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Binding of Monoclonal Antibody (4F2) to its Cell Surface Antigen on Dispersed Adenomatous Parathyroid Cells Raises Cytosolic Calcium and Inhibits Parathyroid Hormone Secretion^*

Endocrine-Hypertension Unit, Department of Medicine, Brigham and Women’s Hospital Boston, Massachusetts 02115
the Joslin Diabetes Center Boston, Massachusetts 02115
Harvard Medical School Boston, Massachusetts 02115
Department of Immunology, Scripps Clinic and Research Foundation LaJolla, California 92037

Address all correspondence and requests for reprints to: James T. Posillico, Ph.D., Endocrine-Hypertension Unit, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115.

In the course of characterizing monoclonal antibodies (MAbs) recognizing cell surface antigens on dispersed human parathyroid cells (dPTCs), we identified one MAb (4F2) that bound avidly to parathyroid cells and had marked effects on parathyroid function. The binding of MAb 4F2 to human adenomatous dPTCs resulted in a marked [53.8 ± 7.9% (±SEM)] reduction in low calcium (Ca)-stimulated PTH secretion to levels equivalent to those in cell suppressed by high extracellular Ca (1.5 mM). Typically, these functional effects were optimal at antibody dilutions of 1:10⁴ to 1:10⁶. Cell viability was confirmed at the conclusion of each experiment by trypan blue exclusion (>90–95%) and cell surface immunofluorescence. Parallel studies using the Ca-sensitive dye Quin-2 showed that inhibition of PTH secretion in 4F2-treated cells was associated with a concomitant increase in cytosolic Ca (Ca;) of 188% in 0.5 mM Ca_i these values also approached Ca; levels in control cells incubated in high Ca. Mab controls, P3x63, which do not bind to dPTCs, and Mab LC7-2, which recognizes a different epitope of the same antigen as 4F2 on dPTCs, did not alter PTH secretion or Ca;. Immunoprecipitation of ¹²⁵I-labeled parathyroid cell extracts with MAb 4F2 demonstrated proteins with mol wt of approximately 145,85, and 45 under nonreducing conditions and 85 and 45 kilodaltons after reduction with 5% mercaptoethanol. These studies suggest that 1) Mab-4F2 binding to its cell surface antigen inhibits PTH secretion by human adenomatous para-thyroid cells in vitro; 2) the alterations in secretory function could be related to by an attendant increase in Ca_i; 3) the 4F2 antigen on dPTCs is a heterodimeric protein of (approximately) 85K and 45K; and 4) the 4F2 antigen may be an important component of the Ca-sensing and/or signal-transducing mechanism in this cell.

^* This work was supported by NIH Grants AM-25778, AM-1378, AM-36801, and AM-36796 and the Juvenile Diabetes Foundation (045C81 and 83F200).

Received March 28, 1986.

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