Elsevier

Biochemical Pharmacology

Volume 56, Issue 12, 15 December 1998, Pages 1615-1624
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Effect of flupirtine on cell death of human umbilical vein endothelial cells induced by reactive oxygen species

https://doi.org/10.1016/S0006-2952(98)00258-5Get rights and content

Abstract

Flupirtine (KATADOLON®), known as a nonopiate centrally acting analgesic drug, was tested as to its potential to prevent apoptosis of human endothelial cells induced by reactive oxygen species (ROS). It was found that Flupirtine displayed no effect on viability and cell proliferation of human umbilical vein endothelial cells (HUVEC) up to a concentration of 10 μg/mL. Apoptosis, induced by ROS and generated by hypoxanthine/xanthine oxidase (EC 1.1.3.22) (HX/XOD) or t-butyl hydroperoxide, was reduced after preincubation with Flupirtine for 3 hr by 35% and 41%, respectively. The maximal cytoprotective effect against apoptosis was observed at a drug concentration of 1 to 3 μg/mL. Flow cytometric studies revealed that Flupirtine was able to decrease the number of necrotic cells as well as of apoptotic cells. Neither the simultaneous administration of Flupirtine with the apoptosis-inducing agent nor the preincubation of HUVEC with Flupirtine influenced the increase in the intracellular Ca2+ concentration [Ca2+]i caused by the production of ROS.

Section snippets

Materials

FBS, fungizone, l-glutamine, and HEPES were obtained from GIBCO BRL and Medium 199 was purchased from PAA Laboratories. Kanamycin monosulfate, t-BOOH, heparin, histamine, collagenase A (EC 3.4.24.3), trypsin (EC 3.4.21.4), proteinase K (EC 3.4.23.6) and RNAse A (EC 3.1.27.5) were purchased from Sigma, and fura-2-AM, fura-red-AM and H2DCF-DA from Molecular Probes. MTT (M2128), collagenase A, and ECGF were obtained from Boehringer, and the annexin-V assay apoptest was purchased from Nexins. XOD

Effect of flupirtine on viability of HUVEC

We first determined the effect of Flupirtine on the viability of confluent HUVEC. As summarised in Table 1, no significant change in cell number in a concentration range of 0.1 to 10 μg/mL of Flupirtine was observed during an incubation period of 24 hr. The amount of protein did not vary between treated and untreated cultures. The MTT assay system was applied as a further test to show that the compound did not affect cell viability. Setting the viability of untreated cultures to 100%,

Discussion

ROS are known inducers of apoptosis in neurons [42] as well as other somatic cells, e.g. endothelial cells [29], and are considered to be a main basis for neurodegenerative disorders such as β-amyloid peptide-mediated Alzheimer’s disease [43] or prion-mediated cell injury [44]. In the present study, two ROS generators, the HX/XOD and t-BOOH/Cu2+ systems, were used to induce apoptosis in HUVEC. The extent of apoptosis in HUVEC was measured both by flow cytometry and by DNA fragmentation.

Acknowledgements

This work was supported by a grant from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF Verbundprojekt “Infektionsforschung”; 01 KI 94863).

References (53)

  • S.C Chow et al.

    Reevaluation of the role of de novo protein synthesis in rat thymocyte apoptosis

    Exp Cell Res

    (1995)
  • G Benzi et al.

    Are reactive oxygen species involved in Alzheimer’s disease?

    Neurobiol Aging

    (1995)
  • U.M Vischer et al.

    Reactive oxygen intermediates induce regulated secretion of von Willebrand factor from cultured human vascular endothelial cells

    Blood

    (1995)
  • K Rupalla et al.

    Flupirtine protects neurons against excitotoxic or ischemic damage and inhibits the increase in cytosolic Ca2+ concentrations

    Eur J Pharmacol

    (1995)
  • J.C Tsai et al.

    Induction of apoptosis by pyrrolidinedithiocarabamate and N-acetylcyteine in vascular smooth muscle cells

    J Biol Chem

    (1996)
  • A.H Wyllie

    Glucocorticoid-induced thymocyte apoptosis is associated with endogenous nuclease activation

    Nature

    (1980)
  • A Alzheimer

    Über eine eigenartige Erkrankung der Hirnrinde

    Zentralbl Nervenheilk Psychiatr

    (1907)
  • S.B Prusiner

    Biology and genetics of prion diseases

    Annu Rev Microbiol

    (1994)
  • A.D Owen et al.

    Oxidative stress and Parkinson’s disease

    Ann NY Acad Sci

    (1996)
  • R.A Schwartzman et al.

    ApoptosisThe biochemistry and molecular biology of programmed cell death

    Endocrine Rev

    (1993)
  • C.B Thompson

    Apoptosis in the pathogenesis and treatment of disease

    Science

    (1995)
  • D.E Brenneman et al.

    Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestine peptide

    Nature

    (1988)
  • G Forloni et al.

    Neurotoxicity of a protein fragment

    Nature

    (1993)
  • D.T Loo et al.

    Apoptosis is induced by β-amyloid in cultured central nervous system neurons

    Proc Natl Acad Sci USA

    (1993)
  • I.B Szelenyi et al.

    Mode of antinociceptive action of Flupirtine in the rat

    Brit J Pharmacol

    (1989)
  • H.A Friedel et al.

    FlupirtineA review of its pharmacological properties and therapeutic efficacy in pain patients

    Drug

    (1993)
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