Salts of the two-coordinate homoleptic manganese(I) dialkyl anion [Mn{C(SiMe3)3}2](-) with quenched orbital magnetism.

The reduction of Mn{C(SiMe3)3}2 with KC8 in the presence of crown ethers yielded the d(6), Mn(I) salts [K2(18-crown-6)3][Mn{C(SiMe3)3}2]2 and [K(15-crown-5)2][Mn{C(SiMe3)3}2], that have near-linear manganese coordination but almost completely quenched orbital magnetism as a result of 4s-3dz(2) orbital mixing which affords a non-degenerate ground state.

A synthetic, structural, magnetic, and spectral study of several [Fe[tris(pyrazolyl)methane]2](BF4)2 complexes: observation of an unusual spin-state crossover.

The complexes [Fe[HC(3,5-Me2pz)3]2](BF4)2 (1), [Fe[HC(pz)3]2](BF4)2 (2), and [Fe[PhC(pz)2(py)]2](BF4)2 (3) (pz = 1-pyrazolyl ring, py = pyridyl ring) have been synthesized by the reaction of the appropriate ligand with Fe(BF4)2.6H2O. Complex 1 is high-spin in the solid state and in solution at 298 K. In the solid phase, it undergoes a decrease in magnetic moment at lower temperatures, changing at ca. 206 K to a mixture of high-spin and low-spin forms, a spin-state mixture that does not change upon subsequent cooling to 5 K. Crystallographically, there is only one iron(II) site in the ambient-temperature solid-state structure, a structure that clearly shows the complex is high-spin. Mössbauer spectral studies show conclusively that the magnetic moment change observed at lower temperatures arises from the complex changing from a high-spin state at higher temperatures to a 50:50 mixture of high-spin and low-spin states at lower temperatures. Complexes 2 and 3 are low-spin in the solid phase at room temperature. Complex 2 in the solid phase gradually changes over to the high-spin state upon heating above 295 K and is completely high-spin at ca. 470 K. In solution, variable-temperature 1H NMR spectra of 2 show both high-spin and low-spin forms are present, with the percentage of the paramagnetic form increasing as the temperature increases. Complex 3 is low-spin at all temperatures studied in both the solid phase and solution. An X-ray absorption spectral study has been undertaken to investigate the electronic spin states of [Fe[HC(3,5-Me2pz)3]2](BF4)2 and [Fe[HC(pz)3]2](BF4)2. Crystallographic information: 2 is monoclinic, P2(1)/n, a = 10.1891(2) A, b = 7.6223(2) A, c = 17.2411(4) A, beta = 100.7733(12) degrees, Z = 2; 3 is triclinic, P1, a = 12.4769(2) A, b = 12.7449(2) A, c = 13.0215(2) A, alpha = 83.0105(8) degrees, beta = 84.5554(7) degrees, gamma = 62.5797(2) degrees, Z = 2.

Lead induced rise in intracellular free calcium is mediated through activation of protein kinase C in osteoblastic bone cells.

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.

Cadmium perturbs calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells; a possible role for protein kinase C.

The mechanism of the toxic effects of Cd2+ on bone cell function is not completely understood at this time. This study was designed to characterize the effect of Cd2+ on Ca2+ metabolism in ROS 17/2.8 cells. Cells were labeled with (45)Ca (1.87 mM Ca) for 20 h in the presence of 0.01, 0.1, or 1.0 microM Cd2+ and kinetic parameters were determined from (45)Ca efflux curves. Three kinetic compartments described the intracellular metabolism of (45)Ca. Cd2+ (0.01 microM) caused an approximate 9 x increase in Ca2+ flux across the plasma membrane and a decrease in the most rapidly exchanging intracellular Ca2+ compartment (S1). However, there was no change in total cell Ca2+, indicating an increased cycling of Ca2+ across the plasma membrane. Flux between S1 and the intermediate Ca2+ compartment (S2) was also increased and S2 increased significantly. All Cd2+ induced changes in Ca2+ homeostasis were obliterated by concurrent treatment with 0.1 microM calphostin C (CC), a potent protein kinase C (PKC) inhibitor. This data suggests that Cd2+ perturbs Ca2+ metabolism via a PKC dependent process.

The effect of cadmium on cytosolic free calcium, protein kinase C, and collagen synthesis in rat osteosarcoma (ROS 17/2.8) cells.

Cadmium affects normal bone growth but the mechanisms of Cd2+ toxicity are not fully understood. Calcium is an integral component of bone growth and a second messenger necessary for the actions of calciotropic hormones. Ca2+ activates protein kinase C (PKC), and PKC is a mediator of [Ca2+]1 and mediator of collagen synthesis in osteoblastic cells. Therefore, PKC is a possible loci of Cd2+ effects on Ca2+ metabolism and Ca(2+)-regulated processes. This work was conducted to determine the effect of Cd2+ on cytosolic free Ca2+ ([Ca2+]i) levels, characterize the activation and/or inhibition of PKC by Cd2+ and Ca2+, and measure the effect of Cd2+ on collagen synthesis in ROS 17/2.8 cells. Cells were treated for 120 min with Cd2+ (0 to 30 microM) and [Ca2+]i was measured. Basal [Ca2+]i was 132 nM and the maximal increase to 268 nM occurred in the presence of 5 microM Cd2+. Treatment with 1 or 5 microM Cd2+ caused an increase in [Ca2+]i at 40 min with return to basal levels at 120 min of treatment. Pretreatment (24 hr) with 0.1 microM calphostin C (CC), a PKC inhibitor, produced no change in [Ca2+]i and prevented any rise in [Ca2+]i in response to Cd2+. Free Cd2+ activates PKC with an activation constant of 7.5 X 10(-11) M, while Ca2+ activates PKC with an activation constant of 3.6 X 10(-7) M. Cd2+ also caused a dose-dependent decrease in collagen synthesis, a PKC-mediated process. These data suggest that Cd2+ affects Ca2+ metabolism and Ca(2+)-mediated processes via unwarranted PKC activation as demonstrated by Cd2+ perturbation of collagen synthesis.

Lead perturbs 1,25 dihydroxyvitamin D3 modulation of intracellular calcium metabolism in clonal rat osteoblastic (ROS 17/2.8) cells.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is known to modulate Ca2+ metabolism in several cell types. 1,25(OH)2D3 causes an increase in Ca2+ influx and probably exerts many of its effects via the Ca2+ messenger system. Lead (Pb2+) interacts with and perturbs normal Ca2+ signalling pathways; hence, the purpose of this work was to determine if Pb2+ perturbs 1,25(OH)2D3 modulation of Ca2+ metabolism in ROS 17/2.8 cells, which express receptors for and respond to 1,25(OH)2D3, and to determine the effect of 1,25(OH)2D3 on Pb2+ metabolism in these cells. In both cases three kinetic compartments described the intracellular metabolism of the isotope. These data show that 1 microM Pb2+ inhibits 1,25(OH)2D3 modulated increases in Ca2+ flux, whereas 5 microM Pb2+ increases membrane fluxes, all intracellular Ca2+ pools, and total cell Ca2+. In the Pb2+ metabolism studies it was found that 10 nM 1,25(OH)2D3 increases intracellular Pb2+. Pb2+ appears to disrupt the modulation of intracellular steady-state Ca2+ homeostasis by 1,25(OH)2D3 in a complex, biphasic manner and may therefore perturb functions that are modulated by 1,25(OH)2D3 via the Ca2+ messenger system.

Lead activation of protein kinase C from rat brain. Determination of free calcium, lead, and zinc by 19F NMR.

Lead (Pb2+) has been reported to activate calcium/phospholipid-dependent protein kinase C (PKC) at subnanomolar concentrations (Markovac, J., and Goldstein, G. W. (1988) Nature 334, 732-734); however, others have failed to find any Pb(2+)-induced activation of PKC (Murakami, K., Feng, G., and Chen, S. G. (1993) J. Pharmacol. Exp. Ther. 264, 757-761). In neither of these studies was the actual free Pb2+ or Ca2+ concentration measured. In this study, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA) was used to buffer Pb2+ and Ca2+ concentrations in the PKC reaction mixture. The specific free ion concentrations of Pb2+ and Ca2+, as well as Zn2+ and other divalent cations contained in the PKC reaction mixtures, were determined by 19F NMR spectroscopy. Using this approach to set and confirm the free Pb2+ and Ca2+ concentrations, we measured the Pb(2+)-dependent and the Ca(2+)-dependent activation of phosphotydylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous acid precipitable proteins in the 100,000 x g supernatant from homogenized rat brain cortex. We found that free Pb2+ activates PKC in the range from 10(-11) to 10(-8) M, Kact = 5.5 x 10(-11) M, while Ca2+ activates PKC in the range from 10(-8) to 10(-5) M, Kact = 2.56 x 10(-7) M. These findings clearly resolve the activation of PKC by subnanomolar concentrations of free Pb2+ from activation induced by Ca2+ or other divalent cations. Furthermore, it documents the utility of 5F-BAPTA as buffer and indicator when resolving the contributions of multiple divalent cations in biochemical processes.

Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism and collagen synthesis in clonal rat osteoblastic (ROS 17/2.8) cells.

EGF, a single-chain polypeptide growth factor important for many cellular functions including glycolysis and protein phosphorylation, is known to modulate calcium metabolism in several cell systems. EGF causes an increase in Ca2+ influx and accumulation of inositol triphosphate and probably exhibits many, if not all, of its effects via the calcium messenger system. Lead is known to interact with and perturb normal calcium signaling pathways; therefore, the purpose of this work was to determine if lead perturbs EGF modulation of calcium metabolism in ROS 17/2.8 cells and if lead impairs collagen synthesis, which is controlled by EGF. To characterize 45Ca kinetics, cells were labelled with 45Ca (1.87 mM Ca) for 20 hr in the presence of 5 microM Pb, 50 ng/ml EGF, or 5 microM Pb and 50 ng/ml EGF. Kinetic parameters were determined from 45Ca efflux curves. Three kinetic compartments described the intracellular metabolism of 45Ca; 5 microM Pb significantly altered the effect of EGF on intracellular calcium metabolism. Calcium distribution was shifted from the fast-exchanging, quantitatively small calcium pools S1 and S2 to the slow-exchanging, quantitatively large S3. There was also a 50% increase in total cell calcium in cells treated with 5 microM Pb and 50 ng/ml EGF over calcium in cells treated with 50 ng/ml EGF alone. Because EGF and phorbol 12-myristate 13-acetate (PMA) have similar effects on protein kinase C (PKC) and collagen metabolism, the transient effects of EGF and PMA on 45Ca and 210Pb were also characterized. EGF caused a rapid transient increase in efflux of both isotopes, which was further increased by the addition of PMA. In contrast, PMA pretreatment, which depletes PKC, significantly attenuated the latter effect of EGF, suggesting that downregulation by PKC of EGF-induced increases in 45Ca and 210Pb efflux. Moreover, collagen synthesis was decreased by lead, EGF, and PMA in a similar manner, further suggesting PKC as the common modulator of these effects. These data show that Pb impairs the normal modulation of intracellular calcium homeostasis and collagen synthesis by EGF. Furthermore, these results provide additional support to the postulate that an early and discrete effect of lead involves perturbation of the calcium messenger system at one or several loci.

Lead intoxication alters basal and parathyroid hormone-regulated cellular calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells.

The skeleton is the major reservoir of lead and calcium in humans, and plays an important role in systemic calcium regulation. Lead perturbs normal calcium transport and second messenger function, directly or indirectly, in virtually all cells studies so far. Therefore, we and others have postulated that an early and discrete toxic effect of lead is perturbation of one or more loci within the calcium messenger system. To understand further the role of lead on calcium homeostasis in bone, we undertook this study to characterize calcium homeostasis and the effect of lead on calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells, which exhibit the osteoblast phenotype. ROS cells were incubated in medium containing 45Ca for 20 hours. Monitoring the efflux of 45Ca from the cultures for 210 minutes allowed for the determination of kinetic parameters defining steady state calcium homeostasis. Three distinct intracellular kinetic calcium pools characterized 45Ca homeostasis. Treatment with either 400 ng parathyroid hormone (PTH)/ml culture medium for 1 hour or 25 microM lead for 20 hours increased total cell calcium. Treatment with PTH caused a larger increase of cell calcium in lead-intoxicated cells than either lead intoxication or PTH treatment alone. This increase suggests that lead may perturb normal calcium-mediated PTH responsiveness of the osteoblast. These experiments further establish a kinetic model for the study of calcium homeostasis in osteoblastic bone cells. The studies also advance the hypothesis that lead-induced perturbations of calcium-mediated processes represent an early effect of lead toxicity at the cellular level.

Cellular and molecular toxicity of lead in bone.

To fully understand the significance of bone as a target tissue of lead toxicity, as well as a reservoir of systemic lead, it is necessary to define the effects of lead on the cellular components of bone. Skeletal development and the regulation of skeletal mass are ultimately determined by the four different types of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. These cells, which line and penetrate the mineralized matrix, are responsible for matrix formation, mineralization, and bone resorption, under the control of both systemic and local factors. Systemic components of regulation include parathyroid hormone, 1,25-dihydroxyvitamin D3, and calcitonin: local regulators include numerous cytokines and growth factors. Lead intoxication directly and indirectly alters many aspects of bone cell function. First, lead may indirectly alter bone cell function through changes in the circulating levels of those hormones, particularly 1,25-dihydroxyvitamin D3, which modulate bone cell function. These hormonal changes have been well established in clinical studies, although the functional significance remains to be established. Second, lead may directly alter bone cell function by perturbing the ability of bone cells to respond to hormonal regulation. For example, the 1,25-dihydroxyvitamin D3-stimulated synthesis of osteocalcin, a calcium-binding protein synthesized by osteoblastic bone cells, is inhibited by low levels of lead. Impaired osteocalcin production may inhibit new bone formation, as well as the functional coupling of osteoblasts and osteoclasts. Third, lead may impair the ability of cells to synthesize or secrete other components of the bone matrix, such as collagen or bone sialoproteins (osteopontin). Finally, lead may directly effect or substitute for calcium in the active sites of the calcium messenger system, resulting in loss of physiological regulation. The effects of lead on the recruitment and differentiation of bone cells remains to be established. Compartmental analysis indicates that the kinetic distribution and behavior of intracellular lead in osteoblasts and osteoclasts is similar to several other cell types. Many of the toxic effects of lead on bone cell function may be produced by perturbation of the calcium and cAMP messenger systems in these cells.

Lead impairs the production of osteocalcin by rat osteosarcoma (ROS 17/2.8) cells.

The serum level of osteocalcin, a bone-specific protein produced by osteoblasts and an index of bone formation, is decreased in lead-intoxicated children. To elucidate the effect of lead on the hormonal regulation of osteocalcin production, ROS 17/2.8 cells were treated with 0, 5, 10, or 25 microM lead acetate for 24 hr, followed by an additional 24-hr lead treatment with or without 100 pg 1,25-dihydroxyvitamin D3/ml medium. At the end of this period a radioimmunoassay was conducted to determine the amount of osteocalcin present in the cells and secreted into the medium. 1,25-Dihydroxyvitamin D3 increased osteocalcin secretion in control cultures, but this increase was prevented by lead in a concentration-dependent manner. Osteocalcin secretion by cultures treated with 10 or 25 microM lead was even lower than in cultures not stimulated with 1,25-dihydroxyvitamin D3. Intracellular levels of osteocalcin were slightly elevated with 1,25-dihydroxyvitamin D3, and there was no lead effect on cellular levels. These data indicate that lead attenuates basal and 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin in ROS 17/2.8 cells. Because osteocalcin appears to play a central role in bone mineralization, altered osteocalcin production may be a key event in the skeletal toxicity of lead.

Cellular lead toxicity and metabolism in primary and clonal osteoblastic bone cells.

A knowledge of bone lead metabolism is critical for understanding the toxicological importance of bone lead, as a toxicant both to bone cells and to soft tissues of the body, as lead is mobilized from large reservoirs in hard tissues. To further understand the processes that mediate metabolism of lead in bone, it is necessary to determine lead metabolism at the cellular level. Experiments were conducted to determine the intracellular steady-state 210Pb kinetics in cultures of primary and clonal osteoblastic bone cells. Osteoblastic bone cells obtained by sequential collagenase digestion of mouse calvaria or rat osteosarcoma (ROS 17/2.8) cells were labeled with 210Pb as 5 microM lead acetate for 20 hr, and kinetic parameters were determined by measuring the efflux of 210Pb from the cells over a 210-min period. The intracellular metabolism of 210Pb was characterized by three kinetic pools of 210Pb in both cell types. Although the values of these parameters differed between the primary osteoblastic cells and ROS cells, the profile of 210Pb was remarkably similar in both cell types. Both types exhibited one large, slowly exchanging pool (S3), indicative of mitochondrial lead. These data show that primary osteoblastic bone cells and ROS cells exhibit similar steady-state lead kinetics, and intracellular lead distribution. These data also establish a working model of lead kinetics in osteoblastic bone cells and now permit an integrated view of lead kinetics in bone.