PurposeIn diseases such as proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy, and age-related macular degeneration, retinal pigment epithelial (RPE) cells proliferate and migrate. Moreover, platelet-derived growth factor (PDGF) has been shown to enhance proliferation and migration of RPE cells in PVR. Even resveratrol can suppress the migration and adhesion of many cell types, its effects on RPE cell migration and adhesion remain unknown. In this study, we investigated the inhibitory effects of resveratrol on RPE cell migration induced by PDGF-BB, an isoform of PDGF, and adhesion to fibronectin, a major ECM component of PVR tissue.MethodsThe migration of RPE cells was assessed by an electric cell-substrate impedance sensing migration assay and a Transwell migration assay. A cell viability assay was used to determine the viability of resveratrol treated-cells. The cell adhesion to fibronectin was examined by an adhesion assay. The interactions of resveratrol with PDGF-BB were analyzed by a dot binding assay. The PDGF-BB-induced signaling pathways were determined by western blotting and scratch wound healing assay.ResultsResveratrol inhibited PDGF-BB-induced RPE cell migration in a dose-dependent manner, but showed no effects on ARPE19 cell adhesion to fibronectin. The cell viability assay showed no cytotoxicity of resveratrol on RPE cells and the dot binding assay revealed no direct interactions of resveratrol with PDGF-BB. Inhibitory effects of resveratrol on PDGF-BB-induced platelet-derived growth factor receptor β (PDGFRβ) and tyrosine phosphorylation and the underlying pathways of PI3K/Akt, ERK and p38 activation were found; however, resveratrol and PDGF-BB showed no effects on PDGFRα and JNK activation. Scratch wound healing assay demonstrated resveratrol and the specific inhibitors of PDGFR, PI3K, MEK or p38 suppressed PDGF-BB-induced cell migration.ConclusionsThese results indicate that resveratrol is an effective inhibitor of PDGF-BB-induced RPE cell migration via PDGFRβ, PI3K/Akt and MAPK pathways, but has no effects on the RPE cell adhesion to fibronectin.

In the present study, we used the N terminus (amino acids 1ϳ160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1ϳ86 of ACVI and 33-51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, Snapin ⌬33-51 , which harbors an internal deletion of amino acids 33-51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33-51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-⌬A87), which lacked the Snapin-interacting region (amino acids 1-86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain. Adenylyl cyclases (ACs)1 are a family of enzymes that produce cyclic AMP (cAMP) from ATP upon extracellular stimulation. To date, at least 9 membrane-bound ACs have been isolated and characterized (1). These enzymes are capable of integrating positive and negative signals that act directly through stimulation of G protein-coupled receptors (GPCRs) or indirectly via intracellular signaling molecules in isozyme-specific patterns. In addition, the regulatory properties and expression patterns of different AC isoforms greatly diverge and may account for the distinctive cell-and tissue-specific responsiveness of ACs. Recently, several different proteins, including RGS2 and the protein associated with Myc (PAM), have been shown to interact and modulate activity of different AC isozymes (2, 3), adding additional dimensions to the isozymespecific regulation of the AC superfamily.Except for the newly identified soluble AC, all membranebound AC members share a primary structure consisting of 12 transmembrane regions and 3 large cytoplasmic domains (N, C1a/b, and C2). The C1a and C2 domains, which form the catalytic core complex, are highly conserved and are homologous to each other. The N-terminal domains of ACs, in contrast, are variable among ACs, and have been demonstrated to play mostly regulatory roles (4, 5). Among the AC isozymes, ACVI is of particular interest, because...

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The SP Er:YAG laser was superior to the Q-switched lasers for removing cosmetic tattoos.

In the present study, we demonstrate that AC5 (type V adenylate cyclase) interacts with Ric8a through directly interacting at its N-terminus. Ric8a was shown to be a GEF (guanine nucleotide exchange factor) for several alpha subunits of heterotrimeric GTP binding proteins (Galpha proteins) in vitro. Selective Galpha targets of Ric8a have not yet been revealed in vivo. An interaction between AC5 and Ric8a was verified by pull-down assays, co-immunoprecipitation analyses, and co-localization in the brain. Expression of Ric8a selectively suppressed AC5 activity. Treating cells with pertussis toxin or expressing a dominant negative Galphai mutant abolished the suppressive effect of Ric8a, suggesting that interaction between the N-terminus of AC5 and a GEF (Ric8a) provides a novel pathway to fine-tune AC5 activity via a Galphai-mediated pathway.

Abstract:To elucidate the regulation of the rat dopamine transporter (rDAT), we established several PC12 variants overexpressing the rDAT. Treating these cells with a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide, 30 M) depolarized the plasma membrane potential from Ϫ31 Ϯ 2 to 43 Ϯ 5 mV and inhibited rDAT activity significantly in a calcium-and protein kinase C-independent manner. Membrane depolarization by a high external K ϩ concentration or two K ϩ channel blockers (tetraethylammonium hydroxide and BaCl 2 ) also resulted in a marked inhibition of rDAT activity. Such inhibition of dopamine uptake is due to a reduction in V max , with no marked effect on the K m for dopamine. The potency of cocaine in inhibiting dopamine uptake was not significantly altered, whereas that of amphetamine was slightly enhanced by membrane depolarization. Removing extracellular Ca 2ϩ or blocking the voltage-sensitive L-type calcium channels using nifedipine did not exert any significant effect on the inhibition of rDAT activity by depolarization. These data confirm that calcium influx on depolarization is not required for inhibition of the rDAT. Collectively, our data suggest that rDAT activity can be altered by a neurotransmitter that modulates the membrane potential, thus suggesting an exquisite mechanism for the fine-tuning of dopamine levels in the synapse. Key Words: Dopamine transporter-Nicotinic acetylcholine receptor-Depolarization-Protein kinase C-Calcium.

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