Specific binding of parathyroid hormone to living osteoclasts

Abstract

We show that osteoclasts bind parathyroid hormone (PTH) in a manner that displays the properties of receptor-dependent hormone binding, that is, saturability, time dependence, temperature dependence, and hormone specificity. Osteoclasts were isolated from the endosteum of 2 to 3 week chick tibiae and maintained in culture for 4-6 days. Bovine PTH-(1-84) was biotinylated with N-hydroxysuccinimidobiotin. Biotinyl-PTH (btPTH, 10(-5)-10(-11) M) was added to the cultured osteoclasts for 2-20 minutes. After rinsing away unbound btPTH, fluorescein isothiocyanate-labeled avidin (FITC-avidin) at a concentration of 66 micrograms/ml was applied. Receptor binding characteristics were assessed: (1) saturation occurred at around 10(-6) M btPTH; (2) competition of excess unlabeled PTH was found, namely, a 10-fold excess abolished fluorescence; (3) specificity was shown by adding other polypeptide hormones (insulin, glucagon, and calcitonin) in 10- to 100-fold excess--no effect on PTH binding was observed; and (4) affinity of btPTH for its binding site was indicated by half-maximal binding approximately equal to 10(-7) M for both osteoclasts and osteoblasts. Biotin (10(-5) M) or FITC-avidin (66 micrograms/ml) alone did not cause fluorescence. The time course of btPTH on the cell exterior was short: at 2 and 5 minutes dots of fluorescence were randomly dispersed over the cell surface, by 10 minutes most of the fluorescence was clustered in one region of the membrane, and by 20 minutes most of the hormone was no longer present on the surface of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine Regulation of Calcium Transport in Epithelia

“…The classical action is based on the initial understanding that the major biological activities of PTH are subserved by the 34‐residues in its N‐terminal domain and that the PTH residues located beyond position 34 are largely irrelevant 79–81 . Non‐classical actions are now known to require additional regions of the peptide hormone and include binding of intact PTH to rat and avian osteoclasts; 82–85 the actions unique to intact PTH are reviewed elsewhere 86 …”

Simulation of generation–collection experiments with homogeneous kinetics: application to electrochemical investigation of superoxide radical anion generation by osteoclasts on bone

“…It is not surprising that a kinetically distinct response is observed compared to PTH for which the osteoclast expresses a membrane receptor. 15,16 The magnitude of the stimulation by pertussis toxin was found to be smaller than that produced by PTH which may be a result of the relative number of NADPH oxidase molecules which are activated, however, the accurate quantification of the initial flux, j 0 , is difficult because it is very sensitive to the rate of disproportionation of superoxide during the transit between the cell and the detector electrode (Fig. 3).…”

“…In view of the significant role of O 2 À in osteoclastic bone resorption, it is important to understand the precise intracellular signaling that is involved in the regulation of free radical production. Although it was originally believed that the modulation of osteoclast activity by major hormones, such as parathyroid hormone (PTH), 1,25-dihydroxycholecalciferol D 3 , and oestrogens is mediated indirectly via cells of osteoblast lineage, several reports have shown that osteoclasts possess receptors for PTH, 15,16 1,25-dihydroxycholecalciferol D 3 17 and oestrogens. 18 Indeed, the exposure of individual avian osteoclasts to PTH has been observed to stimulate hydrogen ion production via a G-protein coupled receptor.…”