Studies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line

Abstract

The expression of melatonin receptors (MR) of the Mel(1a) subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK- 293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I- MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (K(d)) value of 23.8 ± 0.5 pM and a maximum number of binding sites (B(max)) value of 1.17 ± 0.11 fmol/mg protein (n = 5), which are comparable with the reported K(d) and B(max) values in human kidney cortex. Coincubation with GTPyS (10 μM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (K(d) was increased by a factor of 1.5-2.0), with no significant difference in B(max). Melatonin (1 μM) decreased the forskolin (10 μM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel(1a) receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT- PCR, HEK-293 cells express both Mel(1a) and Mel(1b) messenger RNAs, but the messenger RNA level for Mel(1b) is several orders of magnitude lower than for Mel(1a). We conclude that HEK-293 cells express MR predominantly of the Mel(1a) subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.