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The present study reports on the effects of extracellular calcium ([Ca2+]o) elevation and ionomycin on cell spread area of medullary bone osteoclasts freshly isolated from egg-laying Japanese quail. The responses were compared with those demonstrated in osteoclasts cultured for periods of 5-8 days and also to those previously demonstrated in neonatal rat osteoclasts. Freshly isolated medullary bone osteoclasts, unlike rat osteoclasts, were refractory to 20 mM [Ca2+]o, in that they showed no change in cell spread area. They did, however, show a modest (15%) reduction in cell spread area to ionomycin (7-50 microM), applied for 15-30 min. When medullary bone osteoclasts were precultured for 5-8 days, they exhibited a well-developed response to 20 mM [Ca2+]o with a 46% reduction in cell spread area. They also showed a similar reduction in cell spread area in response to ionomycin (4 microM). It is concluded that, unlike freshly isolated neonatal rat osteoclasts, those obtained from quail medullary bone appear refractory to inhibitory factors such as [Ca2+]o. However, when the avian cells are cultured for a few days they appear to recover their ability to respond to [Ca2+]o.

We have performed a set of independent studies on the effects of the circulating pancreatic polypeptide, amylin, on rat osteoclast function, in vitro. Time-lapse video observations, measuring cell protrusions and retraction, showed that 250 nmol l-1 amylin or 250 nmol l-1 beta-calcitonin gene-related peptide (beta-CGRP) inhibited osteoclast motility (quiescence or Q effect). Both amylin and beta-CGRP produced inhibitory responses with a significant first-order regression over time (half-times, 19 and 28 min respectively). In contrast, 250 nmol l-1 amylin or 250 nmol l-1 beta-CGRP produced no change of osteoclast spread area, whilst 300 pmol l-1 calcitonin (CT) application resulted in cell retraction (R effect). Forskolin (10 mumol l-1) mimicked amylin and CGRP in inhibiting osteoclast motility (half-time, 8.6 min), and similarly lacked an effect on cell spread area. Neither amylin nor beta-CGRP (62.5–1250 nmol l-1) elevated cytosolic free calcium levels ([Ca2+]i) in single osteoclasts whilst 300 pmol l-1 salmon calcitonin (sCT) produced a rapid phasic elevation of [Ca2+]i, confirming previous results with asusuberic (1-7) eel calcitonin. The osteoclast-bone resorption assay revealed the following potency difference in direct comparison of the area of resorption per bone slice: beta-CGRP/amylin, 0.1; sCT/amylin, 800 and human CT/amylin, 12. The potency of deamidated amylin approached that of beta-CGRP. Assay precision ranged between 0.3 and 0.8. Amylin (250 nmol l-1) also significantly (P < 0.05) reduced supernatant (tartrate-resistant) acid phosphatase in the bone-osteoclast cultures. These measures independently indicate an effect of amylin on osteoclast motility through mechanisms distinct from those of calcitonin, possibly through different selectivities for receptor subtypes, the cyclic AMP-linked 'amylin subtype' and the [Ca2+]i-linked 'calcitonin subtype'.