A spontaneously arising mutation in connexin32 with repeated passage of FRTL-5 cells coincides with increased growth rate and reduced thyroxine release.

In this study we examine changes in the cellular properties of FRTL-5 cells as a function of passage number, with particular emphasis on gap junction expression, karyotype, morphology, growth rate and thyroxine (T(4)) release. Early passage FRTL-5 follicular cells transfer dye through gap junctions from injected cell(s) to third-order neighboring cells and beyond within their respective follicles and have immuno-detectable connexin32 (Cx32) type gap junctional plaques in their lateral contacting plasma membranes. By contrast, FRTL-5 cells established as monolayers, or as follicles from cultures passed more than 15 times, did not transfer microinjected Lucifer Yellow dye to contiguous neighboring cells and did not express any immuno-detectable rat thyroid specific connexins (Cx43, Cx32 or Cx26). Western blots confirmed that total, membrane and cytosolic Cx32 protein was present only in early pass follicular cultures. To better understand the passage-dependent loss of Cx32 expression, RT-PCR primers were made to the most unique sequences of the rat Cx32 molecule, the cytoplasmic and carboxyl-terminal regions. These primers were used to screen FRTL-5 RNA from cultures of various passage numbers. The results revealed that later passage cultures had a single base deletion in the middle of the Cx32 cytoplasmic loop region at nucleotide position 378. This base deletion was in the middle position of the codon for amino acid 116, which is normally a CAC (histidine) but read with the frame shift was a CCC (proline). The four amino acids that followed this deletion were also altered with the fourth one becoming UAA, the ochre translation stop codon. This premature stopping of translation resulted in a truncation of 60% of the protein, which included the remaining cytoplasmic loop, third and fourth transmembrane regions and the carboxyl-terminus. The later passage cultures did not produce a carboxyl-terminal RT-PCR product, indicating that the mRNA was also truncated. These regions of the Cx32 molecule contain the sequences and epitopes to which probes and antibodies are directed, and as such alterations of these regions with repeated passage explains reports by others that FRTL-5 cells do not express Cx32, and implies that cultures used for these assessments were passed more than 15 times. To determine if genetic or epigenetic abnormalities existed in FRTL-5 cells we performed chromosome spreads from various passage cultures. FRTL-5 cells have been reported to be diploid and more recently non-diploid; however, we found them to be fully tetraploid. This tetraploidy appears to be unstable in that later passes are tetraploid plus two or three extra chromosomes. There were no obvious translocations, breaks or large-scale interstitial deletions of any chromosomes in the FRTL-5 cultures tested. As FRTL-5 cells were repeatedly passed their morphology changed. Monolayer areas spread from beneath the follicles, and the follicles became flattened in appearance. These physical changes were coincident with dramatically increased growth rates. Early cultures (passed 3-12 times) divided on average every 49+/-1 h, whereas later passes (passes 20-25) divided every 28+/-3 h. To correlate these changes with a measure of thyroid function we assayed T(4) output. Early passage follicular cultures incubated for 6 h with sodium iodide, released on average 5.27+/- 0.33 ng/ml of T(4)/100 follicles. Later passes, or early passes treated with heptanol to down-regulate Cx32, released an average of 3.84+/-0.50 ng/ml of T(4)/100 follicles. There was a 27% difference in T(4) release between early follicular cultures, that were coupled by Cx32, and late or down-regulated early follicular cultures, that were uncoupled (P<0.0001). Collectively, the physical changes documented in this study were coincident with the loss of functional Cx32. This implies a relationship between the loss of intercellular communication and changes in morphogenic appearance, growth rate and reduced thyroid function and supports the previously postulated, tumor-suppressor role for Cx32. FRTL-5 cultures from low passage numbers are an excellent model of primary thyroid cells. However, many reports in the literature ascribe features to FRTL-5 cells that are mutually inconsistent. These differences may be resolved in the future by addressing the passage number and the conditional differences of the cultures being studied.

ELF magnetic fields initiate protein tyrosine phosphorylation of the T cell receptor complex.

The human T cell line Jurkat registers a sinusoidal extremely low frequency (ELF), 0.10 mT magnetic fields (MFs) at the level of the plasma membrane. In this study, the protein tyrosine phosphorylation (PY) of two membrane-associated proteins in Jurkat cells were examined following a short-term MFs exposure, the zeta chains and the Src kinases p56lck. These proteins are interesting to study since the earliest biochemical event upon T cell receptor (TcR) activation is PY of the zeta chains. These signalling chains in the TcR complex was assessed using Western blotting and the activation of the p56lck kinase was analysed by in vitro kinase assay. The MFs exposure of Jurkat for 5 min activated p56lck and resulted in PY of zeta. These findings are in line with earlier reports on how MFs exposure affects signal transduction in Jurkat.

Effects of prolactin on osteoblast alkaline phosphatase and bone formation in the developing rat.

The purpose of the current study was to determine whether maternal prolactin (PRL) had any effects on bone formation in the developing rat pup. Because the most prevalent forms of PRL in rats are unmodified and phosphorylated PRL, both recombinant PRL and a molecular mimic of phosphorylated PRL (PP-PRL) were administered to pregnant animals. Blood samples from the dams showed normal estrogen and progesterone and no effect of extra PRL on parathyroid hormone (PTH), calcium, or alkaline phosphatase (AP). In newborn pups, however, there was a 30% decrease in blood AP in both PRL-treated groups, whereas PTH and calcium levels were not different from controls. When primary rat osteoblasts were exposed to both PRLs, AP activity was reduced, with PP-PRL being the more potent form of the hormone. Histological examination of pup bone formation showed reduced calvarial bone and reduced endochondral ossification in pups exposed to PP-PRL. These results are the first to show a direct inhibitory effect of PRL on osteoblast function.

Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines.

Effects of applying extremely low-frequency electromagnetic fields (ELF-EMF) for different durations (24, 48, and 72 h) and different field intensities (0.1-1.0 mT) on micronucleus (MN) formation and induction of apoptosis were examined in a human squamous cell carcinoma cell line (SCL II) and in a human amniotic fluid cell line (AFC). A statistically significant increase of MN frequency and of induction of apoptosis in SCL II cells after 48-h and 72-h continuous exposure to 50 Hz magnetic field (MF) (0.8 and 1.0 mT) was found. However, exposure of AFC cells to EMF of different intensities and for different exposure times showed no statistically significant differences when compared with controls. These results demonstrate that different human cell types respond differently to EMF. Dose-dependent induction of apoptosis and genotoxic effects, resulting in increased micronucleus formation, could be demonstrated in the transformed cell line, whereas the nontransformed cell line did not show statistically significant effects. These findings suggest that EMF could be a promotor but not an initiator of carcinogenic effects.

Redistribution of calbindin-D28k in chick intestine in response to calcium transport.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.

Effects of low-energy electromagnetic fields (pulsed and DC) on membrane signal transduction processes in biological systems.

The vertebrate organism possesses a number of internal processes for signaling and communication between cell types. Hormones and neurotransmitters move from one cell type to another and carry chemical "messages" that modulate the metabolic responses of tissues to the environment. Interaction with these signaling systems is a potential mechanism by which very low-energy electromagnetic fields might produce metabolic responses in the body. Hormone and neurotransmitter receptors are specialized protein molecules that use a variety of biochemical activities to pass chemical signals from the outside of a cell across the plasma membrane to the interior of the cell. Since many low-energy electromagnetic fields have too little energy to directly traverse the membrane, it is possible that they may modify the existing signal transduction processes in cell membranes, thus producing both transduction and biochemical amplification of the effects of the field itself. As an example of the kinds of processes that may be involved in these interactions, one metabolic process in which the physiological effects of low-energy electromagnetic fields is well established is the healing of bone fractures. The process of regulation of bone turnover and healing is reviewed in the context of clinical applications of electromagnetic energy to the healing process, especially for persistent nonunion fractures. A hypothetical molecular mechanism is presented that might account for the observed effects of electromagnetic fields on bone cell metabolism in terms of the fields' interference with signal transduction events involved in the hormonal regulation of osteoblast function and differentiation.

Evidence that pulsed electromagnetic fields inhibit coupling of adenylate cyclase by parathyroid hormone in bone cells.

To investigate the biochemical effects of pulsed electromagnetic fields (PEMF) on bone in particular and on cell membrane-associated activity in general, we have studied the modification by PEMF of cAMP metabolism in primary calvarial bone cells. We report that PEMF inhibited cAMP accumulation stimulated by bovine PTH(1-34) peptide. After a 1-hr PEMF exposure, the cAMP response to PTH (2-7 min) was decreased in exposed cells to 48-70% (p less than 0.05) of the response of unexposed cells; furthermore, this inhibition disappeared after 10-20 min with PTH. This inhibition occurred at submaximal PTH doses (2.4-7.3 nM) and no effect was observed at maximal PTH doses (24 nM). Thus with PEMF, the dose response curve for PTH became 0.5 log unit less sensitive. PEMF did not affect the cAMP response to cholera toxin and forskolin. However, when submaximal doses of both forskolin (0.5-1.0 microM) and PTH (0.24-2.4 nM) were used, forskolin prevented inhibition of cAMP production by PEMF in the range of fields and stimulus epochs which normally inhibit cAMP production. It is proposed that PEMF inhibits PTH-stimulated coupling of the adenylate cyclase system and that this inhibition does not affect the intrinsic activity of the G-protein and the catalytic subunit.

Identification of a 150-kDa membrane component which is modulated by parathyroid hormone.

Monoclonal antibodies have been produced against primary bone cells obtained from the collagenase digestion of mouse cranial bone. Antibodies were selected on the basis of their immunoglobulin class and those which were identified as IgG were further screened for their ability to inhibit cAMP accumulation in response to sub-maximal doses of the 1-34 amino-terminal peptide of bovine parathyroid hormone, bPTH(1-34). Nine hybridoma clones were subsequently characterized as inhibitory with respect to parathyroid hormone (PTH) responses in intact mouse cranial bone and which also identified a variety of membrane components from detergent extracts of surface-labeled primary bone cells. Five of these antibodies immunoprecipitated a membrane component with Mr of 80 000 that appeared to be a major component of the extract susceptible to surface-labeling with 125I. All nine monoclonal antibodies were shown to bind to a suspended-cell preparation of primary bone cells with 2-3 orders of magnitude greater binding than that of control antibodies. Using this assay, one clone, designated 3G12 IgG, was observed to exhibit desensitization effects at the binding level with a time course and dose dependency for PTH pre-incubation that was similar to the establishment of the refractory state in other systems. In addition, the desensitization effect occurred at 37 degrees C but not at 4 degrees C. This antibody was shown to bind saturably to both intact mouse cranial bone and primary bone cells with an apparent affinity constant (Ka) in the range of 10(9) M. Inhibition of bone cAMP accumulation in response to 2.5 nM bPTH(1-34) was directly correlated to the binding of 3G12 IgG to intact mouse calvariae. A maximum inhibition of approximately 85% was observed. 3G12 IgG immunoprecipitated a single membrane component, Mr 150 000, from NP-40 detergent extracts of 125I-labeled primary mouse bone cells. The molecular mass of this component was also 150 000 daltons when run on polyacrylamide gel slabs under non-reducing conditions. Control and PTH-pre-treated bone cells were surface-labeled, detergent-solubilized and immunoprecipitated with 3G12 IgG in order to investigate further the desensitization effect at the molecular level. Incubation of bone cells with 1 microgram/ml bPTH(1-34) for 45 min at 37 degrees C caused an increased susceptibility to surface-labeling with 125I that was approximately three-fold higher in specific activity than that of control cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a monoclonal antibody which interacts with the parathyroid hormone receptor-adenylate cyclase system in murine bone.

We have produced monoclonal antibodies which bind specifically to mouse bone cells and then selected these monoclonal antibodies for their ability to inhibit parathyroid hormone (PTH) responses in mouse cranial bone treated with the (1-34) amino terminal peptide of bovine PTH [bPTH(1-34)]. One clone, designated 3-6, characterized as an IgM(kappa), significantly inhibited the accumulation of cAMP in response to bPTH(1-34) at concentrations of hormone between 10(-9) and 10(-7) M. This antibody was subsequently isolated by gel filtration and shown to bind to intact mouse calvariae, with saturation binding occurring at 3 micrograms/ml IgM. A maximal inhibition of approximately 70% of the cAMP accumulation produced in response to 2.5 X 10(-9) M (100 ng/ml) bPTH(1-34) was obtained with 7 micrograms/ml of the purified 3-6 IgM. At this concentration of 3-6 IgM, the half-maximal dose of PTH for activation of cAMP accumulation was increased from 5 X 10(-9) M to 2 X 10(-8) M with no reduction in maximal levels of cAMP production. The utility of this antibody as an inhibitor was further tested by its ability to block the binding of an iodinated PTH analogue, 125I-[Nle8, Nle18, Tyr34]-bPTH(1-34) to mouse cranial bone. The 3-6 IgM at a concentration of 5 X 10(-8) M inhibited 70% of the specific binding of the 125I-labeled analogue. In the absence of parathyroid hormone, 2 X 10(-8) M 3-6 IgM produced a 4-fold increase in cAMP above basal levels, as compared to 40-fold maximal increases observed with PTH, indicating a partial PTH agonist activity of this antibody. When tested for effects on other hormones, 3-6 IgM did not inhibit cAMP accumulation produced in response to salmon calcitonin, epinephrine, prostaglandin E2 or cholera toxin. We propose that the 3-6 monoclonal IgM is specific for the PTH receptor or a component of the PTH receptor-adenylate cyclase system and that this or similar antibodies will serve as useful reagents for future molecular characterization of this receptor.

A human lymphokine activates macrophage C3 receptors for phagocytosis: studies using monoclonal anti-lymphokine antibodies.

We have identified a human T lymphocyte cell line, the Mo line, that constitutively elaborates a lymphokine that activates macrophage receptors for the third complement component (C3) for phagocytosis. The molecule is physically, functionally, and immunologically very similar, if not identical, to a previously characterized murine lymphokine. Both molecules were inactivated by the same physical and chemical treatments, activated macrophage C3 receptors for phagocytosis, and freed macrophage C3 receptors to move within the cell's plasma membrane. In addition, both the human and the murine products were recognized by the same monoclonal antibodies. Treatment with monoclonal antibodies abolished both the ability of lymphokine-containing supernatants to mobilize macrophage C3 receptors and the supernatants' ability to activate C3 receptor-mediated phagocytosis. The dose-response curves for both effects of the antibodies were identical, strongly suggesting that a single molecule was responsible for both effects and that the lymphokine activated marophage C3 receptors for phagocytosis by freeing the anchored receptors and allowing them to diffuse within the cell's plasma membrane. These findings strengthen the hypothesis that, for a receptor to promote phagocytosis, it must be able to diffuse within the macrophage plasma membrane.

Production of human/mouse hybridomas secreting a human lymphokine, osteoclast activating factor.

A human/mouse hybridoma was developed which has the property of secreting a human bone resorbing factor similar or identical to the osteoclast activating factor (OAF) isolated from human tonsil lymphocytes. Mouse plasmacytoma cells negative for OAF production were fused with an enriched subpopulation of human tonsil lymphocytes that had been activated with phytohemagglutinin (PHA) to produce OAF (G.E. Nedwin, M.A. Mohler, and R.A. Luben, submitted for publication). Culture supernatants from mixed hybridomas contained a bone resorbing protein shown to cause the release of 45Ca from previously labeled mouse calvaria. The bone resorbing activity from these hybridomas was inhibited by the presence of OAF-specific monoclonal antibodies. Several hybridomas retained OAF production following limited dilution cloning. One clone, CD6.20, showed a biphasic dose-response curve for bone resorption similar to that of purified OAF from PHA-activated human tonsil lymphocytes. OAF production in the CD6.20 cell line has been retained for over 100 passages. Karyotype analysis of this cell shows the presence of human chromosomes 10 and 18 and the X chromosome.

Multiple hormonal mechanisms for the control of collagen synthesis in an osteoblast-like cell line, MMB-1.

An isolated osteoblast-like cell line (MMB-1) was used to study the hormonal regulation of collagen synthesis in bone cells. Collagen synthesis was measured by incorporation of [3H]proline into collagenase-digestible and collagenase-non-digestible proteins after exposure of the cells in culture to varying concentrations of PTH, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], osteoclast-activating factor, and insulin. Collagen synthesis was inhibited by 10(-10) M 1,25-(OH)2D3 and 3 X 10(-10) M PTH after 9-12 h of treatment. Osteoclast-activating factor at 10(-10) M also inhibited collagen synthesis. Insulin at 10(-8) M increased collagen synthesis without stimulating proline incorporation into noncollagen proteins. No effect on collagen synthesis was observed with 24,25-(OH)2D3. Inhibition of collagen synthesis was also observed when cells were treated with either 3 X 10(-5) M 8-bromo-cAMP or 3 X 10(-5) M (Bu)2cAMP. For all agents tested, the onset of the effects was gradual, with differences from controls beginning at 4-8 h, and maximal effects occurring only after 24 h or more of treatment. The collagen synthesized by these cells remained associated primarily with the cell monolayer and was estimated to be greater than 90% type I collagen. No detectable changes in the type or composition of collagen synthesized were found with any of the hormonal treatments. These studies indicate that the synthesis of collagen in bone cells is under multihormonal control, with both cAMP-dependent and cAMP-independent mechanisms involved. The MMB-1 cell line offers a suitable model system for studies of the interactions of hormones in the control of bone turnover.

Monoclonal antibodies to hypothalamic growth hormone-releasing factor with picomoles of antigen.

Monoclonal antibodies specific for rat hypothalamic growth hormone-releasing factor (rGRF) have been produced by in vitro immunization of mouse spleen cells with less than 1 nanomole of rGRF in a partially purified preparation. Hybridoma supernatants were screened for anti-rGRF activity by use of a pituitary culture assay system that can detect growth hormone-releasing factor in the femtomole range. Such highly sensitive in vitro techniques permit the use of picomole quantities of an antigen in partially purified preparations for the isolation of monoclonal antibodies, which can in turn be used in biological studies and in immunochemical procedures for large-scale purification and isolation of that antigen.

1,25-Dihydroxyvitamin D receptors in an established bone cell line. Correlation with biochemical responses.

A stable cell line derived from mouse bone (cell line MMB-1) has been used for studies of the cellular receptor for 1,25-dihydroxyvitamin D3 in osteoblasts. Previous studies have demonstrated that collagen synthesis in the MMB-1 cell line is specifically inhibited by 1,25-dihydroxyvitamin D3 as well as by other bone-regulating hormones. Incubation of cell homogenates with [3H]1,25-dihydroxyvitamin D3 indicated the presence of a specific receptor which was located primarily in the chromatin fraction. Optimum conditions for the receptor assay required the inclusion of 500 kallikrein-inactivating units of Trasylol/ml and 10 mM NaMoO4. Under these conditions the receptors were stable for 2 h at 23 degrees C and for 24 h at 4 degrees C. Cellular content of receptors was dependent upon the state of confluency of the cells: fully confluent cells contained minimal concentrations of receptors. In cultures of 70-80% confluency, the 1,25-dihydroxyvitamin D3 receptors demonstrated linear Scatchard plots with Kd = 0.4 nM. Peak receptor activity was found at 3.7 S in linear sucrose gradient fractions of cell homogenates. The synthesis of collagen by MMB-1 cells was inhibited by 1,25-dihydroxyvitamin D3 in direct proportion to the concentration of cellular receptors at varying levels of culture confluence. The data indicate that MMB-1 cells contain cytoplasmic/nuclear receptors for 1,25-dihydroxyvitamin D3 which are similar to the receptors found in other target tissues for this hormone and suggest that these receptors are mediators of the effects of 1,25-dihydroxyvitamin D3 on collagen synthesis.

Effects of electromagnetic stimuli on bone and bone cells in vitro: inhibition of responses to parathyroid hormone by low-energy low-frequency fields.

Low-energy electromagnetic fields pulsed at frequencies of 10-90 Hz significantly increase healing of chronic fracture nonunions in man. These fields are effective at tissue current levels several orders of magnitude lower than those required for transmembrane depolarization of normal cells. We have examined the effects of two clinically used pulsed electromagnetic fields on cultures of the osteoblast-like mouse bone cell line MMB-1. Both fields significantly reduced cellular production of cAMP in response to parathyroid hormone and osteoclast activating factor. Neither basal nor fluoride-activated levels of adenylate cyclase were altered in membranes from cells cultured in the fields; however, the same membrane preparations exhibited markedly inhibited responses to parathyroid hormone. The fields blocked the inhibitory effects of the hormone on collagen synthesis by MMB-1 cells. However, there was no effect on the inhibition of collagen synthesis by 1,25-dihydroxyvitamin D(3), which is believed to act primarily by a nuclear, rather than by a membrane-dependent, mechanism. No significant differences were noted between effects of the two fields, one generating continuous pulse trains (72 Hz) and the other generating recurrent bursts (15 Hz) of shorter pulses. We hypothesize that these field effects are mediated primarily at the plasma membrane of osteoblasts, either by interference with hormone-receptor interactions or by blocking of receptor-cyclase coupling in the membrane. These responses occurred with induced extracellular fields of 1 mV/cm or less, even though transmembrane potential gradients are typically 10(5) V/cm.

Production of hybridomas secreting monoclonal antibodies against the lympholine osteoclast activating factor.

The human lympholine osteoclast activating factor (OAF) is thought to be involved in several bone-destroying diseases. The current studies were designed to produce monoclonal antibodies against OAF for use in the subsequent design of immunoassays for OAF in clinical samples. Spleen cells from mice immunized with purified human OAF were hybridized with mouse plasmacytoma cells in vitro to yield hybridomas. Several clones of these hybridomas secreted into the culture medium antibodies, which neutralized the biological activity of OAF at dilutions as high as 1:100,000 relative to the initial culture medium. These antibodies did not interfere with the activities of parathyroid hormone in the same systems. These results represent the first report of monoclonal antibodies against a human lympholine, and validate the concept that hybridoma production is a useful technique for developing antibodies against weak or scarce antigens.

Effects of fluoride on in vitro calcification of tendon matrix.

Ca2+ and Pi uptake induced in vitro by a collagenous matrix derived from bovine tendon is inhibited by 1 X 10(-6) to 2 X 10(-5) M NaF and stimulated by 2 X 10(-5) to 2 X 10(-3) M NaF. Fluoride uptake occurs only over the latter concentration range. The uptake of Ca2+, Pi, and F-1 progresses toward a limiting extent at which the molar Ca/P and Ca/F values are 1.6 to 1.7 and 4.5 to 5.7, respectively. Although the matrix-bound mineral, previously formed in the absence of NaF, readily undergoes dissolution when exposed to a Ca2+- and P-free medium of pH less than 7.4, the bound mineral phase formed in the presence of NaF does not. We conclude that fluoroapatite is the primary matrix-bound mineral. The uptake of fluoride, Ca2+. amd Pi by both uncalcified and previously calcified matrices is inhibited by methylenediphosphonate and by phosphonoacetate as is calcification in the absence of NaF. Kinetic studies indicate that formation of a CaP complex precedes the uptake of F-1 and suggest that F-1 and OH-1 compete for interaction with that CaP complex during the calcification process. We concluded that fluoroapatite formation induced by the collagenous matrix occurs by a multistep pathway comparable to that proposed previously for hydroxyapatite formation.