title: Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells.
abstract: Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA. bmueller@live.unthsc.edu
authors.forename: Brett H
authors.initials: BH
authors.lastname: Mueller
authors.forename: Yong
authors.initials: Y
authors.lastname: Park
authors.forename: Donald R
authors.initials: DR
authors.lastname: Daudt
authors.forename: Hai-Ying
authors.initials: HY
authors.lastname: Ma
authors.forename: Irina
authors.initials: I
authors.lastname: Akopova
authors.forename: Dorota L
authors.initials: DL
authors.lastname: Stankowska
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
authors.forename: Thomas
authors.initials: T
authors.lastname: Yorio
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D005029:Ethylenediamines / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010423:Pentazocine / Q000494:pharmacology; D010620:Phenazocine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017480:Receptors, sigma / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology; D000097605:Sigma-1 Receptor
chemical_list: D002118:Calcium; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D005029:Ethylenediamines; D016257:Fura-2; D010423:Pentazocine; D010620:Phenazocine; D017480:Receptors, sigma; D014700:Verapamil; D000097605:Sigma-1 Receptor; C049925:fura-2-am; C097963:N-(2-(3,4-Dichlorphenyl)ethyl)-N,N',N'-trimethyl-1,2-ethandiamin; C012134:SK&F 10047