The observation that increases in extracellular Ca2+ or the addition of divalent cations, such as Ba2+, Mg2+, Mn2+, or Sr2+, stimulate the accumulation of inositol trisphosphate (InsP3) and its breakdown products in parathyroid cells strongly supports the idea that polyphosphoinositides are hydrolyzed under these conditions. Since phosphatidic acid is produced as a result of polyphosphoinositide hydrolysis, and it has been proposed that phosphatidic acid may be a second messenger for Ca2+mobilization, we examined the effects of this compound on parathyroid cells. We assessed PTH release, intracellular free Ca2+ ([Ca2+]i), and inositol polyphosphate accumulation in response to phosphatidic acid. Natural phosphatidic acid reduced PTH release at 1.0 mM extracellular Ca2+ by 18 ± 6%, 48 ± 5%, 59 ± 10%, and 79 ± 6% at concentrations of 1, 10, 50, and 100 fig/ml, respectively (n = 5-11). The effect was not dependent on the presence of extracellular Ca2+, since phosphatidic acid (100 Mg/ml) inhibited PTH secretion by 39 ± 3% in medium with no added Ca2+ and 1.0 mM EGTA (n = 3). This agent rapidly and transiently increased [Ca2+]i in a dose-dependent manner, as determined by fura-2 fluorescence. At 1.0 mM extracellular Ca2+, [Ca2+]i rose from 309 ± 8 to a peak of 356 ± 26, 454 ± 22, and 587 ± 57 nM with the addition of 1, 10, and 100 µg/ml phosphatidic acid, respectively (n = 2-14). In the absence of extracellular Ca2+ (i.e. medium with 1 or 2 mM EGTA and no added Ca2+), phosphatidic acid produced a quantitatively smaller peak increment of 38 ± 4% in [Ca2+]i(indicating that this compound could mobilize Ca2+ from intracellular stores (n = 3). At 1.0 mM extracellular Ca2+, phosphatidic acid (200 µg/ml) stimulated the accumulation of Inositol trisphosphate (InsP3), Inositol bisphosphate (InsP2), and Inositol monophosphate (InsP1) by 46 ± 9%, 37 ± 9%, and 59 ± 11% after 60 sec, respectively (n = 5-7). Phosphatidic acid had no significant effect on forskolin-stimulated cAMP accumulation. We further determined whether the specific fatty acid composition of phosphatidic acid might influence its effects in parathyroid cells by testing several synthetic compounds. Dipalmitoyl phosphatidic acid (≥50 µg/ml) inhibited PTH release in a dose-dependent manner without significantly changing [Ca2+]i. Dioleoyl phosphatidic acid had modest biphasic effects on secretion, with 20 ± 5% inhibition observed at lower doses (10 µg/ml) and a 27 ± 8% stimulation of secretion at 100 µg/ml (n = 6). At 1.0 mM extracellular Ca2+, dioleoyl phosphatidic acid increased [Ca2+]isubstantially and in a concentration- dependent manner from 345 ± 15 to 400 ± 49, 436 ± 58, 557 ± 85, and 782 ± 82 nM with the addition of 1, 10, 50, and 100 µg/ml, respectively (n = 4-6). This study shows that phosphatidic acid can mobilize intracellular Ca2+ and increase InsP3 in parathyroid cells and suggests that this agent may also interact with other signalling pathways controlling secretion. The overall effects of exogenous phosphatidic acid on PTH release may be stimulatory or inhibitory depending on the specific fatty acid constituents of the phosphatidic acid and its interaction with other second messengers. © 1990 by The Endocrine Society.
