Studies of the renal action of melatonin: evidence that the effects are mediated by 37 kDa receptors of the Melia subtype localized primarily to the basolateral membrane of the proximal tubule

The pineal hormone melatonin controls circadian behavior of a variety of organs in different species, including humans. However, the precise mechanism (or mechanisms) by which this occurs remains largely unknown. At the cellular level its effects are believed to be mediated via interaction with specific melatonin receptors (MR), which have previously been cloned from human brain (Mel1a) and retina (Mel1b). At the tissue level, MR have been investigated primarily through empirical definition of specific binding sites, but so far there has been little success in biochemical or molecular characterization of native MR. In the kidney, there is strong circumstantial evidence that melatonin affects diurnal variations in renal function, but relatively little is known about the overall glomerular vs. tubular contributions to these effects. The strategy behind the present study was to use a panel of peptide-specific antibodies to identify MR proteins in various tissues, and from a determination of the intrarenal distribution of MR, gain insight into the mechanism by which melatonin might regulate kidney function. We used two peptide-specific antibodies directed against different regions of Mel1a to identify MR. Our results show that the native Mel1a receptor is a 37 kilodalton (kDa) protein in human and rat brain. Further, immunofluorescent studies carried out in guinea pig kidney have revealed that anti-Mel1a antibody is also localized to the basolateral membrane (BLM) of the renal cortical epithelium, especially the early proximal tubule. Immunoblotting of purified BLM fractions from guinea pig renal cortex and small intestine using the two different peptide-specific antibodies reveals the presence of a single peptide-blockable band at 37 kDa. These same BLM fractions also demonstrate the presence of high-affinity 2-[125I]iodomelatonin (125I-MEL) binding sites, with the pharmacological specificity of binding expected of the Mel1a receptor subtype, inhibited by guanosine 5'-O-(3'-thiotriphosphate) (GTPgammaS) and pertussis toxin. We conclude that functional MR in guinea pig kidney and small intestine are of the Mel1a subtype, and are expressed as 37 kDa proteins localized to the BLM and coupled to a pertussis toxin-sensitive G-protein (Gi). This localization strongly suggests that the proximal tubule plays a significant role in mediating the renal action of melatonin.