Multidimensional Biomarker Analysis Including Mitochondrial Stress Indicators for Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is accompanied by a complex and multifactorial pathogenesis with sequential progressions from inflammation to fibrosis and then to cancer. This heterogeneity interferes with the development of precise diagnostic and prognostic strategies for NAFLD. The current approach for the diagnosis of simple steatosis, steatohepatitis, and cirrhosis mainly consists of ultrasonography, magnetic resonance imaging, elastography, and various serological analyses. However, individual dry and wet biomarkers have limitations demanding an integrative approach for the assessment of disease progression. Here, we review diagnostic strategies for simple steatosis, steatohepatitis and hepatic fibrosis, followed by potential biomarkers associated with fat accumulation and mitochondrial stress. For mitochondrial stress indicators, we focused on fibroblast growth factor 21 (FGF21), growth differentiation factor 15 (GDF15), angiopoietin-related growth factor and mitochondrial-derived peptides. Each biomarker may not strongly indicate the severity of steatosis or steatohepatitis. Instead, multidimensional analysis of different groups of biomarkers based on pathogenic mechanisms may provide decisive diagnostic/prognostic information to develop a therapeutic plan for patients with NAFLD. For this purpose, mitochondrial stress indicators, such as FGF21 or GDF15, could be an important component in the multiplexed and contextual interpretation of NAFLD. Further validation of the integrative evaluation of mitochondrial stress indicators combined with other biomarkers is needed in the diagnosis/prognosis of NAFLD.

Association Between Low Muscle Mass and Non-alcoholic Fatty Liver Disease Diagnosed Using Ultrasonography, Magnetic Resonance Imaging Derived Proton Density Fat Fraction, and Comprehensive NAFLD Score in Korea / 예방의학회지

Objectives@#Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent metabolic disease. Muscle is known to influence NAFLD development. Therefore, this study aimed to determine the relationships among low muscle mass, NAFLD, and hepatic fibrosis using various definitions of low muscle mass and NAFLD diagnostic methods, including magnetic resonance imaging-based proton density fat fraction (MRI-PDFF). @*Methods@#This cross-sectional study included 320 participants (107 males, 213 females) from the Korean Genome and Epidemiology Study on Atherosclerosis Risk of Rural Areas in the Korean General Population cohort. Muscle mass was assessed using whole-body dual-energy X-ray absorptiometry and adjusted for the height squared, body weight, and body mass index (BMI). NAFLD was diagnosed using ultrasonography (US), MRI-PDFF, and the comprehensive NAFLD score (CNS). Hepatic fibrosis was assessed using magnetic resonance elastography. Multivariable logistic and linear regression analyses were performed to determine the aforementioned associations. @*Results@#According to US, 183 participants (57.2%) had NAFLD. Muscle mass adjusted for body weight was associated with NAFLD diagnosed using US (odds ratio [OR], 3.00; 95% confidence interval [CI], 1.70 to 5.31), MRI-PDFF (OR, 2.00; 95% CI, 1.13 to 3.53), and CNS (OR, 3.39; 95% CI, 1.73 to 6.65) and hepatic fibrosis (males: β=-0.070, p<0.01; females: β=-0.037, p<0.04). Muscle mass adjusted for BMI was associated with NAFLD diagnosed by US (OR, 1.71; 95% CI, 1.02 to 2.86) and CNS (OR, 1.95; 95% CI, 1.04 to 3.65), whereas muscle mass adjusted for height was not associated with NAFLD. @*Conclusions@#Low muscle mass was associated with NAFLD and liver fibrosis; therefore, maintaining sufficient muscle mass is important to prevent NAFLD. A prospective study and additional consideration of muscle quality are needed to strengthen the findings regarding this association.

Bone Marrow-Derived Mesenchymal Stem Cells Isolated from Patients with Cirrhosis and Healthy Volunteers Show Comparable Characteristics

Background and Objectives@#Autologous or allogeneic bone marrow-derived mesenchymal stem cells (BMSCs) have been applied in clinical trials to treat liver disease. However, only a few studies are comparing the characteristics of autologous MSCs from patients and allogeneic MSCs from normal subjects. @*Methods@#and Results: We compared the characteristics of BMSCs (BCs and BPs, respectively) isolated from six healthy volunteers and six patients with cirrhosis. In passage 3 (P3), senescent population and expression of p53 and p21 were slightly higher in BPs, but the average population doubling time for P3–P5 in BPs was approximately 65.3±11.1 h, which is 18.4 h shorter than that in BCs (83.7±9.2 h). No difference was observed in the expression of CD73, CD90, or CD105 between BCs and BPs. Adipogenic differentiation slightly increased in BCs, but the expression levels of leptin, peroxisome proliferator-activated receptor γ, and CCAAT-enhancer-binding protein α did not vary between differentiated BCs and BPs. While ATP and reactive oxygen species levels were slightly lower in BPs, mitochondrial membrane potential, oxygen consumption rate, and expression of mitochondria-related genes such as cytochrome c oxidase 1 were not significantly different between BCs and BPs. @*Conclusions@#Taken together, there are marginal differences in the proliferation, differentiation, and mitochondrial activities of BCs and BPs, but both BMSCs from patients with cirrhosis and healthy volunteers show comparable characteristics.

Response: Increased Serum Angiopoietin-Like 6 Ahead of Metabolic Syndrome in a Prospective Cohort Study (Diabetes Metab J 2019;43:521-9)

No abstract available.

Increased Serum Angiopoietin-Like 6 Ahead of Metabolic Syndrome in a Prospective Cohort Study

BACKGROUND: Despite being an anti-obesity hepatokine, the levels of serum angiopoietin-like 6 (ANGPTL6) are elevated in various metabolic diseases. Thus, ANGPTL6 expression may reflect metabolic burden and may have compensatory roles. This study investigated the association between serum ANGPTL6 levels and new-onset metabolic syndrome. METHODS: In total, 221 participants without metabolic syndrome were randomly selected from a rural cohort in Korea. Baseline serum ANGPTL6 levels were measured using an enzyme-linked immunosorbent assay. Anthropometric and biochemical markers were analyzed before and after follow-up examinations. RESULTS: During an average follow-up period of 2.75 (interquartile range, 0.76) years, 82 participants (37.1%) presented new-onset metabolic syndrome and had higher ANGPTL6 levels before onset than those without metabolic syndrome (48.03±18.84 ng/mL vs. 64.75±43.35 ng/mL, P=0.001). In the multivariable adjusted models, the odds ratio for the development of metabolic syndrome in the highest quartile of ANGPTL6 levels was 3.61 (95% confidence interval, 1.27 to 10.26). The use of ANGPTL6 levels in addition to the conventional components improved the prediction of new-onset metabolic syndrome (area under the receiver operating characteristic curve: 0.775 vs. 0.807, P=0.036). CONCLUSION: Increased serum ANGPTL6 levels precede the development of metabolic syndrome and its components, including low high density lipoprotein, high triglyceride, and high glucose levels, which have an independent predictive value for metabolic syndrome.

Dr. Sa Young Ahn (1890–1968), A Compassionate Devotion to Humanity

No abstract available.

Expression of Fibroblast Growth Factor 21 and β-Klotho Regulates Hepatic Fibrosis through the Nuclear Factor-κB and c-Jun N-Terminal Kinase Pathways

BACKGROUND/AIMS: Fibroblast growth factor (FGF) 21 is associated with hepatic inflammation and fibrosis. However, little is known regarding the effects of inflammation and fibrosis on the β-Klotho and FGF21 pathway in the liver. METHODS: Enrolled patients had biopsy-confirmed viral or alcoholic hepatitis. FGF19, FGF21 and β-Klotho levels were evaluated using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blotting. Furthermore, we explored the underlying mechanisms for this process by evaluating nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathway involvement in Huh-7 cells. RESULTS: We observed that the FGF19 and FGF21 serum and mRNA levels in the biopsied liver tissue gradually increased and were correlated with fibrosis stage. Inflammatory markers (interleukin 1β [IL-1β], IL-6, and tumor necrosis factor-α) were positively correlated, while β-Klotho expression was negatively correlated with the degree of fibrosis. In Huh-7 cells, IL-1β increased FGF21 levels and decreased β-Klotho levels. NF-κB and JNK inhibitors abolished the effect of IL-1β on both FGF21 and β-Klotho expression. FGF21 protected IL-1β-induced growth retardation in Huh-7 cells. CONCLUSIONS: These results indicate that the inflammatory response during fibrogenesis increases FGF21 levels and suppresses β-Klotho via the NF-κB and JNK pathway. In addition, FGF21 likely protects hepatocytes from hepatic inflammation and fibrosis.

Serum Fibroblast Growth Factor 21 and New-Onset Metabolic Syndrome: KoGES-ARIRANG Study

PURPOSE: Fibroblast growth factor 21 (FGF21) is a crucial metabolic regulator, with multiple favorable effects on glucose homeostasis and lipid metabolism. Since serum FGF21 level has been implicated as a potential marker for the early identification of metabolic syndrome (MetS), we investigated the association between serum FGF21 level and the development of MetS in a population-based prospective study. MATERIALS AND METHODS: We conducted a prospective study of 221 randomly sampled adults without MetS from a general population-based cohort study who were examined from 2005–2008 (baseline) and from 2008–2011 (follow-up). Baseline serum FGF21 levels were analyzed using enzyme-linked immunosorbent assay. RESULTS: During the average 2.8-year follow-up period, 82 participants (36.6%) developed new-onset MetS. Serum FGF21 levels were significantly higher in patients with new-onset MetS than in those without MetS (209.56±226.80 vs. 110.09±81.10, p < 0.01). In multivariate adjusted models, the odds for MetS development were greater in patients with serum FGF21 levels in the highest quartile, compared to those in the lowest quartile (3.84, 95% confidence interval: 1.59–9.28). CONCLUSION: Serum FGF21 level was an independent predictor for new-onset MetS in a population-based prospective study.

Adefovir-induced Fanconi syndrome associated with osteomalacia

Fanconi syndrome is a dysfunction of the proximal renal tubules that results in impaired reabsorption and increased urinary loss of phosphate and other solutes. The pathophysiology of drug-induced Fanconi syndrome is unclear. Here we report the case of a 36-year-old woman who presented with pain in multiple bones and proteinuria. She had a 7-year history of taking adefovir at 10 mg/day for chronic hepatitis B. Three years previously she had received surgery for a nontraumatic right femur neck fracture, after which she continued to complain of pain in multiple bones, and proteinuria, glycosuria, and phosphaturia were noted. The findings of a light-microscope examination of a renal biopsy sample were normal, but mitochondrial damage of the proximal tubules was evident in electron microscopy. Western blot analysis revealed that the level of serum fibroblast growth factor 23 (FGF23) was lower than in normal controls. After 2 months of treatment, hypophosphatemia and proximal tubular dysfunction were reversed, and serum FGF23 had normalized. This case suggests that direct mitochondrial damage in proximal tubules can cause drug-induced Fanconi syndrome associated with osteomalacia.

Oxidative stress and calcium dysregulation by palmitate in type 2 diabetes

Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca²⁺ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca²⁺ release and thereby deplete ER Ca²⁺ stores. The resulting ER Ca²⁺ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca²⁺ release, cytosolic and mitochondrial matrix Ca²⁺ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca²⁺ depletion mediate persistent Ca²⁺ influx, further impairing cytosolic and mitochondrial Ca²⁺ homeostasis. Mitochondrial Ca²⁺ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in β-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.

Klotho plays a critical role in clear cell renal cell carcinoma progression and clinical outcome

Klotho functions as a tumor suppressor predominantly expressed in renal tubular cells, the origin of clear cell renal cell carcinoma (ccRCC). Altered expression and/or activity of growth factor receptor have been implicated in ccRCC development. Although Klotho suppresses a tumor progression through growth factor receptor signaling including insulin-like growth factor-1 receptor (IGF-1R), the role of Klotho acting on IGF-1R in ccRCC and its clinical relevance remains obscure. Here, we show that Klotho is favorable prognostic factor for ccRCC and exerts tumor suppressive role for ccRCC through inhibiting IGF-1R signaling. Our data shows the following key findings. First, in tumor tissues, the level of Klotho and IGF-1R expression are low or high, respectively, compared to that of adjacent non-neoplastic parenchyma. Second, the Klotho expression is clearly low in higher grade of ccRCC and is closely associated with clinical outcomes in tumor progression. Third, Klotho suppresses IGF-1-stimulated cell proliferation and migration by inhibiting PI3K/Akt pathway. These results provide compelling evidence supporting that Klotho acting on IGF-1R signaling functions as tumor suppressor in ccRCC and suggest that Klotho is a potential carcinostatis substance for ccRCC.

A putative pH-dependent nuclear localization signal in the juxtamembrane region of c-Met

The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines. This substitution also decreased the association of c-Met fragment with importin beta. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, an Na+/H+ exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin beta, indicating that low pH itself enhanced nuclear translocation. Consistent with this, nigericin treatment also increased the nuclear level of endogenous c-Met in HeLa cells. The putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a 'pH-dependent' NLS.

Defective Mitochondrial Function and Motility Due to Mitofusin 1 Overexpression in Insulin Secreting Cells

Mitochondrial dynamics and distribution is critical for their role in bioenergetics and cell survival. We investigated the consequence of altered fission/fusion on mitochondrial function and motility in INS-1E rat clonal beta-cells. Adenoviruses were used to induce doxycycline-dependent expression of wild type (WT-Mfn1) or a dominant negative mitofusin 1 mutant (DN-Mfn1). Mitochondrial morphology and motility were analyzed by monitoring mitochondrially-targeted red fluorescent protein. Adenovirus-driven overexpression of WT-Mfn1 elicited severe aggregation of mitochondria, preventing them from reaching peripheral near plasma membrane areas of the cell. Overexpression of DN-Mfn1 resulted in fragmented mitochondria with widespread cytosolic distribution. WT-Mfn1 overexpression impaired mitochondrial function as glucose- and oligomycin-induced mitochondrial hyperpolarization were markedly reduced. Viability of the INS-1E cells, however, was not affected. Mitochondrial motility was significantly reduced in WT-Mfn1 overexpressing cells. Conversely, fragmented mitochondria in DN-Mfn1 overexpressing cells showed more vigorous movement than mitochondria in control cells. Movement of these mitochondria was also less microtubule-dependent. These results suggest that Mfn1-induced hyperfusion leads to mitochondrial dysfunction and hypomotility, which may explain impaired metabolism-secretion coupling in insulin-releasing cells overexpressing Mfn1.

Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used Ca2+ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP (10 microM) elicited strong but transient [Ca2+]i increase in a concentration-dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking [Ca2+]i transients were 2MeS-ATP>ATP>>UTP=alphabeta-MeATP, which was compatible with the subclass of P2Y1 receptor. The [Ca2+]i transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of P2Y1 selective blocker (MRS 2179; 30 microM). P2Y1 receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, P2Y1 receptor is mainly expressed in a retinoblastoma cell, which elicits Ca2+ release from internal Ca2+ storage sites via the phospholipase C-mediated pathway. P2Y1 receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic [Ca2+]i signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

Laboratory Rodents Negatively Affected by Construction Environment / 한국실험동물학회지

This report describes rodents in a laboratory animal facility that was adversely affected by a noisy environment during construction work. There was much noise and vibration as well as dust caused by the drilling and hammering. The noise levels, frequencies, and length of time when occurring in the drilling and hammering, were all measured. The drilling showed noise levels ranging from 50-90 decibels (dB) (A-filter, A), and the hammering presented 60-70 dB (A). Some researchers raised problems regarding animal experiments, including skin injuries resulted from self-mutilation, and increase of mortality. This gives useful information to people who plan to renovate laboratory animal facilities as it is a very rare case.

Changes in Inward Rectifier K+ Channels in Hepatic Stellate Cells During Primary Culture

PURPOSE: This study examined the expression and function of inward rectifier K+ channels in cultured rat hepatic stellate cells (HSC). MATERIALS AND METHODS: The expression of inward rectifier K+ channels was measured using real-time RT-PCR, and electrophysiological properties were determined using the gramicidin-perforated patch-clamp technique. RESULTS: The dominant inward rectifier K+ channel subtypes were K(ir)2.1 and K(ir)6.1. These dominant K+ channel subtypes decreased significantly during the primary culture throughout activation process. HSC can be classified into two subgroups: one with an inward-rectifying K+ current (type 1) and the other without (type 2). The inward current was blocked by Ba2+ (100micrometer) and enhanced by high K+ (140mM), more prominently in type 1 HSC. There was a correlation between the amplitude of the Ba2+-sensitive current and the membrane potential. In addition, Ba2+ (300micrometer) depolarized the membrane potential. After the culture period, the amplitude of the inward current decreased and the membrane potential became depolarized. CONCLUSION: HSC express inward rectifier K+ channels, which physiologically regulate membrane potential and decrease during the activation process. These results will potentially help determine properties of the inward rectifier K+ channels in HSC as well as their roles in the activation process.

Effects of Haloperidol on Ca2+i Change in HIT T-15 Insulinoma Cells / 대한정신약물학회지

OBJECTIVE: The purpose of this study was to investigate the effects of haloperidol on [Ca2+]i in hamster insulinoma cells (HIT T-15). METHODS: [Ca2+]i levels were measured by calcium imaging techniques, and membrane potential ionic currents were recorded using conventional patch-clamp methods. RESULTS: Haloperidol induced a transient [Ca2+]i increase, which was abolished by the removal of extracellular Ca2+ or pretreatment with Ca2+ channel blockers (nimodipine and mibefradil). Haloperidol depolarized the membrane potential and inhibited the ATP-sensitive K+ (KATP) channels. Sigma receptor agonists, (+)-SKF10047 and ifenprodil, induced a transient [Ca2+]i increase similar to haloperidol. BD1047, a sigma receptor antagonist, completely blocked the [Ca2+]i increase induced by haloperidol. Haloperidol inhibited the KCl-induced [Ca2+]i increase and voltage-dependent Ca2+ currents. Sigma receptor agonists [(+)-SKF10047, ifenprodil] also inhibited the KCl-induced [Ca2+]i increase. CONCLUSION: Our results suggest that haloperidol induces depolarization, which increases [Ca2+]i by voltage-gated Ca2+ currents via the closing of KATP channels. Haloperidol also inhibits KCl-induced [Ca2+]i increases in the same manner. These effects of haloperidol seemed to be mediated by sigma receptors, which might be linked to the pathogenesis of haloperidol-induced diabetes mellitus.

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.

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.