MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF THE OXYTOCIN RECEPTOR FROM A RAT PANCREATIC-CELL LINE (RINM5F)

Oxytocin (OT) and vasopressin (AVP) stimulate insulin and glucagon rel ease from the pancreas, and evoke insulin secretion from the rat insul inoma cell line, RINm5F. To determine which AVP/OT receptor subtype is expressed in RINm5F cells, we used PCR with degenerate primers to two transmembrane domains of the AVP (Via, V1b (or V3), V2) and OT recept ors (OTRs). The single PCR fragment identified was used to obtain a fu ll length cDNA from a RINm5F cDNA library. Comparison of the deduced a mino acid sequence of this clone with uterine OTR sequences from sever al species (human, sheep, bovine) and to the pig kidney epithelial cel l (LLC-PK1) OTR reveals a very high degree of homology. After the RIN cell OTR cDNA was stably transfected into CHO cells (CHO-OTR), the cel l membranes bound iodinated oxytocin antagonist with an apparent K-d c omparable to that of RIN cell membranes and those from other OT target cells. Comparison of the ligand specificities of CHO-OTR and RIN cell s membranes showed that the relative K-i values of a series of OT anal ogues were approximately equivalent in both preparations. The rank ord er of apparent K-i values also corresponded to published values for th e rat myometrium, where OT elicits intracellular calcium transients, a nd increases inositol phosphate production. In uterine endometrium and amnion cells, OT stimulates prostaglandin release. Stimulation of CHO -OTR cells with OT caused an increase in cytosolic calcium concentrati on originating from both intracellular and extracellular sources, and a dose-dependent increase in inositol phosphate levels. Arachidonic ac id release and PGE, synthesis were also stimulated by OT These finding s (amino acid sequence homology, binding specificity, and signal trans duction/second messenger production) suggest that OTRs from RINm5F cel ls are indistinguishable from OTRs that have been described in other t issues. The expression of OTR in pancreatic cells implies that OT play s a role in pancreatic function.