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Studies of Melatonin Effects on Epithelia Using the Human Embryonic Kidney-293 (HEK-293) Cell Line¹

The Clarke Institute of Psychiatry (C.W.Y.C., G.M.B.), Toronto, Ontario, M5T 1R8, Canada; Medical Research Council Membrane Biology Group (Y.S., M.S.), Department of Medicine, University of Toronto, Medical Science Building, Toronto, Ontario, M5S 1A8, Canada; Department of Medicine (M.W.), University of Toronto, Toronto, Ontario, M5S 1A8, Canada; and Department of Physiology (S.F.P.), University of Hong Kong, Hong Kong

Address all correspondence and requests for reprint to: Dr. M. Silverman, Medical Research Council Membrane Biology Group, Department of Medicine, University of Toronto, Medical Science Building, Toronto, Ontario M5S 1A8, Canada.

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 GTPS (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_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. 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.

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