TY - JOUR
T1 - Molecular cloning and functional characterization of the oxytocin receptor from a rat pancreatic cell line (RINm5F)
AU - Jeng, Y. J.
AU - Lolait, S. J.
AU - Strakova, Z.
AU - Chen, C.
AU - Copland, J. A.
AU - Mellman, D.
AU - Hellmich, M. R.
AU - Soloff, M. S.
N1 - Funding Information:
We thank Kirk Ives for carrying out the fura-2 imaging studies and Dr David Konkel for reviewing the manuscript. This study was supported in part by grant HD08406 (MSS) from the National Institutes of Health. DM was the recipient of a Summer Intramural Research Training Award (IRTA) from NIH.
PY - 1996
Y1 - 1996
N2 - Oxytocin (OT) and vasopressin (AVP) stimulate insulin and glucagon release from the pancreas, and evoke insulin secretion from the rat insulinoma 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 (V1a, V1b (or V3), V2) and OT receptors (OTRs). The single PCR fragment identified was used to obtain a full length cDNA from a RINm5F cDNA library. Comparison of the deduced amino acid sequence of this clone with uterine OTR sequences from several species (human, sheep, bovine) and to the pig kidney epithelial cell (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 cell membranes bound iodinated oxytocin antagonist with an apparent K(d) comparable to that of RIN cell membranes and those from other OT target cells. Comparison of the ligand specificities of CHO-OTR and RIN cells membranes showed that the relative K(i) values of a series of OT analogues were approximately equivalent in both preparations. The rank order of apparent K(i) values also corresponded to published values for the rat myometrium, where OT elicits intracellular calcium transients, and 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 concentration originating from both intracellular and extracellular sources, and a dose-dependent increase in inositol phosphate fevers. Arachidonic acid release and PGE2 synthesis were also stimulated by OT. These findings (amino acid sequence homology, binding specificity, and signal transduction/second messenger production) suggest that OTRs from RINm5F cells are indistinguishable from OTRs that have been described in other tissues. The expression of OTR in pancreatic cells implies that OT plays a role in pancreatic function.
AB - Oxytocin (OT) and vasopressin (AVP) stimulate insulin and glucagon release from the pancreas, and evoke insulin secretion from the rat insulinoma 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 (V1a, V1b (or V3), V2) and OT receptors (OTRs). The single PCR fragment identified was used to obtain a full length cDNA from a RINm5F cDNA library. Comparison of the deduced amino acid sequence of this clone with uterine OTR sequences from several species (human, sheep, bovine) and to the pig kidney epithelial cell (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 cell membranes bound iodinated oxytocin antagonist with an apparent K(d) comparable to that of RIN cell membranes and those from other OT target cells. Comparison of the ligand specificities of CHO-OTR and RIN cells membranes showed that the relative K(i) values of a series of OT analogues were approximately equivalent in both preparations. The rank order of apparent K(i) values also corresponded to published values for the rat myometrium, where OT elicits intracellular calcium transients, and 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 concentration originating from both intracellular and extracellular sources, and a dose-dependent increase in inositol phosphate fevers. Arachidonic acid release and PGE2 synthesis were also stimulated by OT. These findings (amino acid sequence homology, binding specificity, and signal transduction/second messenger production) suggest that OTRs from RINm5F cells are indistinguishable from OTRs that have been described in other tissues. The expression of OTR in pancreatic cells implies that OT plays a role in pancreatic function.
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U2 - 10.1016/S0143-4179(96)90039-6
DO - 10.1016/S0143-4179(96)90039-6
M3 - Article
C2 - 9004255
AN - SCOPUS:0030471160
SN - 0143-4179
VL - 30
SP - 557
EP - 565
JO - Neuropeptides
JF - Neuropeptides
IS - 6
ER -