Abstract:

Human erythrocytes have been regarded as perfect osmometers, which swell or shrink as dictated by their osmotic environment. In contrast, in most other cells, swelling elicits a regulatory volume decrease (RVD) modulated by the activation of purinic and pyrimidinic receptors (P receptors). For human erythrocytes this modulation has not been tested, and we thus investigated whether P receptor activation can induce RVD in these cells. Further, because ectonucleotidases may scavenge ATP or ADP or act as a source for extracellular adenosine and therefore modulate P receptor activation and RVD, we also determined their activity in intact erythrocytes. We found relatively low ectoATPase but significant ectoADPase and ectoAMPase activities. When erythrocytes were exposed to hypotonic medium alone, they swelled as expected for an osmometric response and showed no RVD. Activation of P2 receptors by exogenous ATP or ADP did not trigger RVD, whereas P1 agonists adenosine and adenosine-5′-N-ethylcarboxamide induced significant RVD. The effect of adenosine-5′-N-ethylcarboxamide was dose-dependent (maximal RVD of 27%; apparent K½ of 1.6 ± 1.7 μM). The RVD induced by adenosine was blocked 80% with the non-selective P1 antagonist 8-(p-sulfophenyl theophylline) or the P1-A2B inhibitor MRS1754, but not by inhibitors of P1 subtypes A1, A2A, and A3. In addition, forskolin (an inducer of intracellular cAMP formation) could mimic the effect of adenosine, supporting the idea of P1-A2B receptor activation. In conclusion, we report a novel P1-A2B receptor-mediated RVD activation in mature human erythrocytes and thus indicate that these long held perfect osmometers are not so perfect after all.

Author affiliation: Pafundo, Diego Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Author affiliation: Alvarez, Cora Lilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Author affiliation: Krumschnabel, Gerhard. Universidad de Innsbruck; Austria

Author affiliation: Schwarzbaum, Pablo Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Abstract:

The modulation of purinergic receptors plays an important role in the regulation of bone formation by the osteoblast. On the other hand, bone morphogenetic proteins (BMPs), members of the transforming growth factor-β superfamily, regulate the differentiation of osteoprogenitor bone cells and stimulate bone formation. In this study, we investigate the effects of several nucleotides on osteoblast differentiation and function, and their relation with the gene expression of osteogenic proteins BMP-2, BMP-4 and BMP-5 as well as of differentiation markers alkaline phosphatase (ALP) and bone sialoprotein (BSP). Our results indicate that 100 μM ATP, ATPγS and UTP, but not ADP, ADPβS or UDP, promote ALP activity in rat primary osteoblasts, showing a peak about day 7 of the treatment. ATP, ATPγS and UTP also increase the mRNA levels of ALP, BMP-2, BMP-4, BMP-5 and BSP. Both the ALP activity and ALP and BMP-4 mRNA increments induced by ATP and UTP are inhibited by Ly294002, a PI3K inhibitor, suggesting the involvement of PI3K/AKT signaling pathway in purinergic modulation of osteoblast differentiation. Furthermore, bone mineralization enhance 1 and 1.5 fold after culturing osteoblasts in the presence of 100 μM ATP or UTP, respectively, but not of ADP or UDP for 22 days. This information suggests that P2Y2 receptors (responsive to ATP, ATPγS and UTP) enhance osteoblast differentiation involving PI3K/AKT signaling pathway activation and gene expression induction of ALP, BMP-2, BMP-4, BMP-5 and BSP. Our findings state a novel molecular mechanism that involves specific gene expression activation of osteoblast function by the purinoreceptors, which would be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Author affiliation: Ayala Peña, Victoria Belen. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina

Author affiliation: Scolaro, Luis Alberto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Santillan, Graciela Edith. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Alpha-hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic Escherichia coli strains, whose expression correlates with the severity of the infections produced. HlyA is synthesized as a protoxin, ProHlyA, that becomes matured to the active form in the cytosol by hemolysin-C-directed fatty acylation at the ε-amino residues of Lys 564 and Lys 690, before export from the toxin-producing bacteria. This posttranslational modification is remarkable because the nature of the protein is changed by the lipidic moiety from a benign protein to a frank toxin.In the present work, we demonstrated for the first time that, despite being hemolitically inactive, ProHlyA induced cellular morphologic changes in rabbit erythrocytes. A discocyte-to-echinocyte transformation was triggered by the protoxin in the absence of any accompanying increase in intracellular-Ca2+ levels. In addition, the Ca2+-influx kinetics in HlyA-treated erythrocytes indicated that the active toxin induced an elevation in intraerythrocyte-Ca2+ content in a biphasic manner, with an initial rise in Ca2+ that depended on the association of the protein with the target membrane and a second increment corresponding to an activation of purinergic channels. The first increase was sufficient to trigger a discocyte-echinocyte-spherocyte transition in erythrocyte shape, though the subsequent rise mediated by purinergic signalling was essential for the occurrence of hemolysis. The results presented here provide new insights into the mechanism of action of this toxin.

Author affiliation: Vazquez, Romina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina

Author affiliation: Maté, Sabina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina

Author affiliation: Bakas, Laura Susana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; Argentina

Author affiliation: Muñoz Garay, Carlos. Universidad Nacional Autónoma de México; México

Author affiliation: Herlax, Vanesa Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina

Abstract:

We present the results of a detailed molecular dynamics study of the closed form of the P2X4 receptor. The fluctuations observed in the simulations were compared with the changes that occur in the transition from the closed to the open structure. To get further insight on the opening mechanism, the actual displacements were decomposed into interchain motions and intrachain deformations. This analysis revealed that the iris-like expansion of the transmembrane helices mainly results from interchain motions that already take place in the closed conformation. However, these movements cannot reach the amplitude required for the opening of the channel because they are impeded by interactions occurring around the ATP binding pocket. This suggests that the union of ATP produces distortions in the chains that eliminate the restrictions on the interchain displacements, leading to the opening of the pore.

Author affiliation: Pierdominici Sottile, Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

Author affiliation: Moffatt, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina

Author affiliation: Palma, Juliana Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina