Co-localization of activating transcription factor 3 and phosphorylated c-Jun in axotomized facial motoneurons / 대한해부학회지

Activating transcription factor 3 (ATF3) and c-Jun play key roles in either cell death or cell survival, depending on the cellular background. To evaluate the functional significance of ATF3/c-Jun in the peripheral nervous system, we examined neuronal cell death, activation of ATF3/c-Jun, and microglial responses in facial motor nuclei up to 24 weeks after an extracranial facial nerve axotomy in adult rats. Following the axotomy, neuronal survival rate was progressively but significantly reduced to 79.1% at 16 weeks post-lesion (wpl) and to 65.2% at 24 wpl. ATF3 and phosphorylated c-Jun (pc-Jun) were detected in the majority of ipsilateral facial motoneurons with normal size and morphology during the early stage of degeneration (1-2 wpl). Thereafter, the number of facial motoneurons decreased gradually, and both ATF3 and pc-Jun were identified in degenerating neurons only. ATF3 and pc-Jun were co-localized in most cases. Additionally, a large number of activated microglia, recognized by OX6 (rat MHC II marker) and ED1 (phagocytic marker), gathered in the ipsilateral facial motor nuclei. Importantly, numerous OX6- and ED1-positive, phagocytic microglia closely surrounded and ingested pc-Jun-positive, degenerating neurons. Taken together, our results indicate that long-lasting co-localization of ATF3 and pc-Jun in axotomized facial motoneurons may be related to degenerative cascades provoked by an extracranial facial nerve axotomy.

The Actions of Adenosine on Voltage-dependent K+ Currents in Neurons of Male Rat Major Pelvic Ganglia / 대한비뇨기과학회지

PURPOSE: The major pelvic ganglia (MPG) function as a relay center for autonomic pathways to the urogenital organs, such as the urinary bladder, vas deference, and penis. It is well known that adenosine acts as an important neuromodulator in various neuronal tissues. Several studies have suggested that some of these actions are coupled with potassium conductances. However, the exact mechanisms are unclear. Therefore, the roles of adenosine on the various potassium channels, in MPG neurons, were investigated. MATERIALS AND METHODS: Single neurons of the MPGs, located on the lateral surfaces of the prostate gland, from male rats were enzymatically dissociated. Ionic currents were recorded using the whole-cell variant patch-clamp technique. RESULTS: Two types of voltage-dependent outward potassium channels were isolated in the MPG neurons using whole-cell voltage protocols. One was the transient outward potassium current (type A-current, IA), the other was the delayed rectifier potassium current (IKDR). The IA and IKDR were recorded in both adrenergic and nonadrenergic neurons, which were distinguished by the existence of T-type calcium currents. Both the adrenergic and nonadrenergic neurons had the same kind of outward potassium currents. Application of adenosine (10(-4)M) increased the IA reversibly. N-cyclopentyladenosine (CPA, 10(-5)M), an A1 selective agonist, produced the same effect. However, the delayed rectifier components were not affected by the adenosine or CPA. The effects of adenosine and CPA on the IA were mostly prevented by pretreatment with DPCPX, an A1 selective antagonist. CONCLUSIONS: Adenosine increased the IA only, via the selective activation of A1 adenosine receptors. The augmentation of A-currents by adenosine may reduce neuronal firings, and then contribute to regulation of neuronal excitability in male rat MPG neurons.

Identification of ATP-sensitive K+ Conductances in Male Rat Major Pelvic Ganglion Neurons

Major pelvic ganglia (MPG) neurons are classified into sympathetic and parasympathetic neurons according to the electrophysiological properties; membrane capacitance (Cm), expression of T-type Ca2+ channels, and the firing patterns during depolarization. In the present study, function and molecular expression of ATP-sensitive K+ (K(ATP)) channels was investigated in MPG neurons of male rats. Only in parasympathetic MPG neurons showing phasic firing patterns, hyperpolarizing changes were elicited by the application of diazoxide, an activator of K(ATP) channels. Glibenclamide (10microM), a K(ATP) channel blocker, completely abolished the diazoxide-induced hyperpolarization. Diazoxide increased inward currents at high K+ (90 mM) external solution, which was also blocked by glibenclamide. The metabolic inhibition by the treatment with mitochondrial respiratory chain inhibitors (rotenone and antimycin) hyperpolarized the resting membrane potential of parasympathetic neurons, which was not observed in sympathetic neurons. The hyperpolarizing response to metabolic inhibition was partially blocked by glibenclamide. RT-PCR analysis revealed that MPG neurons mainly expressed the K(ATP) channel subunits of Kir6.2 and SUR1. Our results suggest that MPG neurons have K(ATP) channels, mainly formed by Kir6.2 and SUR1, with phenotype-specificity, and that the conductance through this channel in parasympathetic neurons may contribute to the changes in excitability during hypoxia and/or metabolic inhibition.

Effects of Fluoxetine on Membrane Potential and Ionic Currents in RINm5F Insulinoma Cells / 대한정신약물학회지

OBJECTIVE: The purpose of this study was to investigate the effects of fluoxetine (Prozac) on membrane potential and ionic currents in RINm5F insulinoma cells. METHODS: Membrane potential and ionic currents in RINm5F cell were recorded by using whole-cell and perforated-patch clamp techniques. RESULTS: Under current clamp conditions, diazoxide (200 microM), an activator of K ATP channels, induced a hyperpolarization of the resting membrane potential (-16.1+/-1.4 mV, n=), which was accompanied by a abolition of action potential firing. This diazoxide-induced hyperpolarization was blocked by glibenclamide (10 microM). Fluoxetine produced significant depolarization of membrane potential (15.9+/-3.1 mV, n=) and blocked diazoxide-induced hyperpolarization. Diazoxide activated inward currents in the presence of high external K + (90 mM) at a holding potential of -60 mV. Fluoxetine suppressed diazoxide-activated currents in a concentration-dependent (IC 50 =.84 microM) manner. However, the inhibitory action of fluoxetine was not specific to K ATP currents because it also inhibited both voltage-activated K + and Ca 2+ currents in a concentration-dependent manner. K ATP currents were more sensitive to fluoxetine block than both voltage-activated K + and Ca 2+ currents. CONCLUSION: Our results indicate that fluoxetine increased excitability of RINm5F cells mainly by the preferential block of K ATP currents. Fluoxetine-induced depolarization may influence insulin secretion in insulinoma cells.

Vascular Reactivity by Purinoceptor Activation in Rat Inferior Vena Cava

BACKGROUND: Extracellular ATP, released from platelets and nerve endings, plays significant roles in the regulation of circulation. The effects of ATP depend on the location of the vessels and the species of experimental animals. Until now, studies were limited to arteries, so we compared the effects of ATP in rat vena cava with those in the aorta and attempted to identify the characteristics of their receptors. METHODS: Vascular rings were isolated from the rat inferior vena cava and descending thoracic aorta. Endothelial cells were preserved or removed by gentle rubbing. The isometric contractions were recorded on polygraph using a force transducer. RESULTS: In the vena cava ring precontracted by 100 nM norepinephrine (NE), ATP elicited relaxations in a dose-dependent manner. These effects were abolished by removal of the endothelium or pretreatment with a nitric oxide synthase inhibitor. Relaxations to ATP in the vena cava (EC50 :9.9 microM) were less potent than those in the aorta (1.7 microM). The relative order of potencies was ADP>ATP>AMP>adenosine, but the maximal relaxation to ADP was smaller than to ATP. ATP-induced vasorelaxation was blocked by suramin, a nonselective antagonist for P2 purinoceptor and reactive blue-2, a P2Y blocker. At basal tension, ATP contracted the vena cava dose-dependently and these effects were potentiated by endothelium-removal. Contractions in the vena cava were also less potent than in the aorta, and the order of potencies was alpha, beta-MeATP>UTP>ATP>ADP>AMP=adenosine. ATP-induced vasoconstriction was blocked by suramin and alpha, beta-MeATP, a desensitizing antagonist of P2X purinoceptor, and potentiated by pretreatment with UTP. CONCLUSION: These results suggest that ADP and ATP acts on P2Y1- and P2Y2-purinoceptor in the endothelium, respectively and induces vasorelaxation of the vena cava, which is mediated by nitric oxide. Since ATP and UTP induced vasoconstriction in endothelium-denuded condition, it may be mediated by the activation of the P2X and P2Y4, 6 purinoceptor on smooth muscles, respectively.

Effects of adenosine tetraphosphate (ATPP) on vascular tone in the isolated rat aorta

Effects of a platelet-released, naturally occurring nucleotide, adenosine 5'-tetraphosphate (ATPP) on vascular tone were analyzed in the isolated rat aorta. Under resting tension ATPP (1 approximately 100 microM) elicited concentration-dependent contractions in endothelium-intact aortic rings in contrast to the concentration-dependent relaxation with ATP. In endothelium-denuded aortic rings, ATPP induced contraction, as ATP did, but with a greater potency. alpha, beta-methylene ATP (APCPP 50 microM), a P2x-purinoceptor antagonist, significantly inhibited ATPP- as well as ATP-induced contractions in the endothelium-denuded preparations suggesting that ATPP acts via P2x-purinoceptors. ATPP (10 approximately 100 microM) relaxed precontracted aortic rings with an intact endothelium in a concentration-dependent manner. This effect of ATPP was 3.7 fold less potent than that of ATP. However, after P2x-purinoceptor blockade, the effect became identical between the two nucleotides. Reactive blue 2, a selective antagonist of P2x-purinoceptors, significantly attenuated the ATPP-induced relaxation with no change in the ATP-induced relaxation. These results indicated that the rat aortic endothelium contains heterogeneous populations of P2-purinoceptors (possibly P2y and nucleotide receptors). Since ATPP shows dual effects depending upon the vascular tension, it may play a significant role in the physiological regulation of vascular tone.