title: Involvement of G protein-coupled receptor kinase 4 and 6 in rapid desensitization of dopamine D1 receptor in rat IEC-6 intestinal epithelial cells.
abstract: Dopamine-induced inhibition of Na(+)-K(+)-ATPase has been suggested to play a role in the regulation of Na(+) absorption at the intestinal level, and these effects were mediated by dopamine D(1)-like receptors. The aim of this work was to evaluate the effect of the activation of the D(1)-like receptors on the activity of the Na(+)/H(+) exchanger (NHE) in the rat intestinal epithelial cell line IEC-6. The presence of D(1) receptors was confirmed by immunoblotting. The dopamine D(1)-like receptor agonist SKF-38393 produced a concentration-dependent inhibition of NHE activity and stimulation of adenylyl cyclase (AC), this being antagonized by the D(1) selective antagonist SKF-83566. Effects of SKF-38393 on NHE and AC activities were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 25 min. Exposure of cells for 25 min to dibutyryl-cAMP (0.5 mM) or to the AC activator forskolin (3 microM) effectively inhibited NHE activity. Pretreatment of cells with heparin (1 microM), a nonselective G protein-coupled receptor kinase (GRK) inhibitor, prevented the loss of effects on NHE activity after 25 min exposure to SKF-38393. The presence of GRK4, GRK6A, and GRK6B was confirmed by immunoblotting. Overnight treatment with the anti-GRK4-6 antibody complexed with Lipofectin was also effective in preventing loss of the effects of SKF-38393 on NHE and AC activities. It is concluded that dopamine D(1) receptors in IEC-6 rapidly desensitize to D(1)-like agonist stimulation and GRK4 and 6 appear to be involved in agonist-mediated responsiveness and desensitization.
authors.affiliation: Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal.
authors.forename: Sónia
authors.initials: S
authors.lastname: Fraga
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Jose
authors.forename: Patrício
authors.initials: P
authors.lastname: Soares-da-Silva
mesh_terms: D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D018491:Dopamine Agonists / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000502:physiology*; D054771:G-Protein-Coupled Receptor Kinase 4; D054768:G-Protein-Coupled Receptor Kinases; D006493:Heparin / Q000494:pharmacology; D007422:Intestines / Q000166:cytology / Q000502:physiology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000262:Adenylyl Cyclases; D019138:Antibodies, Blocking; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D018491:Dopamine Agonists; D004791:Enzyme Inhibitors; D054771:G-Protein-Coupled Receptor Kinase 4; D054768:G-Protein-Coupled Receptor Kinases; D006493:Heparin; D007527:Isoenzymes; D017346:Protein Serine-Threonine Kinases; D017447:Receptors, Dopamine D1; D017923:Sodium-Hydrogen Exchangers; C044846:SKF83566; C089176:G-protein-coupled receptor kinase 6; C517854:Grk4 protein, rat