Dopamine D1-like Receptor-Mediated Inhibition of Cl-/HCO3- Exchanger Activity in Rat Intestinal Epithelial IEC-6 Cells is Regulated by G Protein-Coupled Receptor Kinase 6 (GRK 6)

Abstract

The present study investigated the effect of dopamine D₁-like receptor stimulation on the Cl^-/HCO₃^- exchange activity in rat intestinal epithelial IEC-6 cells. The Cl^-/HCO₃^- exchange activity was found to be a chloride-dependent, DIDS-sensitive and niflumate-insensitive process. The presence of the SLC26A6 anion exchanger was detected by both RT-PCR and immunoblotting analysis in IEC-6 cells, in which three different small interfering RNAs (siRNAs) targeting SLC26A6 markedly inhibited Cl^-/HCO₃^- exchange. Activation of dopamine D₁-like receptors with SKF 38393 inhibited Cl^-/HCO₃^- exchanger activity, this being antagonized by the D₁ selective antagonist SKF 83566. However, effects of SKF 38393 were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 15 min, suggestive of agonist-induced desensitization of D₁-like receptors. Pretreatment of cells with heparin, a non-selective inhibitor of G protein-coupled receptor kinases (GRKs), prevented the observed attenuation of SKF 38393-induced inhibition of Cl^-/HCO₃^- exchange. Overnight pretreatment with anti-GRK6A and anti-GRK6B, but not with anti-GRK4 antibodies, prevented the loss of SKF 38393-mediated effects. Both PKA and PKC signaling pathways participate in SKF 38393-mediated inhibition of Cl^-/HCO₃^- exchange. These findings suggest that SLC26A6 is at least one of the anion exchanger’s family members responsible for Cl^-/HCO₃^- exchange in IEC-6 cells. Dopamine D₁ receptors in IEC-6 rapidly desensitize to D₁-like agonist stimulation and GRK 6, but not GRK 4, appear to be involved in agonist-mediated responsiveness and desensitization.