TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca~(2+)浓度的升高

经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。     

Physiological properties and functions of Ca(2+) sparks in rat intrapulmonary arterial smooth muscle cells

Ca(+) spark has been implicated as a pivotal feedback mechanism for regulating membrane potential and vasomotor tone in systemic arterial smooth muscle cells (SASMCs), but little is known about its properties in pulmonary arterial smooth muscle cells (PASMCs). Using confocal microscopy, we identified spontaneous Ca(2+) sparks in rat intralobar PASMCs and characterized their spatiotemporal properties and physiological functions. Ca(2+) sparks of PASMCs had a lower frequency and smaller amplitude than cardiac sparks. They were abolished by inhibition of ryanodine receptors but not by inhibition of inositol trisphosphate receptors and L-type Ca(2+) channels. Enhanced Ca(2+) influx by BAY K8644, K(+), or high Ca(2+) caused a significant increase in spark frequency. Functionally, enhancing Ca(2+) sparks with caffeine (0.5 mM) caused membrane depolarization in PASMCs, in contrast to hyperpolarization in SASMCs. Norepinephrine and endothelin-1 both caused global elevations in cytosolic Ca(2+) concentration ([Ca(2+)]), but only endothelin-1 increased spark frequency. These results suggest that Ca(2+) sparks of PASMCs are similar to those of SASMCs, originate from ryanodine receptors, and are enhanced by Ca(2+) influx. However, they play a different modulatory role on membrane potential and are under agonist-specific regulation independent of global [Ca(2+)].     

Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension

Chronic hypoxia causes pulmonary hypertension with vascular remodeling, increase in vascular tone, and altered reactivity to agonists. These changes involve alterations in multiple Ca(2+) pathways in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that vanilloid (TRPV)- and melastatin-related transient receptor potential (TRPM) channels are expressed in pulmonary arteries (PAs). Here we found that TRPV4 was the only member of the TRPV and TRPM subfamilies upregulated in PAs of chronic hypoxic rats. The increase in TRPV4 expression occurred within 1 day of hypoxia exposure, indicative of an early hypoxic response. TRPV4 in PASMCs were found to be mechanosensitive. Osmo-mechanical stress imposed by hypotonic solution activated Ca(2+) transients; they were inhibited by TRPV4 specific short interfering RNA, the TRPV blocker ruthenium red, and the cytochrome P450 epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide. Consistent with TRPV4 upregulation, the Ca(2+) response induced by the TRPV4 agonist 4α-phorbol 12,13-didecanoate and hypotonicity was potentiated in hypoxic PASMCs. Moreover, a significant myogenic tone, sensitive to ruthenium red, was observed in pressurized endothelium denuded small PAs of hypoxic but not normoxic rats. The elevated basal intracellular Ca(2+) concentration in hypoxic PASMCs was also reduced by ruthenium red. In extension of these results, the development of pulmonary hypertension, right heart hypertrophy, and vascular remodeling was significantly delayed and suppressed in hypoxic trpv4(-/-) mice. These results suggest the novel concept that TRPV4 serves as a signal pathway crucial for the development of hypoxia-induced pulmonary hypertension. Its upregulation may provide a pathogenic feed-forward mechanism that promotes pulmonary hypertension via facilitated Ca(2+) influx, subsequently enhanced myogenic tone and vascular remodeling.     

Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.     

Hydrogen peroxide-induced Ca2+ mobilization in pulmonary arterial smooth muscle cells

Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca(2+) mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca(2+) response and to delineate the Ca(2+) pathways activated by hydrogen peroxide (H(2)O(2)) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 microM to 1 mM H(2)O(2) elicited concentration-dependent increase in intracellular Ca(2+) concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP(3)) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca(2+) in external solution. The secondary phase was dependent on extracellular Ca(2+) influx. It was unaffected by the voltage-gated Ca(2+) channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La(3+), but inhibited concentration dependently by millimolar Ni(2+), and potentiated by the Na(+)/Ca(2+) exchange inhibitor KB-R 7943. H(2)O(2) did not alter the rate of Mn(2+) quenching of fura 2, suggesting store- and receptor-operated Ca(2+) channels were not involved. By contrast, H(2)O(2) elicited a sustained inward current carried by Na(+) at -70 mV, and the current was inhibited by Ni(2+). These results suggest that H(2)O(2) mobilizes intracellular Ca(2+) through multiple pathways, including the IP(3)- and ryanodine receptor-gated Ca(2+) stores, and Ni(2+)-sensitive cation channels. Activation of these Ca(2+) pathways may play important roles in ROS signaling in PASMCs.     

New mechanisms of pulmonary arterial hypertension: role of Ca²⁺ signaling

Pulmonary arterial hypertension (PAH) is a severe and progressive disease that usually culminates in right heart failure and death if left untreated. Although there have been substantial improvements in our understanding and significant advances in the management of this disease, there is a grim prognosis for patients in the advanced stages of PAH. A major cause of PAH is increased pulmonary vascular resistance, which results from sustained vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness. In addition to other signal transduction pathways, Ca(2+) signaling in pulmonary artery smooth muscle cells (PASMCs) plays a central role in the development and progression of PAH because of its involvement in both vasoconstriction, through its pivotal effect of PASMC contraction, and vascular remodeling, through its stimulatory effect on PASMC proliferation. Altered expression, function, and regulation of ion channels and transporters in PASMCs contribute to an increased cytosolic Ca(2+) concentration and enhanced Ca(2+) signaling in patients with PAH. This review will focus on the potential pathogenic role of Ca(2+) mobilization, regulation, and signaling in the development and progression of PAH.     

T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na(+) currents as well as transient Ca(2+) currents abolished by the external application of Ni(2+). Biophysical and pharmacological data indicated that the Ca(2+) current is predominantly mediated by T-type (Ca(v)3.2) channels. The number of cells expressing T-type channels and Ca(v)3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca(2+) currents with Ni(2+) induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Ca(v)3.2) Ca(2+) channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Ca(v)3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca(2+) entry mediated by Ca(v)3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.     

T-type Ca2+ channels in vascular smooth muscle: multiple functions

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.     

Functional expression of voltage-gated calcium channels in human melanoma

The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.     

TRP channels in hypertension

Pulmonary and systemic arterial hypertension are associated with profound alterations in Ca(2+) homeostasis and smooth muscle cell proliferation. A novel class of non-selective cation channels, the transient receptor potential (TRP) channels, have emerged at the forefront of research into hypertensive disease states. TRP channels are identified as molecular correlates for receptor-operated and store-operated cation channels in the vasculature. Over 10 TRP isoforms are identified at the mRNA and protein expression levels in the vasculature. Current research implicates upregulation of specific TRP isoforms to be associated with increased Ca(2+) influx, characteristic of vasoconstriction and vascular smooth muscle cell proliferation. TRP channels are implicated as Ca(2+) entry pathways in pulmonary hypertension and essential hypertension. Caveolae have recently emerged as membrane microdomains in which TRP channels may be co-localized with the endoplasmic reticulum in both smooth muscle and endothelial cells. Such enhanced expression and function of TRP channels and their localization in caveolae in pathophysiological hypertensive disease states highlights their importance as potential targets for pharmacological intervention.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca (2+) is essential for cardiomyocyte function, and alterations in proteins that regulate Ca (2+) influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca (2+) channels are one pathway of Ca (2+) entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Cav3.1 (α1G) and Cav3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca (2+) channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Cav3.1 and Cav3.2 Ca (2+) channel expression in the pathological setting. The putative association of T-type Ca (2+) channels with Ca (2+) dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

Pulmonary expression of voltage-gated calcium channels: special reference to sensory airway receptors

Studying depolarisation induced calcium entry in our recently developed in situ lung slice model for molecular live cell imaging of selectively visualised pulmonary neuroepithelial bodies (NEBs), exemplified the need for information on the localisation of voltage-gated calcium channels (Ca(v)) in lungs in general, and related to sensory airway receptors more specifically. The present study therefore aimed at identifying the expression pattern of all major classes and subtypes of Ca(v) channels, using multiple immunostaining of rat lung cryosections. Ca(v) channel antibodies were combined with antibodies that selectively label NEBs, nerve fibre populations, smooth muscle, endothelium and Clara cells. Ca(v)2.1 (P/Q-type) was the only Ca(v) channel expressed in NEB cell membranes, and appeared to be restricted to the apical membrane of the slender NEB cell processes that reach the airway lumen. Subpopulations of the vagal but not the spinal sensory nerve fibres that contact NEBs showed immunoreactivity (IR) for Ca(v)1.2 (L-type) and Ca(v)2.1. Ca(v)2.3 (R-type) was selectively expressed by the so-called Clara-like cells that cover NEBs only, and appears to be a unique marker to discriminate this epithelial cell type from the much more extensive group of Clara cells in rat airways. The laminar nerve endings of smooth muscle-associated airway receptors (SMARs) revealed IR for both Ca(v)2.1 and Ca(v)2.2 (N-type). More generally, Ca(v)1.2 was seen to be expressed in vascular smooth muscle, Ca(v)2.3 and Ca(v)3.1 (T-type) in bronchial smooth muscle, Ca(v)3.1 and Ca(v)3.2 (T-type) in endothelial cells, and Ca(v)1.3 (L-type) in a limited number of epithelial cells. In conclusion, the present immunocytochemical study has demonstrated that the various subtypes of Ca(v) channels have distinct expression patterns in rat lungs. Special focus on morphologically/neurochemically characterised sensory airway receptors learned us that both NEBs and SMARs present Ca(v) channels. Knowledge of the identification and localisation of Ca(v) channels in airway receptors and surrounding tissues provides a solid basis for interpretation of the calcium mediated activation studied in our ex vivo lung slice model.     

Testosterone is a potent inhibitor of L-type Ca(2+) channels

Testosterone administration is beneficial in alleviating myocardial ischaemia in men with significant coronary artery disease (CAD), a condition which is associated with hypotestosteronaemia. Infusion of physiological concentrations of testosterone into coronary arteries at angiography results in rapid vasodilatation in patients with CAD. Whilst the cardiovascular benefits of testosterone have long been documented, the underlying mechanism(s) have not yet been revealed. Here, we have investigated whether testosterone might act like widely prescribed antihypertensive dihydropyridines, as an endogenous Ca(2+) channel antagonist. To do this, we used the whole-cell patch-clamp technique to record Ca(2+) currents from the A7r5 smooth muscle cell line and HEK 293 cells stably expressing either L- or T-type Ca(2+) channels. We demonstrate that testosterone directly inhibited both native and human recombinant vascular L-type Ca(2+) channels in a manner that was voltage-independent and, crucially, displayed an IC(50) value of 38 nM, a value within the physiological range. At higher (supraphysiological) concentrations both native and human recombinant T-type channels were also inhibited by testosterone. Our data indicate that testosterone acts like widely prescribed antihypertensive dihydropyridines to reduce Ca(2+) influx into vascular smooth muscle and so promote vasodilation. This effect is likely to account for its beneficial cardiovascular actions.     

Silencing of STIM1 attenuates hypoxia-induced PASMCs proliferation via inhibition of the SOC/Ca2+/NFAT pathway

Background

Stromal interaction molecule 1 (STIM1) is a newly discovered Ca²⁺ sensor on the endoplasmic reticulum which is an indispensable part in the activation of store-operated Ca²⁺ channels (SOC). Recent studies demonstrate that SOC of pulmonary smooth muscle cells (PASMCs) were upregulated by chronic hypoxia which contribute to the enhanced pulmonary vasoconstriction and vascular remodeling. However, the exact role of STIM1 in the development of chronic hypoxic pulmonary hypertension(HPH) remains unclear.

Methods

In this study we investigated the cellular distribution and expression of STIM1 by immunofluorescence, qRTPCR and Western blotting methods in Wistar rat distal intrapulmonary arteries under normal and chronic hypobaric hypoxic conditions. In vitro, Wistar rat PASMCs were isolated and cultured. PASMCs were transfected with siRNA targeting STIM1 gene by liposome. The expression of STIM1 protein was detected by Western blotting. [³H]-thymidine ([³H]-TdR) incorporation were performed to detect PASMCs proliferation. The cell cycle was analyzed by flow cytometry. The SOC-mediated Ca²⁺ influx was calculated by Ca²⁺ fluorescence imaging and the nuclear translocation of NFATc3 was determined by immunofluorescence and Western blot analysis of nuclear extracts.

Results

We found that during the development of HPH and the initiation of vascular remodeling, the mRNA and protein expression levels of STIM1 significantly increased in the distal intrapulmonary arteries. Moderate hypoxia significantly promotes PASMCs proliferation and cell cycle progression. Silencing of STIM1 significantly decreased cellular proliferation and delayed the cell cycle progression induced by hypoxia. Silencing of STIM1 also significantly decreased SOC-mediated Ca²⁺ influx and inhibited the nuclear translocation of NFATc3 in hypoxic PASMCs.

Conclusion

Our findings suggest that chronic hypobaric hypoxia upregulates the expression of STIM1 in the distal intrapulmonary arteries which plays an important role in the hypoxia-induced PASMCs proliferation via SOC/Ca²⁺/NFAT pathway and may represent a novel therapeutic target for the prevention of hypoxia pulmonary hypertension.     

Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.     

Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.