Presence Of Niflumic Acid Research Articles

Determination of potential toxicodynamic differences of pyrethroid insecticides on native voltage-sensitive sodium channels in juvenile versus adult rat brain

Microtransplantation of neurolemma tissue fragments from mammalian brain into the plasma membrane of Xenopus laevis oocytes is a tool to examine the endogenous structure and function of various ion channels and receptors associated with the central nervous system. Microtransplanted neurolemma can originate from a variety of sources, contain ion channels and receptors in their native configuration, and are applicable to examine diseases associated with different channelopathies. Here, we examined potential age-related differences in voltage-sensitive sodium channel (VSSC) expression and concentration-dependent responses to pyrethroids following the microtransplantation of juvenile or adult rat brain tissue (neurolemma) into X. laevis oocytes. Using automated western blotting, adult neurolemma exhibited a 2.5-fold higher level of expression of VSSCs compared with juvenile neurolemma. The predominant isoform expressed in both tissues was Nav1.2. However, adult neurolemma expressed 2.8-fold more Nav1.2 than juvenile and expressed Nav1.6 at a significantly higher level (2.2-fold). Microtransplanted neurolemma elicited ion currents across the plasma membrane of oocytes following membrane depolarization using two electrode voltage clamp electrophysiology. A portion of this current was sensitive to tetrodotoxin (TTX) and this TTX-sensitive current was abolished when external sodium ion was replaced by choline ion, functionally demonstrating the presence of native VSSC. Increasing concentrations of permethrin or deltamethrin exhibited concentration-dependent increases in inward TTX-sensitive current in the presence of niflumic acid from both adult and juvenile tissues following a pulsed depolarization of the oocyte plasma membrane. Concentration-dependent response curves illustrate that VSSCs associated with juvenile neurolemma were up to 2.5-fold more sensitive to deltamethrin than VSSCs in adult neurolemma. In contrast, VSSCs from juvenile neurolemma were less sensitive to permethrin than adult VSSCs at lower concentrations (0.6–0.8-fold) but were more sensitive at higher concentrations (up to 2.4-fold). Nonetheless, because the expected concentrations in human brains following realistic exposure levels are approximately 21- (deltamethrin) to 333- (permethrin) times below the threshold concentration for response in rat neurolemma-injected oocytes, age-related differences, if any, are not likely to be toxicologically relevant.

Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

Cholecystokinin activates a Ca2+‐dependent Cl‐ conductance in intestinal nodose neurons that is inhibited in diet‐induced obesity and may suppress satiety (1107.8)

The cholecystokinin (CCK) induced satiety signaling of vagal afferents is suppressed in diet‐induced obese mice, which contributes to the increased food intake. In this study we tested the hypothesis that a Ca2+‐activated chloride channel (CaCC) mediates the response of normal intestinal vagal afferent nodose neurons to CCK and that excessively high fat diet (HFD) suppresses the channel activation and leads to obesity. We first used Ca2+ imaging techniques and the whole cell patch‐clamp on cultured nodose ganglia neurons isolated from anesthetized C57/BL6 mice. Application of CCK increased [Ca2+]i in a dose‐dependent manner reaching near maximal level at 10 μM. This dose of CCK also induced a large inward current (36.0±11.0 pA/pF, n=7) that was eliminated with the fast Ca2+ chelator BAPTA (1.0±0.5 pA/pF, n=7). The reversal potential of the current was ~0mV with equal concentration of Cl‐ on both sides of the cell membrane and shifted to a more positive direction after lowering the extracellular [Cl‐]o confirming that it is an outward Cl‐ conductance. Moreover, the CCK‐induced response was reduced significantly from 35.2±3.2 pA/pF in untreated control neurons (n=9) to 13.6±2.0 pA/pF in the presence of niflumic acid (100μM, n=8), a blocker of the CaCC channel (p<0.05). DiI labeled proximal intestinal nodose neurons obtained from control mice responded to CCK with an average current of 24.8±4.9 pA/pF (n=7), whereas the response of labeled neurons from obese mice fed a 60% HFD for 10 weeks was significantly lower at 6.1±2.9 pA/pF (n=7) (p<0.01). Our results demonstrate that a CCK‐sensitive CaCC in proximal intestinal nodose neurons is desensitized in mice fed a high fat diet. This CaCC ionic conductance may be responsible for satiety signaling of vagal afferents and its impairment may contribute to excessive food intake and obesity.Grant Funding Source: Supported by NIH HL 14388

124. THE ROLE OF CALCIUM ACTIVATED CHLORIDE CHANNELS AT FERTILISATION

Sperm entry into the oocyte triggers a signal transduction pathway resulting in intracellular calcium [Ca2+] oscillations that coincide with hyperpolarisations in membrane potential (Em). Ca2+ oscillations have been previously described and found to be important for embryo development, yet Em hyperpolarisations and their importance at fertilisation still remains unclear. Thimerosal, a sulfhydryl reagent, has been shown to mimic the physiological changes caused by sperm following fertilisation. It does this by direct sensitisation of the inositol 1,4,5-triphosphate receptor-1 to basal levels of inositol 1,4,5-triphopshate. Previous patch clamp analysis of unfertilised mouse oocytes has shown that thimerosal elicits simultaneous Em hyperpolarisations and Ca2+ oscillations. These results have lead us to hypothesise that hyperpolarisations in Em may be due to the activation of a Ca2+ activated Cl- channel (CaCC) present in the membrane of mouse oocytes. The present study aims to identify this CaCC and assess its role in early development following fertilisation. Hyperpolarisations induced by thimerosal were inhibited by niflumic acid, a selective blocker of CaCCs. The inhibition of Em hyperpolarisations suggests that a CaCC is present and plays an active role in initiating hyperpolarisations. To identify the function of the CaCC at fertilisation, in vitro fertilisation was performed in the presence of niflumic acid. Niflumic acid inhibited polar body extrusion and pronuclei formation; two events that are indicators of fertilisation. Furthermore, Ca2+ imaging experiments with the calcium sensitive dye fura 2-AM, demonstrated that in the presence of niflumic acid, Ca2+ oscillations induced by thimerosal are reduced in size, number and duration. Taken together these data suggest that the activation of a Ca2+ activated Cl– channel in the mouse oocyte may play an important role in the events occurring at fertilisation.

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

Effects of niflumic acid on α 1-adrenoceptor-induced vasoconstriction in mesenteric artery in vitro and in vivo in two-kidney one-clip hypertensive rats

The influence of niflumic acid (3 and 10 μM), a Cl − channel antagonist, on cirazoline-induced vasoconstriction in isolated perfused mesenteric artery (5 ml/min) from two-kidney one-clip (2K1C) hypertensive and sham normotensive rats was examined. In addition, the effect of a single i.v. bolus injection of niflumic acid (3 mg/kg) on cirazoline-mediated reduction in vascular conductance in superior mesenteric artery was determined in pentobarbital-anaesthetized hypertensive and normotensive rats. Bolus injections of cirazoline induced a dose-dependent transient increase in the perfusion pressure in vitro. In the presence of niflumic acid, cirazoline-mediated vasoconstriction was significantly inhibited. Cirazoline-induced vasoconstriction in isolated mesenteric beds was also significantly inhibited following perfusion with Cl −-free buffer. Pre-perfusion of mesenteric blood vessels with Cl −-free buffer resulted in a significantly greater inhibition of cirazoline-mediated vasoconstriction in sham normotensive rats than in hypertensive rats. We found that in Cl −-free buffer, cirazoline-mediated vasoconstriction could be further inhibited by niflumic acid. Intravenous infusion of cumulative doses of cirazoline in vivo caused a dose-dependent decrease in superior mesenteric vascular conductance. Pretreatment with niflumic acid significantly impaired cirazoline-mediated decreases in vascular conductance. Our results indicate that chloride ions play an important role in α 1-adrenoceptor-mediated vasoconstriction in mesenteric blood vessels. In addition, the contribution of chloride ions in α 1-adrenoceptor-mediated vasoconstriction in blood vessels from hypertensive rats appears to be reduced.

Functional expression of voltage dependent sodium channels in Xenopus oocytes injected with mRNA from neonatal or adult rat brain

The two electrode voltage clamp technique was used to study voltage-dependent sodium currents ( I Na) in Xenopus laevis oocytes previously injected with mRNA extracted from adult (A) or neonatal (N, < 5 days old) rat brains. In the presence of niflumic acid (300 μM) to block endogenous Ca 2+-activated Cl − currents, depolarizing voltage steps from a holding potential of −100 mV to various voltages elicited in both groups of oocytes fast inward sodium currents which peaked at ∼ 0 mV and then slowly declined to ∼ 75% of the maximum current at +40 mV. At the peak, A I Na was significantly larger than N I Na (296 ± 59 nA vs. 147 ± 32 nA). Inactivation kinetics of N I Na was best fit with one exponential component whereas A I Na with two exponential components. A significant difference in the voltage dependence of inactivation was found between A I Na or N I Na. The values of V h were −53 ± 0.9 mV or −59.8 ± 0.7 mV for A I Na or N I Na respectively. The recovery from inactivation was fitted in both groups with two exponential functions ( τ f and τ s) whose values were not significantly different. However the ratio between τ f and τ s was significantly higher for N I Na comparing to A I Na (5.7 vs. 2.1). TTX reversibly blocked I Na. The IC 50 value was 58.2 ± 6.3 nM for A I Na and 20.4 ± 2.2 nM for N I Na. These results suggest that different isoforms of TTX-sensitive, voltage-dependent sodium channel subunits are functionally expressed, may be in different proportions in oocytes injected with A or N mRNA.

Depression of ATP-induced Ca2+ signalling by high K+ and low Cl- media in human aortic endothelial cells.

We studied the contribution of the Cl- channel as well as K+ channel in the regulation of Ca2+ signalling in fura-2-loaded cultured human aortic endothelial cells. Low Cl- (20 mM) superfusion did not affect the ATP (10 microM)-induced [Ca2+]i increase at the initial peak (control 309 +/- 30 nM (mean +/- SD, n = 17) versus 20 mM [Cl-]o 308 +/- 40 nM (n = 8)) but depressed it at the sustained phase (at 5 min, 170 +/- 29 nM versus 85 +/- 10 nM). Similar selective depression of the sustained phase occurred also in Ca(2+)-free and 140 mM K+ solutions and in the presence of niflumic acid (300 microM), a blocker of the Cl- channel and Ca2+ permeable cation channel. After application of ATP, the increase of [Cl-]o from 20 to 146 mM resulted in a Ca2+ overshoot. Both Cl- and K+ channels play an important role in the regulation of Ca2+ influx presumably by controlling the membrane potential in vascular endothelial cells.