Role of Endogenous ENaC and TRP Channels in the Myogenic Response of Rat Posterior Cerebral Arteries

Aims

Mechanogated ion channels are predicted to mediate pressure-induced myogenic vasoconstriction in small resistance arteries. Recent findings have indicated that transient receptor potential (TRP) channels and epithelial sodium channels (ENaC) are involved in mechanotransduction. The purpose of this study was to investigate the role of TRP channels and ENaC in the myogenic response. Our previous study suggested that ENaC could be a component of the mechanosensitive ion channels in rat posterior cerebral arteries (PCA). However, the specific ion channel proteins mediating myogenic constriction are unknown. Here we found, for the first time, that ENaC interacted with TRPM4 but not with TRPC6 using immunoprecipitation and confocal microscopy.

Methods and Results

Treatment with a specific βENaC inhibitor, amiloride, a specific TRPM4 inhibitor, 9-phenanthrol, and a TRPC6 inhibitor, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365, resulted in inhibition of the pressure-induced myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with small interfering RNA of βENaC, TRPM4, and TRPC6. Co-treatment with amiloride and 9-phenanthrol showed a similar inhibitory effect on myogenic contraction compared to single treatment with amiloride or 9-phenanthrol. The myogenic response was not affected by 9-phenanthrol or amiloride treatment in PCA transfected with βENaC or TRPM4 siRNA, respectively. However, pressure-induced myogenic response was fully inhibited by co-treatment with amiloride, 9-phenanthrol, and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365, and by treatment with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365 in PCA transfected with βENaC siRNA.

Conclusion

Our results suggest that ENaC, TRPM4, and TRPC6 play important roles in the pressure-induced myogenic response, and that ENaC and TRPM4 interact in rat PCA.     

TRPC通道对PC12细胞缺氧缺糖再灌注后氧化损伤的保护作用

瞬时受体电位C通道(transient receptor potential canonical,TRPC)属于瞬时受体电位（TRP）离子通道家族成员之一,与Ca2+ 调控及氧化应激关系密切．然而,TRPC通道在缺血缺氧性脑损伤中的作用仍有争议．本实验采用MTT法、台盼蓝排斥实验、LDH漏出实验联合Annexin/PI染色流式分析等显示,当采用通道阻断剂-SKF96365特异阻断TRPC通道时,PC12细胞对缺氧缺糖再灌注（oxygen-glucose deprivation／reperfusion,OGD-R）引起的损伤变得更敏感,细胞凋亡增加．尽管同时采用SKF96365、NMDA受体、AMPA受体和L-型钙通道阻断剂可引起细胞损伤和死亡减弱,但仍呈现明显的SKF96365浓度依懒性,提示TRPC通道参与细胞对缺氧缺糖再灌注耐受的调节,具有保护作用．钙指示剂Fluo-3AM荧光标记结合激光共聚焦显微镜分析显示,SKF96365可明显降低缺氧缺糖再灌注后细胞内的Ca2+浓度．总之,实验结果提示,TRPC通道对缺氧缺糖再灌注引起的细胞损伤和凋亡具有保护作用,其机制可能涉及TRPC通道对细胞内钙浓度的调节,详尽机制有待进一步研究．     

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 microM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 microM) and was not observed in Fluo-3-loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl- channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 microM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.     

Group I metabotropic glutamate receptor-dependent TRPC channel trafficking in hippocampal neurons

The group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elicited two phases of synchronized neuronal (epileptiform) discharges in hippocampal slices: an initial phase of short duration discharges followed by a phase of prolonged discharges. We assessed the involvement of transient receptor potential canonical (TRPC) channels in these responses. Pre-treatment of hippocampal slices with TRPC channel blockers, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365) or 2-aminoethoxydiphenyl borate, did not affect the short epileptiform discharges but blocked the prolonged epileptiform discharges. SKF96365 suppressed ongoing DHPG-induced prolonged epileptiform discharges. Western blot analysis showed that the total TRPC4 or TRPC5 proteins in hippocampal slices were unchanged following DHPG. DHPG increased TRPC4 and TRPC5 in the cytoplasmic compartment and decreased these proteins in the plasma membrane. Translocation of TRPC4 and TRPC5 was suppressed when the epileptiform discharges were blocked by ionotropic glutamate receptor blockers. Translocation of TRPC4 and TRPC5 was also prevented in slices from phospholipase C (PLC) beta1 knockout mice, even when synchronized discharges were elicited by the convulsant 4-aminopyridine. The results suggest that TRPC channels are involved in generating DHPG-induced prolonged epileptiform discharges. This function of TRPC channels is associated with a neuronal activity- and PLCbeta1-dependent translocation of TRPC4 and TRPC5 proteins from the plasmalemma to the cytoplasmic compartment.     

Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase

Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca²⁺ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Moreover, Jujuboside B increased intracellular Ca²⁺ concentration dose-dependently, which was inhibited by EGTA and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Besides, Jujuboside B induced a rapid Ca²⁺ influx instantaneously after depleting intracellular Ca²⁺ store, which was significantly inhibited by {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca²⁺ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved endothelium-dependent hyperpolarizaiton through endothelial potassium channels. Jujuboside B is a natural compound with new pharmacological effects on improving endothelial dysfunction and treating vascular diseases.     

TRPC channels and diacylglycerol dependent calcium signaling in rat sensory neurons

Transient receptor potential (TRP) channels of the TRPV, TRPA, and TRPM subfamilies play important roles in somatosensation including nociception. While particularly the Thermo TRPs have been extensively investigated in sensory neurons, the relevance of the subclass of "canonical" TRPC channels in primary afferents is yet elusive. In the present study, we investigated the presence and contribution to Ca(2+) transients of TRPC channels in dorsal root ganglion neurons. We found that six of the seven known TRPC subtypes were expressed in lumbar DRG, with TRPC1, C3, and C6 being the most abundant. Microfluorimetric calcium measurements showed Ca(2+) influx induced by oleylacylglycerol (OAG), an activator of the TRPC3/C6/C7 subgroup. Furthermore, OAG induced rises in [Ca(2+)](i) were inhibited by SKF96365, an inhibitor of receptor and store operated calcium channel. OAG induced calcium transients were also inhibited by blockers of diacylglycerol (DAG) lipase, lipoxygenase or cyclooxygenase and, intriguingly, by inhibitors of the capsaicin receptor TRPV1. Notably, SKF96365 did not affect capsaicin-induced calcium transients. Taken together, our findings suggest that TRPC are functionally expressed in subpopulations of DRG neurons. These channels, along with TRPV1, contribute to calcium homeostasis in rat sensory neurons.     

Paradoxical effect of ethanol on potassium channel currents and cell survival in cerebellar granule neurons

Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K⁺ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K⁺ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I _A (transient K⁺ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I _K (delayed rectifier K⁺ current) current. Pipette dialysis with GTPγS or GDPβS did not modify the effects of EtOH (400 mM) on both membrane potential and I _K current. In contrast, the reversible depolarization and slowly recovering inhibition of I _A induced by EtOH (10 mM) became irreversible in the presence of GTPγS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K⁺ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K⁺ currents partly involved in the control of granule cell death.     

Epidermal Growth Factor Promotes Uncoupling from Adenylyl Cyclase of the Rat D2S Receptor Expressed in GH4C1 Cells

Abstract: In anterior pituitary cells or when transfected into host cell lines, the D₂ dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D₂ receptors, responds to epidermal growth factor (EGF) by expressing a D₂ receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in α subunit of the G protein G₁₃. In this study we have investigated the effects of EGF on the transduction mechanisms of D₂ receptors in GH4C1 cells transfected and permanently overexpressing the rat short D₂ receptor. Activation of D₂ receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D₂ receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of G₁₃ protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.     

Capacitative calcium entry and proliferation of human osteoblast-like MG-63 cells

Abstract. Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca²⁺) control in numerous cellular functions; however, involvement of capacitative Ca²⁺ entry (CCE) in proliferation of bone cells is less well investigated. Objectives: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast‐like MG‐63 cells. Meterials and Methods: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. Results: Intracellular Ca²⁺ store depletion by thapsigargin induced CCE in MG‐63 cells; this was characterized by a rapid transient increase of intracellular Ca²⁺ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet‐derived growth factor. Inhibitors of store‐operated Ca²⁺ channels (2‐APB and SKF‐96365) prevented CCE, while voltage‐dependent Ca²⁺ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage‐dependent Ca²⁺ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2‐APB and SKF‐96395, inhibited it. Cell cycle analysis showed that 2‐APB and SKF‐96395 lengthen the S and G₂/M phases, which would account for the reduction in cell proliferation. Conclusions: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca²⁺ influx involved in osteoblast proliferation.     

Transient receptor potential canonical channels are essential for chemotactic migration of human malignant gliomas

The majority of malignant primary brain tumors are gliomas, derived from glial cells. Grade IV gliomas, Glioblastoma multiforme, are extremely invasive and the clinical prognosis for patients is dismal. Gliomas utilize a number of proteins and pathways to infiltrate the brain parenchyma including ion channels and calcium signaling pathways. In this study, we investigated the localization and functional relevance of transient receptor potential canonical (TRPC) channels in glioma migration. We show that gliomas are attracted in a chemotactic manner to epidermal growth factor (EGF). Stimulation with EGF results in TRPC1 channel localization to the leading edge of migrating D54MG glioma cells. Additionally, TRPC1 channels co-localize with the lipid raft proteins, caveolin-1 and β-cholera toxin, and biochemical assays show TRPC1 in the caveolar raft fraction of the membrane. Chemotaxis toward EGF was lost when TRPC channels were pharmacologically inhibited or by shRNA knockdown of TRPC1 channels, yet without affecting unstimulated cell motility. Moreover, lipid raft integrity was required for gliomas chemotaxis. Disruption of lipid rafts not only impaired chemotaxis but also impaired TRPC currents in whole cell recordings and decreased store-operated calcium entry as revealed by ratiomeric calcium imaging. These data indicated that TRPC1 channel association with lipid rafts is essential for glioma chemotaxis in response to stimuli, such as EGF.     

Insulin increases surface expression of TRPC6 channels in podocytes: role of NADPH oxidases and reactive oxygen species

Insulin receptors in podocytes are essential for normal kidney function. Here, we show that insulin evokes a rapid increase in the surface expression of canonical transient receptor potential-6 channel (TRPC6) channels in cultured podocytes, but caused a decrease in surface expression of TRPC5. These effects are accompanied by a marked increase in outwardly rectifying cationic currents that can be blocked by 10 μM SKF96365 or 100 μM La(3+). Application of oleoyl-2-acetyl-sn-glycerol (OAG) also increased SKF96365- and La(3+)-sensitive cationic currents in podocytes. Importantly, current responses to a combination of OAG and insulin were the same amplitude as those evoked by either agent applied alone. This occlusion effect suggests that OAG and insulin are targeting the same population of channels. In addition, shRNA knockdown of TRPC6 markedly reduced cationic currents stimulated by insulin. The effects of insulin on TRPC6 were mimicked by treating podocytes with H(2)O(2). Insulin treatment rapidly increased the generation of H(2)O(2) in podocytes, and it increased the surface expression of the NADPH oxidase NOX4 in cultured podocytes. Basal and insulin-stimulated surface expression of TRPC6 were reduced by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidases and other flavin-dependent enzymes, by siRNA knockdown of NOX4, and by manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, a membrane-permeable mimetic of superoxide dismutase and catalase. These observations suggest that insulin increases generation of ROS in part through activation of NADPH oxidases, and that this step contributes to modulation of podocyte TRPC6 channels.     

Angiotensin-2-Mediated Ca2+ Signaling in the Retinal Pigment Epithelium: Role of Angiotensin-Receptor- Associated-Protein and TRPV2 Channel

Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca²⁺inhibited by losartan. Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca²⁺response. RPE cells from Atrap^−/− mice showed smaller AngII-evoked Ca²⁺peak (by 22%) and loss of sustained Ca²⁺elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca²⁺-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca²⁺-rise. Application of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca²⁺transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca²⁺transients in the RPE by releasing Ca²⁺from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca²⁺elevation.     

TRPC1 and TRPC5 form a novel cation channel in mammalian brain

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.     

Hydrogen sulfide evokes neurite outgrowth and expression of high-voltage-activated Ca2+ currents in NG108-15 cells: involvement of T-type Ca2+ channels

We investigated if stimulation of T-type Ca²⁺ channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H₂S), could cause neuronal differentiation of NG108-15 cells. Like dibutyryl cyclic AMP (db-cAMP), treatment with NaHS at 1.5–13.5 mM for 16 h enhanced neurite outgrowth in a concentration-dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db-cAMP at subeffective concentrations. Exposure to NaHS or db-cAMP for 2 days resulted in enhancement of expression of high-voltage-activated currents consisting of N-, P/Q-, L- and also other types, but not of T-type currents. Mibefradil, a pan-T-type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db-cAMP. The NaHS-evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca²⁺, and by zinc chloride at a concentration known to selectively inhibit Ca_v3.2 isoform of T-type Ca²⁺ channels, but not Ca_v3.1 or Ca_v3.3. Further, l -ascorbate, recently proven to selectively inhibit Ca_v3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H₂S is a novel inducer of neuronal differentiation in NG108-15 cells, as characterized by neuritogenesis and expression of high-voltage-activated currents, and suggest the involvement of T-type Ca²⁺ channels, especially Ca_v3.2.     

Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.     

Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca²⁺ channel α₁ subunit, α_1H, from a human medullary thyroid carcinoma cell line. The α_1H subunit is structurally similar to previously described α₁ subunits. Northern blot analysis indicates that α_1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba²⁺ currents recorded from human embryonic kidney 293 cells transiently expressing α_1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α_1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni²⁺ (IC₅₀ = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC₅₀ = 167 μ M ). Thus, α_1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca²⁺ channels.     

Antibodies Against Rat Brain Vesicle-Associated Membrane Protein (Synaptobrevin) Prevent Inhibition of Acetylcholine Release by Tetanus Toxin or Botulinum Neurotoxin Type B

Abstract: The Shaw-type K⁺ channel K_v3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K⁺ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to K_v 3.1 channels expressed transiently in Xenopus oocytes. K_v 3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13-dibutyrate decreased K_v 3.1 currents. Based on these results, we find that the stable expression of voltage-gated K⁺ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.     

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K⁺ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K⁺ equilibrium potential (E_K) – membrane potential (V_m), or secondary active by coupling through a carrier to the inward driving force of H⁺ or Na⁺. Known K⁺ channels are permeable to monovalent cations, a permeability order being K⁺ > Rb⁺ > NH₄⁺ > Na⁺≥ Li⁺ > Cs⁺. The macroscopic K⁺ currents across a cell or protoplast surface commonly show rectification, i.e. a V^m-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca²⁺, of K⁺, of H⁺, or by the K⁺ driving force. Analysis by the patch clamp technique reveals that plant K⁺ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K⁺ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.