The glycosyl phosphatidylinositol anchor of human T-cadherin binds lipoproteins

T-cadherin (T-cad) is a Ca(2+)-dependent cell adhesion glycoprotein bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. T-cad expressed on vascular smooth muscle cells (SMC) binds lipoproteins on blot. To analyze the molecular basis for the interaction of T-cad with lipoproteins we expressed recombinant human T-cad in HEK293 cells. Whereas membrane-bound T-cad from SMC and T-cad transfected HEK293 cells bind lipoproteins, T-cadherin proteins cleaved from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) do not. The lipoprotein-binding function is also lacking both for a recombinant human T-cad expressed in HEK293 cells without the GPI signal sequence, and for a human T-cad form expressed in Escherichia coli that contains the signal sequence for GPI attachment but is not modified with a GPI. We conclude that the GPI moiety of T-cadherin is necessary and sufficient to mediate lipoprotein binding.     

TRPM4b channel suppresses store-operated Ca2+ entry by a novel protein-protein interaction with the TRPC3 channel

We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein-protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)-TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca²⁺ entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca²⁺ entry through the TRPC3 channel.     

The Role of TRP Channels in Oxidative Stress-induced Cell Death

The transient receptor potential (TRP) protein superfamily is a diverse group of voltage-independent calcium-permeable cation channels expressed in mammalian cells. These channels have been divided into six subfamilies, and two of them, TRPC and TRPM, have members that are widely expressed and activated by oxidative stress. TRPC3 and TRPC4 are activated by oxidants, which induce Na⁺ and Ca²⁺ entry into cells through mechanisms that are dependent on phospholipase C. TRPM2 is activated by oxidative stress or TNFα, and the mechanism involves production of ADP-ribose, which binds to an ADP-ribose binding cleft in the TRPM2 C-terminus. Treatment of HEK 293T cells expressing TRPM2 with H₂O₂ resulted in Ca²⁺ influx and increased susceptibility to cell death, whereas coexpression of the dominant negative isoform TRPM2-S suppressed H₂O₂-induced Ca²⁺ influx, the increase in [Ca²⁺]_i, and onset of apoptosis. U937-ecoR monocytic cells expressing increased levels of TRPM2 also exhibited significantly increased [Ca²⁺]_i and increased apoptosis after treatment with H₂O₂ or TNFα. A dramatic increase in caspase 8, 9, 3, 7, and PARP cleavage was observed in TRPM2-expressing cells, demonstrating a downstream mechanism through which cell death is mediated. Inhibition of endogenous TRPM2 function through three approaches, depletion of TRPM2 by RNA interference, blockade of the increase in [Ca²⁺]_i through TRPM2 by calcium chelation, or expression of the dominant negative splice variant TRPM2-S protected cell viability. H₂O₂ and amyloid β-peptide also induced cell death in primary cultures of rat striatal cells, which endogenously express TRPM2. TRPM7 is activated by reactive oxygen species/nitrogen species, resulting in cation conductance and anoxic neuronal cell death, which is rescued by suppression of TRPM7 expression. TRPM2 and TRPM7 channels are physiologically important in oxidative stress-induced cell death.     

Regulation of contractile signaling and matrix remodeling by T-cadherin in vascular smooth muscle cells: Constitutive and insulin-dependent effects

Expression of GPI-anchored T-cadherin (T-cad) on vascular smooth muscle cells (VSMC) is elevated in vascular disorders such as atherosclerosis and restenosis which are associated with insulin resistance. Functions for T-cad and signal transduction pathway utilization by T-cad in VSMC are unknown. The present study examines the consequences of altered T-cad expression on VSMC for constitutive and insulin-induced Akt/mTOR axis signaling and contractile competence. Using viral vectors rat (WKY and SHR) and human aortic VSMCs were variously transduced with respect to T-cad-overexpression (Tcad+-VSMC) or T-cad-deficiency (shT-VSMC) and compared with their respective control transductants (E-VSMC or shC-VSMC). Tcad+-VSMC exhibited elevated constitutive levels of phosphorylated Akt^ser473, GSK3β^ser9, S6RP^ser235/236 and IRS-1^ser636/639. Total IRS-1 levels were reduced. Contractile machinery was constitutively altered in a manner indicative of reduced intrinsic contractile competence, namely decreased phosphorylation of MYPT1^{thr696 or thr853} and MLC₂₀ ^thr18/ser19, reduced RhoA activity and increased iNOS expression. Tcad+-VSMC-populated collagen lattices exhibited greater compaction which was due to increased collagen fibril packing/reorganization. T-cad+-VSMC exhibited a state of insulin insensitivity as evidenced by attenuation of the ability of insulin to stimulate Akt/mTOR axis signaling, phosphorylation of MLC₂₀ and MYPT1, compaction of free-floating lattices and collagen fibril reorganization in unreleased lattices. The effects of T-cad-deficiency on constitutive characteristics and insulin responsiveness of VSMC were opposite to those of T-cad-overexpression. The study reveals novel cadherin-based modalities to modulate VSMC sensitivity to insulin through Akt/mTOR axis signaling as well as vascular function and tissue architecture through the effects on contractile competence and organization of extracellular matrix.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

Rational method in the repetitive calcium oscillation measurement in wild type human epithelial kidney cells

Cells stimulated with physiological stimuli usually exhibit oscillations in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), a signal playing central roles in regulation of various cellular processes. For explicating their unknown mechanisms, studies are commonly conducted in single cells from several cell lines, in particular the human epithelial kidney (HEK293) cell line. However, [Ca²⁺]_i oscillating responses to agonists in vitro are found difficult to be induced and varied with different types of cells and agonists. This study shows that treatment of the wild type HEK293 cells with low concentrations of carbachol (1–10 μM), an agonist of the muscarinic receptor, resulted in non-oscillated but sustained [Ca²⁺]_i increase by loading the cells with 1 μM fura2/AM. However, repetitive and long lasting [Ca²⁺]_i oscillations could be induced in 31.1% of the tested cells loaded with 0.1 μM fura2/AM. Additionally, the occurrence of the typical Ca²⁺ spikes further increased to 47.2% and 60.7% when the Ca²⁺ concentration in the bathing medium was decreased from 1.8 mM to 1.5 mM and the medium temperature was set to 35 ± 1°C from 22 ± 2°C. Therefore, this study provides a useful approach for measuring [Ca²⁺]_i oscillatory response to relevant physiological stimulation in a wild type cell line through the adjustments of the concentrations adopted for the Ca²⁺ indicator and extracellular medium Ca²⁺ and of the temperature set for the experiment.     

Antiadhesive molecule T-cadherin is an atypical low-density lipoprotein receptor in vascular cells

Elevated serum LDL level, which results in cholesterol accumulation in vascular wall, is widely accepted as a risk factor in atherosclerosis development. Additionally to metabolic effects, LDL can produce hormone-like effects in a number of cells: activate second messenger systems, regulate gene expression and activate platelets and stimulate cell proliferation. The responses elicited by LDL are rapid, dose-dependent and capable of being saturated, indicating the involvement of specific receptor/binding sites in LDI-stimulated signal transduction. This LDL-binding protein was isolated from human aorta media and identified as T-cadherin. Cadherins are a superfamily of adhesion molecules that mediate Ca2+ -dependent cell-cell adhesion in embryogenesis and in adult organism's solid tissues. Intercellular junctions are formed as a result of interactions between extracellular domains of the neighboring cells' cadherins. Binding of the intercellular domain to the acting cytoskeleton ensures stability of cadherin-mediated adhesive junctions. T-cadherin is a unique member of calcium-dependent adherent proteins; in contrast to classical cadherins T-cadherin is anchored to the cell surface membranes via a glycosyl phosphatidyl inositol (GPI) moiety. Subcellular distribution of T-cadherin is restricted to lipid rafts on the cell membranes where it co-localizes with signal-transducing molecules. The function of T-cadherin has not yet been revealed. It was originally cloned from chicken embryo brain where the spatial-temporally restricted pattern of T-cadherin suggests its role as a negative guidance cue in tegulating the segmental organization of trunk neural crest migration and motor axon projections. Comparative study of the T-cadherin expression in human organs and tissues revealed that T-cadherin content was maximal in cardiovascular system. Its expression in VSMC depends on the cell phenotype and proliferate activity and increases in atherosclerotic lesion and restenosis. T-cadherin seems to play a key role in the regulation of the vascular cell phenotype, migration and growth. We hypothesize that T-cadherin is an anti-adhesive molecule which participates in intercellular interactions informing cells about their environment and regulating migration and proliferation of cells in vascular wall, while LDL interfere with the normal function of T-cadherin.     

Effects of amyloid-β peptides on voltage-gated L-type Ca(V)1.2 and Ca(V)1.3 Ca(2+) channels

Overload of intracellular Ca²⁺ has been implicated in the pathogenesis of neuronal disorders, such as Alzheimer’s disease. Various mechanisms produce abnormalities in intracellular Ca²⁺ homeostasis systems. L-type Ca²⁺ channels have been known to be closely involved in the mechanisms underlying the neurodegenerative properties of amyloid-β (Aβ) peptides. However, most studies of L-type Ca²⁺ channels in Aβ-related mechanisms have been limited to Ca_V1.2, and surprisingly little is known about the involvement of Ca_V1.3 in Aβ-induced neuronal toxicity. In the present study, we examined the expression patterns of Ca_V1.3 after Aβ_25–35 exposure for 24 h and compared them with the expression patterns of Ca_V1.2. The expression levels of Ca_V1.3 were not significantly changed by Aβ_25–35 at both the mRNA levels and the total protein level in cultured hippocampal neurons. However, surface protein levels of Ca_V1.3 were significantly increased by Aβ_25–35, but not by Aβ_35–25. We next found that acute treatment with Aβ_25–35 increased Ca_V1.3 channel activities in HEK293 cells using whole-cell patch-clamp recordings. Furthermore, using GTP pulldown and co-immunoprecipitation assays in HEK293 cell lysates, we found that amyloid precursor protein interacts with β₃ subunits of Ca²⁺ channels instead of Ca_V1.2 or Ca_V1.3 α₁ subunits. These results show that Aβ_25–35 chronically or acutely upregulates Ca_V1.3 in the rat hippocampal and human kidney cells (HEK293). This suggests that Ca_V1.3 has a potential role along with Ca_V1.2 in the pathogenesis of Alzheimer’s disease.     

Single-Channel Characterization of the Rabbit Recombinant RyR2 Reveals a Novel Inactivation Property of Physiological Concentrations of ATP

Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca²⁺ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca²⁺, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5–10 mM. With Ca²⁺ as permeant ion, 5–10 mM ATP consistently inactivated recombinant channels (15/16 experiments). Such inactivation was rarely observed with native RyR2 isolated from cardiac muscle (1 in 16 experiments). However, if the channels were purified, inactivation by ATP was then revealed in all experiments. This action of ATP may be relevant for inactivation of sarcoplasmic reticulum Ca²⁺ release during cardiac excitation–contraction coupling or may represent unnatural behavior that is revealed when RyR2 is purified or expressed in noncardiac systems. Richard Stewart and Lele Song—contributed equally to this work.     

Development of antithrombotic miniribozymes that target peripheral tryptophan hydroxylase

Transient receptor potential (TRP) proteins have been identified as cation channels that are activated by agonist–receptor coupling and mediate various cellular functions. TRPC7, a homologue of TRP channels, has been shown to act as a Ca²⁺ channel activated by G protein-coupled stimulation and to be abundantly expressed in the heart with an as-yet-unknown function. We studied the role of TRPC7 in G protein-activated signaling in HEK293 cells and cultured cardiomyocytes in vitro transfected with FLAG-tagged TRPC7 cDNA and in Dahl salt-sensitive rats with heart failure in vivo. TRPC7-transfected HEK293 cells showed an augmentation of carbachol-induced intracellular Ca²⁺ transient, which was attenuated under a Ca²⁺-free condition or in the presence of SK&F96365 (a Ca²⁺-permeable channel blocker). Upon stimulation with angiotensin II (Ang II), cultured neonatal rat cardiomyocytes transfected with TRPC7 exhibited a significant increase in apoptosis detected by TUNEL staining, accompanied with a decrease in the expression of atrial natriuretic factor and destruction of actin fibers, as compared with non-transfected cardiomyocytes. Ang II-induced apoptosis was inhibited by CV-11974 (Candesartan; Ang II type 1 [AT1] receptor blocker), SK&F96365, and FK506 (calcineurin inhibitor). In Dahl salt-sensitive rats, apoptosis and TRPC7 expression were increased in the failing myocardium, and a long-term treatment with temocapril, an angiotensin-converting enzyme inhibitor, suppressed both. Our findings suggest that TRPC7 could act as a Ca²⁺ channel activated by AT1 receptors, leading to myocardial apoptosis possibly via a calcineurin-dependent pathway. TRPC7 might be a key initiator linking AT1-activation to myocardial apoptosis, and thereby contributing to the process of heart failure.     

Different spatiotemporal organization of GPI-anchored T-cadherin in response to low-density lipoprotein and adiponectin

BackgroundUnlike other cadherins, T-cadherin does not mediate strong cell-cell adhesion. It has two soluble ligands: low density lipoprotein (LDL) and high-molecular-weight (HMW) adiponectin. LDL binding to T-cadherin induces calcium signaling, migration, and proliferation, and has proatherogenic effects, but adiponectin binding promotes antiatherogenic effects. The reasons for this difference and mechanism of signal transduction by glycosylphosphatidylinositol (GPI)-anchored T-cadherin are unknown.MethodsWe compared the ability of LDL and HMW adiponectin to induce calcium signaling, T-cadherin clustering and internalization. We measured calcium signaling in smooth muscle cells and T-cadherin expressing HEK293 using single-cell imaging. To study receptor clustering, we tested three different T-cadherin labeling strategies and then utilized confocal microscopy and flow cytometry assays based on Förster resonance energy transfer (FRET).ResultsEnzymatically labeled T-cadherin retained its cellular localization and physiological activity, features that were otherwise affected by fluorescent proteins and antibodies. This labeling method allowed us to study T-cadherin clustering dynamics at the cell surface. HMW adiponectin induced the formation of stable T-cadherin clusters while LDL induced short-lived clusters. Cellular responses were also different: LDL triggered cholesterol- and actin-dependent calcium signaling without internalization while adiponectin promoted the opposite effect.ConclusionsWe revealed distinct ligand-specific T-cadherin clustering and its ability to induce internalization or intracellular calcium signaling that likely explains the different physiological effects of LDL and HMW adiponectin.General significanceThis work highlights the importance of GPI-anchored receptor clustering dynamics in mediating cellular responses. Different ligands can induce different effects in an identical cell via the same receptor.     

The Influence of Oxidatively Modified Low Density Lipoprotein on Parameters of Energy Metabolism and Contractile Function of Arterial Smooth Muscle

Recently published results provide evidence of the importance of oxidatively modified LDL in the development of atherosclerosis. Several typical characteristics of this disease can be ascribed to the effects of oxidized LDL on the different cells involved in lesion formation. In various cell culture systems oxidized LDL was found to be cytotoxic. Therefore we were interested in its influence on parameters of energy metabolism such as glycogen and ATP content as determined for aortic segments in vitro. The results show that oxidized LDL leads to sharp decreases in both parameters, indicating an activation of cellular energy metabolism. Findings obtained from contraction experiments in which oxidized LDL shows a contractionenhancing effect on arterial segments suggest that the oxidized lipoprotein facilitates cellular Ca²⁺ liberation. This seems to be a common signal leading to its effects on energy metabolism and contraction and could also explain its cytotoxicity if cells are exposed to it for longer periods.     

Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines

Calcium ions (Ca²⁺) play a pivotal role in cellular physiology. Often Ca²⁺-dependent processes are studied in commonly available cell lines. To induce Ca²⁺ signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca²⁺ signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca²⁺ signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca²⁺ in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC₅₀) was in the low micromolar range. In individual cells, transient Ca²⁺ signals and Ca²⁺ oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca²⁺ signaling processes directly and (ii) these cell lines are suitable for calibrating Ca²⁺ biosensors in situ based on histamine receptor evoked responses.     

The role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca²⁺ channels in non-excitable cells has been studied. Receptor- and store-operated Ca²⁺ influxes have been measured using the fluorescent method of detection of cytosolic Ca²⁺ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca²⁺ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels I_max and non-selective channels I_ns in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance I_min channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.     

Ca<Superscript>2+</Superscript> binding protein-1 inhibits Ca<Superscript>2+</Superscript> currents and exocytosis in bovine chromaffin cells

Summary Calcium binding protein-1 (CaBP1) is a calmodulin like protein shown to modulate Ca²⁺ channel activities. Here, we explored the functions of long and short spliced CaBP1 variants (L- and S-CaBP1) in modulating stimulus-secretion coupling in primary cultured bovine chromaffin cells. L- and S-CaBP1 were cloned from rat brain and fused with yellow fluorescent protein at the C-terminal. When expressed in chromaffin cells, wild-type L- and S-CaBP1s could be found in the cytosol, plasma membrane and a perinuclear region; in contrast, the myristoylation-deficient mutants were not found in the membrane. More than 20 and 70% of Na⁺ and Ca²⁺ currents, respectively, were inhibited by wild-type isoforms but not myristoylation-deficient mutants. The [Ca²⁺] _i response evoked by high K⁺ buffer and the exocytosis elicited by membrane depolarizations were inhibited only by wild-type isoforms. Neuronal Ca²⁺ sensor-1 and CaBP5, both are calmodulin-like proteins, did not affect Na⁺, Ca²⁺ currents, and exocytosis. When expressed in cultured cortical neurons, the [Ca²⁺] _i responses elicited by high-K⁺ depolarization were inhibited by CaBP1 isoforms. In HEK293T cells cotransfected with N-type Ca²⁺ channel and L-CaBP1, the current was reduced and activation curve was shifted positively. These results demonstrate the importance of CaBP1s in modulating the stimulus-secretion coupling in excitable cells. M.-L. Chen and Y.-C. Chen contributed equally to this study     

Formyl-peptide receptor like 1: a potent mediator of the Ca2+ release-activated Ca2+ current ICRAC

In electrically non-excitable cells, one major source of Ca²⁺ influx is through the store-operated (or Ca²⁺ release-activated Ca²⁺) channel by which the process of emptying the intracellular Ca²⁺ stores results in the activation of Ca²⁺ channels in the plasma membrane. Using both whole-cell patch-clamp and Ca²⁺ imaging technique, we describe the electrophysiology mechanism underlying formyl-peptide receptor like 1 (FPRL1) linked to intracellular Ca²⁺ mobilization. The FPRL1 agonists induced Ca²⁺ release from the endoplasmic reticulum and subsequently evoked I_CRAC-like currents displaying fast inactivation in K562 erythroleukemia cells which expresses FPRL1, but had almost no effect in K562 cells treated with FPRL1 RNA-interference and HEK293 cells which showed no FPRL1 expression. The currents were impaired after either complete store depletion by the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, or after inhibition of PLC by U73122. Our results present the first evidence that FPRL1 is a potent mediator in the activation of CRAC channels.     

Alterations in calcium homeostasis on lead exposure in rat synaptosomes

The effects of lead on Ca²⁺ homeostasis in nerve terminals was studied. Incubation with leadin vitro stimulated the activity of calmodulin and the maximum effect was observed at 30 M lead, higher concentrations had an inhibitory effect.In vivo exposure to lead increased the activity of calmodulin by 45%. Lead had an inhibitory effect on Ca²⁺ ATPase activity in both calmodulin-rich and calmodulin-depleted synaptic plasma membranes, the IC₅₀ values for inhibition being 13.34 and 16.69 M respectively. Exogenous addition of calmodulin (5 g) and glutathione (1 mM) to calmodulin rich synaptic plasma membranes reversed the inhibition by IC₅₀ concentration of lead.In vivo exposure of lead also significantly reduced the Ca²⁺ ATPase activity, resulting in an increase in intrasynaptosomal calcium. Concomitant with the increase in intrasynaptosomal calcium, lipid peroxidation values also increased significantly in lead-treated animals. In addition lead also had an inhibitory effect on depolarization induced Ca²⁺ uptake and the inhibition was found to be a competitive one. The results sugest that lead exerts its toxic effects by modifications of the intracellular calcium messenger system which would have serious consequences on neuronal functioning.     

Hypoxic Modulation of Ca<Superscript>2+</Superscript> Signaling in Human Venous and Arterial Endothelial Cells

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca²⁺ homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O₂, 24 h). Basal [Ca²⁺] _i and store depletion-mediated Ca²⁺ entry were significantly different between the two cell types, yet agonist (ATP)–mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca²⁺ entry only in venous cells. Clearly, Ca²⁺ signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.     

Identification of Proteins Associating with Glycosylphosphatidylinositol- Anchored T-Cadherin on the Surface of Vascular Endothelial Cells: Role for Grp78/BiP in T-Cadherin-Dependent Cell Survival

There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor α1 subunit, integrin β₃, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin β₃ with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone.T-cadherin (T-cad, or H-cadherin or cadherin-13) is an atypical member of the cadherin superfamily of adhesion molecules. The “classical” cadherins are transmembrane receptors that mediate homophilic Ca²⁺-dependent adhesion between the cells of solid tissues (2). The extracellular domain organization of T-cad is similar to that of classical cadherins, but it lacks transmembrane and cytosolic domains and is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (57). T-cad was shown to mediate weak homophilic cell aggregation in suspensions of transfected cells (45, 57). However, there is a large amount of data suggesting that, in contrast to classical cadherins, atypical T-cad does not primarily function in the maintenance of intercellular adhesion; T-cad is not concentrated at sites of cell-cell contacts, is expressed on the luminal but not the baso-lateral surface of polarized transfected cells, and locates in lipid raft domains of the plasma membrane (29, 42, 43). In the embryonic nervous system T-cad functions as a negative guidance cue regulating motor axon outgrowth and innervation of skeletal muscle (15). Many studies in the cancer field have demonstrated a relationship between T-cad expression levels in tumor cells and tumor progression, although its influence on cell behavior varies in different cancer types, either inhibiting invasion and growth or correlating with a high proliferative and invasive potential (46, 52).In the cardiovascular system T-cad is highly expressed on endothelial cells (ECs), smooth muscle cells, and cardiomyocytes. Its expression level is increased in atherosclerotic lesions from the human aorta (22), in experimental restenosis during neointima formation after balloon catheterization of rat carotid artery (30), and in ECs from tumor vasculature (59). Together, these data suggest that upregulation or/and ligation of T-cad molecules on vascular cells might importantly contribute to progression of vascular pathologies associated with vascular tissue remodelling and stress, such as atherosclerosis, restenosis, and neovascularization of atherosclerotic lesions or tumors. This hypothesis is supported by studies showing that overexpression and/or homophilic ligation of T-cad in ECs stimulates proliferation, migration, and survival under conditions of oxidative stress and promotes angiogenesis in vitro and in vivo (21, 23, 26, 40).Signaling mechanisms underlying T-cad effects on cell growth and motility are poorly studied. We have identified some target signaling pathways activated in cultured vascular ECs by surface T-cad. Changes in cell phenotype during T-cad ligation-induced migration depend on activation of RhoA and Rac GTPases (41). Overexpression and/or ligation of T-cad induces increases in Akt and GSKβ3 phosphorylation levels and activation of β-catenin, and all these effects are blocked by phosphatidylinositol 3-kinase (PI3-kinase) inhibitors (26).There is scant knowledge regarding how cell surface lipid-anchored T-cad transmits signals through the plasma membrane to its intracellular targets. The absence of transmembrane and cytoplasmic domains implies the existence of transmembrane “adaptors” that interact with T-cad on the outer surface of the plasma membrane. Data in the current literature do not allow prediction of membrane associations of T-cad, which from the structural point of view shares homology with two distinct protein groups, namely, cadherins and GPI-anchored proteins. Absence of the cytoplasmic domain excludes any possibility of direct interactions between T-cad and molecular mechanisms utilized by classical cadherins, such as catenins. Recently we have demonstrated that T-cad overexpression results in stimulation of β-catenin signaling in ECs (25). However, this effect is the consequence of Akt/GSK3β pathway activation rather than the result of a direct physical interaction with β-catenin. A requirement for integrin-linked kinase (ILK) upstream of Akt/GSKβ3/β-catenin modulation by T-cad has recently been shown (25). However, ILK is located intracellularly and thus cannot function as a primary molecular adaptor for surface T-cad.The presence of a lipid GPI anchor on the C terminus of the T-cad molecule suggests that T-cad may utilize some of the signaling mechanisms that depend on its localization within lipid rafts, cholesterol- and sphingolipid-rich domains of the plasma membrane that act as signal transduction platforms compartmentalizing, clustering, and facilitating interactions between various lipid-anchored signaling molecules (49). However, the group of GPI proteins is highly heterogeneous both structurally and functionally and includes membrane-associated enzymes, adhesion receptors, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. Likewise, downstream raft-associated signaling is also diverse, including small GTPases, Src kinases, lipid second messengers, and many others (20). Moreover, molecular interactions within lipid rafts have been shown to be determined by many factors, such as the presence of receptor ligands, their precise membrane localization (the leading edge versus overall surface distribution), and lipid composition (ganglioside GM1-enriched versus GM3-enriched lipid rafts), among others (7).This study aimed to identify membrane proteins colocalizing with atypical GPI-anchored T-cad on the surface of cultured ECs and to evaluate the role of identified molecules as adaptors transmitting signals from cell surface T-cad to its intracellular targets. We have identified several candidate proteins with potential functions as membrane adaptors for T-cad, namely, glucose-related protein Grp78/BiP, GABA-A receptor α1 subunit, integrin β₃, and two hypothetical proteins, LOC124245 and FLJ32070. We demonstrate that the interaction between T-cad and surface Grp78 is necessary for T-cad-dependent activation of prosurvival signaling in ECs.