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.

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.

Characteristics of Potassium and Calcium Currents of Hepatic Stellate Cells (Ito) in Rat

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA) -sensitive current, 4-aminopyridine (4-AP) -sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15microM pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.

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.

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.

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.

Comparison of digital radiometric featuresbetween radicular cysts and periapical granulomas / 대한구강악안면방사선학회지

The purpose of this study was to investigate whether a radiometric analysis of radicular cysts and periapical granulomas is useful in the differential diagnosis. In this experiment, twenty-nine periapical radiographs of the radicular cyst and those periapical granuloma were used. The periapical radiography was taken by intraoral paralleling device. The X-ray film was digitized and digitally filtered to reduce film-grain noise. We estimated density difference of the inner/outer area, roundness or circularity, bone profile or scan line of the margin and cumulative percentage frequency curve of radicular cyst & periapical granuloma. The obtained results were as follows ; 1. The differences in density between ROIs of inner and outer area of radicular cysts were smaller than those of periapical granulomas. 2. The equivalent circular diameter was over 6.3mm, there was significant difference between periapical cyst and periapical granuloma. 3. In differential diagnosis of radicular cyst and periapical granuloma using bone profile, sensitivity, spicificity and accuracy were considerably high(0.83, 0.86, 0.86) respectively. 4. Cumulative percentage frequency curve of the radicular cyst was closer to the pseudo-pixel value of 50 than average curve, whereas periapical granuloma was closer to that of 0. Hence we conclude that digital radiometric features might be useful in the differential diagnosis between radicular cyst and periapical granuloma.

Fractal dimension from radiographs of bone as indicatos of possible osteoporosis / 대한구강악안면방사선학회지

The purpose of this study was to investigate wether a radiographic estimate of osseous fractal dimension is useful in the characterization of structural changes in bone. Ten specimens of bone were progressively decalcified in fresh 50ml solutions of 0.1N hydrochloric acid solution at cummulative timed periods of 5, 10, 20, 30, 60 and 90 minutes, and radiographed from 0 degree projection angle controlled by intraoral parelleling device. The test set of 70 radiographs was digitally filtered to reduce film-grain noise. I performed one-dimensional variance and fractal analysis of bony profiles or scan lines, Correlation analysis quantified the relationship between variance and fractal dimension. The obtained results were as follow ; 1. After the first stage of decalcification variance and fractal dimension of scan line pixel intensities generally decreased with a range of 57.94 to 12.64 and 1.59 to 1.36. 2. Correlation coefficient(r) relating variance to fractal dimension was consistantly excellent(range r=0.90 to 0.98). 3. Variance and fractal dimension were much alike in ability to discriminate, at leat on a group basis, between contrl and decalcified specimens.

Effects of taxol and ionizing radiation on cytotoxicity and prostaglandin production in KB, RPMI-2650, SW-13 and L929 / 대한구강악안면방사선학회지

The author evaluated the effects of taxol, a microtubular inhibitor, as a possible radiation sensitizer and the production of prostaglandins on three human cancer cell lines(KB, RPMI-2650 and SW-13) and one murine cell line(L929). Each cell line was divided into four groups(control, taxol only, radiation only and combination of taxol and radiation). The treatment consisted of a single irradiation of 10 Gy and graded doses(5, 50, 100, 200, 300, 500 nM) of taxol for a 24-h period. The cytotoxicity of taxol alone was measured at 1 day after(1-day group) and 4 days after(4-day group) the treatment. The survival ratio of cell was analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyl tetrazolium bromide) test. Prostaglandins(PGE2 and PGI2) were measured in the culture medium by a radioimmunoassay. The results obtained were as follows ; 1. There was a significantly in creased cytotoxicity of KB cells in 4-day group than those in 1-day group. Therr was a high correlation between doses of taxol and cell viability in both groups(1-day group R=0.82741, 4-day group R=0.84655). 2. There was a significantly increased cytotoxicity of RPMI-2650 cells treated with high concentration of taxol in 4-day group than those in 1-day group. Also there was a high correlation between doses of taxol and cell viability in 4-day group(R=0.93917). 3. There was a significantly increased cytotoxicity of SW-13 cell treated with high concentration of taxol in 4-day group than those in 1-day group. However no high correlation was observed between doses of taxol and cell viability in both groups(1-day group R=0.46362, 4-day group R=0.65425). 4. There was a significantly increased cytotoxicity of L929 cells treated with low concentration of taxol in 4-day group than those in 1-day group. At the same time, there was a low correlation between doses of taxol and cell viability in both groups(1-day group R=0.34237, 4-day group R=0.23381). 5. In 1-day group of L929 cells, higher cytotoxicities were observed in the groups treated with 500 nM taxil than given 10 Gy radiation alone showed a radiosensitizing effect by taxol. 6. In addition to L929 cells, all cancer cells treated with a commbinationof taxol and radiation in 4-day group appeared ti have some fragmented nuclei and to float on the medium. In addition, L929 cells appeared to be more confluent. 7. The level of PGE2 production was the highest in the contol KB cells. This appeared to increase in every experimental group of all three cancer except L929 cells. There was a significantly increased production of PGE2 in SW-13 cells treated with a combination taxol and radiation compared to the other experimental groups. 8. The level of PGI2 production in the contol group RPMI-2650 cells was the highest. This appeared to increase in every experimental group of all cells except in SW-13 cells. This also increased signigicantly in RPMI-2650 cells treated with a cimbination of taxol and radiation compared to the other experimental groups.

Characterization of trabecular bone structure using 2D Fourier transformation and fractal analysis / 대한구강악안면방사선학회지

The purpose of this study was to investigate whether a radiographic estimate of osseous fractal dimension and power spectrum of 2D discrete Fourier transform is useful in the characterization of structural changes in bone. Ten specimens of bone were decalcified in fresh 50 ml solutions of 0.1 N hydrochloric acid solution at cummulative timed periods of 0 and 90 minutes, and radiographed from 0 degree projection angle controlled by intraoral parelleling device. I performed one-dimensional variance, fractal analysis of bony profiles and 2D discrete Fourier transform. The results of this study indicate that variance and fractal dimension of scan line pixel intensities decreased significantly in decalcified groups but Fourier spectral analysis didn't discriminate well between control and decalcified specimens.

Comparison of digitized radiographic alveolar features with age / 대한구강악안면방사선학회지

The purpose of the present stydy was to use digital profile image features and digital imge analysis of fixed-dime nsion bone regions, extracted from standardized periapical radiographs of the maxilla, to determine whether differences exist in alveolar bone of younger women(mean age: 59.23+/-7.34 years) and just menopaused women(mean age: 59.23+/-7.34), Periapical films were used from two groups of 20 randomly selected women. None of the subjects had a remarkabel medical history. To simplify protocal, we chose one interproximal bone area between the maxillary right canine and lateral inci sor for study. Each film was digitized into a 1312X1024 pixelX8 bit depth matrix by means of a Nikon 35 mm film scanner(LS-3510AF, Japan) with fixed gain and internal dark current correction to maintain constant illumination. The scanner was interfaced to a Macintosh LC III computer(Apple Computer, Charlotte, N.C.). Area and profile orientation were selecte d with a NIMH Image 1.37(NIH Research Services Brach, Bethesda, Md.). Histogram features were extracted form each profil e and area. The results of this study indicate that mean pixel intensities didn't differ significantly between two group s and there was a high correlation-coefficient between digitized radiographic profile features and area features.

Nonlinear contrast enhancement on subtraction images / 대한구강악안면방사선학회지

This study was performed to demonstrate the effect of linear or nonlinear contrast enhancement on subtraction images. Three different textures were radiograped on dental film. The first radiograph was taken without the presence of an object. the second, which showed trabucular bone, was taken of the molar area of a human. the third radiograph was taken of the coronal part of molars. Each film was digitized into a 1312 X 1024 pixel X 8 bit depth matrix by means of a Nikon 35 mm film scanner(LS-3510AF, Japan) with fixed gain and internal dark current correction to maintain constant illumination. The scanner was interfaced to a Macintosh LC III computer(Apple Computer, Charlotte, N.C.). This resulted in three pairs of images, including different textures-plain, bone and enamel. Digital regular, linearly and nonlinearly enhanced subtraction was performed. Computer software was used to simulate lesions in the shape of a 2D-Gaussian curve on each of a pair of images. The each subtraction images were presented in a random sequence to two groups of 10 observers(students and dentists). ROC analysis was used to compare observer performance. The following results were obtained; 1. All of LCE subtraction, equalized subtraction and regular subtraction images of plain texture were diagnosed the best by far. 2. The data revealed a siginificant LCE effect in both the student group and the expert group. 3. Clinical expertise was a helphul factor for the observers in this study.