This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

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

To investigate the relationship between single nucleotide polymorphisms (SNPs) of CACNA1C (SNPs rs58619945, rs7316246 and rs216008) and susceptibility of chronic spontaneous urticaria (CSU) as well as the curative effect of non-sedating antihistamine drugs.
 Methods: Peripheral blood were extracted from 191 CSU patients to collect DNA. Urticaria Activity Score 7 (UAS7) and Dermatology Life Quality Index (DLQI) changes were collected from these patients with different non-sedating antihistamine drugs. PubMed retrieval system was used to select the 3 SNPs (rs58619945, rs7316246 and rs216008) of CACNA1C. Susceptibility of CSU and curative effect of non-sedating antihistamine drugs (desloratadine, mizolastine, fisofenadine) in 189 CSU patients and 105 controls with different SNPs were compared with Chi-squared test. Data of 105 southern Chinese controls were extracted from the 1 000 genome database.
 Results: Frequency of rs58619945 G allele in the CSU patients was significantly higher than that in the controls [OR(95%CI)=0.660(0.470-0.925), P=0.016]. However, there was no significant differences in rs7316246 and rs216008 between the CSU patients and the controls. Meanwhile there was no significant difference in general curative effect of the 3 drugs in the 3 SNPs (rs58619945: OR=0.843, P=0.454; rs7316246: OR=2.103, P=0.102; rs216008: OR=0.237, P=0.363). There was significant difference in different alleles of rs216008 in the patients administered by desloratadine [OR(95%CI)=0.480(0.247-0.933), P=0.029]. No difference was shown in the 3 SNPs in patients administered by mizolastine.
 Conclusion: The rs58619945 A/G might be related to susceptibility of CSU, and the rs216008 mutation might affect drug response of desloratadine.
Calcium Channels, L-Type ; genetics ; Chronic Disease ; Genetic Predisposition to Disease ; Histamine H1 Antagonists, Non-Sedating ; therapeutic use ; Humans ; Loratadine ; analogs & derivatives ; therapeutic use ; Polymorphism, Single Nucleotide ; Prognosis ; Retrospective Studies ; Urticaria ; drug therapy ; genetics

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

OBJECTIVETo assess the association between single nucleotide polymorphisms (SNPs) of calcium channel β 2 subunit (CACNB2) gene and essential hypertension (EH) in ethnic Han Chinese in Wenzhou area, and to study the influence of rs7069292 alleles on gene expression with luciferase reporter technique.

METHODSSixty hundred and thirty seven Han Chinese with EH and 600 normal controls were enrolled. Genotypes of 6 SNP within CACNB2 gene including rs2228645, rs2357928, rs7069292, rs7099380, rs10764319 and rs11014166 were determined with matrix assisted laser desorption ionization/time of flight mass spectrometer (MALDI-TOF MS). A luciferase reporter gene plasmid containing the fragment flanking rs7069292 (-2831 bp to -2460 bp) in the 5' regulatory region of CACNB2 was constructed.

RESULTSCompared with the control, CT and TT genotypes for the rs7069292 locus were significantly more common in EH group (5.20% vs. 2.17%, 2.59% vs. 1.08%, P< 0.05). CC genotype was not found. Promoter activity for allele C of the rs7069292 locus was significantly increased compared with allele T (P< 0.05). No significant difference was detected for other 5 SNPs in terms of genotypes and allele frequency.

CONCLUSIONThe rs7069292 CT polymorphism of the CACNB2 gene is associated with EH in ethnic Han Chinese from Wenzhou area. A T>C mutation may affect the expression of CACNB2.

Aged ; Alleles ; Base Sequence ; Calcium Channels, L-Type ; genetics ; Case-Control Studies ; Cell Line ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Hypertension ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

Timothy syndrome, long QT syndrome type 8, is highly malignant with ventricular tachyarrhythmia. A 30-month-old boy had sudden cardiac arrest during anesthesia induction before plastic surgery for bilateral cutaneous syndactyly. After successful resuscitation, prolonged QT interval (QTc, 0.58-0.60 sec) and T-wave alternans were found in his electrocardiogram. Starting beta-blocker to prevent further tachycardia and collapse event, then there were no more arrhythmic events. The genes KCNQ1, KCNH2, KCNE1 and 2, and SCN5A were negative for long QT syndrome. The mutation p.Gly406Arg was confirmed in CACNA1C, which maintains L-type calcium channel depolarization in the heart and other systems.
Anesthesia/*adverse effects ; Calcium Channels, L-Type/*genetics ; Death, Sudden, Cardiac/*etiology ; Electroencephalography ; Humans ; Infant ; Long QT Syndrome/*genetics ; Magnetic Resonance Imaging ; Male ; Methyl Ethers/adverse effects ; Nitric Oxide/adverse effects ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; Surgery, Plastic ; Syndactyly/diagnosis/*genetics/surgery

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

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).
Amino Acid Sequence ; Animals ; Antibodies, Blocking ; metabolism ; pharmacology ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; genetics ; immunology ; metabolism ; Guinea Pigs ; Membrane Potentials ; Molecular Sequence Data ; Myocytes, Cardiac ; drug effects ; metabolism ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Sodium-Calcium Exchanger ; antagonists & inhibitors ; genetics ; immunology

Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this study were to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potential mechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension of longitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion with Krebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8 in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. The results showed that the contractions induced by cold stimuli (from 37 degrees C to 12 degrees C stepwise) were inversely proportional to the temperature with a maximum contraction at 17 degrees C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed the expression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced colonic contractions were specially inhibited by TRPA1 blocker, ruthenium red (30 μmol/L), in the proximal and distal colon (P<0.05). The cold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by the pretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 μmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellular calcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 μmol/L) and IP(3) receptor blocker (2-aminoethoxydiphenyl borate, 2-APB, 30 μmol/L) all decreased the contractions evoked by the cooling at 17 degrees C in the proximal and distal colon (P<0.001, P<0.05, P<0.001). Atropine (1 μmol/L) had no effects on these contractions. L-type Ca(2+) channels blocker nifedipine (1 μmol/L) and neurotoxin tetrodotoxin (TTX, 2 μmol/L) decreased the contractile response in the distal colon (P<0.01, P<0.05), but not in the proximal colon. In conclusion, TRPA1 contributes to cold-induced contractions of the rat colon smooth muscle, and the mechanism of TRPA1 activation involves PLC/IP(3)/Ca(2+) pathway. L-type Ca(2+) channel and neurogenic mechanism other than muscarinic receptor might be partially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal colon compared with that of proximal colon.
Animals ; Calcium Channels, L-Type ; metabolism ; Cold Temperature ; Colon ; metabolism ; physiology ; In Vitro Techniques ; Male ; Muscle Contraction ; physiology ; Muscle, Smooth ; metabolism ; physiology ; Physical Stimulation ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; TRPA1 Cation Channel ; TRPC Cation Channels ; genetics ; metabolism