The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone.
The suppression of PTH release by high extracellular calcium (Ca2+) has been associated with secretion of biologically inactive carboxyl-terminal fragments of PTH (C-PTH), while relatively more intact PTH is released under low extracellular Ca2+ conditions. In the presence of high extracellular Ca2+, phorbol myristate acetate (PMA) has been shown to stimulate PTH release to levels observed at low Ca2+, suggesting that protein kinase-C (PKC) is involved in the regulation of PTH secretion. We have examined the effect of PMA on PTH secretion and the release of PTH fragments at high and low calcium concentrations. Primary cultures of bovine parathyroid cells were incubated for 90 min in 0.5 mM (low) or 2.0 mM (high) Ca2+ with or without 1.6 microM PMA. Reverse phase HPLC using an 18-60% gradient of acetonitrile in 0.1% trifluoroacetic acid was performed on the medium from these incubations, and the eluant fractions were analyzed with a carboxyl (C)-terminal-specific PTH RIA. Medium from cultures exposed to low Ca2+ exhibited two large peaks of PTH immunoreactivity, coeluting with intact PTH-(1-84) and a synthetic human C-PTH-(39-84). PMA treatment at low Ca2+ resulted in the secretion of a greatly reduced amount of intact PTH, suggesting that PKC may increase the production of PTH fragment. At high extracellular Ca2+ PMA caused an increase in total immunoreactive PTH release similar to that seen at low Ca2+. However, on HPLC analysis, proportionally more PTH eluted in the position of the C-PTH fragment than was seen with low Ca2+ stimulation of PTH secretion. It, therefore, appears that the degradation of PTH to C-PTH may be linked to activation of PKC and can be separated from the Ca2+ regulation of PTH release occurring at the cell membrane.