Capacitative Ca Entry Research Articles

Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca 2+ entry in familial Alzheimer's disease

Mutations in presenilin 1 (PS1), which are the major cause of familial Alzheimer's disease (FAD), are involved in perturbations of cellular Ca 2+ homeostasis. Attenuation of capacitative Ca 2+ entry (CCE) is the most often observed alteration of Ca 2+ homeostasis in cells bearing FAD PS1 mutations. However, molecular mechanisms underlying this CCE impairment remains elusive. We demonstrate that cellular levels of STIM1 and STIM2 proteins, which are key players in CCE, depend on presenilins. We found increased level of STIM1 and decreased level of STIM2 proteins in mouse embryonic fibroblasts lacking presenilins. Fura-2 ratiometric assays revealed that CCE is enhanced in these cells after Ca 2+ stores depletion by thapsigargin treatment. In turn, overexpression of PS1 with FAD mutations in HEK293 cells led to an attenuation of CCE. Although, no changes in STIM protein levels were observed in these HEK293 cells, FAD mutations in endogenous PS1 in human B lymphocytes resulted in a decreased expression of STIM2 in parallel to an attenuation of CCE. Our experiments showing that knock-out of presenilins in MEF cells and FAD mutations in endogenous PS1 in lymphocytes affect both CCE and the cellular level of STIM proteins open new perspectives for studies on CCE in FAD.

Caffeine inhibits InsP 3 responses and capacitative calcium entry in canine pulmonary arterial smooth muscle cells

Caffeine is a well described and characterized ryanodine receptor (RyR) activator. Previous evidence from independent research studies also indicate caffeine inhibits InsP 3 receptor functionality, which is important to activation of capacitative Ca 2+ entry (CCE) in some cell types. In addition, RyR activation elicits excitatory-coupled Ca 2+ entry (ECCE) in skeletal muscle myotubes. Recent studies by our group show that canine pulmonary arterial smooth muscle cells (PASMCs) have functional InsP 3 receptors as well as RyRs, and that CCE is dependent on InsP 3 receptor activity. The potential for caffeine to activate ECCE as well as inhibit InsP 3 receptor function and CCE was examined using fura-2 fluorescent imaging in canine PASMCs. The data show caffeine causes transient as well as sustained cytosolic Ca 2+ increases, though this is not due to CCE or ECCE activity as evidenced by a lack of an increase in Mn 2+ quench of fura-2. The experiments also show caffeine reversibly inhibits 5-HT elicited—InsP 3 mediated Ca 2+ responses with an IC 50 of 6.87 × 10 − 4 M and 10 mM caffeine fully inhibits CCE. These studies provide the first evidence that caffeine is an inhibitor of InsP 3 generated Ca 2+ signals and CCE in PASMCs.

Stimulation of histamine H 2 receptors activates TRPC3 channels through both phospholipase C and phospholipase D

Histamine plays an important role in many physiological functions; and a change in cytosolic Ca 2+ ([Ca 2+] i) may be an early signal in these processes. In the present study, we investigated the activation mechanism of TRPC3, the Canonical Transient Receptors Potential 3 Channels, by histamine via a non-capacitative Ca 2+ entry pathway. TRPC3 was transfected into HEK293 cells and the cells were treated with thapsigargin to deplete the intracellular Ca 2+ stores; re-addition of Ca 2+ initiated a capacitative Ca 2+ entry (CCE). A subsequent application of histamine evoked another Ca 2+ influx on top of the CCE signal only in the TRPC3-transfected HEK293 cells, indicating that histamine can activate TRPC3 via a non-capacitative Ca 2+ entry pathway (non-CCE). This histamine-induced non-CCE was abolished by cimitidine, a histamine H 2 receptors antagonist, but not by histamine H 1 receptor antagonists pyrilamine and diphenhydramine. KT5720, a protein kinase A (PKA) inhibitor, had no effect on the histamine-induced non-CCE. This histamine-induced non-CCE was partially reduced by U73122, a phospholipase C (PLC) inhibitor, and by butan-1-ol, a phospholipase D (PLD) inhibitor. When both PLC and PLD inhibitors were simultaneously applied, the non-CCE signal was completely abolished. Taken together, our results showed, for the first time, that histamine could activate TRPC3 via histamine H 2 receptors, and both PLC and PLD participated in this process.

Effect of nifedipine on capacitive calcium entry in Jurkat T lymphocytes

The effect of nifedipine—an antagonist of L-type calcium (Ca 2+) channels—on capacitative Ca 2+ entry (CCE) was studied in Jurkat T lymphocytes. CCE was induced by a variety of treatments each of which depleted intracellular Ca 2+ stores. Cells were treated with thapsigargin, ionomycin, anti-CD3 antibodies, and phytohaemagglutinin, or pre-incubated in a Ca 2+-free medium. Activity of CCE was evaluated with a Ca 2+-free/Ca 2+-readmission protocol, in Fluo-3 pre-loaded cells. Nifedipine inhibited CCE in a dose-dependent manner. CCE inhibition was not due to non-specific effects on K + channels. Nifedipine, did not induce any membrane depolarization, as revealed by measurements of the plasma membrane potential with the fluorescent probe bis-oxonol. Moreover, experiments done under depolarizing conditions (i.e. by substituting Na + with K + ions in the medium) revealed that nifedipine could inhibit capacitative Ca 2+ entry independently of plasma membrane depolarization. We also demonstrated the presence in our Jurkat T-cells of transcripts for Ca V1.3 (α 1D) and Ca V1.4 (α 1F) L-type Ca 2+ channels. Verapamil and diltiazem, two unrelated blockers of L-type Ca 2+ channels, were less inhibitory on CCE. Possible mechanisms by which nifedipine interferes with Ca 2+ entry in these cells are discussed.

Maturation of intracellular calcium homeostasis in sheep pulmonary arterial smooth muscle cells

Cytosolic Ca(2+) signaling dynamics are important to pulmonary arterial reactivity, and alterations are implicated in pulmonary vascular disorders. Yet, adaptations in cellular Ca(2+) homeostasis and receptor-mediated Ca(2+) signaling with maturation from fetal to adult life in pulmonary arterial smooth muscle cells (PASMCs) are not known. The present study tested the hypothesis that cytosolic Ca(2+) homeostasis and receptor-generated Ca(2+) signaling adapt with maturation in sheep PASMCs. Digitalized fluorescence microscopy was performed using isolated PASMCs from fetal and adult sheep that were loaded with the Ca(2+) indicator fura 2. The results show that basal cytosolic and sarcoplasmic reticulum Ca(2+) levels are attained before birth. Similarly, Ca(2+) efflux pathways from the cytosol and basal as well as capacitative Ca(2+) entry (CCE) are also developed before birth. However, receptor-mediated Ca(2+) signaling adapts with maturation. Prominently, serotonin stimulation elicited Ca(2+) elevations in very few fetal compared with adult PASMCs; in contrast, phenylephrine elevated Ca(2+) in a similar percentage of fetal and adult PASMCs. Serotonin and phenylephrine elicited Ca(2+) increases of a similar magnitude in reactive cells of fetus and adult, supporting the assertion that inositol trisphosphate signaling is intact. Caffeine and ATP elevated Ca(2+) in equivalent numbers of fetal and adult PASMCs. However, the caffeine-induced cytosolic Ca(2+) increase was significantly greater in fetal PASMCs, whereas the ATP-elicited increase was greater in adult cells. Overall, the results of this study demonstrate selective adaptations in receptor-mediated Ca(2+) signaling, but not in cellular Ca(2+) homeostasis.

P75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction

A member of the TNF receptor family, the p75 neurotrophin receptor (p75(NTR)) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75(NTR) in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75(NTR) in mouse pulmonary arteries and the putative role of p75(NTR) in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75(NTR) knockout (p75(-/-)) mice. Our data indicated that p75(NTR) is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75(-/-) mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca(2+) influx. Furthermore, the contraction due to capacitative Ca(2+) entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca(2+) stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75(-/-) rings. Active tension induced by serotonin, U-46619 (a thromboxane A(2) analog), thrombin, 4-aminopyridine (a K(+) channel blocker), and high extracellular K(+) in p75(-/-) rings was similar to that in WT rings. Deletion of p75(NTR) did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75(NTR) signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion.

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of 109Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd 2+ species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels.

TRPC1 and STIM1 Mediate Capacitative Calcium Entry in Mouse Pulmonary Arterial Smooth Muscle Cells

Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that TRPC1 channel mediates capacitative Ca2+ entry (CCE) but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediate CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured PASMCs loaded with fura‐2, 10μM CPA caused a rise in [Ca2+]i but it was abolished when extracellular Ca2+ was removed. Subsequent addition of 2mM Ca2+ elicited a transient rise in [Ca2+]i that was partially inhibited by 10μM nifedipine, leaving a nifedipine‐insensitive transient and nifedipine‐insensitive sustained rise in [Ca2+]i. The nifedipine‐insensitive sustained, but not transient rise in [Ca2+]i was inhibited in cells pretreated with antibodies raised against extracellular epitope of TRPC1 and STIM1. RT‐PCR revealed TRPC1 and STIM1 mRNAs, whereas Western blot analysis identified TRPC1 and STIM1 proteins in cultured mouse PASMCs. Taken together, store‐depletion causes activation of voltage‐operated Ca2+ entry, CCE and another as yet unidentified voltage‐independent Ca2+ entry pathway. These data suggest that CCE is mediated by TRPC1 channel possibly through activation of STIM1 protein in mouse PASMCs. (Supported by HL49254 and NCRR P20RR15581)

Clearance of store-released Ca2+by the Na+-Ca2+exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase α2-isoform gene-ablated mice

Two alpha-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The alpha 1-isoform is proposed to serve a cytosolic housekeeping role, whereas the alpha 2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase alpha 2-isoform gene-ablated, homozygous null knockout (alpha 2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In alpha 2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the alpha 2-isoform (0.5 microM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not alpha 2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in alpha 2-KO cells. Our findings support the concept of a subsarcolemmal space where the alpha 2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.

Sildenafil Inhibits Human Pulmonary Artery Smooth Muscle Cell Proliferation by Decreasing Capacitative Ca2+ Entry

Ca2+ is a pivotal signal in human pulmonary artery smooth muscle cells (PASMCs) proliferation. Capacitative Ca2+ entry (CCE) via the store-operated channel (SOC), which encoded by the transient receptor potential (TRP) gene, is an important mechanism for regulating intracellular Ca2+ concentration ([Ca2+]i) in PASMCs. Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been proposed as a therapeutic tool to treat or prevent pulmonary arterial hypertension (PAH); however, the mechanism of its antiproliferative effect on PASMCs remains unclear. This study was designed to investigate the possible antiproliferative mechanism of sildenafil on human PASMCs, namely, its effect on the Ca2+-signal pathway. Cultured normal PASMCs were treated with endothelin-1 (ET-1) or ET-1 plus sildenafil separately. Cell number and viability were determined with a hemocytometer or MTT assay. [Ca2+]i was measured by loading PASMCs with fura 2-AM. Expression of the TRPC1 gene and protein was detected by RT-PCR and Western blot, respectively. The results show that sildenafil dose-dependently inhibited the proliferation of PASMCs, the enhancement of basal [Ca2+]i level, increase of CCE, and upregulation of TRPC expression induced by ET-1. These results suggest that sildenafil potently inhibits ET-1–induced PASMCs proliferation and downregulation of CCE and TRPC expression may be responsible for its antiproliferative effect.

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

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. The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast-like MG-63 cells. Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. 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. Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca2+ influx involved in osteoblast proliferation.

Kinetic properties of Ca 2+/calmodulin-dependent phosphodiesterase isoforms dictate intracellular cAMP dynamics in response to elevation of cytosolic Ca 2+

Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca 2+-sensitive ACs have received far greater attention than the Ca 2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca 2+ concentration ([Ca 2+] i ). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater K m for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca 2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E 1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca 2+] i and thereby provide a rationale for the utility of diverse PDE1 species.

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle

Following smooth muscle excitation and contraction, depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE) to replenish stores and sustain cytoplasmic Ca(2+) (Ca(2+)(i)) elevations. The objectives of the present study were to characterize CCE and the Ca(2+)(i) dynamics underlying human colonic smooth muscle contraction by using tension recordings, fluorescent Ca(2+)-indicator dyes, and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca(2+)(i) as well as activation of nonselective cation currents. To deplete Ca(2+)(i) stores, the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca(2+)-free bathing solution. Under these conditions, addition of extracellular Ca(2+) (3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 microM) and lanthanum (100 microM), suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca(2+)-imaging techniques, the CCE-evoked rise of Ca(2+)(i) was associated with inward currents carrying Ca(2+) that were inhibited by SKF-96365. Regional specializations in Ca(2+) influx and handling during CCE were observed. Distinct "hotspot" regions of Ca(2+) rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca(2+) entry occurs in hotspots contributing to localized Ca(2+) elevations in human colonic smooth muscle.

The effect of oxidative stress on Ca 2+ release and capacitative Ca 2+ entry in vascular endothelial cells

Oxidative stress imposed by the accumulation of oxygen free radicals (reactive oxygen species, ROS) has profound effects on Ca 2+ homeostasis in the vascular endothelium, leading to endothelial dysfunctions and the development of cardiovascular pathologies. We tested the effect of the oxidant and ROS generator tert-butyl-hydroperoxide ( tBuOOH) on Ca 2+ signaling in single cultured calf pulmonary artery endothelial (CPAE) cells loaded with the fluorescent Ca 2+ indicator indo-1. Acute brief (5 min) exposures to tBuOOH had no effect on basal cytosolic free Ca 2+ ([Ca 2+] i), agonist (ATP)-induced Ca 2+ release from the endoplasmic reticulum (ER) and on Ca 2+ store depletion-dependent capacitative Ca 2+ entry (CCE). Prolonged (60 min) exposure to tBuOOH did not affect intracellular Ca 2+ release, but caused a profound inhibition of CCE. After 120 min of treatment with tBuOOH not only was CCE further reduced, but also ATP-induced Ca 2+ release due to a slow depletion of the stores that resulted from CCE inhibition. The antioxidant Trolox (synthetic vitamin E analog) prevented the inhibition of CCE by tBuOOH and attenuated the increase of [ROS] i, indicating that inhibition of CCE was due to the oxidant effects of tBuOOH. The data suggest that in vascular endothelial cells oxidative stress primarily affects Ca 2+ influx in response to Ca 2+ loss from internal stores. [Ca 2+] i is an important signal for the production and release of endothelium-derived factors such as nitric oxide (NO). Since CCE is the preferential Ca 2+ source for NO synthase activation, the finding that oxidative stress inhibits CCE may explain how oxidative stress contributes to endothelial dysfunction-related cardiovascular pathologies.

Increased smooth muscle cell expression of caveolin‐1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension

Vasoconstriction and vascular medial hypertrophy, resulting from increased intracellular [Ca2+] in pulmonary artery smooth muscle cells (PASMC), contribute to elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Caveolae, microdomains within the plasma membrane, contain the protein caveolin, which binds certain signaling molecules. We tested the hypothesis that PASMC from IPAH patients express more caveolin-1 (Cav-1) and caveolae, which contribute to increased capacitative Ca2+ entry (CCE) and DNA synthesis. Immunohistochemistry showed increased expression of Cav-1 in smooth muscle cells but not endothelial cells of pulmonary arteries from patients with IPAH. Subcellular fractionation and electron microscopy confirmed the increase in Cav-1 and caveolae expression in IPAH-PASMC. Treatment of IPAH-PASMC with agents that deplete membrane cholesterol (methyl-beta-cyclodextrin or lovastatin) disrupted caveolae, attenuated CCE, and inhibited DNA synthesis of IPAH-PASMC. Increasing Cav-1 expression of normal PASMC with a Cav-1-encoding adenovirus increased caveolae formation, CCE, and DNA synthesis; treatment of IPAH-PASMC with siRNA targeted to Cav-1 produced the opposite effects. Treatments that down-regulate caveolin/caveolae expression, including cholesterol-lowering drugs, reversed the increased CCE and DNA synthesis in IPAH-PASMC. Increased caveolin and caveolae expression thus contribute to IPAH-PASMC pathophysiology. The close relationship between caveolin/caveolae expression and altered cell physiology in IPAH contrast with previous results obtained in various animal models, including caveolin-knockout mice, thus emphasizing unique features of the human disease. The results imply that disruption of caveolae in PASMC may provide a novel therapeutic approach to attenuate disease manifestations of IPAH.

Cyclic stretch decreases capacitative calcium entry in vascular smooth muscle cells from resistance and conduit vessels

An alteration in expression of TRPC channels may affect intracellular Ca2+ homeostasis and cell contractility. We previously showed that cyclic stretch, a model which simulates the elevated vessel strain in hypertension, decreased TRPC4 protein expression in cultured rat aortic smooth muscle cells (RASMC) and rat mesenteric smooth muscle cells (RMSMC). Since TRPC4 proteins have been shown to function as a store-operated channels, we hypothesized that cyclic stretch will also result in a decrease in capacitative Ca2+ entry (CCE). Cells were stretched for 6h using the Flexercell Strain Unit, loaded with fura-2-AM, and fluorescence ratio (340/380nm) was measured. Control cells were not stretched. Baseline Ca2+ levels in the absence of extracellular Ca2+ (0Ca2+) were higher in stretched RASMC (n = 88) than controls (n = 87, P = 0.009). The response to store depletion by cyclopiazonic acid (CPA) in 0Ca2+was decreased in stretched RASMC (P = 0.001). The peak fura-2 ratio and the rate of CCE after reintroduction of extracellular Ca2+ were both decreased by cyclic stretch (P = 0.002 and P < 0.0001). Likewise, stretched RMSMC showed a diminished CPA response (n = 66, P < 0.0001) and a decrease in CCE (P = 0.018) when compared to controls (n = 77). The stretch-induced decrease in TRPC4 expression and CCE may counteract the influx of Ca2+ resulting from increased strain in the vessel wall and prevent an exacerbated myogenic response.

Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+entry

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca(2+) concentration, resulting from enhanced capacitative Ca(2+) entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 muM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 muM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.

Calcium controls smooth muscle TRPC gene transcription via the CaMK/calcineurin-dependent pathways

Transient receptor potential protein family C (TRPC) has been proposed as a candidate for channels involved in capacitative Ca(2+) entry (CCE) mechanisms, but the modulation of their gene expression remains unexplored. In this study we show that guinea pig gallbladder smooth muscle contains mRNA encoding TRPC1, TRPC2, TRPC3, and TRPC4 proteins whose abundance depends on cytosolic Ca(2+) level ([Ca(2+)](i)). Thus lowering the levels of cellular calcium with the chelators EGTA and BAPTA AM results in a downregulation of TRPC1-TRPC4 gene and protein expression. In contrast, activation of Ca(2+) influx through L-type Ca(2+) channels and Ca(2+) release from intracellular stores induced an increase in TRPC1-TRPC4 mRNA and protein abundance. Activation of Ca(2+)/calmodulin-dependent kinases (CaMK) and phosphorylation of cAMP-response element binding protein accounts for the increase in TRPC mRNA transcription in response to L-type channel-mediated Ca(2+) influx . In addition to this mechanism, activation of TRPC gene expression by intracellular Ca(2+) release also involves calcineurin pathway. According to the proposed role for these channels, activation of CCE induced an increase in TRPC1 and TRPC3 mRNA abundance, which depends on the integrity of the calcineurin and CaMK pathways. These findings show for the first time an essential autoregulatory role of Ca(2+) in Ca(2+) homeostasis at the level of TRPC gene and protein 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.

Pregnancy-enhanced Ca2+ responses to ATP in uterine artery endothelial cells is due to greater capacitative Ca2+ entry rather than altered receptor coupling

Uterine artery endothelial cells (UAEC) derived from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes retain pregnancy-specific differences in cell signaling as well as vasodilator production through passage 4. In particular, when P- and NP-UAEC are stimulated with ATP over a 2.5 min recording period, they exhibit similar initial transient peaks in the intracellular free Ca(2+) concentration ([Ca(2+)](i)), but the P-UAEC show a heightened sustained phase. In order to establish whether this was due to an altered subclass of purinergic receptor (P2), both the dose dependency of [Ca(2+)](i) responses to ADP and UTP and the profile of purinergic receptor expression are determined in NP- and P-UAEC. Our findings indicate that while several isoforms of P2X and P2Y receptors are present, it is P2Y2 that is responsible for the ATP-induced initial transient peak in both cell types. We also characterized several key components of the ATP-induced Ca(2+) signaling cascade, including the inositol 1,4,5-trisphosphate receptor and G-proteins, but could not confirm any pregnancy-specific variation in the protein expression that correlated with pregnancy-specific differences in prolonged Ca(2+) signaling. We thus investigated whether such a difference may be inherent to the cell itself rather than specific to the purinergic receptor-signaling pathway. Using thapsigargin (Tg), we were able to demonstrate that the initial Tg-sensitive intracellular pool of Ca(2+)is nearly identical with the capacity in both cell types, but the P-UAEC is nonetheless capable of greater capacitative Ca(2+) entry (CCE) than NP-UAEC. Furthermore, CCE induced by Tg could be dramatically inhibited by 2-aminoethoxydiphenyl borate, suggesting a role for store-operated channels in the ATP-induced [Ca(2+)](i) response. We conclude that changes at the level of capacitative entry mechanisms rather than switching of receptor subtype or coupling to phospholipase C underlies pregnancy adaptation of UAEC at the level of Ca(2+)signaling.