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ABSTRACT: This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y(2), P2Y(4), and P2Y(6)) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca(2+)](i) in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y(6) receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y(6) receptors with MRS 2578 prevented [Ca(2+)](i) rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y(2), P2Y(4), and P2Y(6) receptors. While the expression of P2Y(6) receptors remained fairly constant (7∼21 days), P2Y(2) and P2Y(4) became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y(6) receptors coupled to increases in [Ca(2+)](i) . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.
Journal of Cellular Physiology 09/2011; 227(6):2694-709. · 3.87 Impact Factor
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ABSTRACT: Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.
Journal of Cellular Physiology 10/2010; 226(5):1353-66. · 3.87 Impact Factor
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ABSTRACT: A project for studying the prevalence of hereditary ataxias (HA) and familial spastic paraplegias (FSP) in Portugal was set up in 1993. The ascertainment of patients in previous prevalence studies relied mainly on the information of hospital admissions and out-patient contacts with the neurology and other related departments at central hospitals covering the whole region surveyed. Many patients might be overlooked if large populations were studied using this method, since registers at central hospitals are very incomplete and for most part not yet computerized. On the other hand HA and FSP are rare diseases appearing in family clusters, and it would be unreasonable to undertake a sample survey based upon a suitable frame of the Portuguese population. Therefore we decided to carry out a two-phase prevalence survey at district level, involving the collaboration of all physicians working in the district health institutions and the population, in the screening of eligible subjects in phase 1. All subjects screened as positive were examined by a neurologist in phase 2. This method provided a direct estimate of false positives and false negatives were all patients also examined in phase 2, who came to our knowledge using other sources of information. The prevalence of hereditary ataxias and spastic paraplegias in the pilot district was 6.4 per 100,000 inhabitants. The sensitivity of the screening procedure was 81.2% and the predictive value of a positive screening was 25%. Considering the geographically circumscribed district nature of the populations to be studied, the comprehensive sources of case identification used and the high adherence of the health professionals involved, we believe that this method can be widely used, particularly in countries with similar health care services.
Journal of Clinical Epidemiology 01/1998; 50(12):1377-84. · 4.27 Impact Factor
