ABSTRACT
Ozone is used to treat several medical conditions, while the underlying mechanisms of action are sometimes poorly understood. In the current study, we exposed cultured human epithelial (HeLa) cells acutely and repeatedly to ozone and investigated the effects thereof on cell viability. The involvement of anti-apoptotic pathways in observed adaptive responses to ozone were investigated by employing the Akt inhibitor (-)-deguelin. Cells were exposed to an ozone-saturated physiological solution using various dosing regimens, including acute exposure and various repetitive exposures. Cell viability was determined with Trypan Blue or MTT tests, or by a DNA-fragmentation (comet) assay. Acute ozone exposure compromised cell membrane integrity severely, while adaptation to reverse an initial reduction in mitochondrial activity was observed. Repetitive, short-duration exposures followed by a single long-duration exposure to ozone furnished a protective adaptation that was reversed by Akt inhibition. Extracellular and intracellular damage (and adaptation) occurs differentially. While acute ozone may decrease cell viability, multiple preexposures up-regulates cellular plasticity via induction of anti-apoptotic pathways in a treatment regimen-specific manner.
Subject(s)
Adaptation, Physiological , Apoptosis/drug effects , Cell Survival , DNA/metabolism , Oxidants, Photochemical/pharmacology , Ozone/pharmacology , Cell Membrane/drug effects , HeLa Cells/drug effects , Humans , Mitochondria/drug effects , Mitochondria/metabolism , NF-kappa B/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Rotenone/analogs & derivatives , Rotenone/pharmacology , Trypan BlueABSTRACT
Many irreversible antagonists have been shown to inactivate G protein-coupled receptors (GPCRs) and used to study agonists and spare receptors. Presumably, they bind to primary (agonist) binding sites on the GPCR, although noncompetitive mechanisms of antagonism have been demonstrated but not thoroughly investigated. We studied noncompetitive antagonism by phenoxybenzamine and benextramine at alpha(2A)-adrenoceptors in stably transfected Chinese hamster ovary cells, benextramine and 4-diphenylacetoxy-N-[2-chloroethyl]piperidine hydrochloride (4-DAMP mustard) at endogenous muscarinic acetylcholine (mACh) receptors in human neuroblastoma SH-SY5Y cells, and benextramine at serotonin 5-HT(2A) receptors in stably transfected SH-SY5Y cells. Primary binding sites were protected by reversible competitive antagonists during pretreatment with irreversible antagonists. We conducted appropriate radioligand binding assays by measuring remaining primary binding sites and agonist affinity, functional assays to evaluate agonist-induced responses, and constitutive guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS)-Galpha(o) binding assays to determine remaining G protein activity. Phenoxybenzamine (100 microM; 20 min) and benextramine (10 or 100 microM; 20 min) at alpha(2A)-adrenoceptors, but not 4-DAMP mustard (100 nM; 120 min) at mACh receptors, displayed irreversible noncompetitive antagonism in addition to their known irreversible competitive antagonism. Although agonist binding affinity is not influenced, signal transduction is modulated in a G protein-dependent manner via allotopic interactions. Benextramine noncompetitively inhibits agonist-induced responses at three different GPCR types (alpha(2A), mACh, and 5-HT(2A) receptors) that signal via three families of G proteins (G(i/o), G(s), and G(q/11)). We conclude that, where irreversible antagonists are utilized to study drug-receptor interaction mechanisms, the presence of significant irreversible noncompetitive antagonism may influence the interpretation of results under the experimental conditions used.
