Werner Windischhofer

Phenotypic Heterogeneity of Osteoblast-like MC3T3-E1 Cells: Changes of Bradykinin-Induced Prostaglandin E2 Production During Osteoblast Maturation

We have examined clonal murine calvarial MC3T3‐E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin‐induced production of PGE2). It was found that the sublines investigated showed large variation of the above‐mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above‐mentioned agents. The most striking difference was observed with bradykinin‐induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of3H‐bradykinin to its receptor and also correlated well with the bradykinin‐induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast‐like cell line MC3T3‐E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

Stable Isotope Labelling and Determination of Ketroprofen in Human Plasma and Urine by Gas Chromatography/Negative Ion Chemical Ionization Mass Spectrometry

A method for the quantitative measurement of the non-steroidal anti-inflammatory drug ketoprofen in human plasma and urine is presented. The assay is based on gas chromatography/negative ion chemical ionization mass spectrometry. The preparation of stable isotope-labelled ketoprofen for use as an internal standard is described. After solvent extraction from the acidified matrix, urine samples were analysed as pentafluorobenzyl esters, whereas plasma samples required further derivatization to the hydroxylamine-trimethylsilyl derivatives. The detection limit was found to be 5 pg in both cases. The method was applied to the pharmacokinetics of ketoprofen in man after epidermal application.

On the inhibition of prostanoid formation by SK&F 96365, a blocker of receptor‐operated calcium entry

1 The proposed blocker of receptor‐operated calcium channels, SK&F 96365 was shown to inhibit formation of prostaglandin E2 in two osteoblast‐like cell lines, MC3T3‐E1 and UMR‐106 in a dose‐dependent manner at an IC50 of 3–4 μm. Inhibition was observed with various stimuli (arachidonic acid, bradykinin and calcium ionophore A23187). 2 This effect was also observed in human platelets, where SK&F 96365 dose‐dependently blocked thromboxane biosynthesis and formation of 12‐hydroxy‐heptadecatrienoic acid after stimulation with arachidonic acid (IC50 = 4 μm). 3 The compound had no effect on 12‐hydroxy‐eicosatetraenoic acid production by human platelets. Additionally, linoleic acid oxidation by soybean 15‐lipoxidase was not impaired by SK&F 96365. The results thus provide evidence for cyclo‐oxygenase inhibition by SK&F 96365 at concentrations used to block receptor‐operated calcium influx.

Synthesis of C-5a-substituted derivatives of 4-epi-isofagomine: notable β-galactosidase inhibitors and activity promotors of GM1-gangliosidosis related human lysosomal β-galactosidase mutant R201C.

From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching at C-4 and suitable modification, lipophilic analogs of the powerful β-D-galactosidase inhibitor 4-epi-isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities when compared with the unsubstituted parent compound and may serve as leads toward new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

[3H]Bradykinin Receptor-Binding, Receptor-Recycling, and Receptor-Internalization of the B2 Bradykinin Receptor in the Murine Osteoblast-like Cell Line MC3T3-E1

Bradykinin (BK) has been demonstrated to induce inositol phosphate production, release of intracellular Ca2+, and prostaglandin E2 (PGE2) synthesis in the murine osteoblast‐like cell line MC3T3‐E1. Because cellular response to BK is a function of receptor affinity, receptor coupling, and receptor recycling, we investigated kinetic properties, specificity, and regulation at the BK‐receptor level on intact, BK‐sensitive MC3T3‐E1 cells. Our results clearly demonstrate the existence of a single category of binding sites for [3H]BK (kD = 366 ± 98 pM; Bmax = 45.3 ± 6.6 fmol/mg of protein). Displacement studies with various BK analogs gave a rank order compatible with a B2 BK‐receptor type (BK > Lys‐BK > [Hyp3]‐BK > Met‐Lys‐BK > HOE140 > Tyr‐BK > Tyr8‐BK > D‐Arg, [Hyp3, Thi5,8, D‐Phe7]‐BK > [D‐Phe7]‐BK > des‐Arg9‐BK > des‐Arg9, [Leu8]‐BK = angiotensin II). No atypic high‐affinity binding sites for the B1 receptor agonist des‐Arg9‐BK could be observed. Prestimulation of MC3T3‐E1 cells with BK resulted in the disappearance of accessible B2 receptors at the cell surface by internalization. Postexposure of BK‐pretreated cells to ligand‐free medium resulted in almost complete receptor restoration within 30 minutes, exhibiting an intermediate state of two categories of binding sites (kD1 = 444 ± 37 pM, Bmax1 = 9.2 ± 0.3 fmol/mg of protein and kD2 = 2.7 ± 0.28 pM, Bmax2 = 24.2 ± 0.2 fmol/mg of protein), probably representing coupled and uncoupled B2 receptors. Prolonged stimulation with BK (2.5–5 h) also revealed the temporal occurrence of two categories of binding sites after 2.5 h (kD1 = 228 ± 3.5 pM; Bmax1 = 15.6 ± 0.6 fmol/mg of protein; kD2 = 2.7 ± 0.25 nM; Bmax2 = 40.7 ± 1.5 fmol/mg of protein), whereas low‐affinity binding sites disappeared after 5 h.

Synthesis of C-5a-chain extended derivatives of 4-epi-isofagomine: Powerful β-galactosidase inhibitors and low concentration activators of GM1-gangliosidosis-related human lysosomal β-galactosidase

From an easily available partially protected formal derivative of 1-deoxymannojirimycin, by hydroxymethyl chain-branching and further elaboration, lipophilic analogs of the powerful β-d-galactosidase inhibitor 4-epi-isofagomine have become available. New compounds exhibit improved inhibitory activities comparable to benchmark compound NOEV (N-octyl-epi-valienamine) and may serve as leads towards improved and more selective pharmacological chaperones for GM1-gangliosidosis.

A Morita-Baylis-Hillman based route to C-5a-chain-extended 4-epi-isofagomine type glycosidase inhibitors

By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,β-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of β-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal β-galactosidase mutant R201C.

Potent GH20 N-Acetyl-β-d-hexosaminidase Inhibitors: N-Substituted 3-acetamido-4-amino-5-hydroxymethyl-cyclopentanediols

From 1,2;3,4-di-O-isopropylidene-d-galactopyranose, a preliminary series of highly functionalized amino(hydroxymethyl)cyclopentanes was easily available. These amine-containing basic carbasugars featuring the d-galacto configuration are potent inhibitors of the GH20 β-d-hexosaminidases probed and may bear potential as regulators of N-acetyl-d-hexosaminidase activities in vivo.

A new type of pharmacological chaperone for GM1-gangliosidosis related human lysosomal β-galactosidase: N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of β-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid β-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.

N-Substituted 5-amino-1-hydroxymethyl-cyclopentanetriols: A new family of activity promotors for a GM1-gangliosidosis related human lysosomal β-galactosidase mutant.

From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of β-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid β-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.