Locally formed dopamine stimulates cAMP accumulation in LLC-PK1 cells via a DA1 dopamine receptor

Proximal tubules have been shown to produce dopamine (DA) from (-)-3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) and to express DA1 dopamine (DA1) receptors linked to inhibition of sodium transport. The LLC-PK1 renal epithelial cell line expresses proximal tubule cell-like properties in vitro. Here, we sought to determine whether the LLC-PK1 cell line would be a useful model system to study dopaminergic mechanisms in vitro. LLC-PK1 cells contained high levels of aromatic L-amino acid decarboxylase (AADC) (Km 0.19 +/- 0.08 mM, Vmax 3.69 +/- 0.57 nmol.mg-1.min-1) and converted L-dopa to DA in a nonsaturable fashion up to 1 mM L-dopa. DA production was blocked by the AADC inhibitor carbidopa. Dopamine stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in LLC-PK1 cells in a dose-dependent manner (50% effective concentration, 1.53 +/- 0.38 microM; maximal stimulation, 46.6 +/- 10.88 pmol/mg protein); this effect was blocked by addition of DA1-receptor antagonists. L-Dopa also stimulated cAMP accumulation, and this effect was attenuated by an equimolar concentration of carbidopa and blocked by the DA1 antagonist Sch 23390. These results indicate that LLC-PK1 cells convert L-dopa to DA, which then stimulates cAMP via a DA1 receptor coupled to activation of adenylate cyclase. Moreover, the demonstration that locally formed DA can act as an autocrine/paracrine substance in LLC-PK1 cells in vitro is consistent with a role for DA as an autocrine/paracrine substance in vivo.