PARATHYROID-HORMONE (PTH) SECRETION - STIMULATION OF PTH SECRETION BYA PEPTIDE DERIVED FROM THE ADENOSINE DIPHOSPHATE-RIBOSYLATION FACTOR

Using preparations of dispersed bovine parathyroid cells, we have inve stigated the effect of a 16-residue synthetic peptide, ARF-16, which c orresponds to the N-terminus of the ADP-ribosylation factor, on the se cretion of PTH. We find it to be a very effective secretagogue for PTH secretion, acting in a dose- and time-dependent manner. At concentrat ions in the range of 15-25 mu M, the ARF peptide stimulated PTH secret ion to a greater degree than low extracellular calcium, and at 25 mu M was more effective than isoproterenol. The stimulatory effect of ARF was not dependent on the extracellular calcium concentration over the range of 0.5-3 mM. Upon testing other synthetic peptides of similar si ze we found no effect on PTH secretion, indicating that the ARF-16 eff ect is specific. In an attempt to define the structural elements of AR F that are required for activity, we tested several analogs of ARF wit h amino acids deleted from the N- and C-terminus. Deletion of the 2 N- terminal residues yielded a peptide with substantially reduced activit y. Further deletions from the N-terminus yielded an inactive peptide. Similarly, a peptide with deletions of 3 residues from the C-terminus was inactive. Thus, the activity of ARF-16 requires both the N- and C- terminal sequence, suggesting that the 16-residue peptide is the minim al sequence required for full activity. Measurements of cAMP concentra tions indicate that the stimulatory effect is not mediated via this se cond messenger. The ARF peptide does not alter intracellular calcium, suggesting that its effect is not mediated by calcium. Although cells incubated with ARF are vigorously stimulated to secrete PTH, this effe ct is reversible, as demonstrated by washing cells free of ARF, whereu pon PTH secretion returns to basal levels. These results indicate that the peptide is not entering the cells, but is effecting secretion thr ough a low affinity interaction at the cell surface. Other experiments , in which the capacity for ARF stimulation was abolished after a brie f exposure of the cells to trypsin, support this conclusion. Character istics of the ARF stimulatory effect, such as dose dependency and reve rsibility, lead us to conclude that the peptide is probably acting on the regulated secretory pathway. As the effect is not dependent on ext racellular calcium levels and is not mediated via cAMP, we believe tha t this peptide will be a useful additional tool for future studies of the mechanisms of PTH secretion.