This study aims to validate our hypothesis that acid-sensing ion channels (ASICs) may contribute to the symptom of pain in patients with chronic prostatitis (CP). We first established a CP rat model, then isolated the L5-S2 spinal dorsal horn neurons for further studies. ASIC1a was knocked down and its effects on the expression of neurogenic inflammation-related factors in the dorsal horn neurons of rat spinal cord were evaluated. The effect of ASIC1a on the Ca²⁺ ion concentration in the dorsal horn neurons of rat spinal cord was measured by the intracellular calcium ([Ca²⁺]i) intensity. The effect of ASIC1a on the p38/mitogen-activated protein kinase (MAPK) signaling pathway was also determined. ASIC1a was significantly upregulated in the CP rat model as compared with control rats. Acid-induced ASIC1a expression increased [Ca²⁺]i intensity in the dorsal horn neurons of rat spinal cord. ASIC1a also increased the levels of neurogenic inflammation-related factors and p-p38 expression in the acid-treated dorsal horn neurons. Notably, ASIC1a knockdown significantly decreased the expression of pro-inflammatory cytokines. Furthermore, the levels of p-p38 and pro-inflammatory cytokines in acid-treated dorsal horn neurons were significantly decreased in the presence of PcTx-1, BAPTA-AM, or SB203580. Our results showed that ASIC1a may contribute to the symptom of pain in patients with CP, at least partially, by regulating the p38/MAPK signaling pathway.
Acid Sensing Ion Channel Blockers/pharmacology* ; Acid Sensing Ion Channels/genetics* ; Animals ; Calcium/metabolism* ; Chelating Agents/pharmacology* ; Chronic Disease ; Cytokines/metabolism* ; Disease Models, Animal ; Egtazic Acid/pharmacology* ; Gene Knockdown Techniques ; Imidazoles/pharmacology* ; Inflammation/metabolism* ; MAP Kinase Signaling System/genetics* ; Male ; Pain/genetics* ; Peptides/pharmacology* ; Phosphorylation/drug effects* ; Posterior Horn Cells/metabolism* ; Prostatitis/complications* ; Protein Kinase Inhibitors/pharmacology* ; Pyridines/pharmacology* ; Rats ; Spider Venoms/pharmacology* ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism*

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

OBJECTIVETo explore whether the inhibitory effect of Genipin on uncoupling protein-2 (UCP-2) in mitochondria is involved in energy metabolism of androgen-independent PC3 prostate cancer cells.

METHODSPC3 prostate cancer cells were cultured and treated with Genipin at the concentrations of 40, 80, and 160 μmol/L for 48 hours. Then the proliferation of the cells was detected by MTT assay, the expression of UCP-2 mRNA determined by RT-PCR, and the content of intracellular pyruvic acid (PA) and the activity of succinate dehydrogenase (SDH) in the mitochondria measured by visible spectrophotometry.

RESULTSWith the increased concentration of Genipin, the proliferative activity of the PC-3 cells, the expression level of UCP-2 mRNA, the content of intracellular PA and the activity of SDH in the cells were all decreased, namely, with the enhanced inhibitory effect of Genipin on UCP-2, a trend of reduction was observed in the proliferation of the cells, intracellular PA content, and SDH activity in the mitochondria.

CONCLUSIONGenipin is involved in the energy metabolism of androgen-independent PC3 prostate cancer cells by reducing the content of intracellular PA and the activity of SDH in the mitochondria, which may be associated with its inhibitory effect on UCP-2.

Cell Line, Tumor ; drug effects ; Energy Metabolism ; Humans ; Ion Channels ; metabolism ; Iridoids ; pharmacology ; Male ; Mitochondria ; metabolism ; Mitochondrial Proteins ; metabolism ; Prostatic Neoplasms ; metabolism ; Pyruvic Acid ; metabolism ; RNA, Messenger ; Succinate Dehydrogenase ; metabolism ; Uncoupling Protein 2

OBJECTIVETo investigate activation of brown adipose tissue (BAT) stimulated by medium-chain triglyceride (MCT).

METHODS30 Male C57BL/6J obese mice induced by fed high fat diet (HFD) were divided into 2 groups, and fed another HFD with 2% MCT or long-chain triglyceride (LCT) respectively for 12 weeks. Body weight, blood biochemical variables, interscapular brown fat tissue (IBAT) mass, expressions of mRNA and protein of beta 3-adrenergic receptors (β3-AR), uncoupling protein-1 (UCP1), hormone sensitive lipase (HSL), protein kinase A (PKA), and adipose triglyceride lipase (ATGL) in IBAT were measured.

RESULTSSignificant decrease in body weight and body fat mass was observed in MCT group as compared with LCT group (P<0.05) after 12 weeks. Greater increases in IBAT mass was observed in MCT group than in LCT group (P<0.05). Blood TG, TC, LDL-C in MCT group were decreased significantly, meanwhile blood HDL-C, ratio of HDL-C/LDL-C and norepinephrine were increased markedly. Expressions of mRNA and protein of β3-AR, UCP1, PKA, HSL, ATGL in BAT were greater in MCT group than in LCT group (P<0.05).

CONCLUSIONOur results suggest that MCT stimulated the activation of BAT, possible via norepinephrine pathway, which might partially contribute to reduction of the body fat mass in obese mice fed high fat diet.

Adipose Tissue, Brown ; drug effects ; Adiposity ; drug effects ; Animals ; Dietary Fats ; administration & dosage ; pharmacology ; Ion Channels ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mitochondrial Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Triglycerides ; chemistry ; pharmacology ; Uncoupling Protein 1 ; Weight Loss

Chlorogenic acid (5-caffeoylquinic acid, CGA) is a phenolic compound that is found ubiquitously in plants, fruits and vegetables and is formed via the esterification of caffeic acid and quinic acid. In addition to its notable biological functions against cardiovascular diseases, type-2 diabetes and inflammatory conditions, CGA was recently hypothesized to be an alternative for the treatment of neurological diseases such as Alzheimer's disease and neuropathic pain disorders. However, its mechanism of action is unclear. Voltage-gated potassium channel (Kv) is a crucial factor in the electro-physiological processes of sensory neurons. Kv has also been identified as a potential therapeutic target for inflammation and neuropathic pain disorders. In this study, we analysed the effects of CGA on the two main subtypes of Kv in trigeminal ganglion neurons, namely, the IK,A and IK,V channels. Trigeminal ganglion (TRG) neurons were acutely disassociated from the rat TRG, and two different doses of CGA (0.2 and 1 mmol⋅L(-1)) were applied to the cells. Whole-cell patch-clamp recordings were performed to observe alterations in the activation and inactivation properties of the IK,A and IK,V channels. The results demonstrated that 0.2 mmol⋅L(-1) CGA decreased the peak current density of IK,A. Both 0.2 mmol⋅L(-1) and 1 mmol⋅L(-1) CGA also caused a significant reduction in the activation and inactivation thresholds of IK,A and IK,V. CGA exhibited a strong effect on the activation and inactivation velocities of IK,A and IK,V. These findings provide novel evidence explaining the biological effects of CGA, especially regarding its neurological effects.
Analgesics ; pharmacology ; Animals ; Animals, Newborn ; Cell Culture Techniques ; Chlorogenic Acid ; administration & dosage ; pharmacology ; Ion Channel Gating ; drug effects ; Membrane Potentials ; drug effects ; Neurons ; drug effects ; Neurotransmitter Agents ; administration & dosage ; pharmacology ; Patch-Clamp Techniques ; Potassium Channels, Voltage-Gated ; drug effects ; Rats ; Rats, Sprague-Dawley ; Trigeminal Ganglion ; drug effects

Cardiotrophin-1 (CT-1) activates a distinct form of cardiac muscle cell hypertrophy in which the sarcomeric units are assembled in series. The aim of the study was to determine the expression pattern of sarcomeric contractile protein α-actin, specialized cytoskeletal protein α-actinin and mitochondrial uncoupling protein-2 (UCP2) in myocardial remodeling induced by chronic exposure to CT-1. Kunming mice were intraperitoneally injected with carboxy-terminal polypeptide (CP) of CT-1 (CT-1-CP, 500 μg·kg(-1)· day(-1)) for 1, 2, 3 and 4 week (s), respectively (4 groups obtained according to the injection time, n=10 each, with 5 males and 5 females in each group). Those injected with physiological saline for 4 weeks served as controls (n=10, with 5 males and 5 females). The heart tissues of mice were harvested at 1, 2, 3 or 4 week (s). Immunohistochemistry (IHC) and Western blotting (WB) were used to detect the distribution and expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2 in myocardial tissues. IHC showed that α-actin was mainly distributed around the nuclei of cardiomyocytes, α-actinin concentrated around the striae and UCP2 scattered rather evenly in the plasma. The expression of α-actin was slightly greater than that of α-actinin and UCP2 in the control group (IHC: χ(2)=6.125; WB: F=0.249, P>0.05) and it gradually decreased after exposure to CT-1-CP. There was no significant difference in the expression of α-actin between the control group and the CT-1-CP-treated groups (χ (2)=7.386, P>0.05). But Western blotting revealed significant difference in the expression of α-actin between the control group and the 4-week CT-1-CP-treated group (F=2.912; q=4.203, P<0.05). Moreover, it was found that the expression of α-actinin increased stepwise with the exposure time in CT-1-CP-treated groups and differed significantly between CT-1-CP-treated groups and the control group (ICH: χ (2)=21.977; WB: F=50.388; P<0.01). The expression of UCP2 was initially increased (WB: control group vs. 1- or 2-week group, q values: 5.603 and 9.995, respectively, P<0.01) and then decreased (WB: control group vs. 3-week group, q=4.742, P<0.01; control group vs. 4-week group, q=0.558, P>0.05). It was suggested that long-term exposure to CT-1-CP could lead to the alteration in the expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2. The different expressions of sarcomeric structure proteins and mitochondrial UCP2 may be involved in myocardial remodeling.
Actinin ; biosynthesis ; Actins ; biosynthesis ; Animals ; Cardiomegaly ; chemically induced ; metabolism ; pathology ; Cytokines ; adverse effects ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Ion Channels ; biosynthesis ; Male ; Mice ; Mitochondrial Proteins ; biosynthesis ; Myocardium ; metabolism ; pathology ; Sarcomeres ; metabolism ; pathology ; Uncoupling Protein 2

FXYD6, FXYD domain containing ion transport regulator 6, has been reported to affect the activity of Na(+)/K(+)-ATPase and be associated with mental diseases. Here, we demonstrate that FXYD6 is up-regulated in hepatocellular carcinoma (HCC) and enhances the migration and proliferation of HCC cells. Up-regulation of FXYD6 not only positively correlates with the increase of Na(+)/K(+)-ATPase but also coordinates with the activation of its downstream Src-ERK signaling pathway. More importantly, blocking FXYD6 by its functional antibody significantly inhibits the growth potential of the xenografted HCC tumors in mice, indicating that FXYD6 represents a potential therapeutic target toward HCC. Altogether, our results establish a critical role of FXYD6 in HCC progression and suggest that the therapy targeting FXYD6 can benefit the clinical treatment toward HCC patients.
Animals ; Antibodies, Monoclonal ; pharmacology ; Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Female ; HEK293 Cells ; Humans ; Ion Channels ; antagonists & inhibitors ; metabolism ; Liver Neoplasms ; drug therapy ; metabolism ; Mice, Inbred BALB C ; Mice, Nude ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays

Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in neuropathic pain has not been investigated. In this study, we investigated the effect of the intrathecal amiloride and benzamil in neuropathic pain model, and also examined the role of ASICs on modulation of neuropathic pain. Neuropathic pain was induced by L4-5 spinal nerve ligation in male Sprague-Dawley rats weighing 100-120 g, and intrathecal catheterization was performed for drug administration. The effects of amiloride and benzamil were measured by the paw-withdrawal threshold to a mechanical stimulus using the up and down method. The expression of ASICs in the spinal cord dorsal horn was also analyzed by RT-PCR. Intrathecal amiloride and benzamil significantly increased the paw withdrawal threshold in spinal nerve-ligated rats (87%+/-12% and 76%+/-14%, P=0.007 and 0.012 vs vehicle, respectively). Spinal nerve ligation increased the expression of ASIC3 in the spinal cord dorsal horn (P=0.01), and this increase was inhibited by both amiloride and benzamil (P<0.001 in both). In conclusion, intrathecal amiloride and benzamil display antinociceptive effects in the rat spinal nerve ligation model suggesting they may present an alternative pharmacological tool in the management of neuropathic pain at the spinal level.
Acid Sensing Ion Channels/genetics/metabolism ; Amiloride/*analogs & derivatives/pharmacology/*therapeutic use ; Analgesics/pharmacology/*therapeutic use ; Animals ; Disease Models, Animal ; Male ; Neuralgia/*drug therapy ; RNA, Messenger/metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord/metabolism ; Transcription, Genetic/drug effects

The roles of intermediate conductance Ca(2+)-activated K(+) channel (IKCa1) in the pathogenesis of hepatocellular carcinoma (HCC) were investigated. Immunohistochemistry and Western blotting were used to detect the expression of IKCa1 protein in 50 HCC and 20 para-carcinoma tissue samples. Real-time PCR was used to detect the transcription level of IKCa1 mRNA in 13 HCC and 11 para-carcinoma tissue samples. The MTT assay was used to measure the function of IKCa1 in human HCC cell line HepG2 in vitro. TRAM-34, a specific blocker of IKCa1, was used to intervene with the function of IKCa1. As compared with para-carcinoma tissue, an over-expression of IKCa1 protein was detected in HCC tissue samples (P<0.05). The mRNA expression level of IKCa1 in HCC tissues was 2.17 times higher than that in para-carcinoma tissues. The proliferation of HepG2 cells was suppressed by TRAM-34 (0.5, 1.0, 2.0 and 4.0 μmol/L) in vitro (P<0.05). Our results suggested that IKCa1 may play a role in the proliferation of human HCC, and IKCa1 blockers may represent a potential therapeutic strategy for HCC.
Calcium Channel Blockers ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; physiopathology ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Intermediate-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors ; metabolism ; Ion Channel Gating ; drug effects ; Liver Neoplasms ; pathology ; physiopathology ; Potassium ; metabolism ; Pyrazoles ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo investigate the antiobesity effect of Jueming Prescription (JMP), a Chinese herbal medicine formula, and its influence on mRNA expressions of beta3 adrenergic receptor (beta3-AR) and uncoupling protein-2 (UCP-2) in adipose tissue of diet-induced obese rats.

METHODSFifty male Sprague-Dawley rats were randomly divided into the normal control group (n =8) that was on a standard chow diet, and the obese model group (n =42) that was on a diet of high fat chow. Two weeks after the high fat diet, 29 obese rats in the obese model group were further randomly divided into 3 groups: the untreated obese model group (n =9), the metformin group (n =10, metformin 300 mg kg⁻¹ day)⁻¹, and the JMP group (n =10, JMP 4 g kg⁻¹ day⁻¹). After 8-week treatment, body weight, wet weight of visceral fat, and percentage of body fat (PBF) were measured. The levels of fasting blood glucose, serum lipids, and insulin were assessed, and insulin sensitivity index (ISI) was calculated. The adipose tissue section was stained with hematoxylin-Eosin, and the cellular diameter and quantity of adipocytes were evaluated by light microscopy. The mRNA expressions of beta3-AR and UCP-2 from the peri-renal fat tissue were determined by real-time reverse transcription polymerase chain reaction (RT-PCR).

RESULTSCompared with the obese model group, treatment with JMP resulted in significantly lower body weight, wet weight of visceral fat, PBF, and diameter of adipocytes, and significantly higher level of high-density lipoprotein cholesterol, ISI (all P<0.01), JMP increased the mRNA expressions of beta3-AR and UCP-2 from perirenal fat tissue (P <0.05, P<0.01).

CONCLUSIONSJMP could reduce body weight and adipocyte size; and the effect was associated with the up-regulation of beta3-AR and UCP-2 expressions in the adipose tissue and improvement of insulin sensitivity.

Adipocytes ; drug effects ; metabolism ; pathology ; Adiposity ; drug effects ; Animals ; Blood Glucose ; metabolism ; Body Weight ; drug effects ; Cell Size ; drug effects ; Diet, High-Fat ; Drugs, Chinese Herbal ; pharmacology ; Epididymis ; drug effects ; pathology ; Fasting ; blood ; Gene Expression Regulation ; drug effects ; Insulin ; blood ; Intra-Abdominal Fat ; drug effects ; metabolism ; pathology ; Ion Channels ; genetics ; metabolism ; Lipids ; blood ; Male ; Mitochondrial Proteins ; genetics ; metabolism ; Obesity ; blood ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, beta-3 ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Uncoupling Protein 2 ; Weight Loss ; drug effects