Expression and role of the TRPC family in TGF-β1-induced calcium influx in podocytes / 生理学报

The TRPC family consists of multiple important cationic channels in mammals that participate in a variety of physiological and pathological processes. Our previous studies have shown that transforming growth factor-β1 (TGF-β1) increases the expression of TRPC6 in podocytes, but the roles of other members of the TRPC family in podocytes require further investigation. In this study, we investigated the effect of TGF-β1 on the expression of the TRPC family and the role of the TRPC family in the changes of the intracellular Ca2+ concentration ([Ca2+]i) in podocytes induced by TGF-β1. The model of podocyte injury was established by treatment with TGF-β1 in immortalized glomerular podocytes (MPC5) in vitro. qRT-PCR and Western blot were used to detect the effect of TGF-β1 on the mRNA and protein expression of each TRPC family member. After the expression of each TRPC family member was knocked down by a siRNA-based approach and blocked by SKF96365, respectively, free cytosolic Ca2+ was measured using the fluorescent Ca2+ indicator Fluo-3/AM, and the dynamic change of [Ca2+]i in podocytes was detected by a dynamic high-speed calcium imaging system. The results showed that TGF-β1 increased the protein expression of TRPC1/3/6 in podocytes, but had no effects on the protein expression of TRPC4. The protein expression levels of TRPC5/7 were only affected by 4 ng/mL and 8 ng/mL TGF-β1, respectively. TGF-β1 increased TRPC1/3/6 mRNA levels in podocytes, however had no effects on TRPC4/5/7 mRNA. TGF-β1 significantly increased [Ca2+]i in podocytes. Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the [Ca2+]i induced by TGF-β1, but TRPC3/6 knockdown significantly decreased the [Ca2+]i. There was no significant difference in the [Ca2+]i between the TRPC6 siRNA-treated group and SKF96365-treated group, but the [Ca2+]i of the TRPC3 siRNA-treated group was significantly higher than that of SKF96365-treated group. These results demonstrate that TGF-β1 increases the expression of the TRPC1/3/6 in podocytes. TGF-β1 increases [Ca2+]i in podocytes, which is dependent on the TRPC3/6 expression. Our results also suggest that the effect of TRPC6 on [Ca2+]i in podocytes may be greater than that of TRPC3.

Pharmacological characterization of the calcium influx pathways involved in nitric oxide production by endothelial cells / Caracterização farmacológica das vias de influxo de cálcio envolvidas na produção de óxido nítrico por células endoteliais

ABSTRACT Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors. Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings. Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM). Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.

RESUMO Objetivo: Caracterizar as vias do influxo de cálcio envolvidas no aumento sustentado da concentração intracelular de cálcio na célula endotelial, essencial para a síntese e a liberação de fatores relaxantes. Métodos: Analisamos o efeito de derivados pirazólicos sintetizados recentemente, descritos como inibidores seletivos para canais ORAI (BTP2/Pyr2 e Pyr6) e TRPC3 (Pyr3 e Pyr10), nos relaxamentos dependentes de endotélio e cálcio extracelular, produzidos por acetilcolina e tapsigargina, em anéis pré-contraídos da aorta torácica de rato. Resultados: As respostas de acetilcolina e tapsigargina foram completamente revertidas por Pyr2 e Pyr6 (1 a 3μM). Pyr3 (0,3 a 3μM) produziu reversão rápida dos relaxamentos de acetilcolina (6,2±0,08mg.s−1) e tapsigargina (3,9±0,25mg.s−1), enquanto o bloqueador mais seletivo para TRPC3, Pyr10 (1 a 3μM), não apresentou efeito. ML204 (1 a 3μM), bloqueador seletivo de TRPC4, descrito há pouco tempo, reverteu os relaxamentos induzidos por acetilcolina de forma completa, mas afetou minimamente aqueles produzidos por tapsigargina. Os derivados pirazólicos ou ML204 não afetaram os relaxamentos estimulados com GSK1016790A (TRPV4-agonista). Ainda, após pré-incubação com Pyr2 e Pyr6, acetilcolina e tapsigargina provocaram relaxamentos transitórios semelhantes em magnitude e cinética àqueles observados na ausência de cálcio extracelular. Os relaxamentos do nitroprussiato de sódio e as contrações induzidas pela fenilefrina (aorta sem endotélio) não foram afetados pelos compostos pirazólicos (1 a 3μM). Conclusão: Essas observações revelaram uma complexidade desconhecida das vias de influxo de cálcio no endotélio da aorta de rato, que resultam na produção e na liberação de óxido nítrico. Vias distinguíveis farmacologicamente medeiam o influxo estimulado por acetilcolina (ORAI TRPC, diferentes de TRPC3 TRPC4) e tapsigargina (TRPC4 não requerido).

Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia / 生理学报

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO, 21% O, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO, 5% O, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca]in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca]were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca].

Cannabinoids increase mechanosensitivity of trigeminal ganglion neurons innervating the inner walls of rat anterior chambers via activation of TRPA1 / 华中科技大学学报（医学）（英德文版）

Our previous study found that some trigeminal ganglion (TG) nerve endings in the inner walls of rat anterior chambers were mechanosensitive, and transient receptor potential ankyrin 1 (TRPA1) was an essential mechanosensitive channel in the membrane. To address the effect of cannabinoids on the mechanosensitive TG nerve endings in the inner walls of anterior chambers of rat eye, we investigated the effect of the (R)-(+)-WIN55, 212-2 mesylate salt (WIN), a synthetic cannabinoid on their cell bodies in vitro. Rat TG neurons innervating the inner walls of the anterior chambers were labeled by 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfona (FAST DiI). Whole cell patch clamp was performed to record the currents induced by drugs and mechanical stimulation. Mechanical stimulation was applied to the neurons by buffer ejection. WIN evoked inward currents via TRPA1 activation in FAST DiI-labeled TG neurons. WIN enhanced mechanosensitive currents via TRPA1 activation in FAST DiI-labeled TG neurons. Our results indicate that cannabinoids can enhance the mechanosensitivity of TG endings in the inner walls of anterior chambers of rat eye via TRPA1 activation.

Evaluation of brachial plexus fascicles involvement on infraclavicular block: unfixed cadaver study / Avaliação do envolvimento dos fascículos do plexo braquial no bloqueio por via infraclavicular: estudo em cadáveres não fixados / Evaluación de la implicación de los fascículos del plexo braquial en el bloqueo por vía infraclavicular: estudio en cadáveres no fijos

BACKGROUND AND OBJECTIVES: This study shows how the diffusion of the anesthetic into the sheath occurs through the axillary infraclavicular space and hence proves the efficacy of the anesthetic block of the brachial plexus, and may thereby allow a consolidation of this pathway, with fewer complications, previously attached to the anesthesia. MATERIALS AND METHODS: 33 armpits of adult cadavers were analyzed and unfixed. We injected a solution of neoprene with latex dye in the infraclavicular space, based on the technique advocated by Gusmão et al., and put the corpses in refrigerators for three weeks. Subsequently, the specimens were thawed and dissected, exposing the axillary sheath along its entire length. RESULTS AND DISCUSSION: Was demonstrated involvement of all fasciculus of the plexus in 51.46%. In partial involvement was 30.30%, 18.24% of cases the acrylic was located outside the auxiliary sheath involving no issue. CONCLUSIONS: The results allow us to establish the infraclavicular as an effective and easy way to access plexus brachial, because the solution involved the fascicles in 81.76% partially or totally, when it was injected inside the axillary sheath. We believe that only the use of this pathway access in practice it may demonstrate the efficiency. .

JUSTIFICATIVA E OBJETIVOS: Procuramos demonstrar como ocorre a difusão do anestésico no interior da bainha axilar, quando se utiliza o bloqueio por via infraclavicular, através da fossa infraclavicular e, consequentemente, provar a eficácia dessa via, podendo, com isso, permitir uma consolidação da utilização desse acesso, com redução das complicações. MATERIAS E MÉTODO: Foram utilizadas 33 axilas de cadáveres adultos não fixados. Injetamos uma solução de neoprene látex com corante na fossa infraclavicular, baseando-se na técnica preconizada por Gusmão e col, e colocamos os cadáveres em geladeiras por três semanas. Posteriormente, as peças foram descongeladas e dissecadas, expondo a bainha axilar em toda sua extensão. RESULTADOS E DISCUSSÃO: Foi demonstrado envolvimento de todos os fascículos do plexo em 51,46%. Em 30,30% houve envolvimento parcial, e em 18,24% dos casos o acrílico foi localizado fora da bainha axilar, não envolvendo nenhum fascículo. CONCLUSÕES: Os dados obtidos permitem estabelecer a via infraclavicular como uma via eficaz e de fácil acesso ao plexo braquial, visto que a solução injetada envolveu os fascículos em 81,76% parcialmente ou totalmente, quando era injetada dentro da bainha axilar. Acreditamos que apenas a utilização desta via de acesso na prática poderá demonstrar a eficiência da mesma. .

JUSTIFICACIÓN Y OBJETIVOS: Este estudio intenta demostrar cómo ocurre la difusión del anestésico en el interior de la vaina axilar, cuando se utiliza el bloqueo por vía infraclavicular a través de la fosa infraclavicular, y al mismo tiempo, probar la eficacia de esa vía, pudiendo así permitir una consolidación de la utilización de ese acceso con reducción de las complicaciones. MATERIALES Y MÉTODO: Fueron utilizadas 33 axilas de cadáveres adultos no fijadas. Inyectamos una solución de neopreno látex con colorante en la fosa infraclavicular, con la técnica preconizada por Gusmão et al., y colocamos los cadáveres en frigoríficos durante 3 semanas. Posteriormente, las piezas fueron descongeladas y disecadas, exponiendo la vaina axilar en toda su extensión. RESULTADOS Y DISCUSIÓN: Quedó demostrada la implicación de todos los fascículos del plexo en un 51,46%. En un 30,30% hubo una participación parcial, y en un 18,24% de los casos el acrílico fue ubicado fuera de la vaina axilar sin la participación de ningún fascículo. CONCLUSIONES: Los datos obtenidos permiten establecer la vía infraclavicular como una vía eficaz y de fácil acceso al plexo braquial, visto que la solución inyectada tuvo la participación de los fascículos en un 81,76% parcial o totalmente, cuando se inyectaba dentro de la vaina axilar. Creemos que solamente con la utilización de esta vía de acceso en la práctica podrá quedar demostrada su eficacia. .

Expression of transient receptor potential canonical 1 in ozone-induced inflammatory lung tissues in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect the expression of transient receptor potential canonical 1 (TRPC1) in a mouse model of ozone-induced lung inflammation and explore its role in lung inflammation.</p><p><b>METHODS</b>In a mouse model of lung inflammation established by ozone exposure, the expression of TRPC1 in the inflammatory lung tissues was detected by RT-PCR, Wstern blotting and immunohistochemistry.</p><p><b>RESULTS</b>Compared to the control mice, the mice exposed to ozone showed significantly increased expression level of TRPC1 mRNA and protein in the inflammatory lung tissues (P<0.05). Immunohistochemistry showed increased TRPC1 protein expressions in the alveolar epithelial cells, bronchial epithelial cells, and inflammatory cells in the inflammatory lung tissues (P<0.05). The mRNA and protein expression levels of TRPC1 were positively correlated with the counts of white blood cells, macrophages, neutrophils and lymphocytes in the bronchoalveolar lavage fluid of the exposed mice (P<0.01).</p><p><b>CONCLUSION</b>TRPC1 may play a role in ozone-induced lung inflammation in mice.</p>

Role of transient receptor potential canonical 1 in airway remodeling and effect of budesonide on its pulmonary expression in asthmatic guinea pigs / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of transient receptor potential canonical 1 (TRPC1) in airway remodeling and the effect of budesonide intervention on its expression in the lungs of guinea pigs with ovalbumin-induced asthma.</p><p><b>METHODS</b>Fifty male guinea pigs were randomized into 5 equal groups, including a blank control group, ovalbumin group, ovalbumin+TRPC1 siRNA group, ovalbumin+luciferase siRNA group, and ovalbumin+budesonide group. After corresponding treatments, bronchoalveolar lavage was collected from the guinea pigs for eosinophils analysis and detection of IL-5 and IL-13 levels using ELISA. The lung tissues were stained with HE and Masson's trichrome to observe the bronchial wall thickness, smooth muscle hypertrophy, subepithelial collagen deposition, and lung inflammations. Immunohistochemistry and real-time quantitative PCR were performed to detect TRPC1 protein and mRNA expressions in the lungs, respectively.</p><p><b>RESULTS</b>The guinea pig models of ovalbumin-induced asthma showed significantly increased thickness of the bronchial wall, smooth muscle hypertrophy, collagen deposition and inflammatory cell infiltration, but these pathologies were obviously alleviated by treatment with TRPC1 siRNA or budesonide (P/0.05). Immunohistochemstry showed that TRPC1 protein was distributed mainly on the cell membrane and in the nuclei of the basal cells or columnar epithelial cells.</p><p><b>CONCLUSION</b>The up-regulated expression of TRPC1 ion channel is closely associated with the occurrence and progression of airway remodeling and chronic airway inflammation in asthma. Budesonide can partially suppress airway remodeling and inflammation by regulating the expression of TRPC1.</p>

Expressions of transient receptor potential A1 and related inflammatory factors in the rat model of prostatic inflammation / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanism of pain associated with chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) in the rat model of prostatic inflammation.</p><p><b>METHODS</b>Thirty-six male SD rats were equally randomized to an experimental and a control group, the former injected with 50 μl of 3% λ-carrageenan into the ventral prostate to make the model of non-bacterial prostatic inflammation, while the latter with the same volume of sterile saline solution. At 1, 2 and 4 weeks after modeling, the prostate, L6-S1 dorsal root ganglion (DRG) and spinal cord were harvested for examination of the expressions of the nerve growth factor (NGF), transient receptor potential ankyrin 1 (TRPA1), and calcitonin-gene-related peptide (CGRP) by immunohistochemistry and Western blot.</p><p><b>RESULTS</b>The expressions of NGF, TRPA1 and CGRP in the prostatic tissue were all significantly increased in the experimental group as compared with the control (P <0.05), with a gradual decrease with the prolonging of time (P <0.05). In the L6-S1 DRG and spinal cord, the expressions of NGF, TRPA1 and CGRP exhibited no significant differences between the experimental and control groups at 1 week after modeling (P >0.05) and kept at high levels in the experimental group at 2 and 4 weeks, though not significantly different from those at 1 week (P >0.05). Statistically significant differences were observed in the expressions of the three proteins in the experimental rats among different time points (P <0.05), but not between the two groups at any time point (P >0.05).</p><p><b>CONCLUSION</b>The molecular mechanism of CP/CPPS can be evaluated in the rat model of prostatic inflammation established by injecting λ-carrageenan into the prostate. TRPA1 may play an important role in connecting the upstream and down-stream pathways of CP/CPPS-associated pain.</p>

Roles of STIM2 and TRPC3 in the CaR mediated Ca2+ entry and NO generation in human umbilical vein endothelial cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the roles of stromal interaction molecule 2 (STIM2) and transient receptor potential canonical 3 (TRPC3) in extracellular Ca(2+)-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC).</p><p><b>METHODS</b>(1) The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. (2) The expressions of STIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmids. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. (3) The second to fifth passage of HUVEC were divided into: STIM2-002 short hairpin RNA (STIM2-002 shRNA ) + spermine + Ca2+ group and TRPC3-004 short hairpin RNA (TRPC3-004 shRNA ) + spermine + Ca2+ group; control group (spermine + Ca2+ group) and vehicle+ spermine + Ca2+ group. The four groups of cells were incubated with CaR agonist spermine, the intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, and the production of NO was determined by DAF-FM (NO fluorescent probe) of each group in HUVEC.</p><p><b>RESULTS</b>(1) Immunofluorescence technique results showed that STIM2 and TRPC3 proteinswere present in the cytoplasm of HUVEC. (2) The results of transfection constructed STIM2 and TRPC3 RNA interference plasmids demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRPC3 mRNA levels by 88.2% and 74.0%, respectively (P < 0.05), simultaneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively (P < 0.05). (3) Compared with spermine + Ca2+ group, the [Ca2+]i and the net NO fluorescence intensity of spermine + Ca(2+) + ShSTIM2-002 group, spermine + Ca(2+) + ShTRPC3-004 group and spermine + Ca2+ Vehicle group were not changed (P > 0.05).</p><p><b>CONCLUSION</b>STIM2 and TRPC3 do not participate in CaR-mediated Ca2+ influx and NO production individually.</p>

Recent advances in the research on mechanisms underlying podocyte-specific gene mutation-related steroid-resistant nephrotic syndrome / 中国当代儿科杂志

Steroid-resistant nephrotic syndrome poses a significant clinical challenge. Its pathogenesis has not been fully elucidated. In recent years, numerous studies have shown that podocyte-specific gene mutations may play important roles in the development of steroid-resistant nephrotic syndrome. Among the identified genes mutated in podocytes include NPHS2, NPHS1, WT1, TRPC6, MDR1, PLCE1, LMX1B, and LAMB2. This review aims to summarize the characteristics of these mutated genes in podocytes. The putative role for these podocyte-specific mutated genes in the pathogenesis, diagnosis, treatment and prognosis of steroid-resistant nephrotic syndrome is also discussed.

Effects of TRPC6 on invasibility of low-differentiated prostate cancer cells

OBJECTIVE@#To study the expression of TRPC6 among prostate cancer cells, establish high expression cell lines of TRPC6, and to provide potential cell mode for prostate cancer oncogenesis and development.@*METHODS@#Occurrence and development of prostate cancer cells, PC3, PC-3 m DU145, 22 rv1, LNCaP and normal prostate epithelial cells in the PrEC TRPC6 expression level were detected by QPCR method. Calcium phosphate transfection method was used to package retrovirus pLEGFP-N1-TRPC6 and pLEGFP-N1-vector and infect the prostate cancer cells, a stable high expression of TRPC6 prostate cancer cells. Sable cell lines of TRPC6, matrix metalloproteinase (MMP) 2, MMP9 expression was detected by QPCR and Western blot. Change of cell invasion ability was detected by Transwell.@*RESULTS@#The expression level of prostate cancer cells TRPC6 were higher than control group PrEC cells. Among TPRC6 the expression of cell line PC 3 transfer potential wre the lowest, and high transfer cell line PC-3M express was the highest. Real-time fluorescent quantitative PCR and western blot results showed that after filter, the seventh generation of cell TRPC6 protein and mRNA expression levels were higher than the control group obviously. Transwell experimental results showed that the overexpression of TRPC6 could promote the invasion ability of PC3 prostate cancer cells.@*CONCLUSIONS@#TRPC6 expressed in prostate cancer cells is in disorder, and its action may be associated with the invasion and metastasis of prostate cancer cells; successful establishment of stable high expression of TRPC6 prostate cancer cells primarily confirm the invasion-trigger ability of TRPC6 on prostate cancer, and lay down the Foundation for exploring the TRPC6's role in the occurrence and development of prostate cancer mechanism.

Human umbilical artery smooth muscle exhibits a 2-APB-sensitive capacitative contractile response evoked by vasoactive substances and expresses mRNAs for STIM, Orai and TRPC channels

After depletion of intracellular Ca2+ stores the capacitative response triggers an extracellular Ca2+ influx through store-operated channels (SOCs) which refills these stores. Our objective was to explore if human umbilical artery smooth muscle presented this response and if it was involved in the mechanism of serotonin- and histamine-induced contractions. Intracellular Ca2+ depletion by a Ca2+-free extracellular solution followed by Ca2+ readdition produced a contraction in artery rings which was inhibited by the blocker of Orai and TRPC channels 2-aminoethoxydiphenyl borate (2-APB), suggesting a capacitative response. In presence of 2-APB the magnitude of a second paired contraction by serotonin or histamine was significantly less than a first one, likely because 2-APB inhibited store refilling by capacitative Ca2+ entry. 2-APB inhibition of sarcoplasmic reticulum Ca2+ release was excluded because this blocker did not affect serotonin force development in a Ca2+-free solution. The PCR technique showed the presence of mRNAs for STIM proteins (1 and 2), for Orai proteins (1, 2 and 3) and for TRPC channels (subtypes 1, 3, 4 and 6) in the smooth muscle of the human umbilical artery. Hence, this artery presents a capacitative contractile response triggered by stimulation with physiological vasoconstrictors and expresses mRNAs for proteins and channels previously identified as SOCs.

Essential role of TRPC6 in the proliferation of gastric cancer and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the essential role and mechanism of TRPC6 gene in the development of gastric cancer.</p><p><b>METHODS</b>The expression of TRPC6 protein was assessed in gastric cancer tissues and normal tissues adjacent to the cancer from 30 patients with gastric cancer. The inhibiting effect of TRPC6 activity on cell growth, cell cycle of a human gastric cancer cell line AGS cells, tumor progression and development of xenografted human gastric cancer in a mouse model was tested using dominant-negative mutant TRPC6 (DNC6). The survival of mice bearing xenografted tumors in the GFP and DNC6 was compared using Kaplan-Meier analysis. All statistical tests were two-sided.</p><p><b>RESULTS</b>The TRPC6 protein in the tumor tissues and para-tumor tissues was (21.60 ± 8.32)% versus (7.14 ± 2.24)%. After transfection of DNC6 virus for 24 hours, 48 hours, 72 hours and 96 hours, the growth inhibition rates of gastric cancer cells were (36.90 ± 1.13)%, (44.06 ± 2.17)%, (52.12 ± 2.76)% and (50.89 ± 1.97)%, respectively. The clone formation rates of control group and DNC6 group were (14.70 ± 3.00)% versus (43.80 ± 7.00)%. After transfection with DNC6 virus for 0, 24, 36 and 48 hours, the G(2)/M phase arrest was (20.34 ± 1.98)%, (24.31 ± 2.37)%, (27.70 ± 2.36)%, (35.10 ± 3.0)% in the DNC6 group and (18.40 ± 2.01)%, (18.0% ± 1.72)%, (17.50 ± 1.74)%, (16.80 ± 1.71)% in the control group, respectively. Inhibition of TRPC6 activity also reduced the subcutaneous tumor volume in the mouse models with xenografted human tumors (P < 0.05).</p><p><b>CONCLUSION</b>In the preclinical models tested, TRPC6 channels are essential for gastric cancer development via regulation of G(2)/M phase transition.</p>

Effect of TRPC6 knockdown on puromycin aminonucleoside-induced podocyte injury / 华中科技大学学报（医学）（英德文版）

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Expression of transient receptor potential canonical channel proteins in human non-small cell lung cancer / 中国肺癌杂志

<p><b>BACKGROUND AND OBJECTIVE</b>Transient receptor potential canonical (TRPC) proteins, a group of Ca2' permeable nonselective cation channels, are thought to constitute store-operated calcium channels (SOCC) and mediate store-operated calcium entry (SOCE) in various cell types. Members of TRPC have been found to be involved in abnormal proliferation, differentiation, and growth of cancer cells. The aim of this study is to detect the mRNA and protein expression of TRPC in non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>Real-time quantitative PCRwas performed to screen the expression of TRPC mRNA in NSCLC tissue. Protein expression of TRPC was detected by Western blot.</p><p><b>RESULTS</b>Among the seven family members of TRPC so far identified (TRPC1-7), we detected the expression ofTRPC1, TRPC3, TRPC4, TRPC6 mRNA in 24 cases of NSCLC tissue; TRPC2, TRPC5 and TRPC7 mRNA were not detectable. The relative abundance of the expressed TRPC was TRPC1 approximately equal TRPC6 > TRPC3 > TRPC4. Western blot confirmed the protein expression of TRPC1, TRPC3, TRPC4 and TRPC6 in NSCLC tissue.</p><p><b>CONCLUSION</b>Out of the seven members of TRPC, we found TRPC1, TRPC3, TRPC4, TRPC6 mRNA and protein were selectively expressed in human NSCLC tissue. This study could provide a basis for future exploration of the individual role of these TRPC proteins in mediating SOCE and in the progression of lung cancer.</p>

TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.

TRPC6 mediates the enhancements of pulmonary arterial tone and intracellular Ca2+ concentration of pulmonary arterial smooth muscle cells in pulmonary hypertension rats / 生理学报

Pulmonary arterial hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that canonical transient receptor potential channel 6 (TRPC6) genes, which encode receptor-operated cation channels (ROCC) in PASMCs, play an important role in Ca2+ regulation and cell proliferation. The aim of the present study was to investigate the role of TRPC6 in monocrotaline (MCT)-induced pulmonary artery hypertension. Sprague-Dawley rats were randomly divided into normal control group and MCT group. In MCT group, pulmonary arterial hypertension was induced by a single intraperitoneal injection of MCT at a dose of 60 mg/kg. After 3 weeks, the right ventricular systolic pressure (RVSP) and the right ventricular mass index (RVMI) were measured. The lung sections were stained by HE and observed under light microscope. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot were performed to detect the expression of TRPC6 in rat pulmonary arteries. The 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced contractile tension of pulmonary arteries were measured by vascular ring tension analysis and the intracellular Ca2+ concentration ([Ca2+](i))of PASMCs was monitored using Fluo3-AM assay. The results showed that RVSP and RVMI markedly elevated in MCT group (P<0.01) in comparison to CON group. The thickness of pulmonary vascular smooth muscles was increased and the inner diameter of pulmonary arteries was diminished in MCT group. Though there was no significant difference in the levels of mRNA and protein of TRPC6 between CON and MCT groups, the application of OAG, which can directly activate ROCC, induced greater contraction tension of pulmonary arteries (P<0.01) and more Ca2+ entries in PASMCs (P<0.05) in MCT group compared to those in control group. These results indicate that MCT induces pulmonary artery hypertension and thus remodeling of the right ventricle and pulmonary arteries in rats. The expression of mRNA and protein of TRPC6 is not potentiated by MCT, but the TRPC6/ROCC-mediated Ca2+ entry in PASMCs and vascular tone of pulmonary arteries are significantly increased with MCT treatment.

Transient receptor potential A1 is involved in cold-induced contraction in the isolated rat colon smooth muscle / 生理学报

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.

Heteromerization of TRP channel subunits: extending functional diversity

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.