Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

Many efforts have been made to understand excitotoxicity and develop neuroprotectants for the therapy of ischemic stroke. The narrow treatment time window is still to be solved. Given that the ischemic core expanded over days, treatment with an extended time window is anticipated. Bestrophin 1 (BEST1) belongs to a bestrophin family of calcium-activated chloride channels. We revealed an increase in neuronal BEST1 expression and function within the peri-infarct from 8 to 48 h after ischemic stroke in mice. Interfering the protein expression or inhibiting the channel function of BEST1 by genetic manipulation displayed neuroprotective effects and improved motor functional deficits. Using electrophysiological recordings, we demonstrated that extrasynaptic glutamate release through BEST1 channel resulted in delayed excitotoxicity. Finally, we confirmed the therapeutic efficacy of pharmacological inhibition of BEST1 during 6-72 h post-ischemia in rodents. This delayed treatment prevented the expansion of infarct volume and the exacerbation of neurological functions. Our study identifies the glutamate-releasing BEST1 channel as a potential therapeutic target against ischemic stroke with a wide time window.

Inhibitory effect of CLC-2 chloride channel targeted blocking on fibrosis of human conjunctival fibroblasts / 中华实验眼科杂志

Objective:To investigate the inhibitory effect of CLC-2 chloride channel targeted blocking on fibrosis of human conjunctival fibroblasts (HConF).Methods:HConF were divided into blank control group, lipofectamine 2000 (Lipo2000) group, nonsense small interfering RNA (siRNA) group, and CLC-2 siRNA transfected group.The HConF were cultured in medium containing the corresponding transfection reagents according to grouping.No intervention was given to blank control group.The expression level of CLC-2 mRNA of HConF was detected by real-time fluorescence quantitative PCR; absorbance ( A) value indicating the proliferative ability of HConF was determined by CCK-8 kit; the apoptosis ratio of HConF was tested by flow cytometry; the migration ability of HConF was identified by cell scratch test and Transwell migration assay; the contraction rate of HConF was assayed by collagen contraction test; the expression levels of collagenⅠ, collagen Ⅲ, PI3K, Akt, p-PI3K and p-Akt proteins were measured by Western blot. Results:Significant differences were found in relative expression levels of CLC-2 mRNA and A value among four groups ( F=90.110, 198.680; both at P<0.001). The relative expression level of CLC-2 mRNA and A value were significantly lower in CLC-2 siRNA transfected group than nonsense siRNA group, showing statistically significant differences (both at P<0.001). The proportion of apoptotic HConF in blank control group, Lipo2000 group, nonsense siRNA group, and CLC-2 siRNA transfected group was (4.78±1.10)%, (4.54±1.51)%, (4.82±0.88)% and (28.90±0.91)%, respectively, and a statistically significant difference was found ( F=363.260, P<0.001). The proportion of apoptotic HConF was significantly higher in CLC-2 siRNA transfected group than nonsense siRNA group, with a statistically significant difference ( P<0.001). Statistically significant differences were found in cell migration rate and the number of migrating cells among four groups ( F=74.493, 1 625.431; both at P<0.01). The cell migration rate of HConF in CLC-2 siRNA transfected group was significantly lower and the number of migrating cells was significantly smaller than those of nonsense siRNA group, with statistically significant differences (both at P<0.001). A statistically significant difference in contraction rate was found among four groups ( F=104.692, P<0.001). The contraction rate of HConF was significantly lower in CLC-2 siRNA transfected group than nonsense siRNA group, and the difference was statistically significant ( P<0.001). Statistically significant differences were found in relative expression levels of collagen Ⅰ and collagen Ⅲ proteins, p-PI3K/PI3K ratio, and p-Akt/Akt ratio among four groups ( F=112.073, 456.931, 340.889, 43.021; all at P<0.001). The relative expression levels of collagen Ⅰ and collagen Ⅲ proteins, p-PI3K/PI3K ratio and p-Akt/Akt ratio in CLC-2 siRNA transfected group were significantly lower than those of nonsense siRNA group, showing statistically significant differences (all at P<0.05). Conclusions:Targeted blocking of CLC-2 chloride channel gene expression can inhibit fibrosis of HConF by promoting apoptosis of HConF through PI3K/Akt signaling pathway and inhibit fibrotic processes such as cell migration, collagen synthesis and collagen contraction.

Establishment of a cell model for high-throughput TRPV4 regulator screening based on CaCC / 解放军医学杂志

Objective To construct a high-throughput screening model for transient receptor potential vanilloid 4 (TRPV4) channel modulators based on calcium-activated chloride channels (CaCC). Methods RT-PCR was used to detect the endogenous expression of TRPV4 in Fischer rat thyroid (FRT) cells. The PCR products obtained were subjected to nucleic acid sequencing using gel-recovery technology. Western blotting was employed to detect the expression of TRPV4 protein in FRT cells. The liposome transfection method was applied to construct the FRT cell model that co-expressed anoctamin 1 (ANO1) and YFP-H148Q/ I152L. The expressions of ANO1 and YFP-H148Q/I152L in cells were identified by the inverted fluorescence microscope and the fluorescence quenching kinetics test. After adding TRPV4 activators and inhibitors, the fluorescence quenching kinetics experiment was used to test whether the model could screen TRPV4 modulators. The Fura-2 fluorescent probe method was applied to detect the calcium concentration in cells after adding TRPV4 activators; The Z' factor was calculated to evaluate the sensitivity and specificity of the cell model. Results RT-PCR and Western blotting confirmed the endogenous expression of TRPV4 in FRT cells; ANO1 was clearly expressed on the FRT cell membrane and YFP-H148Q/I152L was clearly expressed in the cytoplasm of FRT cells under the inverted fluorescence microscope. The FRT cell model co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed. Fluorescence quenching kinetics experiments confirmed that the model could screen TRPV4 regulators, and the slope value of fluorescence change and the concentration of TRPV4 regulator concentration were in a dose-dependent manner. The model could sensitively detect changes in intracellular calcium concentration, and the slope value could reflect intracellular calcium concentration. The Z' factor was 0.728, which demonstrates its capacity for high-throughput screening. Conclusions We successfully constructed a high-throughput model that could screen TRPV4 modulators sensitively and efficiently.

Research progress of ClC-3 in endometriosis / 中国医师杂志

Chloride Channel-3 (ClC-3) is a member of the Cl2 channel superfamily,which is encoded by CLCN3 gene.It involves regulating electrical activity,cell volume,proliferation,migration,invasion,apoptosis and intracellular pH,which have become a hot spot of current research.In recent years,the research of ClC-3 in gynecological diseases is increasing day by day.This article will review the research progress of ClC-3 and benign and malignant gynecological diseases,especially the relationship between ClC-3 and the occurrence and development of endometriosis.

Colonic Dysmotility in Murine Partial Colonic Obstruction Due to Functional Changes in Interstitial Cells

BACKGROUND/AIMS: Interstitial cells play important roles in gastrointestinal (GI) neuro-smooth muscle transmission. The underlying mechanisms of colonic dysmotility have not been well illustrated. We established a partial colon obstruction (PCO) mouse model to investigate the changes of interstitial cells and the correlation with colonic motility. METHODS: Western blot technique was employed to observe the protein expressions of Kit, platelet-derived growth factor receptor-α (Pdgfra), Ca²⁺-activated Cl⁻ (Ano1) channels, and small conductance Ca²⁺- activated K⁺ (SK) channels. Colonic migrating motor complexes (CMMCs) and isometric force measurements were employed in control mice and PCO mice. RESULTS: PCO mice showed distended abdomen and feces excretion was significantly reduced. Anatomically, the colon above the obstructive silicone ring was obviously dilated. Kit and Ano1 proteins in the colonic smooth muscle layer of the PCO colons were significantly decreased, while the expression of Pdgfra and SK3 proteins were significantly increased. The effects of a nitric oxide synthase inhibitor (L-NAME) and an Ano1 channel inhibitor (NPPB) on CMMC and colonic spontaneous contractions were decreased in the proximal and distal colons of PCO mice. The SK agonist, CyPPA and antagonist, apamin in PCO mice showed more effect to the CMMCs and colonic smooth muscle contractions. CONCLUSIONS: Colonic transit disorder may be due to the downregulation of the Kit and Ano1 channels and the upregulation of SK3 channels in platelet-derived growth factor receptor-α positive (PDGFRα⁺) cells. The imbalance between interstitial cells of Cajal-Ano1 and PDGFRα-SK3 distribution might be a potential reason for the colonic dysmotility.

EF-hand like Region in the N-terminus of Anoctamin 1 Modulates Channel Activity by Ca²⁺ and Voltage

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca²⁺ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca²⁺ binding. Because the Ca²⁺ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca²⁺ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca²⁺ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca²⁺-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca²⁺ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca²⁺. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca²⁺ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca²⁺ and modulate the activation by Ca²⁺ and voltage.

Calcium Signaling in Salivary Secretion

Calcium has versatile roles in diverse physiological functions. Among these functions, intracellular Ca²⁺ plays a key role during the secretion of salivary glands. In this review, we introduce the diverse cellular components involved in the saliva secretion and related dynamic intracellular Ca²⁺ signals. Calcium acts as a critical second messenger for channel activation, protein translocation, and volume regulation, which are essential events for achieving the salivary secretion. In the secretory process, Ca²⁺ activates K⁺ and Cl⁻ channels to transport water and electrolyte constituting whole saliva. We also focus on the Ca²⁺ signals from intracellular stores with discussion about detailed molecular mechanism underlying the generation of characteristic Ca²⁺ patterns. In particular, inositol triphosphate signal is a main trigger for inducing Ca²⁺ signals required for the salivary gland functions. The biphasic response of inositol triphosphate receptor and Ca²⁺ pumps generate a self-limiting pattern of Ca²⁺ efflux, resulting in Ca²⁺ oscillations. The regenerative Ca²⁺ oscillations have been detected in salivary gland cells, but the exact mechanism and function of the signals need to be elucidated. In future, we expect that further investigations will be performed toward better understanding of the spatiotemporal role of Ca²⁺ signals in regulating salivary secretion.

Chloride channels and ischemic stroke / 国际脑血管病杂志

Ischemic stroke is one of the diseases with the highest morbidity and disability.Hypertension is recognized as the most important independent risk factor for ischemic stroke.Vascular remodeling during the development of hypertension is the pathological basis of causing ischemic stroke.Studies have shown that vascular smooth muscle cell proliferation and apoptosis will lead to vascular remodeling.In addition,cerebral ischemia-reperfusion can result in neuronal damage and apoptosis.Recent research has shown that vascular remodeling and neuronal apoptosis are associated with chloride channels.At least 3 chloride channels including volume regulated chloride channel,calcium activated chloride channel and cystic fibrosis transmembrane conductance regulator are involved in these processes.This article reviews the roles of the 3 chloride channels in vascular remodeling,neuronal apoptosis,and ischemic stroke.

Mechanisms underlying contraction of rat isolated coronary artery induced by acidosis / 中国病理生理杂志

AIM:To explore the mechanisms underlying contraction induced by extracelluar acidosis (pHex6.8) in rat isolated coronary artery (RCA).METHODS:Using the microvessel tension recorder system, the effects of acid-base transporters on RCA contraction induced by pHex6.8 were explored by applying the selective pharmacological inhibitors of Na+-H+ exchanger 1 (NHE-1) and Na+-HCO-3 cotransporter (NBC), HOE-642 and S0859, respectively.The effects of chloride channel on RCA contraction induced by pHex6.8 were explored by applying the inhibitors of chloride channel (NPPB and NFA), and by replacing the extracellular NaCl with equimolar NaBr.RESULTS:pHex6.8 augmented the resting tension of RCA, and the maximum contraction was (3.90±0.95) mN.HOE-642 at 30 μmol/L and S0859 at 100 μmol/L both inhibited the contraction of RCA induced by pHex6.8 (P<0.01).NPPB and NFA both inhibited the contraction of RCA induced by pHex6.8 or KCl (60 mmol/L) in a concentration-dependent manner.NPPB and NFA (100 μmol/L) both inhibited the contraction of RCA induced by U46619 (1 μmol/L).Replacing the extracellular NaCl with equimolar NaBr almost completely inhibited RCA contraction induced by pHex6.8 (P<0.01), but had no obvious effect on the contraction induced by KCl (60 mmol/L) or U46619 (1 μmol/L).CONCLUSION:Extracellular acidosis-induced contraction in RCA may be related to the activated NHE-1 and NBC, and it may be also related to the enhanced chloride transport across the membrane.

Role of chloride channels on apoptosis of CNE-2Z cells induced by arsenic trioxide / 中国病理生理杂志

AIM: To investigate the role of chloride channels in the apoptosis of human poorly differentiated nasopharyngeal carcinoma CNE-2Z cells induced by arsenic trioxide (As2O3).METHODS: The apoptotic rates of CNE-2Z cells induced by As2O3 for 24 h or 48 h were monitored by flow cytometry.The technique of whole-cell patch clamp was used to record the currents activated by As2O3 in the CNE-2Z cells.The inhibition of As2O3-induced apoptosis by chloride channel blocker DIDS in the CNE-2Z cells was analyzed by flow cytometry.RESULTS: As2O3 at 5 μmol/L induced apoptosis of CNE-2Z cells in time-dependent manner.The currents with outward rectification were activated when the cells were exposed to 5 μmol/L As2O3.No obvious time-and voltage-dependent inactivation of the currents was observed.The reverse potential of the currents was close to the equilibrium potential for chloride.The activated currents were inhibited by the chloride channel blockers NPPB and DIDS.The 47% hypertonic solution inhibited the activated currents completely.Chloride channel blocker DIDS inhibited the apoptosis of CNE-2Z cells induced by As2O3.CONCLUSION: As2O3 activates volume-sensitive chloride channels, and chloride channels may play an important role in the apoptosis of CNE-2Z cells induced by As2O3.

Effect of ClC-3 siRNA on cell cycle of HeLa cells / 中国病理生理杂志

AIM:To investigate the roles of ClC-3 chloride channels in the regulation of cell cycle and the re-lationship between ClC-3 chloride channels and the cell cycle regulators , such as cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, P21 and P27 in the HeLa cells.METHODS:ClC-3 genes were silenced by the siRNA technique in the HeLa cells.The transfection efficiency of ClC-3 siRNA was detected by real-time PCR.The cell cycle distribution was ana-lyzed by the flow cytometry .The protein expression of ClC-3, P21, P27, CDK4, CDK6 and cyclin D1 was determined by Western blot .RESULTS:ClC-3 was knocked down by ClC-3 siRNA in the HeLa cells .Transfection of the cells with ClC-3 siRNA arrested the cells at G0/G1 phases, decreased the expression of cyclin D1, CDK4 and CDK6, and increased the expression of P21 and P27.CONCLUSION:ClC-3 plays an important role in the cell cycle of HeLa cells through the G 1-S transition point.ClC-3 may regulate the cell cycle progression by up-regulation of cyclin D1, CDK4 and CDK6 expression and/or by down-regulation of P21 and P27 expression.

Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia / 亚洲男科学杂志(英文版)

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l-1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l-1 ) to a hypotonic solution (290 mOsm l-1 ), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4'-diisothiocyanatostilbene-2,2'- isulfonic acid (DiDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DiDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.

Chloride channels and kidney diseases / 临床儿科杂志

Chloride ion is an important anion in organisms, managing various physiological events. A particular gene mu-tation leads to involved channel deifciency and to develop channelopathy. In kidney, different chloride channels distribute along certain fractions of the renal tubule, located at apical and basolateral membranes of tubular epithelial cells. Previous studies dis-covered that voltage-sensitive chloride channels in kidney are associated with Bartter syndrome and Dent’s disease. In addition, the kidney can be involved by cystic ifbrosis resulting from dysfunction of cystic ifbrosis transmembrane conductance regulator. In this review, the function and mechanism of chloride channels in maintenance of normal renal function, and the renal diseases caused by related gene defects were discussed.

Adult presentation of Bartter syndrome type IV with erythrocytosis / Apresentação tardia de síndrome de Bartter tipo IV com eritrocitose

Abstract Bartter syndrome comprises a group of rare autosomal-recessive salt-losing disorders with distinct phenotypes, but one unifying pathophysiology consisting of severe reductions of sodium reabsorption caused by mutations in five genes expressed in the thick ascending limb of Henle, coupled with increased urinary excretion of potassium and hydrogen, which leads to hypokalemic alkalosis. Bartter syndrome type IV, caused by loss-of-function mutations in barttin, a subunit of chloride channel CLC-Kb expressed in the kidney and inner ear, usually occurs in the antenatal-neonatal period. We report an unusual case of late onset presentation of Bartter syndrome IV and mild phenotype in a 20 years-old man who had hypokalemia, deafness, secondary hyperparathyroidism and erythrocytosis.

Resumo A síndrome de Bartter compreende um grupo raro de doenças autossômicas recessivas perdedoras de sal, decorrentes de mutações em genes expressos na porção ascendente espessa da alça de Henle, com fenótipos distintos, porém fisiopatogenia única, que consiste em redução severa da reabsorção de sódio, e aumento da excreção urinária de hidrogênio e potássio, levando à alcalose hipocalêmica. A síndrome de Bartter tipo IV, causada por mutações com perda de função da bartina, uma subunidade do canal de cloro CLC-Kb expressa no rim e ouvido interno, geralmente se apresenta nos períodos ante e neonatal. No presente relato, descreve-se um caso não usual de síndrome de Bartter tipo IV com apresentação tardia e fenótipo atenuado, diagnosticado por análise molecular, em um homem adulto de 20 anos que se apresentava com hipocalemia, surdez, hiperparatireoidismo secundário e eritrocitose.

Expression and identification of ANO1 in mouse cardiomyocytes / 中国病理生理杂志

AIM:To explore the expression of anoctamin 1 (ANO1), one of calcium-activated chloride chan-nels ( CaCCs) , in mouse cardiomyocytes and its functional properties.METHODS:The cardiomyocytes from the myocar-dial tissues of C57BL/6 mice were isolated with enzyme and purified by the differential adherent method.The cells were stained with monoclonal anti-sarcomeric actin and Cy3 to evaluate the purity of the myocardial cells.RT-PCR was used to detect the mRNA expression of ANO1 in the mouse cardiomyocytes.The protein expression of ANO1 in the mouse cardio-myocytes was determined by Western blotting analysis.The fluorescence quenching kinetics experiment was used to identify the ion transport properties of ANO1 in the mouse cardiomyocytes.RESULTS: The results of RT-PCR confirmed that ANO1 was expressed in freshly isolated myocardial cells.The results of Western blotting clearly demonstrated the protein expression of ANO1 in primarily cultured myocardial cells.Fluorescence quenching kinetics experiment on freshly isolated single myocardial cell revealed a pronounced outward rectifying property of the ANO1.The functional properties were simi-lar to the classic CaCCs.CONCLUSION:ANO1 expression was identified in the mouse myocardial cells.The function of CaCCs was generated by ANO1, suggesting that ANO1 is the molecular basis of CaCCs.

Research progress of transmembrane protein 16a in cardiovascular system / 中华实用儿科临床杂志

Calcium-activated chloride channels(CaCCs) play an important role in cardiovascular system,including participating in a variety of physiological functions and being associated with the pathology progress of many cardiovascular diseases.With the development of research,transmembrane protein 16a (TMEM16A) has recently been identified as the molecular basis of CaCCs.Since the study of TMEM16A has got some achievement,especially in the section of cardiovascular system,its main research progress and key points from previous researches are summarized,in which the expression and physiological function of TMEM16A,as well as its clinical pathological correlations with some cardiovascular diseases are told about.Besides,the research prospect of TMEM16A in targeting therapy of cardiovascular disease is also discussed.

Roles of chloride channels in apoptosis induced by adriamycin in nasopharyngeal carcinoma cells / 中国药理学通报

Aim To investigate the roles of chloride channels in the apoptosis and apoptotic volume de-crease (AVD)induced by adriamycin in nasopharyn-geal carcinoma CNE-2Z cells.Methods Apoptotic rates were detected by flow cytometry,and the volume changes were measured by the time-lapse live cell ima-ging technique.The patch clamp technique was used to record whole-cell chloride currents.Results Adria-mycin induced apoptosis of CNE-2Z cells.An early ap-optotic volume decrease was observed in the cell trea-ted with adriamycin.The cell volume was decreased by about 10% in 2 h.Adriamycin activated a chloride current which showed outward rectification.The chlo-ride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB ) could inhibit the adriamycin-in-duced chloride currents,apoptosis and prevent cell shrinkage.Conclusions Our findings suggest that ad-riamycin causes cell apoptosis by activation of chloride channels.Chloride channels may be involved in the apoptosis and apoptotic volume decrease induced by adriamycin in CNE-2Z cells.

Activation effects of gelsemium alkaloids on chloride channels in hepatic carcinoma cells / 中国药理学通报

Aim To investigate the effect of gelsemium alkaloids on chloride channels and cell volume in he-patic carcinoma cells. Methods The time-lapse live cell imaging and whole-cell patch clamp techniques were used respectively to detect the volume changes and currents induced by gelsemium alkaloids in HepG2 cells. Results It was found that the cell volume was decreased by (12. 48 ± 2. 2) % (P<0. 01) when ex-posed to gelsemium alkaloids for 50 min and this phe-nomenon could be inhibited by the chloride channel blocker tamoxifen. It was shown by whole-cell patch clamping that a chloride current could be evoked by extracellular application of gelsemium alkaloids ( 2μmol·L-1 ) . The current was outward-rectified with-out obvious voltage- and time-dependent inactivation. The reversal potential of the current was ( -3. 21 ± 0. 67) mV ,which was close to the equilibrium poten-tial of chloride. The extracellular application of the chloride blockers, tamoxifen and 5-notro-2-(3-phenyl-propylamino)benzoic acid (NPPB), and 47% hyper-tonic solution inhibited the current significantly ( P <0. 01 ) . Conclusion Gelsemium alkaloids could acti-vate chloride channels and induce a volume decrease ( named apoptotic volume decrease, AVD) , and these effect could be inhibited by chloride channel blockers. The results suggest that the chloride channel can be one of the targets of gelsemium alkaloids in their anti-cancer action.

Multiple transcripts of anoctamin genes expressed in the mouse submandibular salivary gland

PURPOSE: Salivary fluid formation is primarily driven by Ca2+-activated, apical efflux of chloride into the lumen of the salivary acinus. The anoctamin1 protein is an anion channel with properties resembling the endogenous calcium-activated chloride channels. In order to better understand the role of anoctamin proteins in salivary exocrine secretion, the expression of the ten members of the anoctamin gene family in the mouse submandibular gland was studied. METHODS: Total RNA extracted from mouse submandibular salivary glands was reverse transcribed using primer pairs to amplify the full-length coding regions of each anoctamin gene and was subcloned into plasmid vectors for DNA sequencing. Alternative splice variants were also screened by polymerase chain reaction using primer pairs that amplified six overlapping regions of the complementary DNA of each anoctamin gene, spanning multiple exons. RESULTS: Multiple anoctamin transcripts were found in the mouse submandibular salivary gland, including full-length transcripts of anoctamin1, anoctamin3, anoctamin4, anoctamin5, anoctamin6, anoctamin9, and anoctamin10. Exon-skipping splicing in the N-terminal exons of the anoctamins1, anoctamin5, and anoctamin6 genes resulted in multiple alternative splice variants. No expression of anoctamin2, anoctamin7, or anoctamin8 was found. CONCLUSIONS: The predominant anoctamin transcript expressed in the mouse submandibular gland is anoctamin1ac. The chloride channel protein produced by anoctamin1ac is likely responsible for the Ca2+-activated chloride efflux, which is the rate-limiting step in salivary exocrine secretion.

Protective effect of antihypertensive drug NO.1 on brain cortical tissue in spontaneously hypertensive rat / 中华老年医学杂志

Objective To investigate the protective effect of antihypertensive drug NO.1 on brain cortical tissue in spontaneously hypertensive rats.Methods A total of 60 spontaneously hypertensive rats (SHR,30 males and 30 females) and 12 healthy Wistar rats (6 males and 6 females) weighed (200 20) g,were randomly divided into 6 groups.Model group (SHR,n=12) and control group (healthy Wistar rats,n=12) were given the same dose of placebo.Captopril group (SHR,n=12) were given captopril 5 mg · kg-1 · d-1.The low-,median-and high-dose groups of antihypertensive drug NO.1 were given 0.25,0.5 and 1 g · kg 1 · d-1 respectively.After 4 weeks of treatment,carotid artery blood pressure was detected.All rats were sacrificed,and brain tissue samples were taken.The expressions of Bcl-2,Bax,necrosis factor NF-κB P65,chloride channel 2 and 3 (CLC-2 and CLC-3) were determined by RT-PCR and Western blotting.Results After 4 weeks of antihypertensive NO.1 treatment,SHR carotid artery blood pressure was (182.8 ± 7.3)mmHg in low dose group,(170.3±9.4) mmHg in medium dose group,and (163.9±10.6) mmHg in high dose group; and (205.4 ± 11.3)mmHg in the model group.Antihypertensive drug NO.1 significantly reduced blood pressure in spontaneously hypertensive rats,and had a concentrationresponse relationship (P＜0.05).Antihypertensive drug NO.1 decreased the expressions of Bax,CLC-2 and CLC-3,and increased the expressions of Bcl-2 and NF-κB P65.Conclusions Antihypertensive drug NO.1 plays a protective role in hypertension-induced cell injury by changing the metabolic enhancement in hypertension-induced cell volume decrease.