title: Effects of protein kinase C activation on inositol phosphate generation and intracellular Ca2+ mobilization in bovine parathyroid cells.
abstract: Activators of protein kinase C, such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (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 (greater than or equal to 20 min), however, peak and sustained increments in [Ca2+]i were significantly lower at 550 +/- 32 and 394 +/- 16 nM, respectively (P less than 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 less than 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.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Endocrine Research Unit, San Francisco VA Medical Center, California 94121.
authors.forename: D M
authors.initials: DM
authors.lastname: Shoback
authors.forename: T H
authors.initials: TH
authors.lastname: Chen
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D010280:Parathyroid Glands / Q000187:drug effects / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004075:Diglycerides; D007295:Inositol Phosphates; D010281:Parathyroid Hormone; C058807:1,3-dicapryloylglycerol; D015759:Ionomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium