Chung-Soo Lee

Amitriptyline modulation of Na+ channels in rat dorsal root ganglion neurons

The effects of amitriptyline, a tricyclic antidepressant, on tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) currents in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp method. Amitriptyline blocked both types of Na(+)currents in a dose-and holding potential-dependent manner. At the holding potential of -80 mV, the apparent dissociation constants (K(d)) for amitriptyline to block tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) channels were 4.7 and 105 microM, respectively. These values increased to 181 and 193 microM, respectively, when the membrane was held at a potential negative enough to remove the steady-state inactivation. Amitriptyline dose-dependently shifted the steady-state inactivation curves in the hyperpolarizing direction and increased the values of the slope factors for both types of Na(+) channels. The voltage dependence of the activation of both types of Na(+) channels was shifted in the depolarizing direction. It was concluded that amitriptyline blocked the two types of Na(+) channels in rat sensory neurons by modulating the activation and the inactivation kinetics.

Block of sodium currents in rat dorsal root ganglion neurons by diphenhydramine

To elucidate the local anesthetic mechanism of diphenhydramine, its effects on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were examined by the whole-cell voltage clamp method. Diphenhydramine blocked TTX-S and TTX-R sodium currents with K(d) values of 48 and 86 microM, respectively, at a holding potential of -80 mV. It shifted the conductance-voltage curve for TTX-S sodium currents in the depolarizing direction but had little effect on that for TTX-R sodium currents. Diphenhydramine caused a shift of the steady-state inactivation curve for both types of sodium currents in the hyperpolarizing direction. The time-dependent inactivation became faster and the recovery from the inactivation was slowed by diphenhydramine in both types of sodium currents. Diphenhydramine produced a profound use-dependent block when the cells were repeatedly stimulated with high-frequency depolarizing pulses. The use-dependent block was more pronounced in TTX-R sodium currents. The results show that diphenhydramine blocks sodium channels of sensory neurons similarly to local anesthetics.

7-Ketocholesterol enhances 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells

Summary.The present study investigated the promoting effect of oxysterol 7-ketocholesterol against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP+) in differentiated PC12 cells. 7-Ketocholesterol significantly enhanced the MPP+-induced nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, ascorbate, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of MPP+ in the presence of 7-ketocholesterol. The results indicate that 7-ketocholesterol shows a synergistic effect against the cytotoxic effect of MPP+. 7-Ketocholesterol may enhance the MPP+-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of reactive oxygen species and depletion of GSH. The findings suggest that 7-ketocholesterol as a promoting agent for the formation of mitochondrial permeability transition may enhance the toxic neuronal cell injury.

Modulation of 1-methyl-4-phenylpyridinium-induced mitochondrial dysfunction and cell death in PC12 cells by KATP channel block

Summary.The present study investigated the effect of 5-hydroxydecanoate, a selective mitochondrial KATP channel blocker, on the cytotoxicity of neurotoxin 1-methyl-4-phenylpyridinium (MPP+) in differentiated PC12 cells. 5-Hydroxydecanoate and glibenclamide (a cell surface and mitochondrial KATP channel inhibitor) reduced the MPP+-induced cell death and GSH depletion and showed a maximal inhibitory effect at 5 and 10 µM, respectively. Addition of 5-hydroxydecanoate attenuated the MPP+-induced nuclear damage, changes in the mitochondrial membrane permeability and increase in the reactive oxygen species formation in PC12 cells. The results show that 5-hydroxydecanote may prevent the MPP+-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the cytochrome c release and caspase-3 activation. This effect appears to be accomplished by the inhibitory action on the formation of reactive oxygen species and the depletion of GSH. The blockade of mitochondrial KATP channels seems to prevent the MPP+-induced neuronal cell damage.

ATP evokes different currents in TTX-sensitive and TTX-resistant cells of dorsal root ganglia.

The relationship between the level of expression of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents and the occurrence of two kinetically different ATP-induced currents in rat dorsal root ganglion neurons was studied. ATP evoked two distinct types of currents, one with fast activation and desensitization (I-fast) and the other with slow and persistent development (I-slow). In all TTX-S cells which expressed solely TTX-S sodium currents ATP evoked I-fast. The other cells expressed a variable proportion of TTX-S and TTX-R sodium currents. Only 15% of these TTX-R/S cells responded to ATP with I-fast. I-slow was evoked in both cell types but the magnitude of response was much greater in TTX-R/S cells. This result suggests that a different array of ion channels is equipped in different types of sensory neurons.

Effects of ATP on TTX-sensitive and TTX-resistant sodium currents in rat sensory neurons.

Differential effects of ATP on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons were studied using the whole-cell variation of path-clamp technique. Currents were evoked by step depolarizations to 0 mV from a holding potential of −80 mV. ATP suppressed TTX-S sodium currents while it increased TTX-R sodium currents. The effects were concentration-dependent and were reversible upon washout with ATP-free external solution. ATP-γ-S, a hydrolysis-resistant ATP analog, also affected two types of sodium currents similarly to ATP, excluding the possibility that the effects were caused by the products of ATP hydrolysis, namely adenosine. ATP by modulating sodium currents may exert profound effects on the transmission of sensory information such as nociception.