Abnormal Regulation of Parathyroid Cell Secretion and Proliferation in Primary Cultures of Bovine Parathyroid Cells

Abstract

A method was developed for establishing primary monolayer cultures of bovine parathyroid cells, the cells were characterized morphologically and the effects of Ca on PTH [parathyroid hormone] secretion and cellular proliferation were studied. Fresh bovine parathyroid glands were enzymatically and mechanically dispersed, as previously described, using sterile solutions and apparatus. The cells when then plated at a density of 2.5 .times. 105 cells/cm2 into cluster wells and incubated at 37.degree. 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. EM 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 < 0.02), and cellular protein increased from 40.00 .+-. 1.40 or 177.60 .+-. 57.10 .mu.g/well (P < 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/105 cells.cntdot.h, respectively). Unlike normal bovine parathyroid cells, however, high Ca concentrations inhibited PTH secretion only slightly (21.2 .+-. 4.7%) and had no effect on cellular proliferation. Addition of the divalent cation ionophore A23,187 [calcimycin], inhibited PTH release by 50% or more, suggesting that the secretory apparatus is responsive to increases in the cytosolic Ca concentration. 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 Ca on hormonal secretion and cellular proliferation comparable to that of pathological parathyroid tissue. This cell culture system may provide a useful model for investigating the relationship between secretion and proliferation in normal and abnormal tissue.