r-59949 and Hypoxia

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.. We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.. OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV.. These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Diacylglycerol Kinase; Diglycerides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Hypoxia; In Vitro Techniques; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Perfusion; Piperidines; Pulmonary Circulation; Pyrrolidinones; Quinazolinones; Signal Transduction; Time Factors; TRPC Cation Channels; TRPC6 Cation Channel; Type C Phospholipases; Vasoconstriction; Vasoconstrictor Agents

Hypoxia-inducible factor 1 (HIF-1) induces a gene expression program essential for the cellular adaptation to lowered oxygen environments. The intracellular mechanisms by which hypoxia induces HIF-1 remain poorly understood. Here we show that exposure of various cell types to hypoxia raises the intracellular level of phosphatidic acid primarily through the action of diacylglycerol kinase (DGK). Pharmacological inhibition of DGK activity through use of the specific DGK inhibitors and abrogated specifically HIF-1-dependent transcription analyzed with a HIF-1-responsive reporter plasmid. A more detailed analysis revealed that pharmacological inhibition of DGK activity prevented the hypoxia-dependent accumulation of the HIF-1alpha subunit and the subsequent HIF-1-DNA complex formation as well as hypoxia-induced activity of the HIF-1 transactivation domains localized to amino acids 530-582 and 775-826 of the HIF-1alpha subunit. Our results demonstrate for the first time that accumulation of phosphatidic acid through DGK underlines oxygen sensing and provide evidence for the involvement of this lipid kinase in the intracellular signaling that leads to HIF-1 activation.

Topics: Cell Nucleus; Diacylglycerol Kinase; Diglycerides; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; HeLa Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Luciferases; Nuclear Proteins; Oxygen; Phosphatidic Acids; Phospholipids; Piperidines; Plasmids; Pyrimidinones; Quinazolines; Quinazolinones; Recombinant Proteins; Signal Transduction; Thiazoles; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection