STUDIES OF THE RENAL ACTION OF MELATONIN - EVIDENCE THAT THE EFFECTS ARE MEDIATED BY 37 KDA RECEPTORS OF THE MEL(1A) 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 unknow n. At the cellular level its effects are believed to be mediated via i nteraction with specific melatonin receptors (MR), which have previous ly been cloned front human brain (Mel(1n)) and retina (Mel(1b)). At th e 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 af fects diurnal variations in renal function,but relatively little is kn own about the overall glomerular vs. tubular contributions to these ef fects. The strategy behind the present study was to use a panel of pep tide-specific antibodies to identify MR proteins in various tissues, a nd from a determination of the intrarenal distribution of MR, gain ins ight into the mechanism by which melatonin might regulate kidney funct ion. We used two peptide-specific antibodies directed against differen t regions of Mel(1n) to identify MR. Our results show that the native Mel(1a) receptor is a 37 kilodalton (kDa) protein in hunan and rat bra in. Further, immunofluorescent studies carried out hi guinea pig kidne y have revealed that anti-Mel(1a) antibody is also localized to the ba solateral membrane (BLM) of the renal cortical epithelium, especially the early proximal tubule. Immunoblotting of purified BLM fractions fr om 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 th e presence of high-affinity 2-[I-125]iodomelatonin (I-125-MEL) binding sites, with the pharmacological specificity of binding expected of th e Mel(1a) receptor subtype, inhibited by guanosine 5'-O-(3'-thiotripho sphate) (GTP gamma S) and pertussis toxin. We conclude that functional MR in guinea pig kidney and small intestine are of the Mel(1a) subtyp e, and are expressed as 37 kDa proteins localized to the BLM and coupl ed to a pertussis toxin-sensitive G-protein (Gi). This localization st rongly suggests that the proximal tubule plays a significant role in m ediating the renal action of melatonin.