thiourea and zaprinast

Vascular smooth muscle cell (VSMC) migration and proliferation is central to neointima formation in vein graft failure following coronary artery bypass. However, there are currently no pharmacological interventions that prevent vein graft failure through intimal occlusion. It is hence a therapeutic target. Here, we investigated the contribution of GPR35 to human VSMC and endothelial cell (EC) migration, using a scratch-wound assay, and also the contribution to proliferation, using MTS and BrdU assays, in in vitro models using recently characterized human GPR35 ortholog-selective small-molecule agonists and antagonists. Real-time PCR studies showed GPR35 to be robustly expressed in human VSMCs and ECs. Stimulation of GPR35, with either the human-selective agonist pamoic acid or the reference agonist zaprinast, promoted VSMC migration in the scratch-wound assay. These effects were blocked by coincubation with either of the human GPR35-specific antagonists, CID-2745687 or ML-145. These GPR35-mediated effects were produced by inducing alterations in the actin cytoskeleton via the Rho A/Rho kinase signaling axis. Additionally, the agonist ligands stimulated a proliferative response in ECs. These studies highlight the potential that small molecules that stimulate or block GPR35 activity can modulate vascular proliferation and migration. These data propose GPR35 as a translational therapeutic target in vascular remodeling.

Topics: Actin Cytoskeleton; Aminosalicylic Acids; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Endothelial Cells; HEK293 Cells; Humans; Hydrazones; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthols; Purinones; Receptors, G-Protein-Coupled; rho-Associated Kinases; rhoA GTP-Binding Protein; Saphenous Vein; Signal Transduction; Thiazolidines; Thiourea; Time Factors

The role of endogenously produced nitric oxide (NO) in the regulation of basal catecholamine (CA) secretion was studied in chromaffin cells. Treatment of chromaffin cells with nitric oxide synthase (NOS) inhibitors produced a dose-dependent increase in basal catecholamine secretion, which paralleled their ability to inhibit NOS activity. This inhibitory profile was similar to that found in neurons, suggesting the constitutive expression of neuronal NOS (nNOS) in these cells, which was confirmed by Western blot analysis. A study of the kinetics and pharmacology of nNOS activity expressed in chromaffin cells in culture indicated that NOS activity is calcium-dependent, increases with time, and is highly dependent on both intracellular concentrations of L-arginine (K(m) approximately 4 microM, V(max) = 908 +/- 60 pmol/hr x 10(6) cells) and transport of L-arginine into the cells (exhibiting two affinity constants of k(1) = 3.2 +/- 0.3 microM and k(2) = 126 +/- 5.5 microM). The effects of NOS inhibitors on CA secretion were mediated by the L-arginine-NO-cGMP pathway, insofar as exogenous L-arginine was able to partially block the increase in CA secretion evoked by them, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a specific inhibitor of guanylate cyclase, and zaprinast, an inhibitor of the cGMP phosphodiesterase, were able to increase and inhibit, respectively, basal CA secretion in a dose-dependent manner. These results suggest that chromaffin cells exhibit a tonic production of NO by nNOS that keeps the basal CA secretion at low levels, and this could be necessary for maintaining a normotensive state.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Arginine; Blotting, Western; Catecholamines; Cattle; Cell Culture Techniques; Chromaffin Cells; Citrulline; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Guanylate Cyclase; Isoenzymes; Nerve Tissue Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Phosphodiesterase Inhibitors; Purinones; Thiourea; Time Factors

1. In the present work we have characterized the relaxant response induced by light stimulation (LS) in the lower urinary tract from sheep, pig and rat, establishing its relationship with nitrergic neurotransmission. 2. Urethral, but not detrusor, preparations showed pronounced photo-relaxation (PR) which declined progressively following repetitive LS. Sheep urethral PR was again restored either spontaneously or (to a greater extent) by exogenous nitric oxide (NO) addition and by electrical field stimulation (EFS) of intrinsic nitrergic nerves. 3. Greater NO generation was detected from sheep urethral than from detrusor homogenates following illumination. 4. Sheep urethral PR was inhibited by oxyhaemoglobin, but not by methaemoglobin, carboxy-PTIO, extracellular superoxide anion generators or superoxide dismutase. Guanylyl cyclase but not adenylyl cyclase activation mediates urethral relaxation to LS. 5. Urethral PR was more resistant to inhibition by L-thiocitrulline than EFS-induced responses, although this agent prevented PR restoration by high-frequency EFS. 6. Urethral PR was TTX insensitive and partially modified in high-K+ solutions. Cold storage for 24 h greatly impaired urethral PR, although it was restored by high-frequency EFS. 7. Repetitive exposure to LS, EFS or exogenous NO induced changes in the shape of the EFS-induced nitrergic relaxation, possibly by pre-synaptic mechanisms. 8. In conclusion, we suggest the presence of an endogenous, photo-labile, nitro-compound store in the urethra, which seems to be replenished by neural nitric oxide synthase activity, indicating a close functional relationship with the nitrergic neurotransmitter.

Topics: Adenine; Animals; Citrulline; Cold Temperature; Culture Techniques; Electric Stimulation; Enzyme Inhibitors; Female; Light; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Oxadiazoles; Oxyhemoglobins; Phosphodiesterase Inhibitors; Photic Stimulation; Potassium; Purinones; Quinoxalines; Rats; Rats, Wistar; Sheep; Superoxides; Swine; Synaptic Transmission; Tetrodotoxin; Thiourea; Time Factors; Urethra