Parathyroid hormone (PTH) secretion: stimulation of PTH secretion by a peptide derived from the adenosine diphosphate-ribosylation factor.

Abstract

Using preparations of dispersed bovine parathyroid cells, we have investigated the effect of a 16-residue synthetic peptide, ARF-16, which corresponds to the N-terminus of the ADP-ribosylation factor, on the secretion of PTH. We find it to be a very effective secretagogue for PTH secretion, acting in a dose- and time-dependent manner. At concentrations in the range of 15-25 microM, the ARF peptide stimulated PTH secretion to a greater degree than low extracellular calcium, and at 25 microM 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 size we found no effect on PTH secretion, indicating that the ARF-16 effect is specific. In an attempt to define the structural elements of ARF that are required for activity, we tested several analogs of ARF with amino acids deleted from the N- and C-terminus. Deletion of the 2 N-terminal residues yielded a peptide with substantially reduced activity. 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 minimal sequence required for full activity. Measurements of cAMP concentrations indicate that the stimulatory effect is not mediated via this second 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 effect is reversible, as demonstrated by washing cells free of ARF, whereupon PTH secretion returns to basal levels. These results indicate that the peptide is not entering the cells, but is effecting secretion through a low affinity interaction at the cell surface. Other experiments, in which the capacity for ARF stimulation was abolished after a brief exposure of the cells to trypsin, support this conclusion. Characteristics of the ARF stimulatory effect, such as dose dependency and reversibility, lead us to conclude that the peptide is probably acting on the regulated secretory pathway. As the effect is not dependent on extracellular calcium levels and is not mediated via cAMP, we believe that this peptide will be a useful additional tool for future studies of the mechanisms of PTH secretion.