No abstract

SUMMARY1. Osteoclasts are known to secrete acid phosphatase, an iron-containing phosphohydrolase. We have investigated (a) the possibility that acid phosphatase has a functional role in bone resorption and (b) the factors controlling enzyme secretion from isolated rat osteoclasts.2. Osteoclasts were freshly disaggregated from neonatal rat long bones and dispersed at low densities on devitalized cortical bone slices or on plastic substrate. The levels of acid phosphatase in culture medium were measured spectrophotometrically using 4-nitrophenyl phosphate as hydrolysable substrate. The total plan area of bone resorbed was quantified by scanning electron microscopy in combination with image processing and analysis.3. Ninety-three per cent of the total enzyme activity detected in the supernatant exposed to bone-osteoclast preparations was resistant to inhibition by D-tartaric acid and was bound to an antibody known to be highly specific for the osteoclastderived isoenzyme, showing that it originated from osteoclasts.4. A diminution in the level of supernatant enzyme activity achieved by incubating bone-osteoclast preparations with an antiserum specifically binding the osteoclast isoenzyme, or with a non-competitive inhibitor, molybdate or with competitive inhibitors, disphosphonates, led to a marked reduction of osteoclastic bone resorption.5. The rate of the enzyme released into the culture supernatant, whether from resorbing (cultured on bone) or non-resorbing (cultured on plastic) osteoclasts declined gradually over 22 h, but that from the former was significantly depressed within the first 30 min of incubation. The supernatant enzyme concentration increased linearly up to 3 h; the levels released from resorbing osteoclasts remained consistently lower than those from non-resorbing cells. B. S. MOONGA AND OTHERS 7. Exposure of bone-osteoclast preparations to pertussis toxin produced no significant change of acid phosphatase release, while cholera toxin, dibutyryl cyclic AMP and forskolin produced a marked elevation of enzyme secretion. lonomycin was found to inhibit enzyme release and this was less marked when osteoclasts were incubated on plastic substrate.8. There was a significant positive correlation between basal enzyme release and bone resorption (correlation coefficient 0-78; P < 0-01) and between the percentage fall in supernatant enzyme activity and the percentage reduction of measured resorption in the presence of elevated [Ca2+] generated as a result of osteoclastic resorption. This suggests the existence of an important self-regulatory mechanism. (c) The intracellular regulation of acid phosphatase release is effected via the intermediacy of G proteins, one of which is cholera toxin sensitive and causes stimulation of enzyme release via a cyclic AMPdependent pathway.