Alkaline Phosphatases: Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes

Our knowledge of the structure and function of alkaline phosphatases has increased greatly in recent years. The crystal structure of the human placental isozyme has enabled us to probe salient features of the mammalian enzymes that differ from those of the bacterial enzymes. The availability of knockout mice deficient in each of the murine alkaline phosphatase isozymes has also given deep insights into their in vivo role. This has been particularly true for probing the biological role of bone alkaline phosphatase during skeletal mineralization. Due to space constraints this mini-review focuses exclusively on structural and functional features of mammalian alkaline phosphatases as identified by crystallography and probed by site-directed mutagenesis and kinetic analysis. An emphasis is also placed on the substrate specificity of alkaline phosphatases, their catalytic properties as phosphohydrolases as well as phosphodiesterases and their structural and functional relatedness to a large superfamily of enzymes that includes nucleotide pyrophosphatase/phosphodiesterase

Effects of cyclosporin A on rat osteoblasts (ROS 17/2.8 cells) in vitro

The effects of the immunosuppressive drug cyclosporin A (CsA) were evaluated on ROS 17/2.8 cells in vitro . ROS cells were treated with CsA (0, 0.5, 1.0, 5.0 μg/ml) for 3 days with and without bovine parathyroid hormone (bPTH) (1–34) 10 nM. CsA at 0.5, 1.0, 5.0 μg/ml without PTH and at 5.0 μg/ml in the presence of PTH significantly inhibited proliferation, as determined by a tetrazolium colorimetric assay. In addition, ROS cell number was significantly reduced at 3 and 4 days with CsA (5.0 μg/ml) without affecting cell viability. Incorporation of [ 3 H]-thymidine into DNA was significantly reduced by 3.0 and 5.0 μg/ml CsA after 12 and 24 hours exposure. Basal and 1,25-dihydroxyvitamin D 3 -stimulated alkaline phosphatase levels in confluent ROS cells were reduced ( P <0.05) with CsA (1.0 and 3.0 μg/ml). Pretreatment of ROS 17/2.8 cells with CsA did not alter PTH-stimulated cAMP levels or [ 125 I]-PTHrP binding to ROS cells. CsA treatment of ROS 17/2.8 cells induced a spindle-shaped appearance with loss of attachment in confluent cultures. When ROS cells were cultured in CsA-containing media, cellular attachment at 6 and 12 hours was reduced ( P <0.05) compared with untreated ROS cells. These findings indicate that CsA was capable of inhibiting proliferation, cell number, mitogenesis, alkaline phosphatase levels, and cell attachment of ROS cells without affecting PTH binding or cAMP levels. This direct effect of CsA on osteoblasts may be important in changes of bone remodeling observed in CsA-treated humans and animals.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48004/1/223_2004_Article_BF00334490.pd

Induction of a low voltage-activated, fast-inactivating Ca2+ channel in cultured bone marrow stromal cells by dexamethasone

The production of biochemical markers associated with the osteoblastic phenotype, and accompanying changes in the expression of voltage-operated Ca2+ channels, have been examined in rat bone marrow stromal cell cultures treated with dexamethasone (10(-8) M). Whole cell clamp analysis of voltage-operated Ca2+ channels in control cultures (using Ba2+ as the charge carrier) revealed primarily a high voltage-activated (HVA), slowly inactivating current, which was enhanced two- to threefold by treatment of the cells with Bay K 8644 (300 nM) and inhibited by nifedipine (4 mu M). In dexamethasone-treated cultures, the I-V relationship for inward current was shifted to more positive potentials in comparison with control cells. Most cells in these cultures possessed both the HVA current and also a faster inactivating, low-voltage-activated (LVA), nifedepine-resistant current. These two currents could be separated both by nifedipine and by the use of steady state inactivation of the LVA current. The two components of the Ba2+ current varied widely in their relative size. The combination of LVA and HVA currents seen in dex-induced stromal cells resembles records of voltage-operated Ca2+ channels from cultures of calvarial osteoblasts