Abnormal regulation of parathyroid cell secretion and proliferation in primary cultures of bovine parathyroid cells.

Abstract

We developed a method for establishing primary monolayer cultures of bovine parathyroid cells, characterized the cells morphologically, and studied the effects of calcium on PTH secretion and cellular proliferation. Fresh bovine parathyroid glands were enzymatically and mechanically dispersed, as previously described, using sterile solutions and apparatus. The cells were then plated at a density of 2.5 X 10(5) cells/cm2 into cluster wells and incubated at 37 C in a medium containing Dulbecco's Modified Eagle's and Ham's F-12 medium, 15% newborn calf serum, Hepes, antibiotics, and insulin. In 3-4 days, a near-confluent monolayer of polygonal-shaped cells with less than 10% fibroblasts was achieved. Electron microscopy revealed a homogeneous cell population containing electron-dense secretory granules as well as abundant rough endoplasmic reticulum and mitochondria, with no evidence of organelle swelling. Two parameters of cellular proliferation increased significantly in culture; viable cell number increased from 289,000 +/- 63,000 to 530,000 +/- 60,000 per well (P less than 0.02), and cellular protein increased from 40.00 +/- 1.40 to 177.60 +/- 57.10 micrograms/well (P less than 0.035). On an hourly basis, cultured cells showed a greater secretory rate than that of acutely dispersed cells (9.3 vs. 5.0 ng/10(5) cells X h, respectively). Unlike normal bovine parathyroid cells, however, high calcium concentrations inhibited PTH secretion only slightly (21.2 +/- 4.7%) and had no effect on cellular proliferation. Addition of the divalent cation ionophore A23187, on the other hand, inhibited PTH release by 50% or more, suggesting that the secretory apparatus is responsive to increases in the cytosolic calcium concentration. The present study reveals that bovine parathyroid cells in primary culture proliferate and secrete PTH in vitro. These cells display, however, a generalized decrease in sensitivity to the suppressive effects of extracellular calcium on hormonal secretion and cellular proliferation comparable to that of pathological parathyroid tissue. Thus, this cell culture system may provide a useful model for investigating the relationship between secretion and proliferation in normal and abnormal tissue.