thapsigargin and Hypoxia

Endoplasmic reticulum (ER) stress has been reported to play a role in the pathogenesis of intrauterine growth retardation and preeclampsia, especially implantation failure. Although in vitro ER stress studies in human trophoblast cell line have been conducted in recent years, the influence of Thapsigargin on intracellular dynamics on calcium homeostasis has not been proven. Here, the effects of ER stress and impaired calcium homeostasis on apoptosis, autophagy, cytoskeleton, hypoxia, and adhesion molecules in 2D and spheroid cultures of human trophectoderm cells were investigated at gene expression and protein levels. Thapsigargin caused ER stress by increasing GRP78 gene expression and protein levels. Human trophectoderm cells displayed different characterization properties in 2D and spheroids. While it moves in the pathway of EIF2A and IRE1A mechanisms in 2D, it proceeds in the pathway of EIF2A and ATF6 mechanisms in spheroids and triggers different responses in survival and programmed cell death mechanisms such as apoptosis and autophagy. This led to changes in the cytoskeleton, cell adhesion molecules and cell-cell interactions by affecting the hypoxia mechanism.

Topics: Calcium; Endoplasmic Reticulum Stress; Female; Humans; Hypoxia; Pregnancy; Thapsigargin; Trophoblasts

We studied the mechanisms by which carotid body glomus (type 1) cells produce spontaneous Ca

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Carotid Body; Cell Line; Endoplasmic Reticulum; Female; Hypoxia; Male; Nifedipine; Rats; Rats, Sprague-Dawley; Stromal Interaction Molecule 1; Thapsigargin

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Topics: Animals; Brain Ischemia; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Cell Death; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Hypoglycemia; Hypoxia; Male; Neurons; Neuroprotective Agents; Rats, Wistar; Thapsigargin; Tuberous Sclerosis Complex 1 Protein; Tunicamycin

The present study aims to investigate the effect of noscapine (0.5-2.5 μM), an alkaloid from the opium poppy, on primary murine fetal cortical neurons exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia.. Cells were transferred to glucose-free DMEM and were exposed to hypoxia in a small anaerobic chamber. Cell viability and nitric oxide production were evaluated by MTT assay and the Griess method, respectively.. The neurotoxicities produced by all three hypoxia durations tested were significantly inhibited by 0.5 μM noscapine. Increasing noscapine concentration up to 2.5 μM produced a concentration-dependent inhibition of neurotoxicity. Pretreatment of cells with MK-801 (10 μM), a non-competitive NMDA antagonist, and nimodipine (10nM), an L-type Ca(2+) channel blockers, increased cell viability after 30 min OGD, while the application of NBQX (30 μM), a selective AMPA-kainate receptor antagonist partially attenuated cell injury. Subsequently, cells treated with noscapine in the presence of thapsigargin (1 μM), an inhibitor of endoplasmic reticulum Ca(2+) ATPases. After 60 min OGD, noscapine could inhibit the cell damage induced by thapsigargin. However, noscapine could not reduce cell damage induced by 240 min OGD in the presence of thapsigargin. Noscapine attenuated nitric oxide (NO) production in cortical neurons after 30 min OGD.. We concluded that noscapine had a neuroprotective effect, which could be due to its interference with multiple targets in the excitotoxicity process. These effects could be mediated partially by a decrease in NO production and the modulation of intracellular calcium levels.

Topics: Animals; Brain Ischemia; Cell Survival; Dizocilpine Maleate; Dose-Response Relationship, Drug; Glucose; Hypoxia; Mice; Neurons; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nimodipine; Nitric Oxide; Noscapine; Primary Cell Culture; Quinoxalines; Thapsigargin

Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)-transformed retinal pigment epithelial ARPE-19/HPV-16 cells.. Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined.. The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H(2)O(2), and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5' untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress.. These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies.

Topics: Activating Transcription Factor 4; Blotting, Western; Cell Line, Transformed; Cell Transformation, Viral; Chromatin Immunoprecipitation; Endothelium, Corneal; Enzyme Inhibitors; Gene Expression Regulation; Human papillomavirus 16; Humans; Hydrogen Peroxide; Hypoxia; NF-E2-Related Factor 2; Oxidative Stress; Plasmids; Retinal Pigment Epithelium; Thapsigargin; Trabecular Meshwork

AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O(2)) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKβ abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKβ.

Topics: Acetylcysteine; Adenosine Monophosphate; AMP-Activated Protein Kinases; Animals; Blotting, Western; Calcium; Calcium Channels; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cell Hypoxia; Cell Line, Tumor; Cells, Cultured; Endoplasmic Reticulum; Enzyme Activation; Fluorescence Resonance Energy Transfer; Humans; Hypoxia; Lung; Membrane Proteins; Mice; Mice, Knockout; Neoplasm Proteins; ORAI1 Protein; Rats; Reactive Oxygen Species; RNA Interference; Stromal Interaction Molecule 1; Thapsigargin

Hypoxia-induced gene expression is a critical determinant of neuron survival after stroke. Understanding the cell autonomous genetic program controlling adaptive and pathological transcription could have important therapeutic implications. To identify the factors that modulate delayed neuronal apoptosis after hypoxic injury, we developed an in vitro culture model that recapitulates these divergent responses and characterized the sequence of gene expression changes using microarrays. Hypoxia induced a disproportionate number of bZIP transcription factors and related targets involved in the endoplasmic reticulum stress response. Although the temporal and spatial aspects of ATF4 expression correlated with neuron loss, our results did not support the anticipated pathological role for delayed CHOP expression. Rather, CHOP deletion enhanced neuronal susceptibility to both hypoxic and thapsigargin-mediated injury and attenuated brain-derived neurotrophic factor-induced neuroprotection. Also, enforced expression of CHOP prior to the onset of hypoxia protected wild-type cultures against subsequent injury. Collectively, these findings indicate CHOP serves a more complex role in the neuronal response to hypoxic stress with involvement in both ischemic preconditioning and delayed neuroprotection.

Topics: Animals; Apoptosis; Cell Death; Endoplasmic Reticulum; Gene Expression Regulation; Hypoxia; Ischemic Preconditioning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Thapsigargin; Transcription Factor CHOP

Diets high in soy are neuroprotective in experimental stroke. This protective effect is hypothesized to be mediated by phytoestrogens contained in soy, because some of these compounds have neuroprotective effects in in vitro models of cell death. We tested the ability of the soy phytoestrogens genistein, daidzein, and the daidzein metabolite equol to protect embryonic rat primary cortical neurons from ischemic-like injury in vitro at doses typical of circulating concentrations in human populations (0.1-1 microM). All three phytoestrogens inhibited lactate dehydrogenase (LDH) release from cells exposed to glutamate toxicity or the calcium-ATPase inhibitor, thapsigargin. In cells exposed to hypoxia or oxygen-glucose deprivation (OGD), pretreatment with the phytoestrogens inhibited cell death in an estrogen receptor (ER) dependent manner. Although OGD results in multiple modes of cell death, examination of alpha-spectrin cleavage and caspase-3 activation revealed that the phytoestrogens were able to inhibit apoptotic cell death in this model. In addition, blockade of phosphoinositide 3-kinase prevented the protective effects of genistein and daidzein, and blockade of mitogen-activated protein kinase prevented genistein-dependent neuroprotection. These results suggest that pretreatment with dietary levels of soy phytoestrogens can mimic neuroprotective effects observed with estrogen and appear to use the same ER-kinase pathways to inhibit apoptotic cell death.

Topics: Animals; Apoptosis; Cerebral Cortex; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Glucose; Glutamic Acid; Glycine max; Hypoxia; In Vitro Techniques; L-Lactate Dehydrogenase; Neuroprotective Agents; Phytoestrogens; Rats; Rats, Long-Evans; Receptors, Estrogen; RNA, Messenger; Spectrin; Thapsigargin

Closure of the ductus arteriosus (DA) after birth, essential for postnatal adaptation, is initiated by the transition from hypoxia to normoxia. The current study investigated how hypoxia affects the level of cytosolic calcium ([Ca(2+)](i)) in fetal lamb DA smooth muscle cells (DASMCs) and the role of calcium pumps in this process. The [Ca(2+)](i) variation in response to acute hypoxia was determined spectrofluorometrically with fura-3-AM in cultured fetal DASMCs. Interventions using chemicals or solutions including thapsigargin, vanadate, KB-R7943, alkaline PH9.0 solution, or Na(+)-free medium were administered when samples were exposed to acute hypoxia. The results show that [Ca(2+)](i) decreased dramatically under acute hypoxia. This decrease was not attenuated completely by an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+) adenosine triphosphatase (ATPase) (SERCA), a blocker of plasma membrane Ca(2+) ATPase (PMCA), or an inhibitor and activator of the reserve mode of the Na(+)/Ca(2+) exchanger (NCX). In contrast, KT-R9743, an inhibitor of the forward mode of NCX at a high concentration (30 microm), greatly diminished the hypoxia-induced [Ca(2+)](i) decrease in fetal DASMCs. These results suggest that a hypoxia-induced Ca(2+) decrease in fetal DASMCs results from cytosolic Ca(2+) efflux mediated primarily by the forward mode of NCX.

Topics: Analysis of Variance; Animals; Calcium; Cells, Cultured; Cytosol; Ductus Arteriosus; Fetus; Hydrogen-Ion Concentration; Hypoxia; Microscopy, Confocal; Myocytes, Smooth Muscle; Sheep; Sodium-Calcium Exchanger; Thapsigargin; Thiourea; Vanadates

Expression of brain-derived neurotrophic factor (BDNF) is sensitive to changes in oxygen availability, suggesting that BDNF may be involved in adaptive responses to oxidative stress. However, it is unknown whether or not oxidative stress actually increases availability of BDNF by stimulating BDNF secretion. To approach this issue we examined BDNF release from PC12 cells, a well-established model of neurosecretion, in response to hypoxic stimuli. BDNF secretion from neuronally differentiated PC12 cells was strongly stimulated by exposure to intermittent hypoxia (IH). This response was inhibited by N-acetyl-l-cysteine, a potent scavenger of reactive oxygen species (ROS) and mimicked by exogenous ROS. IH-induced BDNF release requires activation of tetrodotoxin sensitive Na+ channels and Ca2+ influx through N- and L-type channels, as well as mobilization of internal Ca2+ stores. These results demonstrate that oxidative stress can stimulate BDNF release and that underlying mechanisms are similar to those previously described for activity-dependent BDNF secretion from neurons. Surprisingly, we also found that IH-induced secretion of BDNF was blocked by dopamine D2 receptor antagonists or by inhibition of dopamine synthesis with alpha-methyl-p-tyrosine. These data indicate that oxidative stress can stimulate BDNF release through an autocrine or paracrine loop that requires dopamine receptor activation.

Topics: Animals; Autocrine Communication; Boron Compounds; Brain; Brain-Derived Neurotrophic Factor; Butaclamol; Cadmium; Caffeine; Calcium Channel Blockers; Cell Differentiation; Cells, Cultured; Dantrolene; Dopamine; Dopamine Antagonists; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Hydrogen Peroxide; Hypoxia; Nerve Growth Factor; Neurons; Nimodipine; omega-Conotoxin GVIA; Oxidative Stress; PC12 Cells; Potassium Chloride; Pregnancy; Rats; Sulpiride; Thapsigargin; Transfection

Hypoxia-Inducible Factor-1 (HIF-1) is the key transcription factor in control of the expression of hypoxia-inducible genes needed by cells to adapt to decreased oxygen availability. Herein, we investigated the HIF-1alpha-mediated gene expression of carbonic anhydrase 9 (CA9) in response to hypoxia and changes of intracellular calcium levels in the neuroblastoma cell line SH-SY5Y. Decreasing the intracellular calcium level by BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) induced HIF-1alpha nuclear accumulation and enhanced HIF-1 DNA binding within 1 h of incubation. Like hypoxia, BAPTA stimulated HIF-1-dependent transcription by increasing the activity of the C-terminal transactivation domain of HIF-1alpha and greatly enhanced expression of the HIF-1 target gene CA9. Detailed analysis of HIF-1alpha accumulation revealed that BAPTA attenuated the interaction of HIF-1alpha with von-Hippel-Lindau protein thus decreasing proteasomal degradation of HIF-1alpha. Knock down of HIF-1alpha mRNA and protein by small interference RNA for HIF-1alpha revealed that both hypoxia and the BAPTA-induced gene expression of CA9 were strictly dependent on HIF-1alpha. In contrast, elevation of cytosolic calcium level by thapsigargin reduced the BAPTA-mediated effects. Measurements of intracellular calcium under hypoxia revealed a change in the cellular calcium distribution. BAPTA-dependent induction of HIF-1 activity was not caused by its in vitro capability to chelate iron. Instead, effective chelation of cellular calcium caused the accumulation of HIF-1alpha protein through inhibition of HIF-prolyl hydroxylases and activated HIF-1-dependent gene expression under normoxic conditions.

Topics: Blotting, Western; Calcium; Carbonic Anhydrases; Cell Line, Tumor; Cell Nucleus; Cell Survival; Cell-Free System; Chelating Agents; Dose-Response Relationship, Drug; Egtazic Acid; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ions; Iron; Lasers; Ligands; Microscopy, Confocal; Microscopy, Fluorescence; Plasmids; Procollagen-Proline Dioxygenase; Protein Binding; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Time Factors; Transcription Factors

The role of endothelium-derived hyperpolarizing factor (EDHF) in regulating the pulmonary circulation and the participation of cytochrome P-450 (CYP450) activity and gap junction intercellular communication in EDHF-mediated pulmonary vasodilation are unclear. We tested whether tonic EDHF activity regulated pulmonary vascular tone and examined the mechanism of EDHF-mediated pulmonary vasodilation induced by thapsigargin in salt solution-perfused normotensive and hypoxia-induced hypertensive rat lungs. After blockade of both cyclooxygenase and nitric oxide synthase, inhibition of EDHF with charybdotoxin plus apamin did not affect either normotensive or hypertensive vascular tone or acute hypoxic vasoconstriction but abolished thapsigargin vasodilation in both groups of lungs. The CYP450 inhibitors 7-ethoxyresorufin and sulfaphenazole and the gap junction inhibitor palmitoleic acid, but not 18alpha-glycyrrhetinic acid, inhibited thapsigargin vasodilation in normotensive lungs. None of these agents inhibited the vasodilation in hypertensive lungs. Thus tonic EDHF activity does not regulate either normotensive or hypertensive pulmonary vascular tone or acute hypoxic vasoconstriction. Whereas thapsigargin-induced EDHF-mediated vasodilation in normotensive rat lungs involves CYP450 activity and might act through gap junctions, the mechanism of vasodilation is apparently different in hypertensive lungs.

Topics: Animals; Biological Factors; Blood Pressure; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Gap Junctions; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Rats; Rats, Sprague-Dawley; Thapsigargin; Vasoconstriction; Vasodilation

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2 sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2 on the cytosolic Ca2+ concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]i in control but not PHTN PA SMC. In control PA SMC, [Ca2+]i increased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+ channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCa blockade had no effect, whereas Kv blockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ ATPase activity caused a greater increase in [Ca2+]i in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]i in PHTN compared with control PA SMC. KCa channel mRNA is decreased and Kv channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2 sensing, alters intracellular Ca2+ homeostasis, and changes the predominant ion channel that determines basal [Ca2+]i from KCa to Kv.

Topics: Animals; Blood Proteins; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Cytoplasm; Enzyme Inhibitors; Female; Fetal Diseases; Fetus; Hypertension, Pulmonary; Hypoxia; Muscle, Smooth, Vascular; Oxygen; Peptides; Potassium; Potassium Channels; Pregnancy; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sheep; Thapsigargin

To investigate whether the retina of the rat exerts a vasodilatory influence by the release of a relaxing factor and to characterize the retinal relaxing factor (RRF).. The relaxing influence of the rat retina was investigated by placing the retina in close proximity with a precontracted isolated rat carotid artery ring segment, mounted for isometric tension measurements.. Application of rat retina relaxed the artery in a reliable and reproducible way. The nitric oxide (NO)-synthase inhibitor N(omega)-nitro-L-arginine (L-NA), the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the removal of the endothelium of the artery all failed to affect the RRF response. The RRF response was not decreased; in contrast, it increased after treatment with a cyclooxygenase (COX) inhibitor (indomethacin or sodium diclofenac). Acute hypoxia largely enhanced retina-induced relaxation. Several potential mediators of hypoxia-induced vasodilation were excluded as candidates for the RRF or for mediating the enhanced response to RRF in hypoxia. Inhibition of the plasma membrane Ca(2+)-adenosine triphosphatase (ATPase) with vanadate significantly affected the RRF response.. The release of an as yet unidentified relaxing factor(s) from the rat retina was demonstrated. Acute hypoxia profoundly enhances the RRF response. None of the known mediators of hypoxia-induced vasodilation nor NO, prostanoids, or endothelial factors mediate the RRF response. Activation of the plasma membrane Ca(2+)-ATPase seems to be involved in the RRF response.

Topics: Acute Disease; Animals; Calcium-Transporting ATPases; Carotid Arteries; Cyclooxygenase Inhibitors; Diclofenac; Endothelium, Vascular; Enzyme Inhibitors; Female; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Indomethacin; Isometric Contraction; Nitroarginine; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Retina; Sarcoplasmic Reticulum; Thapsigargin; Vasodilation; Vasodilator Agents

Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.

Topics: 3T3 Cells; Animals; Cobalt; eIF-2 Kinase; Endoplasmic Reticulum; Enzyme Activation; Eukaryotic Initiation Factor-2; Fibroblasts; Gene Deletion; Genes, Dominant; HeLa Cells; Homozygote; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Kinetics; Methionine; Mice; Models, Biological; Oxygen; Phosphorylation; Plasmids; Protein Biosynthesis; Protein Folding; Proteins; Serine; Thapsigargin; Time Factors; Transcription Factors; Transfection

Studies of thapsigargin, cyclopiazonic acid, and ryanodine in isolated pulmonary arteries and smooth muscle cells suggest that release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))- and/or ryanodine-sensitive sarcoplasmic reticulum Ca(2+) stores is a component of the mechanism of acute hypoxic pulmonary vasoconstriction (HPV). However, the actions of these agents on HPV in perfused lungs have not been reported. Thus we tested effects of thapsigargin and cyclopiazonic acid, inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase, and of ryanodine, an agent that either locks the ryanodine receptor open or blocks it, on HPV in salt solution-perfused rat lungs. After inhibition of cyclooxygenase and nitric oxide synthase, thapsigargin (10 nM) and cyclopiazonic acid (5 microM) augmented the vasoconstriction to 0% but not to 3% inspired O(2). Relatively high concentrations of ryanodine (100 and 300 microM) blunted HPV in nitric oxide synthase-inhibited lungs. The results indicate that release of Ca(2+) from the ryanodine-sensitive, but not the IP(3)-sensitive, store, contributes to the mechanism of HPV in perfused rat lungs and that Ca(2+)-ATPase-dependent Ca(2+) buffering moderates the response to severe hypoxia.

Topics: Animals; Calcium; Enzyme Inhibitors; Hypoxia; Indoles; Male; Nitric Oxide Synthase; Nitroarginine; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Ryanodine; Thapsigargin; Vasoconstriction

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.

Topics: Adenosine Triphosphate; Animals; Antimetabolites; Aorta; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Deoxyglucose; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endothelium, Vascular; Enzyme Inhibitors; Glycolysis; Hypoxia; Ischemia; Macrocyclic Compounds; Oxazoles; Sodium Cyanide; Swine; Thapsigargin

Hypoxic vasoconstriction (HV) is an intrinsic response of mammalian pulmonary and cyclostome aortic vascular smooth muscle. The present study examined the utilization of calcium during HV in dorsal aortas (DA) from sea lamprey and New Zealand hagfish. HV was temporally correlated with increased free cytosolic calcium (Ca2+c) in lamprey DA. Extracellular calcium (Ca2+o) did not contribute significantly to HV in lamprey DA, but it accounted for 38.1 +/- 5.3% of HV in hagfish DA. Treatment of lamprey DA with ionomycin, ryanodine, or caffeine added to thapsigargin-reduced HV, whereas HV was augmented by BAY K 8644. Methoxyverapamil (D600) in zero Ca2+o did not affect HV in lamprey DA, nor did it prevent further constriction when Ca2+o was restored during hypoxia in hagfish DA. Removal of extracellular sodium (Na+o) caused a constriction in both species. Lamprey DA relaxed to prehypoxic tension following return to normoxia in zero Na+o, whereas relaxation was inhibited in hagfish DA. Relaxation following HV was inhibited in lamprey DA when Na+o and Ca2+o were removed. These results show that HV is correlated with [Ca2+]c in lamprey DA and that Na+/Ca2+ exchange is used during HV in hagfish but not lamprey DA. Multiple receptor types appear to mediate stored intracellular calcium release in lamprey DA, and L-type calcium channels do not contribute significantly to constriction in either cyclostome.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Aorta; Caffeine; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Enzyme Inhibitors; Gallopamil; Hagfishes; Hypoxia; In Vitro Techniques; Indoles; Ionomycin; Ionophores; Lampreys; Muscle, Smooth, Vascular; Norepinephrine; Oxygen; Ryanodine; Thapsigargin; Vasoconstriction; Vasodilator Agents

Hypoxia induces bronchodilation in vivo and in vitro, but the mechanisms are still unclear. To evaluate whether an extra- or intracellular free Ca(2+) ion is involved in the mechanisms of hypoxic relaxation, we simultaneously measured cytosolic Ca(2+)levels and tensions in both intact and denuded guinea-pig tracheal strips precontracted with histamine (100 microM), and assessed the effect of hypoxia on guinea-pig tracheal rings precontracted with okadaic acid (10 microM) and calyculin-A (0.1 approximately 10 microM) under an extracellular Ca(2+)-free state. The exposure of tracheal rings to hypoxia induced an immediate decrease of tracheal tension without decrease in intracellular free Ca(2+)levels. In the presence of okadaic acid but not calyculin-A, hypoxic air exposure caused significant transient reductions in tracheal tone. Further, thapsigargin (5 microM or 10 microM) did not affect hypoxic bronchodilation, suggesting that the release of intracellular Ca(2+) does not take a role in hypoxic bronchodilation. Hypoxic dilation decreased ATP content in epithelium-intact rings but not epithelium-denuded rings, indicating a relationship between hypoxic dilation and change of adenine nucleotide in epithelium-intact rings. Our findings indicate that the epithelium dependent mechanisms of hypoxic relaxation of guinea pig tracheal rings preconstricted with histamine may not be related to the mobilization of extra and intra-cellular Ca(2+).

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Enzyme Inhibitors; Guinea Pigs; Hypoxia; Male; Marine Toxins; Muscle Relaxation; Muscle, Smooth; Okadaic Acid; Oxazoles; Oxygen; Thapsigargin; Trachea

It has been proposed that hypoxic pulmonary vasoconstriction (HPV) is mediated via K+ channel inhibition and Ca2+ influx through voltage-gated channels. HPV depends strongly on the degree of preconstriction, and we therefore examined the effect of Ca2+ channel blockade on tension and intracellular [Ca2+] ([Ca2+]i) during HPV in rat intrapulmonary arteries (IPAs), whilst maintaining preconstriction constant. We also investigated the role of intracellular Ca2+ stores. HPV demonstrated a transient constriction (phase I) superimposed on a sustained constriction (phase II). Nifedipine (1 microM) partially inhibited phase I, but did not affect phase II. In arteries exposed to 80 mM K+ and nifedipine or diltiazem the rises in tension and [Ca2+]i were blunted during phase I, but were unaffected during phase II. At low concentrations (< 3 microM), La3+ almost abolished the phase I constriction and rise in [Ca2+]i, but had no effect on phase II, or constriction in response to 80 mM K+. Phase II was inhibited by higher concentrations of La3+ (IC50 approximately 50 microM). IPA treated with thapsigargin (1 microM) in Ca2+-free solution to deplete Ca2+ stores showed sustained constriction upon re-exposure to Ca2+ and an increase in the rate of Mn2+ influx, suggesting capacitative Ca2+ entry. The concentration dependency of the block of constriction by La3+ was similar to that for phase I of HPV. Pretreatment of IPA with 30 microM CPA reduced phase I by > 80 %, but had no significant effect on phase II. We conclude that depolarization-mediated Ca2+ influx plays at best a minor role in the transient phase I constriction of HPV, and is not involved in the sustained phase II constriction. Instead, phase I appears to be mainly dependent on capacitative Ca2+ entry related to release of thapsigargin-sensitive Ca2+ stores, whereas phase II is supported by Ca2+ entry via a separate voltage-independent pathway.

Topics: Animals; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels; Chelating Agents; Diltiazem; Dinucleoside Phosphates; Egtazic Acid; Electric Stimulation; Enzyme Inhibitors; Hypoxia; Imidazoles; In Vitro Techniques; Ion Channel Gating; Lanthanum; Male; Membrane Potentials; Nifedipine; Phosphodiesterase Inhibitors; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Wistar; Ryanodine; Thapsigargin; Vasoconstriction; Verapamil

Rat pulmonary arterial rings (phenylephrine pre-contracted), were relaxed by carbachol or thapsigargin, or were contracted by Nomega-nitro-L-arginine methyl ester (L-NAME). Mild hypoxia (41 mm Hg) attenuated the carbachol-induced relaxation, whereas the relaxant and contractile effects produced by thapsigargin and L-NAME were unaffected. More severe hypoxia (20 mm Hg) abolished thapsigargin-induced relaxation, with no further change in responses to carbachol or L-NAME. At 7 mm Hg, carbachol-induced relaxation was completely inhibited, and the L-NAME-induced contraction was attenuated but not abolished. The present data is consistent with the conclusion that nitric oxide (NO) synthase activity is less susceptible to oxygen deprivation under basal conditions than during activation.

Topics: Animals; Carbachol; Hypoxia; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Phenylephrine; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thapsigargin

A reduction in oxygen tension in the lungs is believed to inhibit a voltage-dependent K+ (Kv) current, which is thought to result in membrane depolarization leading to hypoxic pulmonary vasoconstriction (HPV). However, the direct mechanism by which hypoxia inhibits Kv current is not understood.. Experiments were performed on rat pulmonary artery resistance vessels and single smooth muscle cells isolated from these vessels to examine the role of Ca2+ release from intracellular stores in initiating HPV. In contractile experiments, hypoxic challenge of endothelium-denuded rat pulmonary artery resistance vessels caused either a sustained or transient contraction in Ca2+-containing or Ca2+-free solution, respectively (n=44 vessels from 11 animals). When the ring segments were treated with either thapsigargin (5 micromol/L), ryanodine (5 micromol/L), or cyclopiazonic acid (5 micromol/L) in Ca2+-containing or Ca2+-free solution, a significant increase in pulmonary arterial tone was observed (n=44 vessels from 11 animals). Subsequent hypoxic challenge in the presence of each agent produced no further increase in tone (n=44 vessels from 11 animals). In isolated pulmonary resistance artery cells loaded with fura 2, hypoxic challenge, thapsigargin, ryanodine, and cyclopiazonic acid resulted in a significant increase in [Ca2+]i (n=18 cells from 6 animals) and depolarization of the resting membrane potential (n=22 cells from 6 animals). However, with prior application of thapsigargin, ryanodine, or cyclopiazonic acid, a hypoxic challenge produced no further change in [Ca2+]i (n=18 from 6 animals) or membrane potential (n=22 from 6 animals). Finally, application of an anti-Kv1.5 antibody increased [Ca2+]i and caused membrane depolarization. Subsequent hypoxic challenge resulted in a further increase in [Ca2+]i with no effect on membrane potential (n=16 cells from 4 animals).. In rat pulmonary artery resistance vessels, an initial event in HPV is a release of Ca2+ from intracellular stores. This rise in [Ca2+]i causes inhibition of voltage-dependent K+ channels (possibly Kv1.5), membrane depolarization, and an increase in pulmonary artery tone.

Topics: Animals; Calcium; Hypoxia; In Vitro Techniques; Indoles; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Male; Membrane Potentials; Pulmonary Artery; Rats; Ryanodine; Thapsigargin; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

To test the hypothesis that Ca2+ is released from intracellular store in the carotid body glomus cells during hypoxia, we stimultaneously measured chemosensory discharge and tissue PO2 of perfused-superfused cat carotid body before and during flow interruption in the presence and absence of extracellular [Ca2+] with and without thapsigargin (1-10 microM). Ca(2+)-free solution increased the latency of sensory response, and decreased the rate of rise and peak activity but thapsigargin significantly influenced these responses, without affecting oxygen consumption. Since thapsigargin depletes the intracellular Ca2+ store, and since Ca2+ is needed for the sensory discharge, these results suggest that intracellular release and influx of Ca2+ occur during hypoxia.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Carotid Body; Cats; Chemoreceptor Cells; Extracellular Space; Hypoxia; Intracellular Membranes; Oxygen Consumption; Thapsigargin

This study addressed the controversy of whether endothelium-derived nitric oxide (NO) activity is increased or decreased in the hypertensive pulmonary vasculature of chronically hypoxic rats. Thapsigargin, a receptor-independent Ca2+ agonist and stimulator of endothelial NO production, was used to compare NO-mediated vasodilation in perfused lungs and conduit pulmonary artery rings isolated from adult male rats either kept at Denver's altitude of 5,280 ft (control pulmonary normotensive rats) or exposed for 4-5 wk to the simulated altitude of 17,000 ft (chronically hypoxic pulmonary hypertensive rats). Under baseline conditions, thapsigargin (10(-9)-10(-7) M) caused vasodilation in hypertensive lungs and vasoconstriction in normotensive lungs. Whereas the sustained vasodilation in hypertensive lungs was reversed to vasoconstriction by the inhibitor of NO synthase N(omega)-nitro-L-arginine (L-NNA; 10(-4) M), a transient vasodilation to thapsigargin in acutely vasoconstricted normotensive lungs was potentiated. As measured by a chemiluminescence assay, the recirculated perfusate of hypertensive lungs accumulated considerably higher levels of NO-containing compounds that did normotensive lungs, and thapsigargin-induced stimulation of NO-containing compounds accumulation was greater in hypertensive than in normotensive lungs. Similarly, low concentrations of thapsigargin (10(-10)-10(-9) M) caused greater endothelium-dependent L-NNA-reversible relaxation of hypertensive than of normotensive pulmonary artery rings. The increased sensitivity of hypertensive arteries to thapsigargin-induced relaxation was eliminated in nominally Ca(2+)-free medium and was not mimicked by ryanodine, a releaser of intracellular Ca2+. These results with thapsigargin, which acts on endothelial cells to stimulate Ca2+ influx and a sustained rise in intracellular Ca2+ concentration, support the idea that pulmonary vascular endothelium-derived NO activity is increased rather than decreased in chronic hypoxia-induced pulmonary hypertension in rats.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypertension; Hypoxia; Male; Nitric Oxide; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thapsigargin; Time Factors

The effects of hypoxia on the cytosolic Ca2+ concentration, [Ca2+]i, were characterized in cultured pulmonary arterial smooth muscle (PASM) cells. Reducing O2 tension (PO2) from 150 to < 25 Torr induced a reversible 100-200% increase in [Ca2+]i that was characterized by two components: an early rise in [Ca2+]i that was dependent on the rate, as well as the magnitude, of decline in PO2 and a later, steady-state increase that was independent of the rate at which PO2 changed. Caffeine lowered [Ca2+]i during normoxia and blocked the early component of the response to hypoxia, whereas the steady-state hypoxic response was only partially inhibited. Like hypoxia, thapsigargin (TG) elevated [Ca2+]i, and there was no additional hypoxia-induced elevation in [Ca2+]i at any time after exposure to TG. At steady state, the hypoxic responses were completely reversed by removal of extracellular Ca2+, whereas, on average, verapamil and nifedipine attenuated the hypoxia-induced increases in [Ca2+]i by only 44 and 35%, respectively. These results suggest that hypoxia-induced elevation of [Ca2+]i in PASM cells consists of an early release of Ca2+ from the sarcoplasmic reticulum and a later influx of extracellular Ca2+, in part, through nifedipine- and verapamil-insensitive Ca2+ channels. The results are consistent with the idea that hypoxia and thapsigargin may share common mechanisms for tonically increasing [Ca2+]i.

Topics: Acute Disease; Animals; Caffeine; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cells, Cultured; Cytosol; Fura-2; Hypoxia; Male; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Nifedipine; Oxygen; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Terpenes; Thapsigargin; Verapamil

The ability of hypoxic-reoxygenated cardiomyocytes to recover from severe cytosolic Ca2+ overload was investigated using the fluorescent Ca2+ indicator fura-2 in ventricular cardiomyocytes from adult rats. When the fura-2 ratio (340/380 nm) reached saturation in hypoxic cardiomyocytes, indicating severe Ca2+ overload, they were reoxygenated. The cell then suddenly hypercontracted but reestablished, after a phase of Ca2+ oscillations, a normal Ca2+ control. Because these oscillations could be abolished by ryanodine (50 nM), they seem to depend on the function of the sarcoplasmic reticulum (SR). In the presence of caffeine (5 mM) and thapsigargin (100 nM), i.e., agents impairing Ca2+ sequestration in the SR, reoxygenation did not lead to Ca2+ oscillations or to a stable recovery of cytosolic Ca2+ control. The additional presence of ruthenium red (5 microM), an inhibitor of mitochondrial Ca2+ uptake, restored the ability of cells treated with caffeine or thapsigargin to reestablish a normal cytosolic Ca2+ control. The results show that cardiomyocytes are able to recover from severe hypoxic Ca2+ overload if, first, a closed sarcolemma is retained (as in isolated cardiomyocytes) and, second, the SR is available for rapid Ca2+ storage (impaired by caffeine and thapsigargin). The results also suggest that, in the case of an impairment of SR function, the inhibition of mitochondrial Ca2+ uptake (as by ruthenium red) has a protective effect.

Topics: Animals; Caffeine; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Hypoxia; Male; Myocardial Contraction; Myocardium; Oxygen; Rats; Reference Values; Ruthenium Red; Terpenes; Thapsigargin

We have investigated overlapping activation pathways for two families of stress genes that are expressed in cells exposed to hypoxia. The growth arrest and DNA damage (gadd) genes are induced by DNA damage and irradiation, and their expression is associated with growth arrest. The glucose-regulated proteins (GRPs) are induced by chemical agents that disrupt protein trafficking in the endoplasmic reticulum such as tunicamycin and A23187 and by hypoxia. Here, we demonstrate that the treatment of NIH-3T3 cells with chemical inducers of GRPs results in increased levels of gadd45 and gadd153 mRNA as well as GRP78 mRNA. In addition, hypoxia was also able to increase gadd45, gadd153, and GRP78 mRNA. Therefore the GRP and gadd genes can be activated by similar stimuli (e.g., hypoxia and chemical inducers). However, the mechanisms leading to increased levels of GRP78 and gadd gene mRNA are different and may involve distinct protein kinases. Increased expression of GRPs after treatment with chemical inducers is sensitive to cycloheximide and the protein kinase inhibitors genistein, 2-aminopurine, and H7, whereas the increase in gadd gene mRNA could be blocked by the protein kinase inhibitors H7 and 2-aminopurine but not by genistein or cycloheximide. GRP78 induction occurs by a pathway that requires protein synthesis and is sensitive to genistein, H7, and 2-aminopurine, whereas gadd gene induction is independent of protein synthesis and is inhibited by H7 and 2-aminopurine only.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; DNA Damage; Endoplasmic Reticulum Chaperone BiP; HSP70 Heat-Shock Proteins; Hypoxia; Isoquinolines; Membrane Proteins; Mice; Piperazines; RNA, Messenger; Terpenes; Thapsigargin; Tunicamycin