Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A₂ (PLA₂), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA₂ in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA₂ enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA₂ involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA₂α, cPLA₂γ, iPLA₂β, or iPLA₂γ. Downregulation of the β isoform of iPLA₂ blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.     

Cyclic adenosine monophosphate-dependent activation of transient receptor potential vanilloid 4 (TRPV4) channels in osteoblast-like MG-63 cells

Parathyroid hormone (PTH) directly interacts with bone remodeling osteoblasts and osteocytes expressing the G-protein coupled receptor PTH receptor 1 (PTH1R), and its osteoanabolic effects mostly involve the cAMP/PKA signaling cascade. Considering that PTH-dependent calcium entry in rat enterocytes is reproduced by the adenylate cyclase agonist forskolin or by cAMP analogues, possible involvement of calcium as a second messenger in PTH-dependent cAMP signaling was investigated in MG-63 cells. First, Ca²⁺ influx was confirmed in Fluo3-loaded MG-63 cells treated with a cell-permeable cAMP analog. Second, PTH (1–34) and forskolin promoted calcium influxes that were completely abrogated by the PKA inhibitor H-89. Ca²⁺ entry was not reproduced when PTH (1–34) was combined with the PKC-activating competitor PTH (3–34). Vanilloid transient potential (TRPV) channel inhibitor Ruthenium Red, but not a voltage-dependent calcium channel (VDCC) inhibitor nifedipine, efficiently stunted Ca²⁺ entry, and comparable abrogation was reproduced in cells treated with TRPV4-selective inhibitor RN-1734 or transfected with TRPV4-specific siRNA. Interestingly, PTH-driven Ca²⁺ through TRPV4 significantly inhibited MG63 cell migration through a mechanism requiring extracellular Ca²⁺. In contrast, the inhibitory effects of forskolin on migration were refractory to TRPV4 silencing or to RN-1734. Altogether, our results indicate that single treatment with PTH (1–34) promotes extracellular calcium entry through TRPV4 channels in MG-63 cells through a cAMP/PKA-dependent mechanism, and that this influx affects cell migration.     

Differential cytotoxicity and intracellular calcium-signalling following activation of the calcium-permeable ion channels TRPV1 and TRPA1

Several members of the transient receptor channel (TRP) family can mediate a calcium-dependent cytotoxicity. In sensory neurons, vanilloids like capsaicin induce neurotoxicity by activating TRPV1. The closely related ion channel TRPA1 is also activated by irritants, but it is unclear if and how TRPA1 mediates cell death. In the present study we explored cytotoxicity and intracellular calcium signalling resulting from activation of TRPV1 and TRPA1, either heterologously expressed in HEK 293 cells or in native mouse dorsal root ganglion (DRG) neurons. While activation of TRPV1 by the vanilloids capsaicin, resiniferatoxin and anandamide results in calcium-dependent cell death, activation by protons and the oxidant chloramine-T failed to reduce cell viability. The TRPA1-agonists acrolein, carvacrol and capsazepine all induced cytotoxicity, but this effect is independent of TRPA1. Activation of both TRPA1 and TRPV1 triggers a strong influx of external calcium, but also a strong calcium-release from intracellular stores most likely including the endoplasmic reticulum (ER). Activation of TRPV1, but not TRPA1 also results in a strong increase of mitochondrial calcium both in HEK 293 cells and mouse DRG neurons. Our data demonstrate that activation of TRPV1, but not TRPA1 mediates a calcium-dependent cell death. While both receptors mediate a release of calcium from intracellular stores, only activation of TRPV1 seems to mediate a robust and probably lethal increase in mitochondrial calcium.     

Elucidation of a TRPC6-TRPC5 channel cascade that restricts endothelial cell movement

Canonical transient receptor potential (TRPC) channels are opened by classical signal transduction events initiated by receptor activation or depletion of intracellular calcium stores. Here, we report a novel mechanism for opening TRPC channels in which TRPC6 activation initiates a cascade resulting in TRPC5 translocation. When endothelial cells (ECs) are incubated in lysophosphatidylcholine (lysoPC), rapid translocation of TRPC6 initiates calcium influx that results in externalization of TRPC5. Activation of this TRPC6-5 cascade causes a prolonged increase in intracellular calcium concentration ([Ca(2+)](i)) that inhibits EC movement. When TRPC5 is down-regulated with siRNA, the lysoPC-induced rise in [Ca(2+)](i) is shortened and the inhibition of EC migration is lessened. When TRPC6 is down-regulated or EC from TRPC6(-/-) mice are studied, lysoPC has minimal effect on [Ca(2+)](i) and EC migration. In addition, TRPC5 is not externalized in response to lysoPC, supporting the dependence of TRPC5 translocation on the opening of TRPC6 channels. Activation of this novel TRPC channel cascade by lysoPC, resulting in the inhibition of EC migration, could adversely impact on EC healing in atherosclerotic arteries where lysoPC is abundant.     

The involvement of transient receptor potential channels in mast cell activation by microbubbles

This study was to explore the activation of mast cells by microbubbles, with the focus on transient receptor potential (TRP) channels mediated degranulation and calcium influx. Bone marrow-derived mast cells (BMMCs) were primarily obtained from femurs in mice and induced differentiation for 4 weeks. After the purity identification, BMMCs were contacted by homogeneous microbubbles with the diameter of 1 mm for 1 h. β-hexosaminidase and histamine levels in supernatants were assessed by enzyme-linked immunosorbent assay (ELISA) and the CD63 expression was tested by flow cytometry. The intracellular calcium binding with Fluo-4 AM dyes in BMMCs was observed under the fluorescence microscope and the mean fluorescence intensity was quantitatively measured by flow cytometry. β-hexosaminidase release, histamine concentration, CD63 expression and calcium influx were significantly increased in BMMCs group upon microbubble stimulation compared to the control groups. After preconditioning with the available inhibitors and microbubble contact, only transient receptor potential vanilloid 1 (TRPV1) and TRPV4 inhibitors robustly suppressed the microbubble-induced degranulation. Likewise, the elevated fluorescence intensity of cytosolic calcium level was also significantly weaken. The results demonstrated microbubble stimulus effectively promoted BMMCs degranulation, which could be substantially restrained by inhibitors targeted for blocking TRPV1 or TRPV4 channel. The alternation of intracellular calcium level in BMMCs was consistent with the changes of degranulation capacity. It's suggested that the activation of BMMCs by microbubbles may involve specific TRP calcium dependent channels.     

Lysophosphatidylcholine-induced cellular injury in cultured fibroblasts involves oxidative events

Lysophosphatidylcholine (lysoPC), formed during LDL oxidation and located within atherosclerotic plaques, induces numerous cellular responses, but via unknown mechanisms. Cellular events involved in sublethal lysoPC-induced injury were examined because these are relevant to mechanisms by which lysoPC alters cell behavior. LysoPC evoked transient membrane permeabilization in fibroblasts within 10 min. Cells underwent reversible rounding within 2 h, returning 3 h later to grossly normal appearance and a normal response to growth stimulation. We asked whether this sublethal permeabilization resulted from physical perturbation of the plasma membrane or if it required cellular events. LysoPC induced leakage of fluorescent dye from unilamellar phospholipid vesicles, suggesting physical membrane perturbation was a significant contributor. To characterize this further we increased the cholesterol content of cells and vesicles to stabilize membranes, and found decreased lysoPC-induced permeabilization in both cell and cell-free systems as cholesterol levels increased. Interestingly, vitamin E, a known antioxidant, blunted lysoPC-induced permeabilization and morphological changes in cells. Thus, lysoPC appeared to cause an unexpected oxidant stress-dependent enhancement of cell injury. To confirm this, several structurally distinct antioxidants, including N, N'-diphenyl-1,4-phenylenediamine, Desferal, Tiron, and 4-hydroxy TEMPO, were applied and these also were inhibitory. Oxidant stress was observed by a lysoPC-induced increase in fluorescence of 5- and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, an intracellular marker of reactive oxygen species. Lysophosphatidylethanolamine (lysoPE) caused qualitatively similar morphological changes to cells and induced permeabilization, but injury by lysoPE was not inhibited by antioxidants. These data suggest that generation of intracellular reactive oxygen species follows lysoPC-induced plasma membrane destabilization and that this lysoPC-specific oxidant stress enhances cell injury. This intracellular oxidant stress in response to lysoPC may be an integral part of the multiple influences lysoPC has on gene expression and cell function.     

PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane

Cellular or chemical activators for most transient receptor potential channels of the vanilloid subfamily (TRPV) have been identified in recent years. A remarkable exception to this is TRPV2, for which cellular events leading to channel activation are still a matter of debate. Diverse stimuli such as extreme heat or phosphatidylinositol-3 kinase (PI3-kinase) regulated membrane insertion have been shown to promote TRPV2 channel activity. However, some of these results have proved difficult to reproduce and may underlie different gating mechanisms depending on the cell type in which TRPV2 channels are expressed. Here, we show that expression of recombinant TRPV2 can induce cytotoxicity that is directly related to channel activity since it can be prevented by introducing a charge substitution in the pore-forming domain of the channel, or by reducing extracellular calcium. In stably transfected cells, TRPV2 expression results in an outwardly rectifying current that can be recorded at all potentials, and in an increase of resting intracellular calcium concentration that can be partly prevented by serum starvation. Using cytotoxicity as a read-out of channel activity and direct measurements of cell surface expression of TRPV2, we show that inhibition of the PI3-kinase decreases TRPV2 channel activity but does not affect the trafficking of the channel to the plasma membrane. It is concluded that PI3-kinase induces or modulates the activity of recombinant TRPV2 channels; in contrast to the previously proposed mechanism, activation of TRPV2 channels by PI3-kinase is not due to channel translocation to the plasma membrane.     

TRPV1 stimulation triggers apoptotic cell death of rat cortical neurons

Transient receptor potential vanilloid 1 (TRPV1) functions as a polymodal nociceptor and is activated by several vanilloids, including capsaicin, protons and heat. Although TRPV1 channels are widely distributed in the brain, their roles remain unclear. Here, we investigated the roles of TRPV1 in cytotoxic processes using TRPV1-expressing cultured rat cortical neurons. Capsaicin induced severe neuronal death with apoptotic features, which was completely inhibited by the TRPV1 antagonist capsazepine and was dependent on extracellular Ca²⁺ influx. Interestingly, nifedipine, a specific L-type Ca²⁺ channel blocker, attenuated capsaicin cytotoxicity, even when applied 2-4 h after the capsaicin. ERK inhibitor PD98059 and several antioxidants, but not the JNK and p38 inhibitors, attenuated capsaicin cytotoxicity. Together, these data indicate that TRPV1 activation triggers apoptotic cell death of rat cortical cultures via L-type Ca²⁺ channel opening, Ca²⁺ influx, ERK phosphorylation, and reactive oxygen species production.     

Mechanosensitivity of human osteosarcoma cells and phospholipase C beta2 expression

Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 microstrains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC beta1, beta3, gamma1, gamma2, and delta1 in all cells, but PLC beta2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC beta2 in mechanotransduction. Inducible antisense expression for 24h inhibited the translation of PLC beta1 in U-2/OS cells by 35% and PLC beta2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC beta2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.     

Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels

The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca(2+)-ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.     

Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies

Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.     

The recombinant human TRPV6 channel functions as Ca2+ sensor in human embryonic kidney and rat basophilic leukemia cells

The activation mechanism of the recently cloned human transient receptor potential vanilloid type 6 (TRPV6) channel, originally termed Ca(2+) transporter-like protein and Ca(2+) transporter type 1, was investigated in whole-cell patch-clamp experiments using transiently transfected human embryonic kidney and rat basophilic leukemia cells. The TRPV6-mediated currents are highly Ca(2+)-selective, show a strong inward rectification, and reverse at positive potentials, which is similar to store-operated Ca(2+) entry in electrically nonexcitable cells. The gating of TRPV6 channels is strongly dependent on the cytosolic free Ca(2+) concentration; lowering the intracellular free Ca(2+) concentration results in Ca(2+) influx, and current amplitude correlates with the intracellular EGTA or BAPTA concentration. This is also the case for TRPV6-mediated currents in the absence of extracellular divalent cations; compared with endogenous currents in nontransfected rat basophilic leukemia cells, these TRPV6-mediated monovalent currents reveal differences in reversal potential, inward rectification, and slope at very negative potentials. Release of stored Ca(2+) by inositol 1,4,5-trisphosphate and/or the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin appears not to be involved in TRPV6 channel gating in both cell lines but, in rat basophilic leukemia cells, readily activates the endogenous Ca(2+) release-activated Ca(2+) current. In conclusion, TRPV6, expressed in human embryonic kidney cells and in rat basophilic leukemia cells, functions as a Ca(2+)-sensing Ca(2+) channel independently of procedures known to deplete Ca(2+) stores.     

Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.     

Moderate extracellular acidification inhibits capsaicin-induced cell death through regulating calcium mobilization, NF-κB translocation and ROS production in synoviocytes

We previously show the expression of transient receptor potential vanilloid 1 (TRPV1) in primary synoviocytes from collagen-induced arthritis (CIA) rats. Capsaicin and lowered extracellular pH from 7.4 to 5.5 induce cell death through TRPV1-mediated Ca(2+) entry and reactive oxygen species (ROS) production. However, under the pathological condition in rheumatoid arthritis, the synovial fluid is acidified to a moderate level (about pH 6.8). In the present study, we examined the effects of pH 6.8 on the TRPV1-mediated cell death. Our finding is different or even opposite from what was observed at pH 5.5. We found that the moderate extracellular acidification (from pH 7.4 to 6.8) inhibited the capsaicin-induced Ca(2+) entry through attenuating the activity of TRPV1. In the mean time, it triggered a phospholipse C (PLC)-related Ca(2+) release from intracellular stores. The nuclear translocation of NF-κB was found at pH 6.8, and this also depends on PLC activation. Moreover, the capsaicin-evoked massive ROS production and cell death were depressed at pH 6.8, both of which are dependent on the activation of PLC and NF-κB. Taken together, these results suggested that the moderate extracellular acidification inhibited the capsaicin-induced synoviocyte death through regulating Ca(2+) mobilization, activating NF-κB nuclear translocation and depressing ROS production.     

Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells

In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5-hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca(2+) fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 microM) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni(2+) and to ruthenium red, a TRPV channel blocker, and mimicked by 4alpha-phorbol-12,13-didecanoate (4alpha-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4alpha-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension.     

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: Voltage-gated calcium channels,and/or phospholipase C coupled to a pertussis-sensitive G-protein

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca²⁺]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca²⁺]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 μg/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca²⁺]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca²⁺ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced[Ca²⁺]i but did not block the effect of IGF-2.2)IGF-1, IGF-2, and insulin also induced a Ca²⁺ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhihitors, neomycin, or U-73122 partially blocked the intracellular [Ca²⁺]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca²⁺ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF₁ receptor antibodies diminished the IGF-1-induced Ca²⁺ spike and totally abolished the Ca²⁺ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca²⁺ influx involves the IGF₁ receptor, while part of IGF-1 and all of IGF-2 Ca²⁺ mobilization do not implicate this receptor. J. Cell. Biochem. 64:414–422. © 1997 Wiley-Liss, Inc.     

Two types of store-operated Ca2+ channels with different activation modes and molecular origin in LNCaP human prostate cancer epithelial cells

The one or more coupling mechanisms of store-operated channels (SOCs) to endoplasmic reticulum (ER) Ca2+ store depletion as well as the molecular identity of SOCs per se still remain a mystery. Here, we demonstrate the co-existence of two populations of molecular distinct endogenous SOCs in LNCaP prostate cancer epithelial cells, which are preferentially activated by either active inositol 1,4,5-trisphosphate (IP3)-mediated or passive thapsigargin-facilitated store depletion and have different ER store content sensitivity. The first population, called SOC(CC) (for "conformational coupling"), is characterized by preferential IP3 receptor-dependent mode of activation, as judged from sensitivity to cytoskeleton modifications, and dominant contribution of transient receptor potential (TRP) TRPC1 within it. The second one, called SOC(CIF) (for "calcium influx factor"), depends on Ca(2+)-independent phospholipase A2 for activation with probable CIF involvement and is mostly represented by TRPC4. The previously identified SOC constituent in LNCaP cells, TRPV6, seems to play equal role in both SOC populations. These results provide new insight into the nature of SOCs and their representation in the single cell type as well as permit reconciliation of current SOC activation hypotheses.     

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage‐gated Ca²⁺ channels restores cytosolic Ca²⁺ levels and reduces this neuronal death ( Snider et al. 2002 ). We now show that this reduction in [Ca²⁺]_i is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca²⁺ influx such as activation of voltage‐gated Ca²⁺ channels or stimulation of Ca²⁺ entry via the plasma membrane Na–Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage‐gated Ca²⁺ channels and a less robust increase in [Ca²⁺]_i after depolarization. Levels of endoplasmic reticulum Ca²⁺ were reduced and capacitative Ca²⁺ entry was impaired early in the cell death process. Mitochondrial Ca²⁺ was slightly increased. Preventing the transfer of Ca²⁺ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca²⁺ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca^2+, possibly mediated by dysfunction of voltage‐gated Ca²⁺ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.     

Capacitative calcium entry in the nervous system

Capacitative calcium entry is a process whereby the depletion of Ca(2+) from intracellular stores (likely endoplasmic or sarcoplasmic reticulum) activates plasma membrane Ca(2+) channels. Current research has focused on identification of capacitative calcium entry channels and the mechanism by which Ca(2+) store depletion activates the channels. Leading candidates for the channels are members of the transient receptor potential (TRP) superfamily, although no single gene or gene product has been definitively proven to mediate capacitative calcium entry. The mechanism for activation of the channels is not known; proposals fall into two general categories, either a diffusible signal released from the Ca(2+) stores when their Ca(2+) levels become depleted, or a more direct protein-protein interaction between constituents of the endoplasmic reticulum and the plasma membrane channels. Capacitative calcium entry is a major mechanism for regulated Ca(2+) influx in non-excitable cells, but recent research has indicated that this pathway plays an important role in the function of neuronal cells, and may be important in a number of neuropathological conditions. This review will summarize some of these more recent findings regarding the role of capacitative calcium entry in normal and pathological processes in the nervous system.