IJUP08 - Universidade do Porto

Adenosine regulates differentiation of human osteoblast cells in
culture
A. Barbosa 1 , M.A. Costa 1,2 , T. Magalhães-Cardoso 1 , A. Teixeira 1 , R. Freitas 3 , J.M.
Neves 3 & P. Correia-de-Sá 1
1Laboratório de Farmacologia e Neurobiologia e 2 Departamento de Química, UMIB, Instituto de
Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), and 3 Serviço de
Ortopedia e Traumatologia, Centro Hospitalar de V.N. Gaia (CHVNG), Portugal.
Bone turnover takes place at discrete sites in the remodeling skeleton that are subject to
mechanical loading forces. Extracellular purines are important local modulators of bone cell
function. Adenine nucleotides in bone microenvironment are largely determined by ATP
release from stressed cells and subsequent metabolism by ecto-enzymes, both of which have
been scarcely investigated in the human bone. Breakdown of ATP into adenosine restricts its
action to that of a localized signal and shifts purinergic transmission mediated by P2 receptors
to long-lasting modulatory signals mediated by P1 adenosine receptors. Surprisingly, there are
a few reports of the regulation of cell function by adenosine in bone cells (e.g. Shimegi, 1995,
Calcif. Tissue Int., 58:109). This prompted us to investigate the role of stable adenosine
analogues in the proliferation and differentiation of non-modified human osteoblast cells in
culture.
Human bone marrow was collected in sterile conditions from patients that underwent
orthopaedic surgery. These proceedings had the approval of the Ethics Committees of CHVNG
and ICBAS-UP. First passage bone marrow was cultured in supplemented� α-Minimal Essential
Medium (α-MEM) for up to 28 days in the absence and in the presence of CPA (30 nM),
CGS21680C (10 nM), NECA (0.3 µM) and 2-Cl-IB-MECA (10 nM). Throughout their
lifespan, cultures were characterised for morphology, cell viability/proliferation (MTT assay),
total protein content (method of Lowry), and alkaline phosphatase (ALP) activity. For the
kinetic experiments of ATP catabolism, samples (75 µl) were collected from the bath at
different times up to 30 min for reverse-phase HPLC analysis of the variation of substrate
disappearance and product formation.
Human osteoblast cells in culture, hydrolyse extracellular ATP (30 µM) forming sequentially
ADP, AMP and adenosine whose concentrations increased to 1.99±0.18 µM, 0.69±0.04 µM
and 17.81±0.64 µM after 30-min incubation, respectively. In control cultures, osteoblast cells
proliferated for approximately 2-3 weeks; the maximum values for MTT reduction and total
protein content were observed at day 14 (MTT assay, 0.626±0.112, n=9). During the
proliferation phase, incubation of osteoblasts with stable adenosine analogues, CPA (30 nM),
CGS21680C (10 nM), NECA (300 nM) and 2-Cl-IB.MECA (10 nM), did not significantly
change (P>0.05) their ability to reduce MTT. Osteoblast differentiation measured as the
activity of ALP on day 14 decreased significantly (P