noc-18 and zaprinast

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels couple cellular metabolic status to membrane electrical activity. In this study, we performed patch-clamp recordings to investigate how cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) regulates the function of K(ATP) channels, using both transfected human SH-SY5Y neuroblastoma cells and embryonic kidney (HEK) 293 cells. In intact SH-SY5Y cells, the single-channel currents of Kir6.2/sulfonylurea receptor (SUR) 1 channels, a neuronal-type K(ATP) isoform, were enhanced by zaprinast, a cGMP-specific phosphodiesterase inhibitor; this enhancement was abolished by inhibition of PKG, suggesting a stimulatory role of cGMP/PKG signaling in regulating the function of neuronal K(ATP) channels. Similar effects of cGMP accumulation were confirmed in intact HEK293 cells expressing Kir6.2/SUR1 channels. In contrast, direct application of purified PKG suppressed rather than activated Kir6.2/SUR1 channels in excised, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit were not modulated by zaprinast or purified PKG. Lastly, reconstitution of the soluble guanylyl cyclase/cGMP/PKG signaling pathway by generation of nitric oxide led to Kir6.2/SUR1 channel activation in both cell types. Taken together, here, we report novel findings that PKG exerts dual functional regulation of neuronal K(ATP) channels in a SUR subunit-dependent manner, which may provide new means of therapeutic intervention for manipulating neuronal excitability and/or survival.

Topics: Adenosine Triphosphate; Animals; Carbazoles; Cell Line; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Humans; KATP Channels; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Purinones; Recombinant Fusion Proteins; Signal Transduction

Nitric oxide (NO), which is known to inhibit systemic vascular smooth muscle cell proliferation, is used in the management of neonatal pulmonary hypertension. Our objectives were to determine: (1) if endogenous NO production by neonatal porcine pulmonary artery smooth muscle cells (PASMCs) varied with oxygen tension in vitro, and (2) the effect of exogenous NO and inducible NO synthase (iNOS) stimulators and inhibitors on PASMC proliferation and apoptosis. PASMCs were exposed to different conditions (varying PO(2), NO donors and scavengers, iNOS stimulators and inhibitors) and proliferation, apoptosis, and cyclic guanosine 5(')-monophosphate (cGMP) assessed. PASMCs proliferated best between 5 and 10% O(2) but cGMP levels were similar at all oxygen levels. NO donors (S-nitroso-N-acetyl-penicillamine, NOC-12, NOC-18) inhibited PASMC proliferation in a dose-dependent manner with associated cGMP increases, while NO scavengers (carboxy-PTIO), iNOS stimulators (interleukin-1beta, lipopolysaccharide), and iNOS inhibitors (aminoethylisothiourea) did not affect proliferation or cGMP. No changes in apoptosis were found at the concentrations of NO donors or iNOS stimulators used. These results suggest that while exogenous NO inhibits PASMC proliferation, endogenous NO may not regulate proliferation during changes in oxygen tension or cytokine levels. Endothelial derived and inhaled NO may attenuate smooth muscle hyperplasia and vascular remodeling. Inducible NOS in porcine PASMCs appears resistant to stimulation with interleukin-1beta or lipopolysaccharide. The mechanisms underlying hypoxia-mediated changes in PASMC proliferation require investigation.

Topics: Animals; Animals, Newborn; Apoptosis; Cell Division; Cells, Cultured; Cyclic GMP; DNA Damage; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Developmental; Immunohistochemistry; Interleukin-1; Lipopolysaccharides; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroso Compounds; Oxygen; Penicillamine; Phosphodiesterase Inhibitors; Pulmonary Artery; Purinones; Swine