When bovine parathyroid cells in culture are exposed to the active vitamin D metabolite 1,25(OH)2D3, a significant decrease in the steady-state levels of pre-proparathyroid hormone (pre-proPTH) mRNA occurs. The possibility that the fall in specific mRNA is due to a decrease in rate of transcription of the PTH gene was examined in this study. In the presence of 1,25(OH)2D3, there was a rapid and steady decline in PTH gene transcription rate which fell to a minimum of 10-15% of control at 24 h. The effect was observed at physiological levels (10(-11)M) and was also fully reversible.

To determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] regulates PTH secretion, we have tested its effects in both short term incubations (30-120 min) and long term primary cell cultures (24-96 h) of bovine parathyroid cells. In short term incubations, 10(-11)-10(-7) M 1,25-(OH)2D3 had no consistent effect on PTH secretion. In primary cultures of bovine parathyroid cells, significant suppression of PTH secretion occurred, as measured by both N-terminal and C-terminal PTH assays. Suppression of PTH secretion was dose dependent when 10(-11), 10(-9), and 10(-7) M 1,25-(OH)2D3 were tested for 48 h in culture, and the effects of 10(-7) M, 1,25-(OH)2D3 were noted as early as 24 h. Reversal of suppression of PTH secretion was observed after an additional 48 h in the absence of 1,25-(OH)2D3. Other studies from our laboratory have demonstrated that 1,25-(OH)2D3 suppresses levels of pre-pro-PTH mRNA in cultured bovine parathyroid cells, and we found a strong correlation at 48 h between the decrease in PTH release and that in mRNA. We conclude that 1) 1,25-(OH)2D3 suppresses PTH secretion rates in a dose-dependent manner in cells grown for 24-48 h in culture, but does not have a significant effect on short term PTH release (30-120 min); 2) cultured cells exhibiting suppression by 1,25-(OH)2D3 demonstrate nearly full recovery of PTH secretion after an additional 48 h in the absence of added 1,25-(OH)2D3; and 3) PTH secretion closely parallels levels of pre-pro-PTH mRNA in cultured cells, suggesting that the observed effects of PTH secretion reflect, at least in part, suppression of synthesis of PTH by 1,25-(OH)2D3.

A B S T R A C T DNA complementary to bovine preproparathyroid hormone mRNA was cloned and labeled by nick translation in order to measure mRNA by molecular hybridization. Bovine parathyroid cells were maintained in primary tissue culture for periods up to 96 h at 0.5 mM, 1.25 mM, and 2.5 mM calcium, which was followed by extraction of cellular RNA. Levels of mRNA showed no differences at 0.5 or 1.25 mM calcium, but at high calcium levels, there was a reversible decrease that began at 16 h to a plateau at 30% of control after 72 h. These studies suggest that the glandular capacity to synthesize hormone may be at or near maximal at normal calcium, but at high calcium, there is a decrease over time in steady state levels of mRNA.

Regulation of protein kinase C in the parathyroid gland was investigated by testing the effects of phorbol ester, exogenous phospholipase C, and low and high calcium concentrations on enzyme activity. Treatment of bovine parathyroid cells with phorbol ester, which activates protein kinase C directly, and with phospholipase C, which produces diacylglycerol, an activator of protein kinase C, significantly stimulated protein kinase C activity. Both agents also enhanced the release of parathyroid hormone. Acute exposure of bovine parathyroid cells to low extracellular calcium (0.5 mM) caused a 5-to 6-fold increase in protein kinase C activity associated with the particulate fraction. In contrast, high extracellular calcium (1.75 mM and 2.5 mM) markedly decreased membrane protein kinase C activity. These data suggest that the effects of extraceilular calcium on parathyroid hormone secretion are due, at least in part, to regulation of protein kinase C activity in the parathyroid-cell membrane.Physiologic control of parathyroid hormone (PTH) secretion is regulated by changes in extracellular calcium, with low concentrations of calcium stimulating and high concentrations of calcium inhibiting hormone release (1-4). How extracellular changes in calcium levels are recognized by the parathyroid cell and what transduces signal recognition into intracellular processes controlling exocytosis and PTH secretion are as yet uncertain.Recent studies suggest that protein kinase C, which is activated by calcium, phospholipid, and diacylglycerol, plays an important role in exocytosis (5). Therefore, we examined the role of protein kinase C in regulation by extracellular calcium of PTH secretion by testing the effects of phorbol ester, exogenous phospholipase C (PLC), and low and high calcium concentrations on protein kinase C activity in isolated bovine parathyroid cells.This Dispersed parathyroid cells were prepared by collagenase digestion of minced glands (6). After purification on Percoll gradients, the cells (which equilibrated within a buoyant density of 1.048-1.062) were aspirated, washed, and resuspended in basal Eagle's medium (BME). Protein kinase C activity was measured by a modification of the method of Kikkawa et al. (7). After cells were lysed and sonicated with 3 ml of buffer A (20 mM Tris HCI, pH 7.5, containing 0.25 M sucrose, 10 mM EGTA, and 2 mM EDTA), cytosolic and particulate membrane fractions were prepared by centrifugation at 100,000 x g for 60 min. The centrifuged membrane pellet was homogenized with a glass rod in 1% Nonidet P-40 buffer A. Crude extracts from the membrane and cytosolic fractions were applied to a 1-ml DE-52 column equilibrated with buffer B (20 mM Tris-HCI, pH 7.5, containing 50 mM 2-mercaptoethanol, 5 mM EGTA, and 2 mM EDTA). Columns were washed with 15 ml of buffer B, 15 ml of buffer C (20 mM Tris-HCI, pH 7.5, containing 50 mM 2-mercaptoethanol, 1 mM EGTA, and 1 mM EDTA), and 0.5 ml of buffer C containing 0.1 M NaCl. An additional 1.5 ml of 0.1 M NaCl/buffer C eluate was then colle...

Previous studies have shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases levels of mRNA for prepro-PTH as well as PTH secretion after chronic exposure (24-48 h) of parathyroid cells in tissue culture. We have now extended these studies to determine the effects of the vitamin D3 metabolite on parathyroid secretory protein (PSP) gene expression. Primary cultures of bovine parathyroid cells were incubated with 10(-8) M 1,25-(OH)2D3 for periods of time ranging from 24-72 h. As observed in earlier experiments, prepro-PTH mRNA decreased to less than 50% of the control value after 72 h. In marked contrast, PSP mRNA showed a 2.5-fold increase by 24 h and greater than 7-fold stimulation by 72 h. In the same studies, PTH secretion was suppressed (to 60% of control), while PSP secretion was increased by 40% over control values. Exposure to high (2.5 mM) or low (0.5 mM) calcium had no effect on PSP mRNA, even though low calcium stimulated the secretion of PSP while high calcium suppressed secretion. These studies showed that 1,25-(OH)2D3 has opposite effects on the gene expression of PSP and PTH in bovine parathyroid cells in tissue culture.