Erythropoietin increases expression and function of transient receptor potential canonical 5 channels

Hypertension is a common complication in hemodialysis patients during erythropoietin (EPO) treatment. The underlying mechanisms of EPO-induced hypertension still remain to be determined. Increased transient receptor potential canonical (TRPC) channels have been associated with hypertension. Now, TRPC gene expression was investigated using quantitative real-time RT-PCR and immunoblotting in cultured human endothelial cells and in monocytes from hemodialysis patients. EPO dose-dependently increased TRPC5 mRNA in endothelial cells. EPO increased TRPC5 mRNA stability, that is, EPO prolonged the half-life period for TRPC5 mRNA from 16 hours (control) to 24 hours (P<0.05). The poly(A) tail length was measured by rapid amplification of cDNA ends-poly(A) test. Increased TRPC5 mRNA stability was attributed to longer 3' poly(A) tail lengths after EPO administration. EPO also significantly increased TRPC5 channel protein abundance by 70% (P<0.05). Whole-cell patch clamp showed that angiotensin II-induced, TRPC5-mediated currents were dramatically increased in endothelial cells treated with EPO. Fluorescent dye techniques confirmed that increased calcium influx after EPO treatment was abolished after TRPC5 knockdown (P<0.05). EPO also significantly increased intracellular reactive oxygen species production. Knockdown of TRPC5 alleviated EPO-induced reactive oxygen species generation in endothelial cells (P<0.05). In vivo, EPO-treated hemodialysis patients showed significantly increased amounts of TRPC5 mRNA in monocytes compared with EPO-free hemodialysis patients (6.0±2.4 [n=12] versus 1.0±0.5 [n=9]; P<0.01). Patients undergoing EPO treatment also showed significantly elevated systolic blood pressure (160±7 versus 139±6 mm Hg; P<0.05). Our findings suggest that upregulated functional TRPC5 gene may be one cause of EPO-induced hypertension in patients with chronic kidney disease.     

瞬时受体电位C通道和大电导钙离子激活钾通道复合体与血管张力调节

瞬时受体电位C通道(transient receptor potential canonical channel,TRPC通道)和大电导钙离子激活钾通道(1arge conductance Ca2+ -activated K+channel,BK通道)是血管平滑肌细胞上两类重要的离子通道,在血管收缩和舒张的过程中具有重要的调节作用[1-2].目前,人们对TRPC通道和BK通道各自的分子结构和功能已有一定的认识,但两者之间是否存在紧密联系,尚未完全清楚.     

The canonical transient receptor potential 6 (TRPC6) channel is sensitive to extracellular pH in mouse platelets

The canonical transient receptor potential-6 (TRPC6) is a receptor-activated non-selective Ca^2 + channel regulated by a variety of modulators such as diacylglycerol, Ca^2 +/calmodulin or phosphorylation. The present study is aimed to investigate whether different situations, such as acidic pH, exposure to reactive oxygen species (ROS) or hypoxic-like conditions modulate TRPC6 channel function. Here we show normal aggregation and Ca^2 + mobilization stimulated by thrombin in TRPC6 KO platelets; however, OAG (1-oleoyl-2-acetyl-sn-glycerol)-evoked Ca^2 + entry was attenuated in the absence of TRPC6. Exposure of mouse platelets to acidic pH resulted in abolishment of thrombin-evoked aggregation and attenuated platelet aggregation induced by thapsigargin (TG) or OAG. Both OAG-induced Ca^2 + entry and platelet aggregation were greatly attenuated in cells expressing TRPC6 channels. Exposure of platelets to H₂O₂ or deferoxamine did not clearly alter thrombin, TG or OAG-induced platelet aggregation. Our results indicate that TRPC6 is sensitive to acidic pH but not to exposure to ROS or hypoxic-like conditions, which might be involved in the pathogenesis of the altered platelet responsiveness to DAG-generating agonists in disorders associated to acidic pH.     

Capsaicin-induced inhibition of platelet aggregation is not mediated by transient receptor potential vanilloid type 1

Capsaicin is an agonist of transient receptor potential vanilloid type 1 (TRPV1), in which it can act as a neuronal stimulant and result in nociception. Capsaicin also affects a variety of nonneuronal tissues, in which its mechanisms of action are less certain. The present study investigated whether the inhibitory effects of capsaicin on platelet aggregation are mediated via TRPV1. Venous whole blood obtained from beagle dogs (n = 6) was preincubated with capsaicin and/or the potent and selective competitive TRPV1 antagonist, A-993610 and then exposed to collagen (2 μg/ml). An aggregometer was used to quantify the platelet response. Capsaicin exposure inhibited collagen-induced platelet aggregation in a concentration-dependent manner, with significant effects at 10 and 30 μg capsaicin per millilitre. A-993610 alone (0.1-1.0 μg/ml) had no effects on collagen-induced platelet aggregation, nor did it have any effects on capsaicin's ability to inhibit platelet aggregation. The current results agree with previous findings that capsaicin can inhibit platelet aggregation. In addition, the present study demonstrates that capsaicin's inhibitory effect on collagen-induced canine platelet aggregation is not mediated by TRPV1.     

Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G

Canonical transient receptor potential (TRPC) channels are Ca2+-permeable nonselective cation channels that are widely expressed in numerous cell types. Seven different members of TRPC channels have been isolated. The activity of these channels is regulated by the filling state of intracellular Ca2+ stores and/or diacylglycerol and/or Ca2+/calmodulin. However, no evidence is available as to whether TRPC channels are regulated by direct phosphorylation on the channels. In the present study, TRPC isoform 3 (TRPC3) gene was overexpressed in HEK293 cells that were stably transfected with protein kinase G (PKG). We found that the overexpressed TRPC3 mediated store-operated Ca2+ influx and that this type of Ca2+ influx was inhibited by cGMP. The inhibitory effect of cGMP was abolished by KT5823 or H8. Point mutations at two consensus PKG phosphorylation sites (T11A and S263Q) of TRPC3 channel markedly reduced the inhibitory effect of cGMP. In addition, TRPC3 proteins were purified from HEK293 cells that were transfected with either wild-type or mutant TRPC3 constructs, and in vitro PKG phosphorylation assay was carried out. It was found that wild-type TRPC3 could be directly phosphorylated by PKG in vitro and that the phosphorylation was abolished in the presence of KT5823. The phosphorylation signal was greatly reduced in mutant protein T11A or S263Q. Taken together, TRPC3 channels could be directly phosphorylated by PKG at position T11 and S263, and this phosphorylation abolished the store-operated Ca2+ influx mediated by TRPC3 channels in HEK293 cells.     

INAF, a protein required for transient receptor potential Ca(2+) channel function

The trp gene of Drosophila encodes a subunit of a class of Ca(2+)-selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate "store-operated Ca(2+) entry," which is important in Ca(2+) homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Here, we report on the Drosophila inaF gene, which encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The inaF mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the trp gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that inaF mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function.     

Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity

We tested the hypothesis that activation of transient receptor potential vanilloid type-1 (TRPV1) by capsaicin prevents adipogenesis. TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans were detected by immunoblotting and quantitative real-time RT-PCR. The effect of TRPV1 on cytosolic calcium was determined fluorometrically in 3T3-L1-preadipocytes and in human visceral fat tissue. Adipogenesis in stimulated 3T3-L1-preadipocytes was determined by oil red O-staining of intracellular lipid droplets, triglyceride levels, expression of peroxisome proliferator-activated receptor-gamma, and expression of fatty acid synthase. Long-term feeding experiments were undertaken in wild-type mice and TRPV1 knockout mice. We detected TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans. In vitro, the TRPV1 agonist capsaicin dose-dependently induced calcium influx and prevented the adipogenesis in stimulated 3T3-L1-preadipocytes. RNA interference knockdown of TRPV1 in 3T3-L1-preadipocytes attenuated capsaicin-induced calcium influx, and adipogenesis in stimulated 3T3-L1-preadipocytes was no longer prevented. During regular adipogenesis TRPV1 channels were downregulated which was accompanied by a significant and time-dependent reduction of calcium influx. Compared with lean counterparts in visceral adipose tissue from obese db/db and ob/ob mice, and from obese human male subjects we observed a reduced TRVP1 expression. The reduced TRPV1 expression in visceral adipose tissue from obese humans was accompanied by reduced capsaicin-induced calcium influx. The oral administration of capsaicin for 120 days prevented obesity in male wild type mice but not in TRPV1 knockout mice assigned to high fat diet. We conclude that the activation of TRPV1 channels by capsaicin prevented adipogenesis and obesity.     

Increased store-operated and 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx in monocytes is mediated by transient receptor potential canonical channels in human essential hypertension

OBJECTIVE: Activation of nonselective cation channels of the transient receptor potential canonical (TRPC) family has been associated with hypertension. Whether store-operated channels, which are activated after depletion of intracellular stores, or second-messenger-operated channels, which are activated by 1-oleoyl-2-acetyl-sn-glycerol, are affected in essential hypertension is presently unknown. METHODS: Using a polymerase chain reaction, an in-cell western assay and the fluorescent dye technique we studied TRPC3, TRPC5, and TRPC6 expression and store-operated and 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx into human monocytes in 19 patients with essential hypertension and in 17 age-matched and sex-matched normotensive control individuals. RESULTS: We observed a significantly increased expression of TRPC3 and TRPC5, but not TRPC6, in essential hypertension. Store-operated calcium influx was significantly elevated in essential hypertension. Store-operated calcium influx was reduced by the inhibitor 2-aminoethoxydiphenylborane, specific TRPC3 and TRPC5 knockdown, but not TRPC6 knockdown using gene silencing by RNA interference. 1-Oleoyl-2-acetyl-sn-glycerol-induced calcium influx and barium influx were also significantly elevated in essential hypertension. The 1-oleoyl-2-acetyl-sn-glycerol-induced cation influx was reduced by TRPC3 and TRPC5 knockdown. CONCLUSION: We demonstrated an increased TRPC3 and TRPC5 expression and a subsequently increased store-operated calcium influx and increased 1-oleoyl-2-acetyl-sn-glycerol-induced cation influx in monocytes of patients with essential hypertension. This increased activation of monocytes through TRPC channels in patients with essential hypertension may promote vascular disease in these patients.     

Involvement of transient receptor potential canonical 1 (TRPC1) in angiotensin II-induced vascular smooth muscle cell hypertrophy

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy as one of the major events leading to atherosclerosis. Increased Ca(2+) entry is an important stimulus for VSMC hypertrophy, but the association with Ang II remains to be determined. Transient receptor potential canonical 1 (TRPC1) forms store-operated Ca(2+) (SOC) channels that are involved in Ca(2+) homeostasis. Our aim was to ascertain the potential involvement of TRPC1 in Ang II-induced VSMC hypertrophy. For this purpose, we used cultured human coronary artery smooth muscle cells (hCASMCs). Store-operated Ca(2+) entry (SOCE) increased in the Ang II-induced hypertrophied cells, and SOC channel blocker inhibited the Ang II-induced hypertrophic response. Although hCASMCs constitutively expressed TRPC1, C3, C4, C5, and C6, only TRPC1 increased in response to Ang II stimulation. TRPC1 siRNA decreased SOCE and prevented Ang II-induced hypertrophy. We found NF-kappaB binding sites in the 5'-regulatory region of the human TRPC1 gene. An electrophoretic mobility shift assay showed that Ang II increased the TRPC1 promoter's NF-kappaB binding activity. Co-treatment with NF-kappaB decoy oligonucleotides not only reduced TRPC1 expression, but also inhibited the hypertrophic responses. In conclusion, our data suggest that Ang II and subsequent NF-kappaB activation induces hCASMC hypertrophy through an enhancement of TRPC1 expression.     

Pregnancy-enhanced store-operated Ca2+ channel function in uterine artery endothelial cells is associated with enhanced agonist-specific transient receptor potential channel 3-inositol 1,4,5-trisphosphate receptor 2 interaction

We have previously shown that endothelial cells (EC) derived from the uterine artery (UA) of both pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes show a biphasic intracellular free Ca(2+) ([Ca(2+)](i)) response after ATP stimulation. In each case, the initial transient peak, caused by the release of Ca(2+) from the intracellular Ca(2+) stores, is mediated by purinergic receptor-Y2 and is very similar in both cell types. However, the sustained phase in particular, caused by the influx of extracellular Ca(2+), is heightened in the P-UAEC, and associates with an increased ability of the cells to demonstrate enhanced capacitative Ca(2+) entry (CCE) via store-operated channels (SOCs). Herein we demonstrated that the difference in the sustained [Ca(2+)](i) response is maintained for at least 30 min. When 2-aminoethoxydiphenyl borate (2APB) (an inhibitor of the inosital 1,4,5-trisphosphate receptor (IP3R) and possibly SOC) was used in conjunction with ATP, it was capable of completely inhibiting CCE. Since 2APB can inhibit SOC in some cell types and 2APB was capable of inhibiting CCE in the UAEC model, the role of SOC in CCE was first evaluated using the classical inhibitor La(3+). The ATP-induced sustained phase was inhibited by 10 microM La(3+), implying a role for SOC in the [Ca(2+)](i) response. Since canonical transient receptor potential channels (TRPCs) have recently been identified as putative SOCs in many cell types, including EC, the expression levels of several isoforms were evaluated in UAEC. Expression of TRPC3 and TRPC6 channels in particular was detected, but no significant difference in expression level was found between NP- and P-UAEC. Nonetheless, we were able to show that IP3R2 interacts with TRPC3 in UAEC, forming a protein complex, and that this interaction is considerably enhanced in an agonist sensitive manner by pregnancy. Thus, while IP3R and TRPC isoforms are not altered in their expression by pregnancy, enhanced functional interaction of TRPC3 with IP3R2 may underlie pregnancy-enhanced CCE in the UAEC model and so explain the prolonged [Ca(2+)](i) sustained phase seen in response to ATP.     

Cytoplasmic LEK1 is a regulator of microtubule function through its interaction with the LIS1 pathway

LIS1 and nuclear distribution gene E (NudE) are partner proteins in a conserved pathway regulating the function of dynein and microtubules. Here, we present data that cytoplasmic LEK1 (cytLEK1), a large protein containing a spectrin repeat and multiple leucine zippers, is a component of this pathway through its direct interaction with NudE, as determined by a yeast two-hybrid screen. We identified the binding domains in each molecule, and coimmunoprecipitation and colocalization studies confirmed the specificity of the interaction between cytLEK1 and NudE. Confocal deconvolution analysis revealed that cytLEK1 exhibits colocalization with endogenous NudE and with the known NudE binding partners, LIS1 and dynein. By localizing the NudE-binding domain of cytLEK1 to a small domain within the molecule, we were able to disrupt cytLEK1 function by using a dominant negative approach in addition to LEK1 knockdown and, thus, examine the role of the cytLEK1-NudE interaction in cells. Consistent with a defect in the LIS1 pathway, disruption of cytLEK1 function resulted in alteration of microtubule organization and cellular shape. The microtubule network of cells became tightly focused around the nucleus and resulted in a rounded cell shape. Additionally, cells exhibited a severe inability to repolymerize their microtubule networks after nocodazole challenge. Taken together, our studies revealed that cytLEK1 is essential for cellular functions regulated by the LIS1 pathway.     

Expression and regulation of transient receptor potential cation channel,subfamily M,member 2 (TRPM2) in human endometrium

To identify estrogen-responsive genes, we previously isolated estrogen receptor (ER)-binding DNA fragments from human genomic DNA using a recombinant ER protein. Six DNA fragments, each including a perfect palindromic estrogen response element (ERE), were obtained. The nucleotide sequence of one of the six fragments (E1 fragment) showed that the ERE of the E1 fragment is located in the 3′-untranslated region (UTR) of transient receptor potential cation channel, subfamily M, member 2 (TRPM2). Here, we confirmed the estrogen-dependent enhancer activity of the ERE of the E1 fragment by chloramphenicol acetyltransferase assay. TRPM2 mRNA expression was investigated in human endometrium, cultured human endometrial stromal cells (ESCs), and cultured human endometrial epithelial cells (EECs) using RT-PCR. Quantitative RT-PCR revealed that TRPM2 mRNA expression in ESCs increased after 17β-estradiol (E2) treatment. This study demonstrated for the first time that TRPM2 is an estrogen-responsive gene expressed in human endometrial cells.