chloramine-t and Neuroblastoma

Methionine sulfoxide reductases A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. Recently, attention has turned to MsrA in coupling with the pathology of Parkinson's disease, which is closely related to neurotoxins that cause dopaminergic neuron degeneration. Here, we firstly provided evidence that pretreatment with a natural polyphenol resveratrol (RSV) up-regulated the expression of MsrA in human neuroblastoma SH-SY5Y cells. It was also observed that the expression and nuclear translocation of forkhead box group O 3a (FOXO3a), a transcription factor that activates the human MsrA promoter, increased after RSV pretreatment. Nicotinamide , an inhibitor of silent information regulator 1 (SIRT1), prevented RSV-induced elevation of FOXO3a and MsrA expression, indicating that the effect of RSV was mediated by a SIRT1-dependent pathway. RSV preconditioning increased methionine sulfoxide(MetO)-reducing activity in SH-SY5Y cells and enhanced their resistance to neurotoxins, including chloramine-T and 1-methyl-4-phenyl-pyridinium. In addition, the enhancement of cell resistance to neurotoxins caused by RSV preconditioning can be largely prevented by MsrA inhibitor dimethyl sulfoxide. Our findings suggest that treatment with polyphenols such as RSV can be used as a potential regulatory strategy for MsrA expression and function.

Topics: 1-Methyl-4-phenylpyridinium; Antioxidants; Cell Line, Tumor; Chloramines; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Methionine; Methionine Sulfoxide Reductases; Models, Biological; Neuroblastoma; Neurotoxins; Oxidative Stress; Resveratrol; Sirtuin 1; Stilbenes; Tosyl Compounds; Up-Regulation

1. The actions of the neuroprotective agent, lifarizine (RS-87476-190), on voltage-dependent Na+ currents have been examined in the neuroblastoma cell line, N1E-115, using the whole-cell variant of the patch clamp technique. 2. At a holding potential of -80 mV, lifarizine reduced the peak Na+ current evoked by a 10 ms depolarizing step with an IC50 of 1.3 microM. At holding potentials of -100 and -60 mV the IC50 concentrations of lifarizine were 7.3 microM and 0.3 microM, respectively. 3. At a holding potential of -100 mV, most channels were in the resting state and the IC50 value for inhibition of Na+ current should correspond to the dissociation constant of lifarizine for resting channels (KR). KR was therefore estimated to be 7.3 microM. 4. In the absence of lifarizine, recovery from inactivation following a 20 s depolarization from -100 mV to 0 mV was complete within 2 s. However, in the presence of 3 microM lifarizine recovery took place in a biexponential fashion with time constants of 7 s and 79 s. 5. Lifarizine (1 microM) had no effect on steady-state inactivation curves when conditioning pre-pulses of 1 s duration were used. However, when pre-pulse durations of 1 min were used the curves were shifted to the left by lifarizine by about 10 mV. Analysis of the shifts induced by a range of lifarizine concentrations revealed that the apparent affinity of lifarizine for the inactivated state of the channel (K1) was 0.19 microM. 6. Lifarizine (1 microM) had no effect on chloramine-T-modified Na+ currents, suggesting no significant open channel interaction. 7. Taken together, these data show that lifarizine is a potent voltage-dependent inhibitor of Na+currents in NIE-115 cells and that the voltage-dependence arises from an interaction of the compound with the inactivated state of the channel. The possible contribution of Na+ current inhibition to the neuroprotective actions of lifarizine is discussed.

Topics: Animals; Cell Line; Cells, Cultured; Chloramines; Dose-Response Relationship, Drug; Imidazoles; Membrane Potentials; Mice; Neuroblastoma; Patch-Clamp Techniques; Piperazines; Sodium Channels; Tosyl Compounds

Patch-clamp experiments were done on sodium channels of neuroblastoma cells (N1E-115) in the presence of tetraethylammonium ions to block potassium channels. In Ringer solution whole-cell records revealed a diphasic INa inactivation with the fast (tau 0) component. being clearly larger than the slow (tau 1 approximately 3 tau 0) component. In single-channel studies on inside-out patches the mean open time, to, turned out to be only a fraction of tau 0 and almost independent of membrane potential. After external application of chloramine-T INa inactivation of whole cells was delayed with both tau 0 and tau 1 increased, and incomplete, i.e. a persistent current component emerged. The latter was maximal at a more positive membrane potential than the peak current. Also, after chloramine-T treatment the peak INa increased, particularly at weak depolarizations. In inside-out patches the equally effective internal application of chloramine-T led to bursting channel openings with mean burst times (tb) approximately 6 ms, and gap times (tg) approximately 20 ms, where gap is defined as a closure of greater than or equal to 1.5 ms. Within the bursts to was approximately 2 ms, again clearly shorter than tau 0; the mean close time, tc was approximately 0.5 ms. The single-channel conductance was approximately 13 pS and unaffected by chloramine-T. Diphasic INa inactivation and the fact that to less than tau 0 led to an extension of the model of Aldrich and Stevens [J Neurosci 7:418-431 (1987)], in which overall kinetics is determined by the openings rather than closures of the sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Chloramines; Electric Conductivity; Isotonic Solutions; Kinetics; Neuroblastoma; Ringer's Solution; Sodium; Sodium Channels; Tosyl Compounds; Tumor Cells, Cultured

The steady-state effects and rate of action of 4-aminopyridine (4-AP) on normal and chloramine-T (CL-T)-modified voltage-dependent potassium (K) currents were studied in neuroblastoma cells with the whole-cell voltage-clamp current recording technique. 4-AP apparently slows both the activation and inactivation of the normal current but does not modify the time course of the CL-T-modified current. These differential effects of 4-AP are interpreted as resulting from the existence of two types of K channels with different 4-AP sensitivities under normal conditions and similar 4-AP sensitivities after CL-T, which furthermore slows their inactivation [8, 9]. While the onset of 4-AP action on the normal current is delayed and can be described by the difference of two exponentials, the onset of 4-AP action on CL-T-modified current starts immediately after the external application of the drug and can be described by the sum of two exponentials. The 4-AP-induced block of the normal current exhibits use-dependent features and is relieved by long conditioning depolarizations. In contrast, the block of the CL-T-modified current is not use-dependent. At high 4-AP concentrations (1-10 mM), the steady-state block of the normal current reaches a saturating value of 95%, while the steady-state block of the CL-T-modified current and the "unblocked" normal current only reaches a saturating value of 35%. The results suggest that CL-T inhibits a channel or membrane constituent which contributes to the inactivation of channels and increases their apparent affinity for 4-AP when they are in closed or open states.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Aminopyridine; Chloramines; Dose-Response Relationship, Drug; Electrophysiology; Neuroblastoma; Potassium Channels; Time Factors; Tosyl Compounds; Tumor Cells, Cultured

Currents through sodium channels of neuroblastoma cells were measured using patch technique in outside-out configuration. Scorpion toxin (ScTX) produced 3 to 4 fold prolongation of mean open time and increased number of reopenings. The mean open times showed slow fluctuations around some average values. The distribution of channel open times for ScTX-modified channels required more than one exponential to be fitted. Chloramine-T (ChT) produced qualitatively similar, though weaker, prolongation of open times.

Topics: Animals; Anti-Infective Agents, Local; Cell Line; Chloramines; Electric Conductivity; Kinetics; Membrane Potentials; Neuroblastoma; Probability; Scorpion Venoms; Sodium Channels; Tosyl Compounds; Tumor Cells, Cultured

We have studied the behaviour of single sodium channels in cell-attached patches in neuroblastoma cell line N1E 115 at 8-15 degrees C modified by chloramine-T (CT). Application of the agent to the external surface of the membrane cause different effects: CT binds to the membrane and cause irreversible effects leading to a decrease of open channel lifetime at 10 or 20 mV relative potentials without affecting the amplitude of single channel current. Channels modified by CT show a tendency to occur in burst (they can open and close several times during depolarization). Therefore, the slower decay of the average current must be explained by the flickering behaviour of the modified channels. We also examined the effect of CT on the relationship between open time of the channels and membrane potential. On the other hand, a small decrease in the mean delay time as a function of membrane voltages was observed which probably is related to the three state model of inactivation. Our results suggest that CT modifies sodium channel inactivation.

Topics: Animals; Chloramines; Kinetics; Membrane Potentials; Mice; Neuroblastoma; Sodium Channels; Tosyl Compounds; Tumor Cells, Cultured

Topics: Animals; Chloramines; Electrophysiology; Mice; Neuroblastoma; Potassium Channels; Tosyl Compounds

Single channel currents of chloramine-T (Chl-T) and sea anemone toxin (ATX-II) modified sodium channels were studied in neuroblastoma cells. With both substances similar subconductance states have been observed. The conductances of the sublevels were multiples of the "unit" step which was about one-forth of the most frequently occurring main conductance. Thus, the current levels observed were one fourth, half and five-fourths of the main current size. Both substances caused a slower decay of the averaged current compared to the current of the native channels. The main single-channel conductance was 15.2 pS (T = 16 degrees C) for the Chl-T and 10.8 pS (T = 12 degrees C) for the ATX-II modified channels. The channel open time was doubled by ATX-II, but was not increased significantly by Chl-T. The existence of the subconductance states suggests that the native channels may also have multiple open conformations.

Topics: Animals; Anti-Infective Agents, Local; Cell Line; Chloramines; Cnidarian Venoms; Ion Channels; Kinetics; Mice; Neuroblastoma; Neurotoxins; Tosyl Compounds

Effect of chloramine-T, the specific reagent on methionine residues, on gating of sodium channels was studied in neuroblastoma cell membrane. After the chloramine treatment the inactivation became slower, incomplete and the steepness of its voltage dependence considerably decreased; the inactivation curve was shifted towards depolarization. Time course of activation did not change. The activation curve shifted towards negative potentials, its slope being insignificantly decreased. Effective charge of activation as determined from the limiting logarithmic slope of the activation decreased by a factor of 1.17. Possible explanations of the phenomena observed are discussed.

Topics: Animals; Chloramines; Hydrogen-Ion Concentration; Indicators and Reagents; Ion Channels; Membrane Potentials; Mice; Neuroblastoma; Sodium; Time Factors; Tosyl Compounds; Tumor Cells, Cultured