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Characterisation of the osteoclast calcium “receptor”: evidence for a novel receptor-operated calcium channel and intracellular mobilisation
48 Characterisation of the osteoclast caicium ‘receptor’: evidence for a novel receptor-operated calcium channel and intracellular mobilisation M. Zaidi, V. S. Shankar, B. S. Moonra, A.S.M.T. Alam, 8. E. 8ax. F. Avaldi, R. Soncini, C. L.-H. Huang. 3. Bax. Department of C&far and Molecular Scicnccs, St. George’s Hospital Medical School, London. The inhibition of ostcoclastic bone resorption, caused by an elevation in the extracellular calcium concentra\ion (ICa2+)& is thought to be mediated by a calcium “receptor”. Activation of this putative “receptor” transduces a rise in the cytosolic free calcium concentration ([Ca2+]&)by a voltage-insensitive process. WC have attempted to dctcrmine whether the transduction process involves (a) the direct gating, by the putative calcium “receptor”, of a unique reccptoroperated calcium channel (ROCC) and/or (b) the redistribution of [Ca2+]i. Single ccl1 lCa2*li and IMg2+11levels were measured, rcsDcctivclv, bv fura 2 and Mae-fura. Evidence far receutor-ooerated Ca;+ entry:.& found that d
49 CARBOXYL-TERMINAL PARATHVHOID HORMONE-RELATED PSOTEIN INHIBITS BONE RESORPTION AND OSTEOCLAST GENERATION IN LONG-TERM CULTURES WITHOUT
EVIDENCE University 0 We have erminal fragment of parathyroid HrP[lO?-1391 is a potent direct inhibitor of osteoclastic bone resorption. We have found that osteoclasts isolated from the long bones of neonatal rats remain viable in culture for up to 144 hours. TRACP-positive, multinucleated cell numbers double in the first 48 hours of culture and remain stable until 96-144 hours when a decline in the population number is noted. Resorption in control cultures increases over the duration of the incubation period, with the greatest increase occuring between 24 and 46 hours. In culture:, of isolated rat osteoclasts, salmon CT (f@%f) acls as a potent inhibitor of osteoclastic bone resorution. However, this eifect is progressively lost after 48 hours a& the osteoclasts “escaoe” from the sCT-induced inhibition. Thir “escaoe” phendmenon has been noted in..qther culture systems, In contrast, PTHrP[? 07-f 39) (IO-sM) persisf@ntly inhibits bone resorption for at least 144 hours without evidence of “escape”. Inhibition of both recruitment and fusion of mononuclear TRACP,.positive cells by sCT is evident by 48 hours in culture. The number of TRACP-positive ,nultinucleated cells in cultures treated with PTHrP[107-1391 does not differ significantly from control cultures. However, the number of TRACP-positive mononucleated cells decreases dramatically at 96 hours, compared to control. These results . suggest that PTHrPIlO7-1391 inhibits recruitment of osteoclast-like cell precurs‘ors but does not inhibit the fusion of mononuclear cells. In summary, PTHrP[107-1391 is a potent long-term inhibitor of osteoclastic bone resorption. In addition, it acts to inhibit the recruitment of osteoclast precursors in long-term cultures of isolated osteoclasts.
based upon iaid down criteria for the prediction of ROCCs (Rink, 1988; Nature 334: 649-650). WC propose the existence of a novel calcium “rcccptor”-operatedcalcium channel. Bvidcnce for mobilisation of [CaL+lI and its desensitisation: We found cvidcnce of divalent cation
sensitivitv of thp uutative calcium “rcccBtor”. Ni2* was found to mimic the cffeciof ICd$ clcvalion causing a fonccntration-dependent (50 pM to 5 mM) clcvation in ICa2*1~. The response to low [Niz’l was monophasic, but at highcrconrontrations (2 mM and at~vc! a sustained rlsc of [C$*]I followed the initial monophasic transient. The latter was indcpcndcnt of [Cal*]c and was thus; not abolished in [Caz’].-free medium. Prctrcatmcnt with a range of concentrations of Ni2* led to a concentration-dependent inhibition of the subsequent response to a high [Ni2+l(5 n&l), suggesting dcscnsitisation. Conclusion: We suggest that the putative calcium “receptor” rcgulatcs osteoclast [Ca2+l,by causing the activation of a novel ROCC, as well as by intracellular calcium mobilisation that can be dcscnsitised on rcpeatcd exposure. 50
DISPLACEMENT OF OSTEOBLASTS BY OSTEOCLASTS IS A SIGNIFKANT FACTORIN OSTE~k~STIC BONE RESORZO; M$$ -he. E w, M Schabe s . JE Aubin and J Fe ‘e.
g;;~;
Penodontal Physiology, University of Toronto, Toronto,
Isolated rabbit asteoclasts plated onto devitalized bone slices or onto plastic surfaces and then c&ured appear to move randomly. When cultured on bone slices, the arcas where bone matrix is resorbed also appear to be selected randomly (Kanehisa and Heersche? 1988). The resorption in devitalized bone slice systems, however, differs from in viva resorption in that the bone surface is not covered by a continuous layer of osteoblasts and/or lining ceils. It has been hypothesized that in vivo osteoblasts or lining cells retract at sites to be resorbed and thus enable osteoclasts to access the bone surface arrd initiaic resorption (Rodan and Martin, 1981). The mechanisms or factors triggering this (presumed) local osteoblast contraction are not known. In the present series of experiments, we have plated rabbit osteoclasts and rabbit o$teoblast-like stromal cells (OB cells) onto plastic surfaces and artalysed the migration patterns of individual osteoclasts using timelapse recording of their activity (Kanehisa and Heersche. 1988). The 08 cells respond to PTH with an increase in intracellular CAMP. We were particularly interested in observing and analysing the interactions between the migrating osteoclasts and the osteoblast-like stromal cells. Observations were made on osteoclasts interacting with single OB cells or small OB colonies (n = 50) and on osteoclasts migrating towards a monolayer of OB cells (n = 35). These observations have provided clear evidence that osteoclasts can displace OB cells from plastic surfaces. With single OB cells and small OB colonies, sut!den retraction of OB cells uoon contact with the osteoclasts is the predominant mechanism whereby these cells begin to move out nl‘ the oath of the osteoclast. Following this, osteoclasts frequently appear to push the 08 cells at speeds cha&cristic of osteoclasi motiiitj. When osteoclasts displace OB cells that are part of a monolayer, the mechanib:mappears to involve migration of osteoclnsts underneath the osteoblast-like cell layer, followed by partial retraction of the OB cells involved. These mechanisms imply osteoclast-mediated partial detachment of OB cells. On the basis of these observations, we propose as a working hypothesis that osteoclasts obtain access to the bone surface by actively displacing osteobiasts or lining cells at sites eimnxked for resorption.
83
49 CARBOXYL-TERMINAL PARATHVHOID HORMONE-RELATED PSOTEIN INHIBITS BONE RESORPTION AND OSTEOCLAST GENERATION IN LONG-TERM CULTURES WITHOUT
EVIDENCE University 0 We have erminal fragment of parathyroid HrP[lO?-1391 is a potent direct inhibitor of osteoclastic bone resorption. We have found that osteoclasts isolated from the long bones of neonatal rats remain viable in culture for up to 144 hours. TRACP-positive, multinucleated cell numbers double in the first 48 hours of culture and remain stable until 96-144 hours when a decline in the population number is noted. Resorption in control cultures increases over the duration of the incubation period, with the greatest increase occuring between 24 and 46 hours. In culture:, of isolated rat osteoclasts, salmon CT (f@%f) acls as a potent inhibitor of osteoclastic bone resorution. However, this eifect is progressively lost after 48 hours a& the osteoclasts “escaoe” from the sCT-induced inhibition. Thir “escaoe” phendmenon has been noted in..qther culture systems, In contrast, PTHrP[? 07-f 39) (IO-sM) persisf@ntly inhibits bone resorption for at least 144 hours without evidence of “escape”. Inhibition of both recruitment and fusion of mononuclear TRACP,.positive cells by sCT is evident by 48 hours in culture. The number of TRACP-positive ,nultinucleated cells in cultures treated with PTHrP[107-1391 does not differ significantly from control cultures. However, the number of TRACP-positive mononucleated cells decreases dramatically at 96 hours, compared to control. These results . suggest that PTHrPIlO7-1391 inhibits recruitment of osteoclast-like cell precurs‘ors but does not inhibit the fusion of mononuclear cells. In summary, PTHrP[107-1391 is a potent long-term inhibitor of osteoclastic bone resorption. In addition, it acts to inhibit the recruitment of osteoclast precursors in long-term cultures of isolated osteoclasts.
based upon iaid down criteria for the prediction of ROCCs (Rink, 1988; Nature 334: 649-650). WC propose the existence of a novel calcium “rcccptor”-operatedcalcium channel. Bvidcnce for mobilisation of [CaL+lI and its desensitisation: We found cvidcnce of divalent cation
sensitivitv of thp uutative calcium “rcccBtor”. Ni2* was found to mimic the cffeciof ICd$ clcvalion causing a fonccntration-dependent (50 pM to 5 mM) clcvation in ICa2*1~. The response to low [Niz’l was monophasic, but at highcrconrontrations (2 mM and at~vc! a sustained rlsc of [C$*]I followed the initial monophasic transient. The latter was indcpcndcnt of [Cal*]c and was thus; not abolished in [Caz’].-free medium. Prctrcatmcnt with a range of concentrations of Ni2* led to a concentration-dependent inhibition of the subsequent response to a high [Ni2+l(5 n&l), suggesting dcscnsitisation. Conclusion: We suggest that the putative calcium “receptor” rcgulatcs osteoclast [Ca2+l,by causing the activation of a novel ROCC, as well as by intracellular calcium mobilisation that can be dcscnsitised on rcpeatcd exposure. 50
DISPLACEMENT OF OSTEOBLASTS BY OSTEOCLASTS IS A SIGNIFKANT FACTORIN OSTE~k~STIC BONE RESORZO; M$$ -he. E w, M Schabe s . JE Aubin and J Fe ‘e.
g;;~;
Penodontal Physiology, University of Toronto, Toronto,
Isolated rabbit asteoclasts plated onto devitalized bone slices or onto plastic surfaces and then c&ured appear to move randomly. When cultured on bone slices, the arcas where bone matrix is resorbed also appear to be selected randomly (Kanehisa and Heersche? 1988). The resorption in devitalized bone slice systems, however, differs from in viva resorption in that the bone surface is not covered by a continuous layer of osteoblasts and/or lining ceils. It has been hypothesized that in vivo osteoblasts or lining cells retract at sites to be resorbed and thus enable osteoclasts to access the bone surface arrd initiaic resorption (Rodan and Martin, 1981). The mechanisms or factors triggering this (presumed) local osteoblast contraction are not known. In the present series of experiments, we have plated rabbit osteoclasts and rabbit o$teoblast-like stromal cells (OB cells) onto plastic surfaces and artalysed the migration patterns of individual osteoclasts using timelapse recording of their activity (Kanehisa and Heersche. 1988). The 08 cells respond to PTH with an increase in intracellular CAMP. We were particularly interested in observing and analysing the interactions between the migrating osteoclasts and the osteoblast-like stromal cells. Observations were made on osteoclasts interacting with single OB cells or small OB colonies (n = 50) and on osteoclasts migrating towards a monolayer of OB cells (n = 35). These observations have provided clear evidence that osteoclasts can displace OB cells from plastic surfaces. With single OB cells and small OB colonies, sut!den retraction of OB cells uoon contact with the osteoclasts is the predominant mechanism whereby these cells begin to move out nl‘ the oath of the osteoclast. Following this, osteoclasts frequently appear to push the 08 cells at speeds cha&cristic of osteoclasi motiiitj. When osteoclasts displace OB cells that are part of a monolayer, the mechanib:mappears to involve migration of osteoclnsts underneath the osteoblast-like cell layer, followed by partial retraction of the OB cells involved. These mechanisms imply osteoclast-mediated partial detachment of OB cells. On the basis of these observations, we propose as a working hypothesis that osteoclasts obtain access to the bone surface by actively displacing osteobiasts or lining cells at sites eimnxked for resorption.
83