Dexmedetomidine attenuates the positive chronotropic effects of intravenous atropine in patients with bradycardia during spinal anaesthesia: a retrospective study.

INTRODUCTION: Dexmedetomidine is a sedative used during spinal anaesthesia. However, it frequently induces bradycardia. Although intravenous atropine is often used for treating bradycardia during regional anaesthesia, the response to atropine might be attenuated by concomitantly administering sedatives. METHODS: We examined the effects of atropine used for treating bradycardia during spinal anaesthesia among patients receiving dexmedetomidine (D group), propofol (P group), or neither (nonDnonP group) for sedation, retrospectively. RESULTS: A total of 108 patients were included. Heart rate was significantly slower at all time points in the D group (n = 69) than in the nonDnonP group (n = 14) (p <  0.025 for all). On the other hand, heart rate was significantly slower only 60 min after administration of atropine in the P group (n = 25) than in the nonDnonP group (p = 0.002). There were differences in the overall values of heart rate (including all the values from time 0 to 60 min) among the three groups (p = 0.026). CONCLUSIONS: The positive chronotropic effects of atropine might be attenuated with the use of dexmedetomidine or propofol during spinal anaesthesia. Although atropine may be administered when bradycardia occurs, a dose of atropine might result in an insufficient effect against the bradycardia. The sufficient number of subjects may change the results of the investigation, and large-scale randomised controlled trials will be necessary.

[Thyroid Storm Precipitated by Intracranial Surgery in a Woman with No History of Thyroid Disease.]

Severe medical stress can trigger thyroid storm, an endocrine emergency that affects consciousness. This case report describes a 43-year-old female patient transferred to our hospital with right motor hemipare- sis. Her medical history included untreated hyperten- sion, but no history of thyroid disease. Emergency magnetic resonance imaging (MRI)of the head revealed bilateral moyamoya disease and ischemic stroke in the right watershed region of the brain. The right superfi- cial temporal artery was anastomosed to the middle cerebral artery on day 71 of hospitalization due to repeated brain infarctions after admission. Although anesthesia was without incident, the patient gradually developed postoperative disturbed consciousness, hypertension and tachycardia, which we considered were the result of craniotomy or insufficient brain per- fusion, until a nurse found a swelling on the patient's neck. Hyperthyroidism was confirmed by laboratory data and an endocrinologist concluded that thyroid storm had caused her symptoms. Thyroid storm should be considered in the differential diagnosis of patients who present with disturbed consciousness after intracranial surgery.

Differential roles of prostaglandin E-type receptors in activation of hypoxia-inducible factor 1 by prostaglandin E1 in vascular-derived cells under non-hypoxic conditions.

Prostaglandin E1 (PGE1), known pharmaceutically as alprostadil, has vasodilatory properties and is used widely in various clinical settings. In addition to acute vasodilatory properties, PGE1 may exert beneficial effects by altering protein expression of vascular cells. PGE1 is reported to be a potent stimulator of angiogenesis via upregulation of VEGF expression, which is under the control of the transcription factor hypoxia-inducible factor 1 (HIF-1). However, the molecular mechanisms behind the phenomenon are largely unknown. In the present study, we investigated the mechanism by which PGE1 induces HIF-1 activation and VEGF gene expression in human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs), both vascular-derived cells. HUVECs and HASMCs were treated with PGE1 at clinically relevant concentrations under 20% O2 conditions and HIF-1 protein expression was investigated. Expression of HIF- 1α protein and the HIF-1-downstream genes were low under 20% O2 conditions and increased in response to PGE1 treatment in both HUVECs and HASMCs in a dose- and time-dependent manner under 20% O2 conditions as comparable to exposure to 1% O2 conditions. Studies using EP-receptor-specific agonists and antagonists revealed that EP1 and EP3 are critical to PGE1-induced HIF-1 activation. In vitro vascular permeability assays using HUVECs indicated that PGE1 increased vascular permeability in HUVECs. Thus, we demonstrate that PGE1 induces HIF- 1α protein expression and HIF-1 activation under non-hypoxic conditions and also provide evidence that the activity of multiple signal transduction pathways downstream of EP1 and EP3 receptors is required for HIF-1 activation.

Hydrogen sulfide inhibits hypoxia- but not anoxia-induced hypoxia-inducible factor 1 activation in a von hippel-lindau- and mitochondria-dependent manner.

AIMS: In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H(2)S) is an endogenously synthesized gaseous molecule that acts as an important signaling molecule in the living body. Transcription factor hypoxia-inducible factor 1 (HIF-1) is known to respond to intracellular reduced oxygen (O(2)) availability, which is regulated by an elaborate balance between O(2) supply and demand. However, the effect of H(2)S on HIF-1 activity under hypoxic conditions is largely unknown in mammalian cells. In this study, we tried to elucidate the effect of H(2)S on hypoxia-induced HIF-1 activation adopting cultured cells and mice. RESULTS: The H(2)S donors sodium hydrosulfide and sodium sulfide in pharmacological concentrations reversibly reduced cellular O(2) consumption and inhibited hypoxia- but not anoxia-induced HIF-1α protein accumulation and expression of genes downstream of HIF-1 in established cell lines. H(2)S did not affect HIF-1 activation induced by the HIF-α hydroxylases inhibitors desferrioxamine or CoCl(2). Experimental evidence adopting von Hippel-Lindau (VHL)- or mitochondria-deficient cells indicated that H(2)S did not affect neosynthesis of HIF-1α protein but destabilized HIF-1α in a VHL- and mitochondria-dependent manner. We also demonstrate that exogenously administered H(2)S inhibited HIF-1-dependent gene expression in mice. INNOVATION: For the first time, we show that H(2)S modulates intracellular O(2) homeostasis and regulates activation of HIF-1 and the subsequent gene expression induced by hypoxia by using an in vitro system with established cell lines and an in vivo system in mice. CONCLUSIONS: We demonstrate that H(2)S inhibits hypoxia-induced HIF-1 activation in a VHL- and mitochondria-dependent manner.

Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice under non-hypoxic conditions in a µ-opioid receptor-dependent manner.

Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and ß antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-µM concentration of fentanyl and other opioids, including 1 µM morphine and 4 µM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia.

Unexpectedly difficult intubation caused by subglottic stenosis in Wegener's granulomatosis.

A 76-year-old woman was scheduled to undergo abdominal aortic repair for progressive abdominal aortic aneurysm. After inducing general anesthesia, the 7.5-mm internal diameter (ID) tracheal tube could not be advanced below the level of the vocal cords because of resistance, and intubation was re-attempted several times using smaller tubes. An otolaryngologist was consulted and subglottic stenosis of unknown origin was suggested. The aortic repair was cancelled and tracheostomy was performed instead. She was diagnosed with Wegener's granulomatosis 46 days after the operation because she developed symptoms of renal dysfunction, hemoptysis, gastrointestinal bleeding, and presence of anti-neutrophil cytoplasmic autoantibodies (c-ANCA). The patient was treated with steroids but died 89 days after the operation because of pulmonary bleeding and renal dysfunction. Tracheal stenosis is a rare presenting feature of Wegener's granulomatosis that usually occurs late in the disease; however, anesthesiologists around the world need to bear in mind that the disease can present airway symptoms and can be the cause of airway obstruction.

The intravenous anesthetic propofol inhibits lipopolysaccharide-induced hypoxia-inducible factor 1 activation and suppresses the glucose metabolism in macrophages.

PURPOSE: Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of propofol on HIF-1 activity is not well documented. In this study, we investigated the effect of propofol on HIF-1 activation using macrophage-differentiated THP-1 cells. METHODS: Cells were exposed to lipopolysaccharide (LPS) under 20 or 1% O(2) conditions with or without propofol treatment. The cell lysate was subjected to Western blot analysis using anti-HIF-1alpha and HIF-1beta antibodies. HIF-1-dependent gene expression was investigated by quantitative real-time reverse-transcriptase PCR analysis and luciferase assay. The amount of cellular lactate and ATP was assayed. RESULTS: Propofol suppressed HIF-1alpha protein accumulation induced by LPS, but not by hypoxia in the THP-1 cells in a dose-dependent manner by inhibiting the neo-synthesis of HIF-1alpha protein. Induction of the HIF-1 downstream gene expression including glucose transporter 1, enolase 1, lactate dehydrogenase A, pyruvate dehydrogenase kinase-1 and vascular endothelial growth factor was inhibited by propofol. Propofol suppressed LPS-induced lactate accumulation and ATP content in THP-1 cells. CONCLUSION: Our experimental results indicate that propofol inhibits HIF-1 activation and downstream gene expression induced by LPS and suppressed HIF-1-dependent glucose metabolic reprogramming. HIF-1 suppression by propofol in macrophages may explain molecular mechanisms behind the inhibitory effect of propofol on cellular inflammatory responses.

The intravenous anesthetics barbiturates inhibit hypoxia-inducible factor 1 activation.

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of barbiturates on HIF-1 activity has not been reported. In this study, we investigated the effect of thiopental and thiamylal on HIF-1 activity using the neuronal SH-SY5Y cells, the non-neuronal HEK293 cells, and the macrophage-differentiated THP-1 cells. Cells were exposed to 20% or 1% O(2) conditions with or without thiopental or thiamylal treatment. The cell lysate were subjected to Western blot analysis using anti-HIF-1alpha and -HIF-1beta antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time RT-PCR and luciferase assay. Hydroxylation of HIF-1alpha protein was evaluated by in vitro pulldown assay using recombinant protein. Both thiopental and thiamylal reversibly suppressed hypoxia-induced HIF-1 activation in the neuronal and the non-neuronal cells in a dose-dependent manner. Moreover, the barbiturates inhibited lipopolysaccharide-induced HIF-1alpha expression in THP-1 cells. The HIF-1-downstream gene expression was also inhibited by the barbiturates. HIFalpha-hydroxylases activity and HIF-1alpha stability were not affected but the HIF-1alpha protein neosynthesis was inhibited by the barbiturates. Our experimental results indicate that barbiturates inhibit induced HIF-1 activation and downstream genes expression.

The calcium channel blocker cilnidipine selectively suppresses hypoxia-inducible factor 1 activity in vascular cells.

Calcium ion is one of the most important second messengers of cellular signal transduction including hypoxia-elicited signals. In this study, we investigated the effects of the L-type calcium channel blockers such as nifedipine, efonidipine cilnidipine, diltiazem, and verapamil, on the activity of hypoxia-inducible factor-1 (HIF-1), a key transcription factor in control of hypoxia-induced gene expression. Using the lung carcinoma cell line A549 cells, human aortic smooth muscle cells, and human umbilical vein endothelial cells, we demonstrated that cilnidipine exclusively suppressed HIF-1 activity and the expressions of downstream genes in a cell-type specific manner. We also demonstrated that cilnidipine blocked the synthesis of the HIF-1alpha protein not by affecting activity of the intracellular hypoxia-sensing element prolyl hydroxylases but inhibiting activity of Akt and mitogen-activated protein kinase and that the inhibition is not dependent on the effect on calcium homeostasis.

Macrophage migration inhibitory factor activates hypoxia-inducible factor in a p53-dependent manner.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner. METHODS/PRINCIPAL FINDINGS: The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly. CONCLUSION/SIGNIFICANCE: MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF's potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1alpha protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.

LPS induces hypoxia-inducible factor 1 activation in macrophage-differentiated cells in a reactive oxygen species-dependent manner.

A prominent feature of various inflamed and diseased tissue is the presence of low oxygen tension (hypoxia). Effector cells of the innate immune system must maintain their viability and physiologic functions in a hypoxic microenvironment. Monocytes circulating in the bloodstream differentiate into macrophages. During this process, cells acquire the ability to exert effects at hypoxic sites of inflammation. The transcription factor hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability. In this study, we demonstrated that lipopolysaccharide (LPS) induces HIF-1 activation by enhancing both HIF-1alpha protein expression through a translation-dependent pathway and HIF-1alpha transcriptional activity in THP-1 human myeloid cells that have undergone macrophage differentiation but not in undifferentiated monocytic THP-1 cells. LPS-induced HIF-1 activation was blocked by treatment with antioxidant (N-acetylcysteine or thioredoxin-1), NADPH oxidase inhibitor (diphenyleneiodonium), indicating that reactive oxygen species generated in response to LPS are essential in this process. LPS-mediated activation of HIF-1 was independent of NF-kappaB activity. LPS-induced ROS generation and HIF-1 activation required the expression of Toll-like receptor 4 or myeloid differentiation factor (MyD) 88, thus providing a molecular basis for the selective activation of HIF-1 in differentiated THP-1 cells.

n-Propyl gallate activates hypoxia-inducible factor 1 by modulating intracellular oxygen-sensing systems.

HIF-1 (hypoxia-inducible factor 1) is a master regulator of cellular adaptive responses to hypoxia. The expression and transcriptional activity of the HIF-1alpha subunit is stringently controlled by intracellular oxygen tension through the action of prolyl and asparaginyl hydroxylases. In the present study we demonstrate that PG (n-propyl gallate) activates HIF-1 and expression of its downstream target genes under normoxic conditions in cultured cells and in mice. The stability and transcriptional activity of HIF-1alpha are increased by PG. PG treatment inhibits the interaction between HIF-1alpha and VHL (von Hippel-Lindau protein) and promotes the interaction between HIF-1alpha and p300, indicating that PG inhibits the activity of both prolyl and asparaginyl HIF-1alpha hydroxylases. We conclude that PG activates HIF-1 and enhances the resultant gene expression by directly affecting the intracellular oxygen sensing system in vitro and in vivo and that PG represents a lead compound for the development of a non-toxic activator of HIF-1.

Activation of hypoxia-inducible factor 1 during macrophage differentiation.

Monocytes/macrophages of the myeloid lineage are the main cellular effectors of innate immunity. Hypoxia-inducible factor 1 (HIF-1) is essential for myeloid cell activation in response to inflammatory stimuli. However, it has not been established whether HIF-1 activity is induced during differentiation from monocyte to macrophage. We demonstrate that macrophage differentiation of THP-1 cells or monocytes from peripheral blood induces increased expression of both HIF-1alpha and HIF-1beta as well as increased HIF-1 transcriptional activity leading to increased expression of HIF-1 target genes. The increased HIF-1 activity in differentiated THP-1 cells resulted from the combined effect of increased HIF-1alpha mRNA levels and increased HIF-1alpha protein synthesis. Differentiation-induced HIF-1alpha protein and mRNA and HIF-1-dependent gene expression was blocked by treating cells with an inhibitor of the protein kinase C or MAP kinase signaling pathway. THP-1 cell differentiation was also associated with increased phosphorylation of the translational regulatory proteins p70 S6 kinase, S6 ribosomal protein, eukaryotic initiation factor 4E, and 4E binding protein 1, thus providing a possible mechanism for the modulation of HIF-1alpha protein synthesis. RNA interference studies demonstrated that HIF-1alpha is dispensable for macrophage differentiation but is required for functional maturation.

Opioid receptor stimulation does not affect cellular hypoxia-induced gene responses mediated by hypoxia-inducible factor 1 in cultured cell lines.

Hypoxia induces a series of adaptive physiological responses including gene inductions. Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that regulates hypoxia-induced gene expression to maintain homeostasis in the living body. Opioids are potent analgesic agents that are widely used in clinical practice. Therefore, we investigated the effect of opioids on HIF-1 activity. SH-SY5Y human neuronal cells, which express opioid receptors intrinsically, were cultured under 1% or 20% O2 conditions with or without treatment by DAGO, DPDPE, or U-50488, which are the selective agonists of mu-, delta-, and kappa-opioid receptors, respectively. Expression of subunits of HIF-1, HIF-1alpha, and HIF-1beta were examined by Western blot using specific antibodies. Expression of the HIF-1-dependent gene were investigated by reporter assay. None of the selective agonists of opioid receptors tested affected HIF-1 activation by hypoxia. Therefore, it is suggested that opioid receptor-mediated signals do not affect HIF-1-dependent cellular hypoxia-induced gene responses.

The effects of local anesthetics on cellular hypoxia-induced gene responses mediated by hypoxia-inducible factor 1.

PURPOSE: Hypoxia (reduced oxygen availability) induces a series of adaptive physiological responses. At the cellular level, the adaptation includes a switch of energy metabolism from oxidative phosphorylation to anaerobic glycolysis, increased glucose uptake, and the expression of stress proteins related to cell survival. One of the most important transcription factors that activate the expression of oxygen-regulated genes is hypoxia-inducible factor 1 (HIF-1). We previously reported that halothane inhibits the hypoxia-induced HIF-1 activation. In this study, we investigated the effect of local anesthetics on HIF-1 activation and its downstream gene expression. METHODS: The established cell line Hep3B and SK-N-MC cells were exposed to 1% O2 with or without treatment by either lidocaine or bupivacaine. Expression of subunits of HIF-1, HIF-1alpha, and HIF-1beta was examined by Western blot using specific antibodies. Expression of mRNA of HIF-1 and the HIF-1-dependent genes was investigated by RT-PCR and reporter assay. RESULTS: Neither of the local anesthetics tested affected the accumulation of HIF-1alpha induced by hypoxia, nor did they affect NOC18-induced HIF-1alpha accumulation. Moreover, they had no effects on HIF-1-mediated hypoxia-induced gene expression. CONCLUSION: The local anesthetics lidocaine and bupivacaine did not affect the HIF-1-dependent cellular hypoxia-induced gene responses.

The intravenous anesthetic propofol inhibits hypoxia-inducible factor 1 activity in an oxygen tension-dependent manner.

Hypoxia elicits a wide range of responses that occur at different organizational levels in the body. Hypoxia is not only a signal for energy conservation and metabolic change, but triggers expression of a select set of genes. The transcription factor hypoxia-inducible factor 1 (HIF-1) is now appreciated to be a master factor of the gene induction. Although knowledge on molecular mechanisms of HIF-1 activation in response to hypoxia is accumulating, the molecular mechanism of maintenance of HIF-1 activity under normoxic conditions remains to be elucidated. We demonstrate that the intravenous anesthetic propofol reversibly inhibits HIF-1 activity and the gene expression mediated by HIF-1 by blocking the synthesis of the HIF-1alpha subunit under 20% or 5% O2 conditions, but not under 1% O2 conditions.

The inhibitory effect of sodium nitroprusside on HIF-1 activation is not dependent on nitric oxide-soluble guanylyl cyclase pathway.

Adaptation to hypoxia and maintenance of O(2) homeostasis involve a wide range of responses that occur at different organizational levels in the body. One of the most important transcription factors that activate the expression of O(2)-regulated genes is hypoxia-inducible factor 1 (HIF-1). Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels and cytotoxicity of activated macrophages. We investigated the effect of the clinically used nitrates nitroglycerin (NTG), isosorbide dinitrate (ISDN), and sodium nitroprusside (SNP) on HIF-1-mediated transcriptional responses to hypoxia. We demonstrate that among the three nitrates, only SNP inhibits HIF-1 activation in response to hypoxia. In contrast, NTG or ISDN does not affect HIF-1 activity. SNP inhibits the accumulation of HIF-1alpha, the regulatory subunit of HIF-1, and the transcriptional activation of HIF-1alpha via a mechanism that is not dependent on either NO or soluble guanylate cyclase.

Induction of hypoxia-inducible factor 1 activity by muscarinic acetylcholine receptor signaling.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular adaptive responses to hypoxia. Levels of the HIF-1alpha subunit increase under hypoxic conditions. Exposure of cells to growth factors, prostaglandin, and certain nitric oxide donors also induces HIF-1alpha expression under non-hypoxic conditions. We demonstrate that muscarinic acetylcholine signals induce HIF-1alpha expression and transcriptional activity in a receptor subtype-specific manner using HEK293 cells transiently overexpressing each of M1-M4 muscarinic acetylcholine receptors. The muscarinic signaling pathways inhibited HIF-1alpha hydroxylation and degradation and induced HIF-1alpha protein synthesis that was confirmed by pulse labeling studies. Muscarinic signal-induced HIF-1alpha protein and HIF-1-dependent gene expression were blocked by treating cells with inhibitors of phosphatidylinositol 3-kinase, MAP kinase kinase, or tyrosine kinase signaling pathways. Dominant-negative forms of Ras and/or Rac-1 significantly suppressed HIF-1 activation by muscarinic signaling. Signaling via M1- and M3- but not M2- or M4-AchRs promote accumulation and transcriptional activation of HIF-1alpha. We conclude that muscarinic acetylcholine signals activate HIF-1 by both stabilization and synthesis of HIF-1alpha and by inducing the transcriptional activity of HIF-1alpha.

Nitric oxide induces hypoxia-inducible factor 1 activation that is dependent on MAPK and phosphatidylinositol 3-kinase signaling.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular adaptive responses to hypoxia. Levels of the HIF-1alpha subunit increase under hypoxic conditions. Exposure of cells to certain nitric oxide (NO) donors also induces HIF-1alpha expression under nonhypoxic conditions. We demonstrate that exposure of cells to the NO donor NOC18 or S-nitrosoglutathione induces HIF-1alpha expression and transcriptional activity. In contrast to hypoxia, NOC18 did not inhibit HIF-1alpha hydroxylation, ubiquitination, and degradation, indicating an effect on HIF-1alpha protein synthesis that was confirmed by pulse labeling studies. NOC18 stimulation of HIF-1alpha protein and HIF-1-dependent gene expression was blocked by treating cells with an inhibitor of the phosphatidylinositol 3-kinase or MAPK-signaling pathway. These inhibitors also blocked NOC18-induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1alpha protein synthesis. In addition, expression of a dominant-negative form of Ras significantly suppressed HIF-1 activation by NOC18. We conclude that the NO donor NOC18 induces HIF-1alpha synthesis under conditions of NO formation during normoxia and that hydroxylation of HIF-1alpha is not regulated by NOC18.