EFFECTS OF PROTEIN KINASE-C ACTIVATION ON INOSITOL PHOSPHATE GENERATION AND INTRACELLULAR CA-2+ MOBILIZATION IN BOVINE PARATHYROID CELLS

Activators of protein kinase C, such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol dioc- tanoylglycerol (diC8), either stimulate or inhibit PTH release depending on the extracellular Ca2+ concentration. By increasing PTH release at high extracellular Ca2+, these agents, in effect, block high Ca2+-induced inhibition of secretion. Since raising extracellular Ca2+ increases intracellular free Ca2+ ([Ca2+]i) and inositol trisphosphate (InsP3) formation in parathyroid cells, we assessed the effects of PMA pretreatment on [Ca2+]i and InsP3 to ascertain whether these second messengers might be altered by protein kinase C activation. Preincubation of parathyroid cells with PMA (10-6 M) significantly lowered the intracellular Ca2+ response to raising extracellular Ca2+ from 0.5-2.0 mM. The peak increase in [Ca2+]i averaged 475 ±11 nM in PMA- treated cells compared to 703 ± 44 nM in control cells. High extracellular Ca2+-induced InsP3 accumulation was also reduced after incubating the cells with PMA. To determine whether intracellular Ca2+ stores and/or transmembrane Ca2+ uptake were affected by activating protein kinase C, we examined intracellular Ca2+ responses to the Ca2+ ionophore ionomycin after PMA pretreatment. At 0.5 mM Ca2+, ionomycin (10-6 M) increased [Ca2+]i to an initial peak of 738 ± 49 nM followed by a sustained increase to 501 ± 30 nM in control cells (n = 15). After exposure to PMA (>20 min), however, peak and sustained increments in [Ca2+]i were significantly lower at 550 ± 32 and 394 ± 16 nM, respectively (P < 0.02, n = 8). In the absence of extracellular Ca2+, basal [Ca2+]i was 197 ± 5 and peaked at 323 ± 15 nM with ionomycin (10-6 M) in PMA-treated cells (n = 16). The latter value was significantly less than the peak increase in [Ca2+]i to 461 ± 19 nM observed with ionomycin (10-6 M) in control cells (P < 0.001, n = 15). With respect to secretion, either of the protein kinase C agonists (i.e. PMA or diC8) or the Ca2+ ionophore ionomycin inhibited PTH release at 0.5 mM Ca2+. To determine whether the concomitant activation of protein kinase C- and Ca2+-dependent pathways could additively suppress PTH release, we assessed the effects of ionomycin and either PMA or diC8 on secretion. PTH release at 0.5 mM Ca2+ was reduced in an additive manner by either of these protein kinase C agonists plus ionomycin. At 2 mM Ca2+, protein kinase C agonists stimulated PTH release. Ionomycin did not, however, reverse the stimulatory effects of these agents on secretion despite its ability to raise [Ca2+]i in PMA-treated cells at this Ca2+. We conclude that activation of protein kinase C may modify signal transduction in parathyroid cells by altering the coupling of the putative Ca2+-sensing mechanism to phospholipase C and to intracellular Ca2+ mobilization. The ability of protein kinase C agonists to stimulate PTH release at high Ca2+ concentrations is not reversed by pharmacologically raising [Ca2+]i with ionomycin. This suggests that activation of protein kinase C may block other signalling pathways responsible for high extracellular Ca2+-induced inhibition of PTH release or may inhibit high Ca2+-mediated intracellular degradation of PTH. © 1990 by The Endocrine Society.