VEGF Stimulates RCAN1.4 Expression in Endothelial Cells via a Pathway Requiring Ca2+/Calcineurin and Protein Kinase C-δ

Background

Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.

Methodology/Principal Findings

We show that VEGF is able to induce RCAN1.4 expression during cellular proliferation and differentiation, and that VEGF-mediated expression of RCAN1.4 was inhibited by the use of inhibitors to protein kinase C (PKC) and calcineurin. Further analysis revealed that siRNA silencing of PKC-delta expression partially inhibited VEGF-stimulated RCAN1.4 expression. Knockdown of RCAN1.4 with siRNA resulted in a decrease in cellular migration and disrupted tubular morphogenesis when HDMECs were either stimulated with VEGF in a collagen gel or in an endothelial/fibroblast co-culture model of angiogenesis. Analysis of intracellular signalling revealed that siRNA mediated silencing of RCAN1.4 resulted in increased expression of specific nuclear factor of activated T-cells (NFAT) regulated genes.

Conclusions/Significance

Our data suggests that RCAN1.4 expression is induced by VEGFR-2 activation in a Ca²⁺ and PKC-delta dependent manner and that RCAN1.4 acts to regulate calcineurin activity and gene expression facilitating endothelial cell migration and tubular morphogenesis.     

Chloroquine Stimulates Cl? Secretion by Ca2+ Activated Cl? Channels in Rat Ileum

Chloroquine (CQ), a bitter tasting drug widely used in treatment of malaria, is associated gastrointestinal side effects including nausea or diarrhea. In the present study, we investigated the effect of CQ on electrolyte transport in rat ileum using the Ussing chamber technique. The results showed that CQ evoked an increase in short circuit current (I_SC) in rat ileum at lower concentration (≤5×10⁻⁴ M ) but induced a decrease at higher concentrations (≥10⁻³ M). These responses were not affected by tetrodotoxin (TTX). Other bitter compounds, such as denatoniumbenzoate and quinine, exhibited similar effects. CQ-evoked increase in I_SC was partly reduced by amiloride(10⁻⁴ M), a blocker of epithelial Na⁺ channels. Furosemide (10⁻⁴ M), an inhibitor of Na⁺-K⁺ -2Cl⁻ co-transporter, also inhibited the increased I_SC response to CQ, whereas another Cl⁻ channel inhibitor, CFTR(inh)-172(10⁻⁵M), had no effect. Intriguingly, CQ-evoked increases were almost completely abolished by niflumic acid (10⁻⁴M), a relatively specific Ca²⁺-activated Cl⁻ channel (CaCC) inhibitor. Furthermore, other CaCC inhibitors, such as DIDS and NPPB, also exhibited similar effects. CQ-induced increases in I_SC were also abolished by thapsigargin(10⁻⁶M), a Ca²⁺ pump inhibitor and in the absence of either Cl⁻ or Ca²⁺ from bathing solutions. Further studies demonstrated that T2R and CaCC-TMEM16A were colocalized in small intestinal epithelial cells and the T2R agonist CQ evoked an increase of intracelluar Ca²⁺ in small intestinal epithelial cells. Taken together, these results demonstrate that CQ induces Cl⁻ secretion in rat ileum through CaCC at low concentrations, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.     

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca²⁺ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca²⁺ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca²⁺ current (I_{Ca, T}) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of I_{Ca, T} and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of I_{Ca, T} mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in I_{Ca, T} and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.     

17β-Estradiol Elevates cGMP and,via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes

Rapid non-genomic effects of 17β-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17β-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17β-estradiol, but not 17α-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Iα, stimulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17β-estradiol-BSA conjugate, suggesting that 17β-estradiol acts at the extracellular face of the plasma membrane. We also show that 17β-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative γ-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17β-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the γ-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.     

Ca2+-Activated Cl? Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons

The Ca²⁺-activated Cl⁻ channel is considered a key constituent of odor transduction. Odorant binding to a specific receptor in the cilia of olfactory sensory neurons (OSNs) triggers a cAMP cascade that mediates the opening of a cationic cyclic nucleotide-gated channel (CNG), allowing Ca²⁺ influx. Ca²⁺ ions activate Cl⁻ channels, generating a significant Cl⁻ efflux, with a large contribution to the receptor potential. The Anoctamin 2 channel (ANO2) is a major constituent of the Cl⁻ conductance, but its knock-out has no impairment of behavior and only slightly reduces field potential odorant responses of the olfactory epithelium. Likely, an additional Ca²⁺-activated Cl⁻ channel of unknown molecular identity is also involved. In addition to ANO2, we detected two members of the ClCa family of Ca²⁺-activated Cl⁻ channels in the rat olfactory epithelium, ClCa4l and ClCa2. These channels, also expressed in the central nervous system, may correspond to odorant transduction channels. Whole Sprague Dawley olfactory epithelium nested RT-PCR and single OSNs established that the mRNAs of both channels are expressed in OSNs. Real time RT-PCR and full length sequencing of amplified ClCa expressed in rat olfactory epithelium indicated that ClCa4l is the most abundant. Immunoblotting with an antibody recognizing both channels revealed immunoreactivity in the ciliary membrane. Immunochemistry of olfactory epithelium and OSNs confirmed their ciliary presence in a subset of olfactory sensory neurons. The evidence suggests that ClCa4l and ClCa2 might play a role in odorant transduction in rat olfactory cilia.     

Neuron–Astrocyte Interactions,Pyruvate Carboxylation and the Pentose Phosphate Pathway in the Neonatal Rat Brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-¹³C]glucose and were sacrificed 30 min later. Brain extracts were analysed using ¹H- and ¹³C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-¹³C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-¹³C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-¹³C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.     

Protective Effects of Notoginsenoside R1 via Regulation of the PI3K-Akt-mTOR/JNK Pathway in Neonatal Cerebral Hypoxic–Ischemic Brain Injury

Notoginsenoside R1 (NGR1) is a predominant phytoestrogen extracted from Panax notoginseng that has recently been reported to play important roles in the treatment of cardiac dysfunction, diabetic kidney disease, and acute liver failure. Studies have suggested that NGR1 may be a viable treatment of hypoxic-ischemic brain damage (HIBD) in neonates by reducing endoplasmic reticulum stress via estrogen receptors (ERs). However, whether NGR1 has other neuroprotective mechanisms or long-term neuroprotective effects is unclear. In this study, oxygen-glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons and unilateral ligation of the common carotid artery (CCL) in 7-day-old postnatal Sprague Dawley (SD) rats followed by exposure to a hypoxic environment were used to mimic an HIBD episode. We assessed the efficacy of NGR1 by measuring neuronal damage with MTT assay and assessed brain injury by TTC staining and brain water content detection 24–48 h after OGD/HIE. Simultaneously, we measured the long-term neurophysiological effects using the beam walking test (5 weeks after HI) and Morris water maze test 5–6 weeks after HI. Expression of PI3K-Akt-mTOR/JNK (24 h after HI or OGD/R) proteins was detected by Western blotting after stimulation with HI, NGR1, LY294002 (PI3K inhibitor), 740Y-P (PI3K agonist), or ICI 182780(estrogen receptors inhibitor). The results indicated that NGR1 exerted neuroprotective effects by inhibiting neuronal apoptosis and promoting cell survival via the PI3K-Akt-mTOR/JNK signaling pathways by targeting ER in neonatal hypoxic–ischemic injury.     

New Molecular Determinant for Inactivation of the Human L-Type α1C Ca2+ Channel

Molecular cloning of the human fibroblast Ca²⁺ channel pore-forming α_1C subunit revealed (Soldatov, 1992. Proc. Natl. Acad. Sci. USA 89:4628-4632) a naturally occurring mutation g²²⁵⁴→ a that causes the replacement of the conservative alanine for threonine at the position 752 at the cytoplasmic end of transmembrane segment IIS6. Using stably transfected HEK293 cell lines, we have compared electrophysiological properties of the conventional α_1C,77 human recombinant L-type Ca²⁺ channel with those of its mutated isoform α_1C,94 containing the A752T replacement. Comparative quantification of steady-state availability of the current carried by α_1C,94 and α_1C,77 showed that A752T mutation prevented a large (≈25%) fraction of the current carried by Ca²⁺ or Ba²⁺ from fully inactivating. This mutation, however, did not appear to alter significantly the Ca²⁺-dependence and kinetics of decay of the inactivating fraction of the current or its voltage-dependence. The data suggests that Ala752 at the cytoplasmic end of IIS6 might serve as a molecular determinant of the Ca²⁺ channel inactivation, possibly regulating the voltage-dependence of its availability. Received: 14 January 2000/Revised: 20 June 2000     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).     

Both Intra- and Extracellular Ca2+ Participate in the Regulation of the Lateral Diffusion of the PDGF-β2 Receptor

When the receptors for platelet-derived growth factor (PDGF) are activatedthey aggregate, become tyrosine-phosphorylated and elicit a cascade ofdown-stream signals, including mobilization of Ca²⁺ from intra- andextracellular stores. Receptor mobility in the plane of the membrane isa prerequisite for receptor aggregation and further signalling. Using humanforeskin fibroblasts (AG 1523) and fluorescence recovery afterphotobleaching (FRAP), we therefore assessed the lateral mobilitycharacteristics of PDGF-2 receptors by their diffusioncoefficient (D), and fraction of mobile receptors (R). This was done oncells stimulated with either normal human serum (NHS) or PDGF underdifferent Ca²⁺-conditions.The results suggest that both intra- and extracellular free Ca²⁺influence the mobility characteristics of the PDGF-2receptor. Interestingly, the extracellular Ca²⁺ seems to imposegeneral restrictions on the mobility of receptors, since R increased whenextracellular Ca²⁺ was quenched with EGTA, whereas intracellularclamping of Ca²⁺ transients with MABTAM (BAPT/AM) primarily affectedD. When both intra- and extracellular Ca²⁺ were quenced, D remainedlow and R high, further supporting the proposition that they achievedistinct effects. Inhibition of tyrosine phosphorylation with Erbstatin,partly inhibited the NHS effects and released PDGF-induced receptorimmobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGFinduced changes in intracellular free [Ca²⁺]. In view of the presentdata it might have important effects on the state of the receptor in themembrane, for instance by regulating its lateral mobility, communicationwith other receptors and signalling functions in the membrane.     

Regulated Extracellular Choline Acetyltransferase Activity— The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer’s disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer’s disease and multiple sclerosis in particular.     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Are B-type Ca2+ Channels of Cardiac Myocytes Akin to the Passive Ion Channel in the Plasma Membrane Ca2+ Pump?

The present study demonstrates that B-type Ca²⁺ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca²⁺-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca²⁺ channels were completely blocked by internal application of PMCA pump inhibitors, namely La³⁺ (100 μm), eosin (10 μm) and AIF₃ (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba²⁺-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba²⁺, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca²⁺ channels are some form of the PMCA pump. Received: 24 July 2000/Revised: 5 October 2000     

Orexin-A Promotes Cell Migration in Cultured Rat Astrocytes via Ca2+-Dependent PKCα and ERK1/2 Signals

Orexin-A is an important neuropeptide involved in the regulation of feeding, arousal, energy consuming, and reward seeking in the body. The effects of orexin-A have widely studied in neurons but not in astrocytes. Here, we report that OX1R and OX2R are expressed in cultured rat astrocytes. Orexin-A stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and then induced the migration of astrocytes via its receptor OX1R but not OX2R. Orexin-A-induced ERK1/2 phosphorylation and astrocytes migration are Ca²⁺-dependent, since they could be inhibited by either chelating the extracellular Ca²⁺ or blocking the pathway of store-operated calcium entry (SOCE). Furthermore, both non-selective protein kinase C (PKC) inhibitor and PKCα selective inhibitor, but not PKCδ inhibitor, prevented the increase in ERK1/2 phosphorylation and the migration of astrocytes, indicating that the Ca²⁺-dependent PKCα acts as the downstream of the OX1R activation and mediates the orexin-A-induced increase in ERK1/2 phosphorylation and cell migration. In conclusion, these results suggest that orexin-A can stimulate ERK1/2 phosphorylation and then facilitate the migration of astrocytes via PLC-PKCα signal pathway, providing new knowledge about the functions of the OX1R in astrocytes.     

Lipopolysaccharide Up-regulates IL-6Rα Expression in Cultured Leptomeningeal Cells via Activation of ERK1/2 Pathway

To clarify the response of leptomeningeal cells to immune stimulation, the effect of lipopolysaccharide (LPS) on expression of IL-6 receptors in the cultured leptomeningeal cells was investigated. The results showed that the expression of IL-6Rα was invisible in the purified leptomeningeal cells while it was seen in the cells when they were co-cultured with astrocytes. On the other hand, GP130 was moderately expressed in both conditions. Following incubation with different doses of LPS, IL-6Rα expression in purified leptomeningeal cells was increased in a time- and dose-dependent manner, while GP130 level remained unchanged. Concomitantly, phosphorylated ERK1/2 level was increased following LPS stimulation and its inhibition by PD98059 attenuated the LPS-induced increase of IL-6Rα expression. These data indicate that leptomeningeal cells can respond to immunogenic stimuli as manifested by expression of cytokine receptors. Moreover, ERK1/2 pathway seems to be involved in the process of LPS-induced IL-6Rα up-regulation in leptomeningeal cells.     

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca2+ Channels

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (Ca_V) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP₂). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of Ca_V channels by PIP₂. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP₂ sensitivity of Ca_V2.2 and Ca_V1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.     

The Influence of Fluoride on the Expression of Inhibitors of Wnt/β-Catenin Signaling Pathway in Rat Skin Fibroblast Cells

The effective therapy of fluoride-induced bone diseases requires an understanding of the mechanism of the disorders. Changes in the inhibitors of the Wnt/β-catenin pathway, Dickkopf-1 (Dkk-1) and Sclerostin (SOST),were studied in supernatants harvested from rat skin fibroblasts cultured with varied doses of fluoride. The contents of SOST and Dkk-1 in fibroblast supernatants were assessed at four exposure time-points and investigated by using the method of ELISA. Compared to the relevant controls(0 mg F(?)/L), a significant decrease of the concentrations of SOST and Dkk-1 was observed as the fluoride concentration increased. Compared to the relevant time controls (24 h), a significant decrease of the concentrations of SOST and Dkk-1 was observed with the extension of time. Our results suggest that the Wnt/β-catenin pathway inhibitors Dkk-1 and SOST play an important role in skeletal fluorosis. They can be used as important indications for diagnosing bone metabolism changes caused by fluoride exposure and therapeutic targets in diseases resulting from fluoride exposure.