ARC 118925XX stimulates cation influx in bEND.3 endothelial cells.

In a previous publication when we studied the purinergic receptor with which ATP interacted in mouse brain bEND.3 endothelial cells, we observed addition of 3 µm ARC 118925XX (ARC; selective P2Y2 antagonist) strongly suppressed ATP-triggered Ca2+ release, suggesting the response was mediated via P2Y2 receptors. We here report ARC unexpectedly promoted substantial Ca2+ influx even when ATP-triggered Ca2+ release was largely inhibited. Since this large Ca2+ influx may have important pharmacological significance, we proceeded to investigate its mechanism. ARC did not trigger intracellular Ca2+ release thus suggesting Ca2+ influx triggered by ARC was not store-operated. ARC-triggered Ca2+ influx could be blocked by 1 mm Ni2+ , a general Ca2+ channel blocker, but not by SK&F 96365, a nonselective TRP channel blocker. Unexpectedly, ARC promoted influx of Na+ and La3+ , but not Mn2+ . This is a surprising finding, since Mn2+ is conventionally used as a Ca2+ surrogate ion (as it permeates Ca2+ channel), and La3+ is classically used as a potent Ca2+ channel antagonist. Electrophysiological examination showed ARC did not stimulate any cation currents. Therefore, ARC opened, rather than a cation channel pore, an unidentified Ca2+ influx pathway which was Na+ - and La3+ -permeable but Mn2+ -impermeable.

Propofol may increase caspase and MAPK pathways, and suppress the Akt pathway to induce apoptosis in MA­10 mouse Leydig tumor cells.

In the western world, there is an increasing trend of occurrence in testicular cancer. Treatment of malignant testicular cancer is primarily combined surgery with various chemical drugs. Propofol has been frequently used as an anesthetic and sedative induction agent, which could modulate different γ­aminobutyric acid receptors in the central nervous system. Studies demonstrated that propofol activates endoplasmic reticulum stress to induce apoptosis in lung cancer. However, it remains elusive whether propofol regulates caspase and/or mitogen­activated protein kinase (MAPK) pathways to induce apoptosis in Leydig tumor cells. In the present study, MA­10 mouse Leydig tumor cells were treated with propofol, and possible signal pathways associated with apoptosis were investigated. Results demonstrated that increasing dosage of propofol (300­600 µM) for 24 h significantly decreased cell viability in MA­10 cells (P<0.05). In flow cytometry analysis, the amount of sub­G1 phase cell numbers in MA­10 cells was significantly increased by propofol (P<0.05). Additionally, Annexin V/propidium iodide double staining further confirmed that propofol could induce MA­10 cell apoptosis. Furthermore, cleaved caspase­8, ­9 and ­3, and/or poly(ADP­ribose) polymerase were significantly activated following treatment of propofol in MA­10 cells. In addition, c­Jun N­terminal kinase, extracellular signal­regulated kinase 1/2, and p38 were significantly activated by propofol in MA­10 cells (P<0.05), indicating that propofol may induce apoptosis through the MAPK pathway. Additionally, propofol diminished the phosphorylation of Akt to activate apoptosis in MA­10 cells. In conclusion, propofol may induce MA­10 cell apoptosis by activating caspase and MAPK pathways, and inhibiting the Akt pathway in MA­10 cells, demonstrating that propofol may be a potential anticancer agent against Leydig cell cancer.

Antagonism of Ca2+-sensing receptors by NPS 2143 is transiently masked by p38 activation in mouse brain bEND.3 endothelial cells.

Ca2+-sensing receptors (CaSR) are G protein-coupled receptors which are activated by a rise in extracellular Ca2+. CaSR activation has been known to inhibit parathyroid hormone release and stimulate calcitonin release from parathyroid glands and thyroid parafollicular C cells, respectively. The roles of CaSR in other cell types including endothelial cells (EC) are much less understood. In this work, we demonstrated protein and functional expression of CaSR in mouse cerebral EC (bEND.3). Unexpectedly, CaSR response (high Ca2+-elicited cytosolic [Ca2+] elevation) was unaffected by edelfosine or U73122 but strongly suppressed by SK&F 96365, ruthenium red, and 2-aminoethoxydiphenyl borate (2-APB), suggesting involvement of TRPV and TRPC channels but not Gq-phospholipase C. Acute application of NPS2143, a negative allosteric modulator of CaSR, suppressed CaSR response. However, a 40-min NPS2143 pre-treatment surprisingly enhanced CaSR response. After 4-24 h of application, this enhancement faded away and suppression of CaSR response was observed again. Similar results were obtained when La3+ and Sr2+ were used as CaSR agonists. The transient NPS 2143 enhancement effect was abolished by SB203580, a p38 inhibitor. Consistently, NPS 2143 triggered a transient p38 activation. Taken together, results suggest that in bEND.3 cells, NPS 2143 caused acute suppression of CaSR response, but then elicited a transient enhancement of CaSR response in a p38-dependent manner. NPS 2143 effects on CaSR in bEND.3 cells therefore depended on drug exposure time. These findings warrant cautious use of this agent as a CaSR modulator and potential cardiovascular drug.

Lysophosphatidylcholine-induced cytotoxicity and protection by heparin in mouse brain bEND.3 endothelial cells.

A pathological feature in atherosclerosis is the dysfunction and death of vascular endothelial cells (EC). Oxidized low-density lipoprotein (LDL), known to accumulate in the atherosclerotic arterial walls, impairs endothelium-dependent relaxation and causes EC apoptosis. A major bioactive ingredient of the oxidized LDL is lysophosphatidylcholine (LPC), which at higher concentrations causes apoptosis and necrosis in various EC. There is hitherto no report on LPC-induced cytotoxicity in brain EC. In this work, we found that LPC caused cytosolic Ca2+ overload, mitochondrial membrane potential decrease, p38 activation, caspase 3 activation and eventually apoptotic death in mouse cerebral bEND.3 EC. In contrast to reported reactive oxygen species (ROS) generation by LPC in other EC, LPC did not trigger ROS formation in bEND.3 cells. Pharmacological inhibition of p38 alleviated LPC-inflicted cell death. We examined whether heparin could be cytoprotective: although it could not suppress LPC-triggered Ca2+ signal, p38 activation and mitochondrial membrane potential drop, it did suppress LPC-induced caspase 3 activation and alleviate LPC-inflicted cytotoxicity. Our data suggest LPC apoptotic death mechanisms in bEND.3 might involve mitochondrial membrane potential decrease and p38 activation. Heparin is protective against LPC cytotoxicity and might intervene steps between mitochondrial membrane potential drop/p38 activation and caspase 3 activation.

Enhancing tetrandrine cytotoxicity in human lung carcinoma A549 cells by suppressing mitochondrial ATP production.

ATP depletion induced by inhibiting glycolysis or mitochondrial ATP production has been demonstrated to cause cancer cell death. Whether ATP depletion can enhance the efficacy and potency of anti-cancer effects of herbal compounds is so far unknown. We examined the enhancing effect of ATP depletion on anti-cancer actions of tetrandrine (TET) in human lung carcinoma A549 cells. A 24-h incubation of A549 cells with tetrandrine caused a concentration-dependent cytotoxic effect (LC50 = 66.1 µM). Co-incubation with 20 mM 2-deoxyglucose (2-DG, glycolysis inhibitor) caused only a very slight enhancement of tetrandrine cytotoxicity. By contrast, inhibiting mitochondrial ATP production with oligomycin (10 µM, ATP synthase inhibitor) and FCCP (30 µM, uncoupling agent) (thus, oligo-FCCP) on its own caused only slight cell cytotoxicity but strongly potentiated tetrandrine cytotoxicity (tetrandrine LC50 = 15.6 µM). The stronger enhancing effect of oligo-FCCP than 2-DG on TET toxicity did not result from more severe overall ATP depletion, since both treatments caused a similar ATP level suppression. Neither oligo-FCCP nor 2-DG synergized with tetrandrine in decreasing mitochondrial membrane potential. TET on its own triggered reactive oxygen species (ROS) production, and oligo-FCCP, but not 2-DG, potentiated TET in causing ROS production. Taken together, our results suggest that inhibiting ATP production from mitochondria, but not from glycolysis, appears to be a very effective means in augmenting TET-triggered ROS production and hence toxicity in A549 cells.

Valproic acid inhibits ATP-triggered Ca2+ release via a p38-dependent mechanism in bEND.3 endothelial cells.

Valproic acid (VA) is currently used to treat epilepsy and bipolar disorder. It has also been demonstrated to promote neuroprotection and neurogenesis. Although beneficial actions of VA on brain blood vessels have also been demonstrated, the effects of VA on brain endothelial cell (EC) Ca2+ signaling are hitherto unreported. In this report, we examined the effects of VA on agonist-triggered Ca2+ signaling in mouse cortical bEND.3 EC. While VA (100 µm) did not cause an acute inhibition of ATP-triggered Ca2+ signaling, a 30-min VA treatment strongly suppressed ATP-triggered intracellular Ca2+ release; however, such treatment did not affect Ca2+ release triggered by cyclopiazonic acid, an inhibitor of SERCA Ca2+ pump, suggesting there was no reduction in Ca2+ store size. VA-activated p38 signaling, and VA-induced inhibition of ATP-triggered Ca2+ release was prevented by SB203580, a p38 inhibitor, suggesting VA caused the inhibition by activating p38. Remarkably, VA treatment did not affect acetylcholine-triggered Ca2+ release, suggesting VA may not inhibit inositol 1,4,5-trisphosphate-induced Ca2+ release per se, and may not act directly on Gq or phospholipase C. Taken together, our results suggest VA treatment, via a p38-dependent mechanism, led to an inhibition of purinergic receptor-effector coupling.

Midazolam activates caspase, MAPKs and endoplasmic reticulum stress pathways, and inhibits cell cycle and Akt pathway, to induce apoptosis in TM3 mouse Leydig progenitor cells.

BACKGROUND: Midazolam (MDZ) has powerful hypnosis, amnesia, anti-anxiety and anticonvulsant effects. Studies have shown that prenatally developmental toxicity of diazepam can be observed in many organs/tissues. However, it remains elusive in male reproductive system. MATERIALS AND METHODS: TM3 mouse Leydig progenitor cell line was used to determine whether MDZ has any unfavorable effects. RESULTS: Midazolam significantly decreased cell viability in dose- and time-dependent manners in TM3 cells. In flow cytometry analysis, midazolam significantly increased subG1 phase cell numbers, and annexin V/PI double staining assay further confirmed that MDZ induced apoptosis in TM3 cells. Moreover, MDZ significantly induced the expression of caspase-8 and -3 proteins and the phosphorylation of JNK, ERK1/2 and p38. Besides, MDZ didn't activate Akt pathway in TM3 cells. Furthermore, the expressions of p-EIF2α, ATF4, ATF3 and CHOP were induced by midazolam, suggesting that midazolam could induce apoptosis through endoplasmic reticulum (ER) stress in TM3 cells. Additionally, the expressions of cyclin A, cyclin B and CDK1 were inhibited by midazolam through the regulation of p53 in TM3 cells, indicating that midazolam could regulate cell cycle to induce apoptosis. CONCLUSION: Midazolam could activate caspase, MAPKs and ER stress pathways and impede Akt pathway and cell cycle to induce apoptosis in TM3 mouse Leydig progenitor cells.

Gossypol stimulates opening of a Ca2+ - and Na+ -permeable but Ni2+ - and Co2+ -impermeable pore in bEND.3 endothelial cells.

Gossypol, a polyphenolic dialdehyde toxin isolated from cotton seed, has anti-cancer properties and has recently shown some success in the treatment of glioma. Its effects on brain neurons and blood vessels are poorly understood. In this work we examined the effects of gossypol on cytosolic Ca2+ concentration ([Ca2+ ]i ) of mouse brain bEND.3 endothelial cells. Cell viability tests revealed that after 3 hour and 18 hour exposures, 10 µmol/L gossypol caused 23% and 65% cell death, respectively; 3 µmol/L gossypol caused no and 21% cell death, respectively. [Ca2+ ]i was raised concentration-dependently by 1-10 µmol/L gossypol. We then explored the Ca2+ signalling triggered by 3 µmol/L gossypol, which inflicted minimal toxicity: the Ca2+ signal was composed largely of Ca2+ influx and to a small extent, intracellular Ca2+ release. Such Ca2+ influx was much larger than store-operated Ca2+ influx triggered by maximal Ca2+ pool depletion. The Ca2+ influx triggered by 3 and 10 µmol/L gossypol caused NO release and cell death, respectively. Gossypol also triggered influx of Mn2+ and Na+ , but not Ni2+ and Co2+ . Gossypol-triggered Ca2+ signal was inhibited only by 14% and 37% by 100 µmol/L La3+ and 10 µmol/L nimodipine, respectively; and not suppressed at all by 5 mmol/L Ni2+ . Gossypol-triggered Ca2+ signal was suppressed by 78% by 30 µmol/L ruthenium red, suggesting gossypol may act on TRPV channels. Our results suggest gossypol triggered opening of a non-selective cation pore, possibly a member of the TRPV family.

Eicosapentaenoic acid triggers Ca2+ release and Ca2+ influx in mouse cerebral cortex endothelial bEND.3 cells.

Eicosapentaenoic acid (EPA), an omega-3 fatty acid abundant in fish oil, protects endothelial cells (EC) from lipotoxicity and triggers EC NO release. The latter is related to an elevation of cytosolic Ca2+. Although EPA has been shown to cause human EC cytosolic Ca2+ elevation, the mechanism is unclear. Microfluorimetric imaging was used here to measure free cytosolic Ca2+ concentration. EPA was shown to cause intracellular Ca2+ release in mouse cerebral cortex endothelial bEND.3 cells; interestingly, the EPA-sensitive intracellular Ca2+ pool(s) appeared to encompass and was larger than the Ca2+ pool mobilized by sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition by cyclopiazonic acid. EPA also opened a Ca2+ influx pathway pharmacologically distinct from store-operated Ca2+ influx. Surprisingly, EPA-triggered Ca2+ influx was Ni2+-insensitive; and EPA did not trigger Mn2+ influx. Further, EPA-triggered Ca2+ influx did not involve Na+-Ca2+ exchangers. Thus, our results suggest EPA triggered unusual mechanisms of Ca2+ release and Ca2+ influx in EC.

Voltage-gated K+ channels promote BT-474 breast cancer cell migration.

OBJECTIVE: A variety of ion channels have been implicated in breast cancer proliferation and metastasis. Voltage-gated K+ (Kv) channels not only cause repolarization in excitable cells, but are also involved in multiple cellular functions in non-excitable cells. In this study we investigated the role of Kv channels in migration of BT474 breast cancer cells. METHODS: Transwell technique was used to separate migratory cells from non-migratory ones and these two groups of cells were subject to electrophysiological examinations and microfluorimetric measurements for cytosolic Ca2+. Cell migration was examined in the absence or presence of Kv channel blockers. RESULTS: When compared with non-migratory cells, migratory cells had much higher Kv current densities, but rather unexpectedly, more depolarized membrane potential and reduced Ca2+ influx. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of Kv1.1, Kv1.3, Kv1.5, Kv2.1, Kv3.3, Kv3.4 and Kv4.3 channels. Cell migration was markedly inhibited by tetraethylammonium (TEA), a delayed rectifier Kv channel blocker, but not by 4-aminopyridine, an A-type Kv channel blocker. CONCLUSIONS: Taken together, our results show that increased Kv channel expression played a role in BT474 cell migration, and Kv channels could be considered as biomarkers or potential therapeutic targets for breast cancer metastasis. The mechanism(s) by which Kv channels enhanced migration appeared unrelated to membrane hyperpolarization and Ca2+ influx.

Perturbation of Akt Signaling, Mitochondrial Potential, and ADP/ATP Ratio in Acidosis-Challenged Rat Cortical Astrocytes.

Cells switch to anaerobic glycolysis when there is a lack of oxygen during brain ischemia. Extracellular pH thus drops and such acidosis causes neuronal cell death. The fate of astrocytes, mechanical, and functional partners of neurons, in acidosis is less studied. In this report, we investigated the signaling in acidosis-challenged rat cortical astrocytes and whether these signals were related to mitochondrial dysfunction and cell death. Exposure to acidic pH (6.8, 6.0) caused Ca2+ release and influx, p38 MAPK activation, and Akt inhibition. Mitochondrial membrane potential was hyperpolarized after astrocytes were exposed to acidic pH as soon as 1 h and lasted for 24 h. Such mitochondrial hyperpolarization was prevented by SC79 (an Akt activator) but not by SB203580 (a p38 inhibitor) nor by cytosolic Ca2+ chelation by BAPTA, suggesting that only the perturbation in Akt signaling was causally related to mitochondrial hyperpolarization. SC79, SB203580, and BAPTA did not prevent acidic pH-induced cell death. Acidic pH suppressed ROS production, thus ruling out the role of ROS in cytotoxicity. Interestingly, pH 6.8 caused an increase in ADP/ATP ratio and apoptosis; pH 6.0 caused a further increase in ADP/ATP ratio and necrosis. Therefore, astrocyte cell death in acidosis did not result from mitochondrial potential collapse; in case of acidosis at pH 6.0, necrosis might partly result from mitochondrial hyperpolarization and subsequent suppressed ATP production. J. Cell. Biochem. 118: 1108-1117, 2017. © 2016 Wiley Periodicals, Inc.

Repressed Ca(2+) clearance in parthenolide-treated murine brain bEND.3 endothelial cells.

Parthenolide is a sesquiterpene lactone compound isolated from the leaves and flowerheads of the plant feverfew (Tanacetum parthenium). The anticancer effects of parthenolide have been well studied and this lactone compound is currently under clinical trials. Parthenolide is also a protective agent in cardiac reperfusion injury via its inhibition of nuclear factor-κB (NF-κB). Not much is known if this compound affects signal transduction in non-tumor cells. We investigated whether parthenolide affected Ca(2+) signaling in endothelial cells, key components in regulating the vascular tone. In this work using mouse cortical microvascular bEND.3 endothelial cells, we found that a 15-h treatment with parthenolide resulted in amplified ATP-triggered Ca(2+) signal; the latter had a very slow decay rate suggesting suppression of Ca(2+) clearance. Evidence suggests parthenolide suppressed Ca(2+) clearance by inhibiting the plasmalemmal Ca(2+) pump; such suppression did not result from decreased expression of the plasmalemmal Ca(2+) pump protein. Rather, such suppression was possibly a consequence of endoplasmic reticulum (ER) stress, since salubrinal (an ER stress protector) was able to alleviate parthenolide-induced Ca(2+) clearance suppression. Given the current deployment of parthenolide as an anti-cancer drug in clinical trials and the potential usage of this lactone as a cardioprotectant, it is important to examine in details the perturbing effects of parthenolide on Ca(2+) homeostasis in endothelial cells and neighboring vascular smooth muscle cells, activities of which exert profound effects on hemodynamics.

Palmitic acid-induced lipotoxicity and protection by (+)-catechin in rat cortical astrocytes.

BACKGROUND: Astrocytes do not only maintain homeostasis of the extracellular milieu of the neurons, but also play an active role in modulating synaptic transmission. Palmitic acid (PA) is a saturated fatty acid which, when being excessive, is a significant risk factor for lipotoxicity. Activation of astrocytes by PA has been shown to cause neuronal inflammation and demyelination. However, direct damage by PA to astrocytes is relatively unexplored. The aim of this study was to identify the mechanism(s) of PA-induced cytotoxicity in rat cortical astrocytes and possible protection by (+)-catechin. METHODS: Cytotoxicity and endoplasmic reticulum (ER) markers were assessed by MTT assay and Western blotting, respectively. Cytosolic Ca(2+) and mitochondrial membrane potential (MMP) were measured microfluorimetrically using fura-2 and rhodamine 123, respectively. Intracellular reactive oxygen species (ROS) production was assayed by the indicator 2'-7'-dichlorodihydrofluorescein diacetate. RESULTS: Exposure of astrocytes to 100µM PA for 24h resulted in apoptotic cell death. Whilst PA-induced cell death appeared to be unrelated to ER stress and perturbation in cytosolic Ca(2+) signaling, it was likely a result of ROS production and subsequent MMP collapse, since ascorbic acid (anti-oxidant, 100µM) prevented PA-induced MMP collapse and cell death. Co-treatment of astrocytes with (+)-catechin (300µM), an anti-oxidant found abundantly in green tea, significantly prevented PA-induced ROS production, MMP collapse and cell death. CONCLUSION: Our results suggest that PA-induced cytotoxicity in astrocytes may involve ROS generation and MMP collapse, which can be prevented by (+)-catechin.

Midazolam induces apoptosis in MA-10 mouse Leydig tumor cells through caspase activation and the involvement of MAPK signaling pathway.

PURPOSE: The present study aims to investigate how midazolam, a sedative drug for clinical use with cytotoxicity on neuronal and peripheral tissues, induced apoptosis in MA-10 mouse Leydig tumor cells. METHODS: The apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods. RESULTS: Data showed that midazolam induced the accumulation of the MA-10 cell population in the sub-G1 phase and a reduction in the G2/M phase in a time- and dose-dependent manner, suggesting an apoptotic phenomenon. Midazolam could also induce the activation of caspase-8, -9, and -3 and poly (ADP-ribose) polymerase proteins. There were no changes in the levels of Bax and cytochrome-c, whereas Bid was significantly decreased after midazolam treatment. Moreover, midazolam decreased both pAkt and Akt expression. In addition, midazolam stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase but not extracellular signal-regulated kinase. CONCLUSION: Midazolam could induce MA-10 cell apoptosis through the activation of caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways.

Suppression of voltage-gated Na(+) channels and neuronal excitability by imperatorin.

Imperatorin is a naturally occurring furocoumarin compound isolated from plants such as Angelica archangelica and Cnidium monnieri. It has multiple pharmacological effects including anticonvulsant effects. Here we determined the effects of imperatorin on voltage-gated Na(+) channels (VGSC) using whole-cell patch clamp techniques in differentiated neuronal NG108-15 cells. We showed that imperatorin inhibited VGSC; such inhibition did not show state-dependence. Imperatorin caused a left shift in the steady-state inactivation curve without affecting activation gating. The inhibition of VGSC by imperatorin displayed a mild frequency-dependence. Imperatorin was also shown to inhibit VGSC and action potential amplitude without affecting voltage-gated K(+) channels in rat hippocampal CA1 neurons. In conclusion, our results suggest that imperatorin dampens neuronal excitability by inhibiting VGSC.

Intravenous mannitol does not increase blood-brain barrier permeability to inert dyes in the adult rat forebrain.

Intravenous mannitol (IV-M) is widely administered in the clinic to lower intracranial pressure in patients with brain trauma and stroke. However, intracarotid arterial mannitol (ICA-M) is known to potently open the blood-brain barrier (BBB) to serum protein tracers such as the Evans blue dye (EBD). In this study, we aimed to determine the potential effect of IV-M on BBB permeability to EBD and a small molecular tracer sodium fluorescein dye (NaF). Rats received intravenous EBD/NaF injections, and after a 30-min equilibration time, they received mannitol (20%, 0.5 g/kg) through either route of administration. At 90 min after the mannitol injection, the rats were perfused to rid their circulations of the tracers, and the tracers extravasated into the brain parenchyma were measured by photospectrometry. As expected, ICA-M considerably increased EBD extravasation into the rat forebrain regions, including the motor cortex (P=0.0069), the striatum (P=0.0097), and the hippocampus (P=0.0281; student's t-test). In marked contrast, IV-M exerted no effect on EBD extravasation into these forebrain regions. To increase the power of the IV-M study, we repeated the experiments in two independent trials of experiments (n=6-9/group/trial) and found the same result. Finally, consistent with no effect on EBD extravasation, IV-M had no effect on NaF extravasation into the rat forebrain. In conclusion, we report direct evidence that IV-M, at a dose used clinically, in contrast to the same dose of ICA-M, exerted no effect on BBB permeability to protein and small molecular tracers.

Inhibition of voltage-gated K+ channels and Ca2+ channels by diphenidol.

BACKGROUND: Although diphenidol has long been deployed as an anti-emetic and anti-vertigo drug, its mechanism of action remains unclear. In particular, little is known as to how diphenidol affects neuronal ion channels. Recently, we showed that diphenidol blocked neuronal voltage-gated Na(+) channels, causing spinal blockade of motor function, proprioception and nociception in rats. In this work, we investigated whether diphenidol could also affect voltage-gated K(+) and Ca(2+) channels. METHODS: Electrophysiological experiments were performed to study ion channel activities in two neuronal cell lines, namely, neuroblastoma N2A cells and differentiated NG108-15 cells. RESULTS: Diphenidol inhibited voltage-gated K(+) channels and Ca(2+) channels, but did not affect store-operated Ca(2+) channels. CONCLUSION: Diphenidol is a non-specific inhibitor of voltage-gated ion channels in neuronal cells.

Increase in Evans blue dye extravasation into the brain in the late developmental stage.

The development of the blood-brain barrier (BBB) against permeability to inert tracers, such as Evans blue dye (EBD), occurs quite early on at embryonic stages (before E13-E15), and the BBB remains resistant to EBD between E15 and early adulthood (P20-P30). Here, we aimed to examine the changes in EBD permeability at a later stage in development, specifically comparing young rats (P20) with adult rats (P86). We found markedly higher EBD extravasation into the forebrains of adult rats compared with those of the young rats (P=0.0132; Student's t-test). In contrast, there was no difference in EBD extravasation to the liver, suggesting no change in vascular permeability in peripheral tissues. Furthermore, EBD extravasation into the cerebellum was less prominent than that into the forebrain, suggesting that the disruption of the BBB was brain-region specific. In conclusion, we found a specific increase in EBD extravasation in the mature forebrain, and the protocol that we used may be a good template for studying developmental disruption of the BBB.

Inhibitory effects of magnolol on voltage-gated Na+ and K+ channels of NG108-15 cells.

Magnolol, a polyphenolic compound isolated from Houpu, a Chinese herb from the bark of Magnolia officinalis, has been reported to have in vitro and in vivo neuroprotective effects. In spite of these reported beneficial effects, studies on the direct impact of magnolol on neuronal ion channels have been scarce. Whether magnolol affects voltage-gated Na(+) channels (VGSC) and voltage-gated K(+) (Kv) channels is unknown. Using the whole-cell voltage-clamp method, we studied the effects of magnolol on voltage-gated ion channels in neuronal NG108-15 cells. Magnolol inhibited VGSC channels with mild state-dependence (IC(50) of 15 and 30 µM, at holding potentials of -70 and -100 mV, respectively). No frequency-dependence was observed in magnolol block. Magnolol caused a left-shift of 18 mV in the steady-state inactivation curve but did not affect the voltage-dependence of activation. Magnolol inhibited Kv channels with an IC(50) of 21 µM, and it caused a 20-mV left-shift in the steady-state inactivation curve without affecting the voltage-dependence of activation. In conclusion, magnolol is an inhibitor of both VGSC and Kv channels and these inhibitory effects may in part contribute to some of the reported neuroprotective effects of magnolol.

Vallecular cyst and laryngomalacia in infants: report of six cases and airway management.

IMPLICATIONS: This report describes difficulties encountered in the airway management of six infants with concurrent vallecular cyst and laryngomalacia. It is hoped that our experience will assist others in the management of such patients.