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 ± 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.     

KB-R7943, an inhibitor of the reverse Na+ /Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I

BACKGROUND AND PURPOSE

An isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na⁺/Ca²⁺ exchanger (NCX_rev), was instrumental in establishing the role of NCX_rev in glutamate-induced Ca²⁺ deregulation in neurons. Here, the effects of KB-R7943 on N-methyl-D-aspartate (NMDA) receptors and mitochondrial complex I were tested.

EXPERIMENTAL APPROACH

Fluorescence microscopy, electrophysiological patch-clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca²⁺ flux measurements were made in isolated rat brain mitochondria.

KEY RESULTS

KB-R7943 inhibited NCX_rev with IC₅₀= 5.7 ± 2.1 µM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca²⁺ with IC₅₀= 13.4 ± 3.6 µM but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca²⁺-independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca²⁺ in the bath solution. KB-R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB-R7943 inhibited 2,4-dinitrophenol-stimulated respiration of cultured neurons with IC₅₀= 11.4 ± 2.4 µM. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca²⁺ uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate.

CONCLUSIONS AND IMPLICATIONS

KB-R7943, in addition to NCX_rev, blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB-R7943 and a better understanding of its neuroprotective action.