Intracellular regulation of enzyme secretion from rat osteoclasts and evidence for a functional role in bone resorption.

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.

Structure, function, and regulation of tartrate-resistant acid phosphatase

“…Addition of anti-TRAP antibodies reduced the in vitro resorption of bone, 84,123 an observation we have reproduced using antiserum raised against recombinant murine TRAP (A.I.C., unpublished observations). Addition of bisphosphonate drugs or molybdate to the culture medium was also found to reduce both TRAP activity and bone resorption.…”

“…Addition of bisphosphonate drugs or molybdate to the culture medium was also found to reduce both TRAP activity and bone resorption. 84 In the somewhat more complex mouse calvarial calcium-release assay, hydroxynaphthalene-phosphonate inhibitors of TRAP also inhibited bone resorption. 110 Mice with even a heterozygous null mutation introduced into the TRAP gene showed evidence of mild osteopetrosis, which was presumed to be due to defective osteoclastic remodeling activity.…”

Calcium Signalling and Calcium Transport in Bone Disease

“…Thus, such extracellular [Ca 2+ ] alterations alter bone resorptive activity in isolated rat osteoclasts [Malgaroli et al, 1989 following rapid and sustained changes in micro-spectrofluometrically determined cytosolic [Ca 2+ ], cell retraction ('R' effect) and longer-term inhibition of enzyme release and bone resorption [Datta et al, 1989acorrect to 1989, Moonga et al, 1990Zaidi, 1990]. These findings suggest an existence of long and short term feedback mechanisms on both enzyme release Moonga et al, 1990] and osteoclastic bone resorptive activity controlled by the increases in extracellular [Ca 2+ ] that result from the latter process.…”

Design, synthesis and testing of purple acid phosphatase inhibitors for the development of anti-osteoporotic drugs

“…1 Furthermore, some cases of long-term use of bisphosphonates have been associated with osteonecrosis of the jaw (ONJ). [36][37] Osteoporosis patients prescribed with bisphosphonates have also been shown to have higher risk of atrial fibrillation [38][39][40] and, peculiarly, higher incidence of atypical femoral shaft fractures. [40][41][42][43][44][45] 58,70 .…”

“…There has been evidence that suggests a strong link between PAP and the development of osteoporosis [30][31][32] in the literature, due to mammalian PAP's postulated role in bone resorption. 16,30,[33][34][35][36][37][38][39][40][41][42][43][44][45] Therefore, this work aims to develop new PAP inhibitors for the development of anti-osteoporotic chemotherapeutics using the approaches discussed in Section 1.5.…”