PARATHYROID HORMONE-RELATED PEPTIDE PRODUCTION AND ACTION IN A MYOEPITHELIAL CELL-LINE DERIVED FROM NORMAL HUMAN BREAST

Physiological roles for PTH-related peptide (PTHrP) appear varied, but remain to be clarified. The peptide is present in large amounts in mi lk, and PTHrP mRNA has been shown to be present in high amounts in lac tating mammary gland. Because PTHrP can cause smooth muscle relaxation , we hypothesized that the peptide might affect the contractility of t he breast myoepithelial cell and thereby affect milk ejection. To test this idea, we asked whether PTHrP might affect second messenger respo nses in a human mammary gland myoepithelial cell line (Hs578Bst) deriv ed originally from normal breast tissue. To verify the presumed origin of these cells, we also examined the effects of oxytocin. Cells were grown in culture in multiwell plates and exposed to test peptides for 15 min in buffer containing 1 mm isobutylmethylxanthine, and intracell ular cAMP was measured by RIA. Both PTHrP-(1-34) and PTH-(1-34) increa sed cAMP in a dose-related fashion (ED50, 5 nm), with a maximal effect (3-fold) occurring at 100 nm. The ability of PTHrP to stimulate cAMP was inhibited by a 10- to 100-fold molar excess of the specific inhibi tors, PTH-(3-34) or PTHrP-(7-34). Inhibitors alone did not alter cAMP. Oxytocin also produced an increase in cAMP, but the effect was incons istent and occurred only with high doses (0.1-1 muM). Using cells grow n on cover-slips and loaded with fura-2AM, intracellular Ca2+ was moni tored in cells exposed to test peptides. Oxytocin (0.2-20 nm) produced rapid dose-related increases in intracellular Ca2+, with a peak and p lateau characteristic of initial mobilization of intracellular Ca2+, f ollowed by entry of extracellular Ca. The plateau was eliminated by th e Ca channel antagonist La3+ or by perfusion of cells with Ca-free med ium. PTHrP (10-100 nm) altered the intracellular Ca2+ response to oxyt ocin in 66% of 39 preparations tested. PTHrP inhibited the Ca2+ respon se when given before oxytocin or transiently decreased the plateau pha se of the Ca2+ response when given after oxytocin. Analysis of cellula r mRNA using reverse transcription polymerase chain reaction indicated that these cells express the gene for PTHrP, and immunohistochemistry using antiserum to PTHrP revealed positive staining of cells. Measure ment of immunoreactive PTHrP in conditioned medium confirmed that thes e cells can synthesize and secrete the peptide. The finding of a respo nse of this cell line to oxytocin provides functional evidence of thei r myoepithelial derivation. The facts that the cells produce PTHrP and show cAMP and intracellular Ca2+ responses to PTHrP-(1-34) support th e notion that PTHrP may serve an autocrine or paracrine role in the re gulation of milk ejection during lactation.