The aim of this study was to investigate the effect of interleukin 6 (IL-6) on the contraction of colon longitudinal muscle strips in rats with acute pancreatitis (AP) and its underlying mechanism. Rat AP model was established by combined injection (i. p.) of ceruletide and lipopolysaccharide. The effect of IL-6 on spontaneous contraction of longitudinal smooth muscle strips of rat colon was observed by biological function experiment system. The level of serum IL-6 was detected by ELISA, the expression and distribution of IL-6 in colon were observed by histochemical staining, and the effect of IL-6 on L-type calcium channel in colon smooth muscle cells was observed by whole cell patch clamp technique. The results showed that, compared with the control group, AP group exhibited reduced contractile amplitude and longer contraction cycle of colon smooth muscle strips. IL-6 prolonged the contraction cycle of colon smooth muscle strips, but did not affect their spontaneous contraction amplitude. Serum IL-6 concentration in AP group was significantly higher than that in control group (P > 0.05). IL-6 was diffusely distributed in the colon of the control group, but the expression of IL-6 was significantly up-regulated in the colon gland, mucosa and submucosa of the AP group. IL-6 significantly decreased the peak current density of L-type calcium channel in rat colon smooth muscle cells. These results suggest that the colon motility of AP rats is weakened, and the mechanism may be that up-regulated IL-6 inactivates L-type voltage-dependent calcium channels, and then inhibits the contraction of colon longitudinal smooth muscle.
Animals ; Calcium Channels, L-Type ; metabolism ; Colon ; Interleukin-6 ; metabolism ; Muscle Contraction ; Muscle, Smooth ; physiopathology ; Pancreatitis ; physiopathology ; Rats

The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca concentration ([Ca]) in ASM cells. The results showed that EA significantly inhibited the high K (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) μmol/L and (56.22 ± 7.62) μmol/L, respectively. EA decreased the K and ACh-induced elevation of [Ca] from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca influx and decreases of [Ca], leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; Enzyme Inhibitors ; pharmacology ; Ethacrynic Acid ; pharmacology ; Mice ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; Respiratory System ; cytology ; drug effects

Calcium Channels, L-Type ; genetics ; metabolism ; Esophageal Achalasia ; genetics ; metabolism ; Esophagus ; metabolism ; Humans ; Nitric Oxide Synthase ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Calcium-Sensing ; genetics ; metabolism

Voltage-gated sodium (Na) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na channels, with Na1.1 and Na1.5 each harboring more than 400 mutations. Na channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca) channel Ca1.1 provides a template for homology-based structural modeling of the evolutionarily related Na channels. In this Resource article, we summarized all the reported disease-related mutations in human Na channels, generated a homologous model of human Na1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na channels, the analysis presented here serves as the base framework for mechanistic investigation of Na channelopathies and for potential structure-based drug discovery.
Animals ; Calcium Channels, L-Type ; chemistry ; genetics ; metabolism ; Channelopathies ; genetics ; metabolism ; Humans ; Mutation ; NAV1.1 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; Protein Domains ; Rabbits ; Structure-Activity Relationship

This study was amid to construct the pharmacophore model of L-type calcium channel antagonist in the application of screening Drugbank and TCMD. This paper repositions the approved drugs resulting from virtual screening and discusses the relocation-based drug discovery methods, screening antihypertensive drugs with L-type calcium channel function from TCMD. Qualitative hypotheses wre generated by HipHop separately on the basis of 12 compounds with antagonistic action on L-type calcium channel expressed in rabbit cardiac muscle. Datebase searching method was used to evaluate the generated hypotheses. The optimum hypothesis was used to search Drugbank and TCMD. This paper repositions the approved drugs and evaluates the antihypertensive effect of the chemical constituent of traditional Chinese medicine resulting from virtual screening by the matching score and literature. The results showed that optimum qualitative hypothesis is with six features, which were two hydrogen-bond acceptors, four hydrophobic groups, and the CAI value of 2.78. Screening Drugbank achieves 93 approved drugs. Screening TCMD achieves 285 chemical constituents of traditional Chinese medicine. It was concluded that the hypothesis is reliable and can be used to screen datebase. The approved drugs resulting from virtual screening, such as pravastatin, are potentially L-type calcium channels inhibitors. The chemical constituents of traditional Chinese medicine, such as Arctigenin III and Arctigenin are potentially antihypertensive drugs. It indicates that Drug Repositioning based on hypothesis is possible.
Animals ; Antihypertensive Agents ; chemistry ; pharmacology ; Calcium Channel Blockers ; chemistry ; pharmacology ; Calcium Channels, L-Type ; genetics ; metabolism ; Drug Repositioning ; methods ; Molecular Structure ; Myocardium ; metabolism ; Rabbits

OBJECTIVE: To learn the influence the gentamycin on C57BL/6J mice hear and cochlear hair cell ribbon synapses CaV1.3 calcium protein amount. To explore the relationship between hear loss and its dosage correlation change and significance. METHOD: The fixed amino glucoside to C57BL/6J mice was used to make abdominal cavity injection mold every day. The auditory brain-stem response ABR was used to measure the hear of mice in 7th, 14th, 28th after the injection. Immunofluorescence method was used to observe cochlear basement membrane of hair ribbon synapse CaV1.3 calcium channel proteins in the distribution and expression. Inner hair cells synaptic membrane was immune fluorescent tags with CtbP2 and CaV1. 3. RESULT: With the growth of the injected drugs, ABR threshold increased,but all the hair cells and shape had no obvious change. However the amount of hair rib bon synapse CaV1.3 calcium ion channel proteins in the expression had significant differences (P < 0.01). CaV1.3 calcium ion channel proteins increased slightly lower than normal at 7th day, significantly decreased at 14th day, had increased, increased quantity compare with 14th day, but at 28th day after intraperitoneal injection of gentamicin. CONCLUSION The increasing,decreasing and increasing trend of cochlear hair cells CaV1.3 proteins in the environment of amino glucoside drug toxicity showed that the increase of hair ribbon synapse CaV1.3 proteins may have a compensatory effect on the drug toxicity. With the increase of the drug toxicity effect, this kind of decompensated function could be the listening decline, which may be one of the mechanism of damage to hearing.
Animals ; Calcium Channels, L-Type ; metabolism ; Evoked Potentials, Auditory, Brain Stem ; Gentamicins ; pharmacology ; Hair Cells, Auditory, Inner ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Proteomics

OBJECTIVETo investigate the molecular and functional changes in L-type Ca2+ channel of hypertrophied cardiomyocytes in neonatal rats induced by angiotensin II (Ang II).

METHODSThe in vitro model of cardiomyocyte hypertrophy was established in cultured cardiomyocytes from neonatal rats. Whole cell patch clamp was used to measure the L-type Ca2+ currents. Semi-quantitative RT-PCR was used to determine the mRNA expression of L-type Ca2+ channel alpha1C subunits.

RESULTSIn the hypertrophied cardiomyocytes induced by Ang II, I(Ca, L) densities were increased, whereas the features of I(Ca,L) activation, inactivation or recovery from inactivation were not affected. Meanwhile, Ang II increased the mRNA expression of L-type Ca2+ channel alpha1C subunits in cardiomyocytes. All these actions of Ang II could be blocked by the angiotensin II 1 type receptor blocker losartan.

CONCLUSIONDuring cardiomyocyte hypertrophy induced by Ang II, there are significant changes in the molecule and function of L-type Ca2+ channels, which are mediated by the angiotensin II 1 type receptor.

Angiotensin II ; adverse effects ; Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; metabolism ; Female ; Hypertrophy ; metabolism ; Male ; Membrane Potentials ; Myocytes, Cardiac ; metabolism ; pathology ; physiology ; Rats ; Rats, Sprague-Dawley

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca2+ channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca2+ current (ICa,L) in a concentration dependent manner with a IC50 of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 microM). DNP did not affect the voltage dependence of activation and inactivation of ICa,L. The alpha1c subunit of cardiac L-type Ca2+ channel proteins was phosphorylated by the treatment of DNP (1 microM), which was completely blocked by KT5823 (1 microM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 +/- 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 microM). These results clearly indicate that DNP inhibits the L-type Ca2+ channel activity by phosphorylating the Ca2+ channel protein via PKG activation.
Action Potentials/drug effects ; Animals ; Biological Transport/drug effects ; Calcium/metabolism ; Calcium Channels, L-Type/*metabolism ; Carbazoles/pharmacology ; Cells, Cultured ; Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors ; Elapid Venoms/*metabolism/pharmacology ; Enzyme Activation ; Heart ; Heart Ventricles/drug effects ; Myocytes, Cardiac/drug effects ; Patch-Clamp Techniques ; Peptides/*metabolism/pharmacology ; Phosphorylation/drug effects ; Rabbits

BACKGROUNDEndometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium ([Ca(2+)](c)) was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-1A cells.

METHODSCells were cultured with nifedipine (10 µmol/L) and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine (3-MA) (2.5 mmol/L) for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alpha1D (Cav1.3) protein was detected. At last, cells were cultured and assigned to four groups with different treatment: untreated (control group), 10 µmol/L nifedipine (N group), 2.5 mmol/L 3-MA (3-MA group), and 10 µmol/L nifedipine plus 2.5 mmol/L 3-MA (N+3MA group). Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins (LC3, Beclin1 and P70s6K) by Western blotting and monodansylcadaverine (MDC) labeled visualization.

RESULTSProliferation of Hec-1A cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours (P = 0.000 for each day). The suppression of migration ability of the nifedipine-treated cells (94.0 ± 8.2) was significantly different from that of the untreated cells (160.00 ± 9.50, P = 0.021). The level of early period cell apoptosis induced by nifedipine was (2.21 ± 0.19)%, which was (2.90 ± 0.13)% in control group (P = 0.052), whereas the late period apoptosis level reached (10.38 ± 0.96)% and (4.40 ± 0.60)% (P = 0.020), respectively. The 3-MA group induced a slight increase in the Cav1.3 levels within 15 minutes, but significantly attenuated the Cav1.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group (20.63 ± 3.36) than the control group (6.29 ± 0.16, P = 0.015). GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N+3MA group, 3-MA group were 2.80 ± 0.29, 2.30 ± 0.17, and 1.80 ± 0.21, respectively. Cells in the N group showed significant augmentation of autophagy (P < 0.05). Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group (0.85 ± 0.21) and N+3MA group (1.21 ± 0.12) compared with nifedipine treatment (2.64 ± 0.15, P < 0.05). The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N+3-MA group ((11.22 ± 0.91)%) differed significantly from that of the control group ((2.51 ± 0.70)%) and N group ((3.47 ± 0.39)%). Similarly, the late period level of the N+3-MA group ((55.19 ± 2.51)%) differed significantly from that of the control group ((15.81 ± 1.36)%) and the N group ((22.09 ± 2.48)%, P < 0.05). The down-regulated expression of P70s6k and up-regulated expression of the Beclin1 revealed significant differences between the N+3-MA group and control group (P = 0.025; Beclin1: P = 0.015).

CONCLUSIONSProliferation and migration in vitro of endometrial carcinoma Hec-1A cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-1A cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cav1.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclin1 and mTOR pathways.

Adenine ; analogs & derivatives ; pharmacology ; Apoptosis Regulatory Proteins ; physiology ; Autophagy ; drug effects ; Beclin-1 ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; physiology ; Cell Line, Tumor ; Endometrial Neoplasms ; drug therapy ; pathology ; Female ; Humans ; Membrane Proteins ; physiology ; Nifedipine ; pharmacology ; Signal Transduction ; physiology ; TOR Serine-Threonine Kinases ; physiology

OBJECTIVETo investigate the negative regulation of microRNA-1 (miR-1) on L-type calcium channel beta2 subunit (Cavbeta 2) during cardiomyocyte hypertrophy and its mechanism.

METHODSCardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (HJ2000). The targets of miR-1 were predicted by online database microCosm. The 3' untranslated region sequence of Cavbeta 2 was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured to verify the expression of luciferase reporter vector. The expression of atrial natriuretic peptide (ANP), beta-myosin heavy chain (beta-MHC), miR-1 and the Cavbeta 2 mRNA were detected by qRT-PCR. The protein expression of Cavbeta 2 was detected by Western blot. The level of miR-1 was up-regulated by miR-1 mimic transfection and the expression level of Cavbeta 2 was down-regulated by RNAi, then effects of which on cardiomyocyte hypertrophy were investigated.

RESULTS(1) The expression of miR-1 was significantly reduced in cardiomyocyte hypertrophy. Upregulating the miR-1 level could suppress the increase of cell surface area, the expression of ANP and beta-MHC mRNA (P < 0.05). (2) Cavbeta 2 was the one of potential targets of miR-1 by prediction using online database microCosm. The luciferase activities of HEK293 cells with the plasmid containing miR-1 and wide type Cavbeta 3' UTR sequence was significantly decreased when compared with that of control group (P < 0.01). Up-regulation of the miR-1 level could suppress the protein expression of Cavbeta 2. (3) The expression of Cavbeta 2 was significantly increased in cardiomyocyte hypertrophy induced by ISO. Downregulation of Cavbeta by RNAi could markedly inhibit the increase of cell surface area, the expression of ANP and beta-MHC mRNA.

CONCLUSIONCavbeta2 is one of potential targets of miR-1 by bioinformatics prediction. The experiment data confirms that Cavbeta2 is truly the target of miR-1. MiR-1 can negatively regulate the expression of Cavbeta 2, resulting in the decrease of intracellular Ca2+ content and the attenuation of cardiomyocyte hypertrophy.

Animals ; Atrial Natriuretic Factor ; metabolism ; Calcium Channels, L-Type ; genetics ; Cardiomegaly ; genetics ; Gene Expression Regulation ; HEK293 Cells ; Humans ; MicroRNAs ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection ; Ventricular Myosins ; metabolism