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The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [1251] bovine PTH(I-34). Binding was initiated by adding [12Sl]-bPTH(I-34) to bisected calvariae at 30°C. Steady state binding was achieved at 90 min at which time 10 mg dry wt of calvaria specifically bound 17% of the added [12Sl]bPTH (I-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was <2%. Insulin, glucagon, and calcitonin (I/~g/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(I-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(I-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [12Sl]bPTH(I-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.Parathyroid hormone (PTH) acts directly on bone acutely to increase bone resorption and decrease bone formation (1, 2) and chronically to increase both bone resorption and bone formation (3). These actions are mediated by the ceUular elements of bone by mechanisms involving, at least in part, the stimulation of cyclic Y,5' adenosine monophosphate (cAMP) production (4, 5). The evidence available indicates that PTH directly or indirectly influences all bone cells (osteogenic precursors, osteoblasts, osteocytes, and osteoclasts) (6-9); however, recent reports (10, 11) which describe the effect of PTH on cAMP production in osteoblastlike or osteoclastlike cells in monolayer culture implicate the osteoblast as the major osseous target cell of PTH.During the past five years, major advances in the production of biologically active, high specific-activity, radioiodinated preparations of PTH have made it possible to investigate the previously elusive PTH receptor in kidney (12). It is only recently that similar progress has been made in the identification and characterization of PTH receptors in bone: using electrolytically [L2SI]-labeled, receptor-purified, synthetic bo-
The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [1251] bovine PTH(I-34). Binding was initiated by adding [12Sl]-bPTH(I-34) to bisected calvariae at 30°C. Steady state binding was achieved at 90 min at which time 10 mg dry wt of calvaria specifically bound 17% of the added [12Sl]bPTH (I-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was <2%. Insulin, glucagon, and calcitonin (I/~g/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(I-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(I-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [12Sl]bPTH(I-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.Parathyroid hormone (PTH) acts directly on bone acutely to increase bone resorption and decrease bone formation (1, 2) and chronically to increase both bone resorption and bone formation (3). These actions are mediated by the ceUular elements of bone by mechanisms involving, at least in part, the stimulation of cyclic Y,5' adenosine monophosphate (cAMP) production (4, 5). The evidence available indicates that PTH directly or indirectly influences all bone cells (osteogenic precursors, osteoblasts, osteocytes, and osteoclasts) (6-9); however, recent reports (10, 11) which describe the effect of PTH on cAMP production in osteoblastlike or osteoclastlike cells in monolayer culture implicate the osteoblast as the major osseous target cell of PTH.During the past five years, major advances in the production of biologically active, high specific-activity, radioiodinated preparations of PTH have made it possible to investigate the previously elusive PTH receptor in kidney (12). It is only recently that similar progress has been made in the identification and characterization of PTH receptors in bone: using electrolytically [L2SI]-labeled, receptor-purified, synthetic bo-
We characterized the binding of 125I-[Nle8, Nle18, Tyr34]parathyroid hormone-(1-34) amide [125I-nlPTH-(1-34)] to renal plasma membranes prepared from chicks to determine the effects of secondary hyperparathyroid states on renal PTH receptors. This radioligand exhibited specific binding to membranes with high affinity (Kd, 2-3 X 10(-9) M). Agonists or competitive antagonists of PTH were effective in competing for binding sites labeled with 125I-nlPTH-(1-34), whereas an inactive fragment of PTH, salmon calcitonin, and bovine growth hormone did not compete with the radioligand for renal PTH receptors. Newly hatched chicks raised on control diet with adequate vitamin D and calcium or diets deficient in either vitamin D or calcium were used to study the regulation of renal PTH receptors in experimental models of secondary hyperparathyroidism. We found that both experimental diets resulted in marked hypocalcemia and progressive loss of renal cyclic AMP responsiveness to PTH in vitro. Associated with this refractoriness to the hormone was a marked reduction in PTH receptors in membranes from both vitamin D-deficient and calcium-deficient chick kidney. No change in the affinity of the PTH receptors was found. Vitamin D3, in a single dose of 250 micrograms, partially restored serum calcium of vitamin D-deficient birds toward normal by 72 h and also partly restored renal cyclic AMP responsiveness to PTH and the PTH receptor number toward control values. We conclude that renal refractoriness to PTH observed in experimentally hyperparathyroid animals models is due to a marked loss of plasma membrane receptor sites for PTH without an apparent change in the affinity of the receptors for the hormone.
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