cyclic-gmp and fluorexon

The aim of the present study was to evaluate the effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) on multidrug resistance. Expression of human P-glycoprotein was assessed by realtime quantitative RT-PCR and western blot. The efflux function of P-glycoprotein was evaluated by rhodamine 123 accumulation and calcein-AM uptake models. The mechanisms of action of YC-1 on different signaling pathways were studied using series of antagonists and the kinetics was also assessed. Cytotoxicity was evaluated by MTT assay. The results demonstrated that increased intracellular accumulation of rhodamine 123 and increased fluorescence of calcein were observed after YC-1 treatment. Furthermore, increased YC-1 concentration resulted in significant decrease in Vmax while K(M) remained unchanged suggested that YC-1 acted as a noncompetitive inhibitor of P-glycoprotein. Moreover, the inhibition of Pgp efflux function by YC-1 was significantly reversed by NO synthase inhibitor, (L)-NAME, the sGC inhibitor, ODQ, the PKG inhibitor, Rp-8-Br-PET-cGMPS, and the PKG inhibitor KT5823. In addition, ERK kinase inhibitor PD98059 also significantly restored YC-1 inhibited Pgp efflux function. These results indicated that YC-1 inhibited Pgp efflux via the NO-cGMP-PKG-ERK signaling pathway through noncompetitive inhibition. The present study revealed that YC-1 could be a good candidate for development as a MDR modulator.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Western; Cell Line; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Extracellular Signal-Regulated MAP Kinases; Fluoresceins; Fluorescent Dyes; Gene Expression Regulation; Humans; Indazoles; Nitric Oxide; Reverse Transcriptase Polymerase Chain Reaction; Rhodamine 123; Signal Transduction

Increased hemichannel opening induced by oxygen glucose deprivation (OGD) was reported in the hippocampal pyramidal neuron. It was suggested that the pannexin1 hemichannel opening could mediate ionic flux dysregulation, anoxic depolarization, and energy-depleting efflux of glucose and ATP for ischemic neurons. However, the regulatory mechanisms of pannexin1 hemichannel opening have been poorly understood. Here we showed that excessive generation of nitric oxide (NO) during ischemia could induce the calcein leakage from neurons, which was markedly reduced by NO synthase inhibitor. The calcein leakage from neurons during OGD was also attenuated by the application of N-ethylmaleimide (NEM), an SH-alkylating agent, and dithiothreitol (DTT), a reducer of oxidized sulfhydryl groups. However, the soluble guanylyl cyclase (sGC) inhibitor had a minor effect on the calcein leakage during OGD. Furthermore, the elevated intracellular but not extracellular levels of glutathione could also inhibit the calcein leakage during OGD. Similar results were observed in metabolic inhibition (MI), which is another ischemic-like condition. Finally, immunocytochemical and immunoblotting analysis revealed that, after 1 hr of OGD stimulation, the distribution and expression of pannexin1 showed no significant difference compared with control. However, the pannexin1 mRNA expression was elevated after 1 hr of OGD and a sustained increase was maintained during reperfusion. These results implied that the reactive oxygen species (ROS), especially NO, might be involved in the enhanced pannexin1 hemichannel opening and that the S-nitrosylation but not the NO/cGMP pathway played a more important role in this event.

Topics: Animals; Blotting, Western; Cell Hypoxia; Cell Membrane Permeability; Connexins; Cyclic GMP; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Glucose; Hippocampus; Immunohistochemistry; Nerve Tissue Proteins; Neurons; Nitric Oxide; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction