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 gonad otropes, and demonstrated that SCT is a potent inhibitor of lactotrope func tion. 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 revea led greater than 99% homology between the sequences. The pit-CT cDNA was li gated 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) w ere obtained by selection in G418. Subsequent Sl-nuclease protection assay and immunocytochemistry results have shown that: (1) pit-CT peptide express ed by CT.U6 cell lines immunoreacted with GCT1-anti-SCT serum; (2) secretio ns 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 ribopro be from the cloned pit-CT cDNA, and localized CT mRNA expression in gonadot ropes of rat AP gland by bi situ hybridization histochemistry. These results demonstrate that pit-CT mRNA is closely homologous to mouse C T mRNA; it is expressed by gonadotropes of the rat AP gland, and the peptid e may significantly affect lactotrope function by inhibiting PRL release an d cell proliferation.