PKC-alpha-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-α in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Go-6976 and 2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether, indicating a role for PKC-α. Overexpression of a kinase deficient PKC-α(K368R) but not wild-type PKC-α significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-α-green fluorescent protein with the membrane fraction within 10-20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-α(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-α in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg- 1) attenuated protein degradation and increased protein synthesis in skeletal muscle. © 2007 Elsevier Inc. All rights reserved.

Mechanism of induction of muscle protein degradation by angiotensin II

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-κB (NF-κB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-κB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-κB. Both angiotensin I and II induced an early decrease in cytoplasmic I-κB levels followed by nuclear accumulation of NF-κB. Using an NF-κB luciferase construct this was shown to increase transcriptional activation of NF-κB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-κB, confirming that activation of NF-κB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-κB. © 2005 Elsevier Inc. All rights reserved.

Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin-proteasome proteolytic pathway

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 μM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome α-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E214k, also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-κBα, and increased nuclear accumulation of nuclear factor-κB (NF-κB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-κB activation by resveratrol (30 μM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-κB. © 2005 Elsevier Inc. All rights reserved.

Modulation of CYP1A1 by PKC Inhibitors and TPA Pre-Treatments in MH1C1 Rat Hepatoma Cells Exposed to 3 -Methylcholanthrene

Cytochrome P4501A1 (CYP1A1), an enzyme known to metabolize polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR). The involvement of protein kinase C (PKC) in the regulation of AhR signal transduction pathway, has been widely studied but the role of specific PKC isoform(s) involved in this process it is not well clarified. To study which PKC isoform(s) is implicated in the regulation of CYP1A1, in the poorly tumorigenic MH1C1 rat hepatoma cells, we examined the effects of some PKC pharmacological inhibitors, Calphostin C (CAL), Staurosporine (STA) and H7, and of 12-0-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, on basal and 3- methylcholanthrene (MC)-induced CYP1A1 protein expression and mediated ethoxyresorufin O-deethylation (EROD) activity. In parallel, the activities of PKC-α, -βI, -δ and -ε isoforms, the most expressed in MH1C1 cells, were monitored. After pre-treatment with CAL, STA and H7, the MC-induced CYP1A1 protein and EROD activity were rapidly reduced with temporal profile similar to the profile of the activity of α and β1 PKC isoforms. Moreover, TPA pre-treatment induced a biphasic effect on EROD activity, and a decline of PKC -βI and -α, in first instance, and -δ and -ε activities later on. These findings clearly show that, in MH1C1 cells, PKC is involved in CYP1A1 regulation and that α and βI classic PKC isoforms play an active role in modulating this process.

Trans-resveratrol inhibits early blood vessel formation (Vasculogenesis) without impairment of embryonic growth

Resveratrol is a stilbene compound found in grapes and other sources. In this study we examined the effects of trans-resveratrol (4.38-438 mu M/implant) in the vasculogenesis of yolk-sac membranes and its capacity to improve chick embryo growth. High concentrations of the stilbene (43.8-438 mu M) significantly inhibited early vessel formation, decreasing the percentage vitelline vessels of 3.5-day embryos by 50% compared to the control. In addition, basic fibroblast growth factor-stimulated vasculogenesis (140% of vessels as compared to control) was partially reversed by t-resveratrol (35% of inhibition) and treatments with cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) as well a protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate) decreased the vessel number to 60%, 50%, and 44%, respectively. Treatments with t-resveratrol (4.38-43.8 mu M/implant) significantly increased the body length of embryos incubated in vitro uncoupled from any impairment in the body shape or detectable embryotoxic effect. We suggest that the antivasculogenic activity and the enhancement in embryonic growth promoted by non acute treatments with t-resveratrol were, at least in part, due to PKC inhibition. We suggest that t-resveratrol can be usable not only as a reliable functional nutriment, but also is useful for the development of prophylactic and/or therapeutic agents for treatment of angiogenic-degenerative diseases.

The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel

In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 μM; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 μM, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites.

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway. We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with phosphorylation in a 33-amino acid-long segment of the receptor carboxyl-terminal cytoplasmic tail. Here, we determined that the in vivo sites of phosphorylation are four serine doublets present at positions 431/432, 441/442, 444/445, and 451/452. In vitro phosphorylation of fusion proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosphorylated on fusion protein but not in intact cells. This indicated that in vivo a different PKC isoform or a PKC-activated kinase may phosphorylate the receptor. The role of phosphorylation on receptor desensitization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doublet to alanines reduced the extent of phorbol 12-myristate 13-acetate-induced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-like peptide-1 receptor can be phosphorylated in response to phorbol 12-myristate 13-acetate on four different sites within the cytoplasmic tail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achieved when all four sites were phosphorylated.

The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity

Recent work has demonstrated that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron-transport chain triggers several pathways of injury [(protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product formation (AGE)] involved in the pathogenesis of diabetic complications by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. On the other hand, PARP activation results in inhibition of GAPDH by poly-ADP-ribosylation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC and AGE formation are prevented by inhibition of PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in rodent models of cardiomyopathy, nephropathy, neuropathy, and retinopathy. PARP activation is also present in microvasculature of human diabetic subjects. The present review focuses on the role of PARP in diabetic complications and emphasizes the therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.

Mechanisms of hydrogen peroxide-induced vasoconstriction in the isolated perfused rat kidney.

The vasoconstrictor effect of hydrogen peroxide (H(2)O(2)) on isolated perfused rat kidney was investigated. H(2)O(2) induced vasoconstriction in the isolated rat kidney in a concentration-dependent manner. The vasoconstrictor effects of H(2)O(2) were completely inhibited by 1200 U/ml catalase. Endothelium-removal potentiated the renal response to H(2)O(2). The H(2)O(2) dose-response curve was not significantly modified by administration of the NO inhibitor L-NAME (10(-4) mol/l), whereas it was increased by the non-specific inhibitor of K+-channels, tetraethylammonium (3.10(-3) mol/l). Separately, removal of extracellular Ca(2+), administration of a mixture of calcium desensitizing agents (nitroprusside, papaverine, and diazoxide), and administration of a protein kinase C (PKC) inhibitor (chelerythrine, 10(-5) mol/l) each significantly attenuated the vasoconstrictor response to H(2)O(2), which was virtually suppressed when they were performed together. The pressor response to H(2)O(2) was not affected by: dimethyl sulfoxide (7.10(-5) mol/l) plus mannitol (3.10(-5) mol/l); intracellular Ca(2+) chelation using BAPTA (10(-5) mol/l); calcium store depletion after repeated doses of phenylephrine (10(-5) g/g kidney); or the presence of indomethacin (10(-5) mol/l), ODYA (2.10(-6) mol/l) or genistein (10(-5) mol/l). We conclude that the vasoconstrictor response to H(2)O(2) in the rat renal vasculature comprises the following components: 1) extracellular calcium influx, 2) activation of PKC, and 3) stimulation of pathways leading to sensitization of contractile elements to calcium. Moreover, a reduced pressor responsiveness to H(2)O(2) in female kidneys was observed.

Impaired glucose metabolism in the heart of obese Zucker rats after treatment with phorbol ester.

OBJECTIVE: To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. DESIGN: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. MEASUREMENTS: Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. RESULTS: PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. CONCLUSION: Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.