Relationship between Diacylglycerol Levels and Extracellular Ca2+in Dispersed Bovine Parathyroid Cells*

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Abstract
In parathyroid cells, high extracellular Ca2+ promotes a rapid increase in inositol trisphosphate (IP3), suggesting activation of phospholipase C. Available data, however, indicate a high Ca2+-induced decrease in sn-1,2-diacylglycerol (DG), rather than the increase expected with hydrolysis of phosphoinositides. To explore this apparent discrepancy between IP3 and DG, we used three methods to quantify DG levels in parathyroid cells in response to high Ca2+ over the time course when IP3 levels increase. A simple enzymatic method was developed for the quantitation of the mass of DG present in crude lipid extracts. The assay employed rat brain DG kinase and defined mixed micellar conditions to solubilize the DG present and allow its quantitative conversion to [32P]phosphatidic acid. [32P]Phosphatidic acid formed in the assay was directly proportional to the amount of DG added over the range of 25 pmol to 25 nmol or to the number of parathyroid cells (5 .times. 105 to 2 .times. 106 cells). Parathyroid cells were also labeled with [3H]glycerol (24 h) or [3H]arachidonic acid (2 or 18 h) and exposed to various extracellular Ca2+ concentrations for different times. The total lipids were then extracted and separated by TLC. Using each of the three methods to measure DG, parathyroid cells showed a rapid increase in DG when extracellular Ca2+ was increased from 0.5 to 2.0 or 3.0 mM. The maximal icreases occurred at 5-20 s. The levels of DG at high Ca2+ then decreased to levels 20-50% higher than those at 0.5 mM Ca2+ from 60 sec to 10 min. DG levels remained higher at 2-3 mM Ca2+ than at 0.5 mM Ca2+ even at 30 min. Similar results were obtained in 10 independent experiments with the kinase method, 7 independent experiments with the [3H]glycerol method, and 12 independent experiments with the [3h]arachidonic acid method. These results show the high Ca2+ rapidly increases intracellular levels of DG as well as IP3 in bovine parathyroid cells, consistent with activation of phospholipase C.Thus, the initial rapid decrease in PTH release at high Ca2+ is not caused by a concomitant decrease in DG, but is presumably related to additional inhibitory mechanisms that override the high Ca2+-induced increases in DG and cytosolic Ca2+.