d-609 and Hypoxia

The aim of the study was to investigate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in hypoxic pulmonary vasoconstriction (HPV) and elucidate its possible interactions within HPV mechanism. Inhibition of PC-PLC with D609 (30μM) resulted in partial reduction of the transient phase and almost complete abolition of the sustained phase of HPV in isolated rat intrapulmonary arteries (IPAs). Intravenous injection of D609 (5mg/kg) 30min before the onset of hypoxia prevented the development of acute hypoxic pulmonary hypertension (AHPH) in rats. D609 also inhibited pulmonary vasoconstriction induced with a generator of superoxide anions LY83583, but not the one induced with hydrogen peroxide. Protein kinase C (PKC) inhibition with Ro-31-8220 partially diminished the transient phase of hypoxic contraction in IPA while the sustained phase remained unchanged. Phosphocholine, known to be released due to phosphatidylcholine breakdown by PC-PLC, induced sustained contraction in isolated IPA and also transient pulmonary and systemic hypertension if administered intravenously (70mg/kg). We conclude that PC-PLC plays an important role in sustained HPV possibly through the activation of PKC-independent mechanism, which may be coupled with phosphocholine release.

Topics: Aminoquinolines; Animals; Bridged-Ring Compounds; Dose-Response Relationship, Drug; Hydrogen Peroxide; Hypoxia; Indoles; Injections, Intravenous; Male; Norbornanes; Phosphorylcholine; Protein Kinase C; Pulmonary Artery; Rats; Rats, Wistar; Thiocarbamates; Thiones; Type C Phospholipases; Vasoconstriction

Cardiac myocytes coexpress multiple protein kinase C (PKC) isoforms which likely play distinct roles in signaling pathways leading to changes in contractility, hypertrophy, and ischemic preconditioning. Although PKC has been reported to be activated during myocardial ischemia, the effect of ischemia/hypoxia on individual PKC isoforms has not been determined. This study examines the effect of hypoxia on the subcellular distribution of individual PKC isoforms in cultured neonatal rat ventricular myocytes. Hypoxia induces the redistribution of PKC alpha and PKC epsilon from the soluble to the particulate compartment. This effect (which is presumed to represent activation of PKC alpha and PKC epsilon) is detectable by 1 h, sustained for up to 24 h, and reversible within 1 h of reoxygenation. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) prevents the hypoxia-induced redistribution of PKC alpha and PKC epsilon, whereas chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) blocks the redistribution of PKC alpha, but not PKC epsilon; D609 and BAPTA do not influence the partitioning of PKC alpha and PKC epsilon in normoxic myocytes. Hypoxia, in contrast, decreases the membrane association of PKC delta via a mechanism that is distinct from the hypoxia-induced translocation/activation of PKC alpha/PKC epsilon, since the response is slower in onset, slowly reversible upon reoxygenation, and not blocked by D609 or BAPTA. The hypoxia-induced shift of PKC delta to the soluble compartment does not prevent subsequent 4-beta phorbol 12-myristate-13-acetate-dependent translocation/activation of PKC delta. Hypoxia does not alter the abundance of any PKC isoform nor does it alter the subcellular distribution of PKC lambda. The selective hypoxia-induced activation of PKC isoforms through a pathway involving phospholipase C (PKC alpha/PKC epsilon) and intracellular calcium (PKC alpha) may critically influence cardiac myocyte contractility, gene expression, and/or tolerance to ischemia.

Topics: Animals; Bridged-Ring Compounds; Calcium; Cell Membrane; Cells, Cultured; Egtazic Acid; Hypoxia; Immunoblotting; Isoenzymes; Myocardium; Norbornanes; Phosphodiesterase Inhibitors; Protein Kinase C; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate; Thiocarbamates; Thiones; Type C Phospholipases