title: Activation of c-SRC underlies the differential effects of ouabain and digoxin on Ca(2+) signaling in arterial smooth muscle cells.
abstract: Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 μg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 μM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Zulian
authors.forename: Cristina I
authors.initials: CI
authors.lastname: Linde
authors.forename: Maria V
authors.initials: MV
authors.lastname: Pulina
authors.forename: Sergey G
authors.initials: SG
authors.lastname: Baryshnikov
authors.forename: Italia
authors.initials: I
authors.lastname: Papparella
authors.forename: John M
authors.initials: JM
authors.lastname: Hamlyn
authors.forename: Vera A
authors.initials: VA
authors.lastname: Golovina
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002316:Cardiotonic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D002478:Cells, Cultured; D004077:Digoxin / Q000008:administration & dosage / Q000494:pharmacology*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008297:Male; D008638:Mesenteric Arteries / Q000166:cytology; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D010042:Ouabain / Q000008:administration & dosage / Q000494:pharmacology*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D050052:TRPC Cation Channels / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C412373:AG 1879; D015220:Calcium Channels; D002316:Cardiotonic Agents; D011743:Pyrimidines; D019831:Sodium-Calcium Exchanger; D050052:TRPC Cation Channels; C492145:Trpc6 protein, rat; C110374:sodium-calcium exchanger 1; C096351:transient receptor potential cation channel, subfamily C, member 1; D010042:Ouabain; D004077:Digoxin; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D009543:Nifedipine