STUDIES OF MELATONIN EFFECTS ON EPITHELIA USING THE HUMAN EMBRYONIC KIDNEY-293 (HEK-293) CELL-LINE

The expression of melatonin receptors (MR) of the Mel(1a) subtype in b asolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investi gate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell line s originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK-293) were each tested for the presence of MR us ing 2-[I-125]iodomelatonin (I-125-MEL) as a radioligand. The HEK-293 c ell line exhibited the highest specific I-125-MEL binding. By intermed iate filament characterization, the HEK-293 cells were determined to b e of epithelial origin. Binding of I-125-MEL in HEK-293 cells demonstr ated saturability, reversibility, and high specificity with an equilib rium dissociation constant (K-d) value of 23.8 +/- 0.5 pM and a maximu m number of binding sites (B-max) value of 1.17 +/- 0.11 fmol/mg prote in (n = 5), which are comparable with the reported K-d and B-max value s in human kidney cortex. Coincubation with GTP gamma S (10 mu M) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affi nity (K-d was increased by a factor of 1.5-2.0), with no significant d ifference in B-max. Melatonin (1 mu M) decreased the forskolin (10 mu M) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine DIA receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamin e stimulated an increase in the level of cAMP. Similarly, transient tr ansfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 recepto rs, each resulted in an hormone inducible increase in cAMP levels. Sur prisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopami ne D1-induced increase in cAMP was completely inhibited by pertussis t oxin (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(1a) 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, indi rect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody dire cted against the cytoplasmic domain required prior cell permeabilizati on. By RT PCR, HEK-293 cells express both Mel(1a) and Mel(1b) messenge r RNAs, but the messenger RNA level for Mel(1b) is several orders of m agnitude lower than for Mel(1a). We conclude that HEK-293 cells expres s MR predominantly of the Mel(1a) subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is throu gh modulation of cellular dopaminergic responses.