Functional crosstalk between the adenosine transporter CNT3 and purinergic receptors in the biliary epithelia.

BACKGROUND & AIMS: Both hepatocytes and cholangiocytes release ATP into the bile, where it acts as a potent autocrine/paracrine stimulus that activates biliary secretory mechanisms. ATP is known to be metabolized into multiple breakdown products, ultimately yielding adenosine. However, the elements implicated in the adenosine-dependent purinergic regulation of cholangiocytes are not known. METHODS: Normal rat cholangiocytes (NRCs) were used to study the expression of adenosine receptors and transporters and their functional interactions at the apical and basolateral membrane domains of polarized cholangiocytes. RESULTS: We found that: (1) cholangiocytes exclusively express two concentrative nucleoside transporters (CNT) known to be efficient adenosine carriers: CNT3, located at the apical membrane, and CNT2, located at apical and basolateral membrane domains; (2) in both domains, NRCs also express the high affinity adenosine receptor A2A, which modulated the activity of apical CNT3 in a domain-specific manner; (3) the regulation exerted by A2A on CNT3 was dependent upon the cAMP/PKA/ERK/CREB axis, intracellular trafficking mechanisms and AMPK phosphorylation; (4) secretin increased the activity of the apically-located CNT3, and promoted additional basolateral CNT3-related activity; and (5) extracellular ATP (a precursor of adenosine) was able to exert an inhibitory effect on the apical activity of both CNT3 and CNT2. CONCLUSIONS: This study uncovered the functional expression of nucleoside transporters in cholangiocytes and provides evidence for direct crosstalks between adenosine transporters and receptors for adenosine and its natural extracellular precursor, ATP. Our data anticipate the possibility of adenosine playing a major role in the physiopathology of the biliary epithelia.

Functional outcome of a novel SLC29A3 mutation identified in a patient with H syndrome.

The H syndrome (OMIM 612391) is an autosomal recessive disorder characterized by hyperpigmentation, hypertrichosis, histiocytosis and short stature. It is caused by mutations in the SLC29A3 gene, which encodes for the equilibrative nucleoside transporter 3 protein (ENT3), of still uncertain subcellular localisation. Here we report a new case of H syndrome with the novel mutation c.243delA, which has been concomitantly described by others [A. Bolze, A. Abhyankar, A.V. Grant, B. Patel, R. Yadav, M. Byun, D. Caillez, J.F. Emile, M. Pastor-Anglada, L. Abel, A. Puel, R. Govindarajan, L. de Pontual, J.L. Casanova, A mild form of SLC29A3 disorder: a frameshift deletion leads to the paradoxical translation of an otherwise noncoding mRNA splice variant, PLoS ONE 7 (2012) e29708]. Patient-derived primary skin fibroblasts and B-lymphoblastoid cell lines (B-LCL) were obtained and, although no differences were found in mRNA levels of ENT3, a significant increase in plasma membrane equilibrative transport activity was found in fibroblasts from the patient. Loss of function of key proteins implicated in nucleoside metabolism can lead to mitochondrial DNA (mtDNA) depletion syndromes (MDS). Measurement of respiratory chain complex activity revealed that mitochondrial function was unaltered. Neither fibroblasts nor B-LCL showed mtDNA depletion when compared with controls. Fibroblasts and B-LCL from the patient were not particularly protected when mitochondrial damage was induced using nucleoside-derived drugs susceptible to being transported by ENT3. Analysis of mtDNA amounts in tissues obtained at autopsy proved inconclusive with respect to mitochondrial involvement in the pathogenesis of this syndrome. Overall, the data do not support the inclusion of H syndrome among the MDS and these findings are compatible with its recent inclusion among the lysosomal storage diseases.

Expression and distribution of nucleoside transporter proteins in the human syncytiotrophoblast.

The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NTs) were investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry, and subcellular localization (basal, heavy, and light apical membranes as well as raft-enriched membranes from the apical domain). In contrast with other epithelia, in this epithelium, we have identified the high-affinity pyrimidine-preferring human concentrative nucleoside transporter (hCNT) 1 as the only hCNT-type protein expressed at both the basal and apical membranes. hCNT1 localization in lipid rafts is also dependent on its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this result favors the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides, and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including intrauterine growth retardation, have been reported.

Lipid rafts and cytoskeletal proteins in placental microvilli membranes from preeclamptic and IUGR pregnancies.

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are leading causes of perinatal and maternal morbidity and mortality. Previously we reported the expression of lipid rafts in classical microvillous membrane (MVM) and light microvillous membrane (LMVM), two subdomains in apical membrane from the human placental syncytiotrophoblast (hSTB), which constitute the epithelium responsible for maternal-fetal transport. Here the aim was to study the raft and cytoskeletal proteins from PE and IUGR. Microdomains from MVM and LMVM were tested with raft markers (placental alkaline phosphatase, lipid ganglioside, and annexin 2) and a nonraft marker (hTf-R). No changes were detected with those markers in whole purified apical membranes in normal, PE, and IUGR pregnancies; however, their patterns of distribution in lipid rafts were different in PE and IUGR. Cholesterol depletion modified their segregation, confirming their presence in lipid rafts, although unlike normal placenta, in these pathologies there is only one type of microdomain. Additionally, the cytoskeleton proteins actin, ezrin, and cytokeratin-7 showed clear differences between normal and pathological membranes. Cytokeratin-7 expression decreased to 50% in PE, and the distribution between LMVM and MVM (~43 and 57%, respectively) changed in both PE and IUGR, in contrast with the asymmetrical enrichment obtained in normal LMVM (~62%). In conclusion, lipid rafts from IUGR and PE have different features compared to rafts from normal placentae, and this is associated with alterations in the expression and distribution of cytoskeletal proteins.

Distinct lipid rafts in subdomains from human placental apical syncytiotrophoblast membranes.

We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal-fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and beta-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.

Distribuiçäo de bactérias planctônicas, colônias bacterianas e biofilmes microbianos em dentes decíduos com pulpite e ou necrose pulpar / Distribution of bacterial plankton, bacterial colonies and microbial biofilm on deciduous teeth with pulpitis and pulp necrosis

As bactérias constituem fatores primordiais da contaminaçäo da polpa dentária de dentes decíduos resultando em implicaçöes clínicas e terapêuticas. Com a finalidade de analisar a presença de bactérias planctônicas, colônias e biofilmes microbianos nas estruturas de dentes decíduos portadores de pulpite e necrose pulpar, utilizaram-se 32 dentes decíduos com cárie profunda. Dezesseis dentes foram seccionados no sentido longitudinal e o restante no sentido transversal. Os espécimes foram corados pela hematoxilina-eosina de Harris e Brown e Brenn para análise pela microscopia óptica. A partir da metodologia empregada pôde-se verificar que: 1) Em dentes decíduos com pulpite decorrente de cárie dentária, as bactérias podem ser encontradas nos túbulos dentinários e especialmente no interior do tecido pulpar na área inflamada. Colônias bacterianas e biofilmes microbianos säo visualisados na superfície cariosa e nos focos de liquefaçäo dentinária; 2) As bactérias plactônicas, colônias bacterianas e biofilmes microbianos estäo abundantemente presentes nas câmaras pulpares e canais radiculares de dentes decíduos com necrose pulpar, localizando-se nas superfícies do teto, do soalho da câmara pulpar, nas paredes laterais do canal radicular e, por extensäo, nas paredes externas apicais...