Regulation of endothelial cell barrier function by store-operated calcium entry

Transient receptor potential (TRP) channels fulfill important and diverse signaling functions, and are generally conserved among species. The canonical subfamily of TRP proteins, TRPC channels, possesses 7 isoforms that combine in various ways to form heteromultimers. In endothelium, TRPC1 and TRPC4 form subunits of a channel that selectively conducts calcium. This channel is activated by calcium depletion in the endoplasmic reticulum, and thus TRPC1/TRPC4 forms the molecular basis of a store operated calcium entry pathway. TRPC4 interacts with protein 4.1, which tethers the channel to the membrane skeleton and represents a gating mechanism required for calcium permeation. In response to inflammatory agonists such as thrombin and bradykinin, the generation of inositol 1,4,5-trisphosphate transiently depletes endoplasmic reticulum calcium and activates the TRPC1/TRPC4 channel. Calcium permeation through this channel triggers cytoskeletal reorganization that is necessary to disrupt the endothelial cell barrier and increase permeability. Thus, inhibition of the TRPC1/TRPC4 channel provides a putative anti-inflammatory strategy.     

Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinases

Background and purpose:

TRPC5 is a Ca²⁺-permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant-derived isoflavone, on TRPC5 activity, and the mechanism(s) involved.

Experimental approach:

Effects of genistein on TRPC5 channels were investigated in TRPC5-over-expressing human embryonic kidney 293 (HEK) cells and bovine aortic endothelial cells (BAECs) using fluorescent Ca²⁺ imaging and electrophysiological techniques.

Key results:

In TRPC5-over-expressing HEK cells, genistein stimulated TRPC5-mediated Ca²⁺ influx, concentration dependently (EC₅₀= 93 µM). Genistein and lanthanum activated TRPC5 channels synergistically. Effects of genistein on TRPC5 channels were mimicked by daidzein (100 µM), a genistein analogue inactive as a tyrosine kinase inhibitor, but not by known tyrosine kinase inhibitors herbimycin (2 µM), PP2 (20 µM) and lavendustin A (10 µM). Action of genistein on TRPC5 channels was not affected by an oestrogen receptor inhibitor ICI-182780 (50 µM) or a phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 µM), suggesting genistein did not act through oestrogen receptors or phospholipase C. In BAECs, genistein (100 µM) stimulated TRPC5-mediated Ca²⁺ influx. In patch clamp studies, both genistein (50 µM) and daidzein (50 µM) augmented TRPC5-mediated whole-cell cation current in TRPC5 over-expressing HEK cells. Genistein stimulated TRPC5 channel activity in excised inside-out membrane patch, suggesting that its action was relatively direct and did not require cytosolic factors.

Conclusions and implications:

The present study is the first to demonstrate stimulation of a TRP channel by isoflavones. Genistein is a lipophilic compound able to stimulate TRPC5 activity in TRPC5-over-expressing HEK cells and in native vascular endothelial cells.     

Targeting podocyte-associated diseases

Injury to the podocytes is the initiating cause of many renal diseases, leading to proteinuria with possible progression to end-stage renal disease. Podocytes are highly specialized cells, with an important role in maintaining the glomerular filtration barrier and producing growth factors for both mesangial cells and endothelial cells. With their foot processes they cover the glomerular basement membrane, and form slit diaphragms with neighboring podocytes.Human podocytopathies include focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy, collapsing glomerulopathy and diabetic nephropathy. Research in the last two decades has demonstrated great progress in understanding the molecular mechanisms leading to podocytopathies. These include single gene defects in slit diaphragm proteins, but also discovery of apoptotic, enzymatic and other pathways involved in podocyte injury. With this progress, a great number of animal models is now available to study either specific podocytopathies, e.g. in mouse models with single gene mutations, or more general podocyte injury patterns, such as the lipopolysaccharide or protamine sulfate model of foot process effacement.In this review, the morphology of the glomerulus will be discussed, with a focus on the podocyte, its interactions with surrounding cells, and the highly differentiated slit diaphragm separating the apical from the basal membrane. We also provide an overview of human podocytopathies and animal models to study these diseases. In the last part we discuss targeted therapies addressing pathways and proteins affected in podocyte injury.     

TRPC channels as signal transducers

The study of the TRPC cation channels as signal transducers in sensory neurons is in its infancy. Mechanoreceptors that monitor arterial pressure are prime candidates for the involvement of TRPC channels as either primary mechanical transducers or as modulators of the transduction process. Their activity patterns can be regulated by growth factors such as BDNF and by a variety of ligands that activate Gq-coupled receptors, mechanisms that have been shown in heterologous expression systems to activate TRPC channels. We investigated the distribution of TRPC1 and TRPC3–7 in nodose sensory neurons and in their peripheral axons that terminate as mechanosensitive receptors in the aortic arch of the rat. Using immunocytochemical techniques we identified these six TRPC proteins in the soma of the nodose neurons but only TRPC1 and TRPC3–5 were found to distribute to the peripheral axons and the mechanosensory terminals. TRPC1 and TRPC3 extended into the low threshold complex sensory endings with very strong labeling. In contrast, TRPC4 and TRPC5 were found primarily in major branches of the receptor but immunoreactivity was weak in the region where mechanotransduction is presumed to occur. Terminals arising from unmyelinated fibers also expressed TRPC1 and TRPC3–5 but not all fibers expressed all of the channels suggesting that specific TRPC protein may be aligned with previously described subclasses of the unmyelinated C-fibers.     

Stim, ORAI and TRPC channels in the control of calcium entry signals in smooth muscle

Ca2+ entry signals are crucial in the control of smooth muscle contraction. Smooth muscle cells are unusual in containing plasma membrane (PM) Ca2+ entry channels that respond to voltage changes, receptor activation and Ca2+ store depletion. Activation of these channel subtypes is highly coordinated. The TRPC6 channel, widely expressed in most smooth muscle cell types, is largely non-selective to cations and is activated by diacylglycerol arising from receptor-induced phosholipase C activation. Receptor activation results largely in Na+ ion movement through TRPC6 channels, depolarization and subsequent activation of voltage-dependent L-type Ca2+ channels. The TRPC6 channels also appear to be activated by mechanical stretch, resulting again in depolarization and L-type Ca2+ channel activation. Such a coupling may be crucial in mediating the myogenic tone response in vascular smooth muscle. The emptying of stores mediated by inositol 1,4,5-trisphosphate receptors triggers the endoplasmic reticulum (ER) Ca2+ sensing protein stromal-interacting molecule (STIM) 1 to translocate into defined ER-PM junctional areas in which coupling occurs to Orai proteins, which serve as highly Ca2+-selective low-conductance Ca2+ entry channels. These ER-PM junctional domains may serve as crucial sites of interaction and integration between the function of store-operated, receptor-operated and voltage-operated Ca2+ channels. The STIM, Orai and TRPC channels represent highly promising new pharmacological targets through which such control may be induced.     

Histopathological analysis of the olfactory epithelium of zebrafish (Danio rerio) exposed to sublethal doses of urea

Chronic renal disease is known to alter olfactory function, but the specific changes induced in olfactory organs during this process remain unclear. Of the uraemic toxins generated during renal disease, high levels of urea are known to induce hyposmic conditions. In this study, the effects of environmental exposure to elevated concentrations of urea (7, 13.5 and 20 g L^?1) on the sensory mucosa of zebrafish in acute toxicity and chronic toxicity tests were described. It was observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced, but only following exposure to the highest concentrations of urea. Pan‐neuronal labelling with anti‐Hu revealed a negative correlation with levels of urea, leading to investigation of whether distinct neuronal subtypes were equally sensitive. Using densitometric analysis of immunolabelled tissues, numbers of G_{α olf}‐, TRPC2‐ and TrkA‐expressing cells were compared, representing ciliated, microvillous and crypt neurons, respectively. The three neuronal subpopulations responded differently to increasing levels of urea. In particular, crypt cells were more severely affected than the other cell types, and G_{α olf}‐immunoreactivity was found to increase when fish were exposed to low doses of urea. It can be concluded that exposure to moderate levels of urea leads to sensory toxicity directly affecting olfactory organs, in accordance with the functional olfactometric measurements previously reported in the literature.     

TRPC6在结节性硬化症癫痫患者皮层脑组织中的表达

目的 研究瞬时受体电位通道蛋白C6 (TRPC6)在结节性硬化症(TSC)癫痫患者皮层脑组织的表达与分布,探讨TRPC6通道参与结节性硬化致痫的可能机制.方法 收集手术切除并经病理检测证实是TSC癫痫患者的皮层脑组织标本18例,与7例正常对照皮层脑组织比较,通过采用Western blot、免疫组化、免疫荧光双标,检测TRPC6在正常脑组织与TSC病理标本中的表达分布情况.结果 Western blot蛋白检测结果显示,在TSC和对照大脑皮质(CTX) TRPC6均在相应的分子量94KDa处有特异性蛋白条带,与CTX比较,TSC组TRPC6条带与内参GAPDH条带灰度值之比显著增高(P＜0.05);免疫组化及免疫荧光双标结果显示,TRPC6在TSC病灶中免疫强度明显增高,且特异性高表达于皮层损伤区致痫灶中的异构神经元.包括特征性的巨形细胞(GCs)、异形神经元(DNs).结论 TRPC6在结节性硬化症患者致痫皮层脑组织中表达异常增高,特异性的细胞分布模式可能与癫痫发病密切相关.     

阿托伐他汀对ApoE基因敲除小鼠动脉粥样硬化病变中TRPC5表达的影响

目的:观察载脂蛋白E基因敲除(Apo E-/-)小鼠动脉粥样硬化斑块形成过程中血管平滑肌细胞瞬时受体电位通道5(TRPC5)蛋白的表达变化,以及阿托伐他汀药物干预对TRPC5的影响并探讨其作用机制。方法:将40只6周龄雄性Apo E-/-小鼠随机分为模型组和他汀干预组,高脂饲料喂养建立动脉粥样硬化模型。他汀干预组给予阿托伐他汀(20 mg·kg-1·d-1)灌胃,模型组给予等量生理盐水灌胃。20只同龄雄性野生型C57BL/6J小鼠给予普通饲料喂养作为正常对照组。各组小鼠分别喂养至20和30周龄,分别取10只取血并处死。取主动脉根部做石蜡切片行HE染色形态学观察,测量并计算斑块相对面积;免疫组织化学染色检测各组小鼠TRPC5蛋白表达变化。取胸腹段主动脉行实时荧光定量PCR,检测主动脉中TRPC5通道蛋白mRNA的表达水平。结果:与模型组相比,阿托伐他汀干预组的血脂明显下降,斑块总面积明显减小,TRPC5蛋白水平及mRNA含量明显下降;30周龄模型组的TRPC5蛋白表达稍高于20周龄模型组,但差异无统计学意义;30周干预组较20周干预组相比,TRPC5水平有降低趋势且差异有统计学意义。结论:阿托伐他汀可能通过下调TRPC5蛋白表达从而延缓动脉粥样硬化进程。