The Effect of Alcohol in the Nicotinic Acetylcholine Receptor / 대한정신약물학회지

OBJECTIVE: It has been well known that alcohol can modulate several ligand-gated ion channel and voltage-gated ion channels. But the roles of alcohol in the autonomic neurons still remain unclear. In this study, thus we characterized the neuronal acetylcholine receptor (nnAChRs) and investigated the modulation of nnAChRs by ethanol (EtOH). METHODS: We used whole-cells which were acutely dissociated male rat major pelvic ganglion (MPG) neurons, and used gramicidin perforated patch clamp techniques. RESULTS: MPG neurons can be classified on the basis of the response of the soma membrane to depolarizing current pulses ; either tonic or phasic neurons. Sympathetic neurons expressing T-type Ca(2+) channels showed tonic firing pattern, while parasympathetic neurons lacking T-type Ca(2+) channels phasic firing to depolarizing current pulses. When hyperpolarizing currents were injected, sympathetic neurons produced post-anodal rebound spikes, while parasympathetic neurons were silent. Under current clamp mode, Acetylcholine (ACh) evoked significant membrane depolarization and produced subsequently marked membrane hyperporization. Under whole-cell mode, application of ACh-induced inward currents held at holding potentials below 0 mV and reversal potential was close to 0 mV, an equilibrium potential of nonselective cation channel. The ACh-activated current was blocked by methyllycaconitine (MLA ; 10 micrometer), hexamethonium (100 micrometer) and alpha-bungarotoxin (alpha-BuTx ; 100 nM), nAChRs antagonists. EtOH (40 mM) potentiated ACh-induced depolarization and hyperpolarization. EtOH also increased both alpha-BuTx-sensitive and -insensitive ACh-activated currents. Futhermore, EtOH potentiated 5-HT-activated current but had a little effect on GABA-activated current. CONCLUSION: These results suggest that EtOH modulates nnAChRs and 5-HT receptors in MPG neurons.

Modulatory Role of Adenylyl Cyclase and Protein Kinase A (PKA) in 5-hydroxytriptamine3 Induced Intracellular Calcium Increase in Parasympathetic Neurons of Rat Major Pelvic Ganglia / 대한비뇨기과학회지

PURPOSE: Serotonin has effects on the bladder contraction or urethral sphincter tone. Different subtypes of 5-hydroxytriptamine (5-HT) receptors appear to mediate the effects of serotonin on voiding. 5-HT1 and 5-HT2, metamorphic receptors, are examined well. However 5-HT3, ionotrophic receptors, are not examined well. Pelvic ganglia provide the majority of the innervation of the lower urinary tract. Major pelvic ganglia (MPG) in rats are autonomic ganglia, containing both sympathetic and parasympathetic neurons related with voiding. We examined the modulatory role of adenylyl cyclase (AC) and protein kinase A (PKA) in 5-HT3 induced intra cellular calcium increase in rat MPG. MATERIALS AND METHODS: The regulatory effects by AC and PKA were investigated in a single neuron of male rat major pelvic ganglia using patch clamp and fluorescence Ca2+ measurement techniques. RESULTS: Inward currents were induced by 5-HT (10microM) at only parasympathetic neurons of MPG. MDL7222 (10(-6)M), selective 5-HT3 receptor antagonists, completely abolished the 5-HT induced inward currents. 5-HT (10microM) induced intracelluar increases of calcium. These increases were blocked by an AC inhibitor SQ22536 (2x10(-5M)) and myristoylated PKA inhibitor (10(-7)M). Furthermore, foskolin (10(-6)M), AC activator, augmented the 5-HT induced intracellular calcium increase. CONCLUSIONS: The activation of AC/PKA-dependent pathway could enhance 5-HT3 induced intacellular calcium increase in parasympathetic neurons of rat MPG and these is helpful for the better understanding the mechanisms of the bladder contraction or urethral sphincter tone.

Rapid, Non-genomic Reduction of IntracelluarCa2+ and Mucin Secretion by Steroid Hormones in Human Nasal Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Airway mucus hypersecretion is one of the major clinical manifestation of patients suffering from various upper or lower respiratory tract diseases. But unfortunately, no drugs are yet available for controlling the airway mucus hypersecretion. Although pharmacological approaches for controlling airway mucus production is currently limited, among the few useful drugs, steroid hormones seem to be the most effective. The effect of steroid hormones is classically described as that of a genomic mechanism involving nucleus transcription. but there is a growing evidence for rapid, non-genomic effect of steroid hormones working through various second messenger systems and ion transporters. MATERIALS AND METHOD: In order to investigate a possible rapid, non-genomic effect of dexamethasone and aldosterone in cultured normal human nasal epithelial (NHNE) cell, the effect of dexamethasone and aldosterone was tested for intracellular calcium response (delta[Ca2+]i) and mucin secretion to external ATP, a known secretagogue in airway epithelial cells, with fluorescence imaging system and ELISA, respectively. Also, we demonstrated the effect of dexamethasone on the mRNA expression of MUC5AC mucin gene for evidence of the existence of non-genomic mechanism of steroid hormones in NHNE cells. RESULTS: Pretreatment with dexamethasone or aldosterone with various concentrations and duration caused a reduction in the delta[Ca2+]i and mucin secretion to ATP. In RT-PCR, Ten minutes dexamethasone pretreatment did not attenuate the mRNA expression of MUC5AC mucin gene, but 24 hours of dexamethasone pretreatment did so. These data indicate that a few minutes of steroid hormone pretreatment can decrease the delta[Ca2+]i and mucin secretion via a non-genomic mechanism. CONCLUSION: In this study, we confirmed the rapid, non-genomic effect of steroid hormones in NHNE cells and suggest this study as a research model for developing antisecretory drugs that have rapid effect.

Characteristics of Nicotinic Receptor Expressed in Human Retinoblastoma

PURPOSE: To identify the characteristics and physiological function of the nicotinic receptor expressed in human retinoblastoma cells. METHODS: We measured possible nicotinic signaling in WERI-Rb-1 cells using the Ca2+ imaging technique and the patch clamp method. RESULTS: 1) Nicotine-induced [Ca2+]i rise arose entirely through Ca2+ influx, which was completely abolished by hexamethonium (100 micro M). 2) Nicotine also induced remarkable depolarization from -56.6 +/- 3.7 mV to -29.6 +/- 3.6 mV (n=4) under current clamp mode, but it failed to directly activate the T-type Ca2+ channel expressed in retinoblastoma cells. CONCLUSIONS: Nicotinic activation can increase the intracellular calcium level through calcium influx in the undifferentiated retinoblastoma cells, which may play important roles in cell proliferation, differentiation, and cell death.

Effect of Fluoxetine on Calcium or Potassium Channels in the Neuron of Rat Major Pelvic Ganglia / 대한비뇨기과학회지

PURPOSE: The major pelvic ganglia (MPG) provide the majority of the innervations to the lower urinary tract. The pelvic ganglia are unique autonomic ganglia that contain both sympathetic and parasympathetic neurons. It has been known that the low-threshold voltage-gated (T-type) Ca2 channels are only expressed only in the sympathetic neurons, whereas these channels are absent in parasympathetic neurons. In the present study, we examined the effect of fluoxetine, a world-wide used antidepressant, on the voltage-dependent Ca2 and K currents in the adrenergic neurons of the MPG. MATERIALS AND METHODS: The effect of fluoxetine on the voltage-dependent Ca2 and K currents in the adrenergic neurons of the MPG were examined using the whole-cell patch-clamp technique. RESULTS: Fluoxetine inhibited the voltage-activated Ca2 currents in the adrenergic neurons of the MPG. Both high-threshold (HVA) and low- threshold (LVA, T-type) Ca2 currents were inhibited by fluoxetine with an IC50 of 5.3 and 10.8microM, respectively. Fluoxetine also decreased the both the peak amplitude and the plateau of the outward K currents. The inhibition of the peak K currents by fluoxetine was concentration- dependent with an IC50 of 3.2microM. The inhibitions of the Ca2 and K currents were quickly reversible upon washout of the fluoxetine. CONCLUSIONS: These results provide evidence for the direct inhibition of the voltage dependant Ca2 and K currents by fluoxetine and these inhibitory effects could modify the synaptic transmission in adrenergic neurons of the MPG.

Neuronal Excitatory Action of GABA on the Pelvic Ganglia

OBJECTIVE: In the central nervous system, gamma-aminobutyric acid (GABA) is well known to act as an inhibitory neurotransmitter by hyperpolarizing postsynaptic neurons through gating GABA-activated Cl- channels. To date, however, the functional roles of GABA remain unclear in the autonomic nervous system. In the present study, we characterize GABA-activated Cl- currents in the neurons of major pelvic ganglia (MPG). METHODS: MPG neurons, located on the lateral surfaces of the prostate gland, from male rats were enzymatically dissociated. Ionic currents were recorded using whole-cell variant patch-clamp technique. Membrane potential was recorded under current clamp mode. Current traces were filterd at 2kHz by using 4-pole Bassel filter in the amplifier. RESULTS: Application of GABA (100micrometer) induced inward currents in the neurons, with holding potentials being maintained below the Cl- equilibrium potential (ECl). The GABA response was concentration-dependent and its reversal potential was close to the theoretical ECl. The GABA-induced Cl- currents were largely blocked by bicuculline (10micrometer, n=5), a GABAA receptor antagonist, but were not affected by 9-AC and niflumic acid, chloride channel blockers. GABA also produced significant membrane depolarization (19mV, n=28). As in the case of the Cl- currents, the GABA-induced depolarizations were largely blocked by bicuculline(10micrometer, n=6), but not by DIDS(50micrometer, n=4), another chloride channel blocker. CONCLUSION: The data suggest that GABAergic roles may be due to it's activation of excitatory GABAA receptors, which are expressed in MPG neurons.

Characterization of Acetylcholine-induced Currents in Male Rat Pelvic Ganglion Neurons

The pelvic ganglia provide autonomic innervations to the various urogenital organs, such as the urinary bladder, prostate, and penis. It is well established that both sympathetic and parasympathetic synaptic transmissions in autonomic ganglia are mediated mainly by acetylcholine (ACh). Until now, however, the properties of ACh-induced currents and its receptors in pelvic ganglia have not clearly been elucidated. In the present study, biophysical characteristics and molecular nature of nicotinic acetylcholine receptors (nAChRs) were studied in sympathetic and parasympathetic major pelvic ganglion (MPG) neurons. MPG neurons isolated from male rat were enzymatically dissociated, and ionic currents were recorded by using the whole cell variant patch clamp technique. Total RNA from MPG neuron was prepared, and RT-PCR analysis was performed with specific primers for subunits of nAChRs. ACh dose-dependently elicited fast inward currents in both sympathetic and parasympathetic MPG neurons (EC50; 41.4microliterM and 64.0microliterM, respectively). ACh-induced currents showed a strong inward rectification with a reversal potential near 0 mV in current-voltage relationship. Pharmacologically, mecamylamine as a selective antagonist for alpha3beta4 nAChR potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons (IC50; 0.53micrometer and 0.22micrometer, respectively). Conversely, alpha- bungarotoxin, alpha-methyllycaconitine, and dihydro-beta-erythroidine, which are known as potent and sensitive blockers for alpha7 or alpha4beta2 nAChRs, below micromolar concentrations showed negligible effect. RT-PCR analysis revealed that alpha3 and beta4 subunits were predominantly expressed in MPG neurons. We suggest that MPG neurons have nAChRs containing alpha3 and beta4 subunits, and that their activation induces fast inward currents, possibly mediating the excitatory synaptic transmission in pelvic autonomic ganglia.

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.

Surgical Experience with Posterior Atlantoaxial Transarticular Screw Fixation in Atlantoaxial Instability

No abstract available.

The Result of Mitochondrial DNA Deletion from PCR Analysis of Peripheral Blood for Parkinson's Disease

Parkinson's disease(PD) is a neurodegenerative disorder characterised clinically by bradykinesia, rigidity, tremor, and pathologically by neuronal cell death in substantia nigra. The cause of dopaminergic neuronal cell death in PD remains unknown. Recently, decreased mitochondrial complex I activities have been reported in platelets, muscles, substantia nigra of the PD patients. Blood samples were lysed with lysis buffer, and incubated 1 hour with 20mg/ml proteinase K at 37degreesC. DNA was extracted with phenol and chloroform(1: ). The long and accurate polymerase chain reaction(LA PCR) was performed by mitochondrial specific primers. The mitochondrial ND1, ND2, CO I, CO II and 1/3ATPase 6/8, CO III, genes as well as parts of ND3 and 3/4ND5 subunit coding regions were analysed by LA PCR. In this study, it is observed not only 4,977 bp mtDNA deletion but a partial mtDNA deletion of the ND1, ND2, CO I~III genes in blood from patients with PD. The result of this study cannot rule out the possibility of point mutation. It is possible that such a deletion would cause mitochondrial dysfuncton, and as a result of mitochondrial dysfunction, Parkinson's disease could progress.