Calcitonin is expressed in gonadotropes of the anterior pituitary gland: its possible role in paracrine regulation of lactotrope function.
Previous studies from this laboratory have shown that salmon (S) calcitonin (CT)-like immunoreactive peptide (CTI) is synthesized and secreted by the anterior pituitary (AP) gland. These studies also co-localized CTI to gonadotropes, and demonstrated that SCT is a potent inhibitor of lactotrope function. However, the molecular structure of putative gonadotrope-derived CTI that inhibits lactotrope function has not been defined. The present studies cloned CT cDNA (pit-CT cDNA) from a mouse gonadotrope L beta T2 cell line using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. Alignment of nucleotide sequences of pit-CT and mouse CT revealed greater than 99% homology between the sequences. The pit-CT cDNA was ligated into a mammalian expression vector, and the construct was transfected into L beta T2 cells. Two stable transfectant cell lines (CT.U6/A and B) were obtained by selection in G418. Subsequent S1-nuclease protection assay and immunocytochemistry results have shown that: (1) pit-CT peptide expressed by CT.U6 cell lines immunoreacted with GCT1-anti-SCT serum; (2) secretions of CT.U6 cells inhibited prolactin (PRL) release, PRL mRNA abundance and DNA synthesis of PRL-secreting GGH3 cells; and (3) CT.U6-induced inhibition was abolished by GCT1-anti-SCT serum. The studies also generated a riboprobe from the cloned pit-CT cDNA, and localized CT mRNA expression in gonadotropes of rat AP gland by in situ hybridization histochemistry. These results demonstrate that pit-CT mRNA is closely homologous to mouse CT mRNA; it is expressed by gonadotropes of the rat AP gland, and the peptide may significantly affect lactotrope function by inhibiting PRL release and cell proliferation.
Ren
Y
Y
Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, 1300 Coulter, Amarillo, Texas 79106, USA.
Chien
J
J
Sun
Y P
YP
Shah
G V
GV
D000818:Animals; D001483:Base Sequence; D002116:Calcitonin / Q000032:analysis* / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D006065:Gonadotropins, Pituitary / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization; D007986:Luteinizing Hormone / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D019899:Paracrine Communication; D010903:Pituitary Gland, Anterior / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011388:Prolactin / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014162:Transfection / Q000379:methods
D006065:Gonadotropins, Pituitary; D011994:Recombinant Proteins; D011388:Prolactin; D007986:Luteinizing Hormone; D002116:Calcitonin