Physiological roles for PTH-related peptide (PTHrP) appear varied, but remain to be clarified. The peptide is present in large amounts in milk, and PTHrP mRNA has been shown to be present in high amounts in lactating mammary gland. Because PTHrP can cause smooth muscle relaxation, we hypothesized that the peptide might affect the contractility of the breast myoepithelial cell and thereby affect milk ejection. To test this idea, we asked whether PTHrP might affect second messenger responses in a human mammary gland myoepithelial cell line (Hs578Bst) derived 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 mil isobutylme-thylxanthine, and intracellular cAMP was measured by RIA. Both PTHrP-(l-34) and PTH-(l-34) increased 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 inhibitors, PTH-(3-34) or PTHrP-(7-34). Inhibitors alone did not alter cAMP. Oxytocin also produced an increase in cAMP, but the effect was inconsistent and occurred only with high doses (0.1-1 μM). Using cells grown on cover-slips and loaded with fura-2AM, intracellular Ca2+ was monitored in cells exposed to test peptides. Oxytocin (0.2-20 nM) produced rapid dose-related increases in intracellular Ca2+, with a peak and plateau characteristic of initial mobilization of intracellular Ca2+, followed by entry of extracellular Ca. The plateau was eliminated by the Ca channel antagonist La3+ or by perfusion of cells with Ca-free medium. PTHrP (10-100 nM) altered the intracellular Ca2+ response to oxytocin in 66% of 39 preparations tested. PTHrP inhibited the Ca2+ response when given before oxytocin or transiently decreased the plateau phase of the Ca2+ response when given after oxytocin. Analysis of cellular 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. Measurement of immunoreactive PTHrP in conditioned medium confirmed that these cells can synthesize and secrete the peptide. The finding of a response of this cell line to oxytocin provides functional evidence of their myoepithelial derivation. The facts that the cells produce PTHrP and show cAMP and intracellular Ca2+ responses to PTHrP-(l-34) support the notion that PTHrP may serve an autocrine or paracrine role in the regulation of milk ejection during lactation.
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