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ABSTRACT: The rat aortic smooth muscle cell line A-10 was used to investigate the effect of dipyridamole on the gap junction coupling of smooth muscle cells. The scrape loading/dye transfer (SL/DT) technique revealed that dipyridamole concentrations between 5 μM and 100 μM significantly increased gap junction coupling. The adenosine receptor antagonist MRS 1754, as well as the PKA inhibitors Rp-cAMPS and H-89 were able to inhibit the dipyridamole-related increase in coupling, while forskolin and Br-cAMP also induced an enhancement of the gap junction coupling. Regarding the time-dependent behaviour of dipyridamole, a short-term effect characterised by an oscillatory reaction was observed for application times of less than 5 h, while applications times of at least 6 h resulted in a long-term effect, characterised by a constant increase of gap junction coupling to its maximum levels. This increase was not altered by prolonged presence of dipyridamole. In parallel, a short application of dipyridamole for at least 15 min was found to be sufficient to evoke the long-term effect measured 6 h after drug washout. We propose that in both the short-term and long-term effect, cAMP-related pathways are activated. The short-term phase could be related to an oscillatory cAMP effect, which might directly affect connexin trafficking, assembly and/or gap junction gating. The long-term effect is most likely related to the new expression and synthesis of connexins. With previous data from a bovine aortic endothelial cell line, the present results show that gap junction coupling of vascular cells is a target for dipyridamole.
Journal of Cell Communication and Signaling 03/2013;
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Willem Bintig,
Daniela Begandt,
Barbara Schlingmann,
Linda Gerhard,
Maria Pangalos, Lutz Dreyer,
Natalija Hohnjec,
Pierre-Olivier Couraud,
Ignacio A. Romero,
Babette B. Weksler,
Anaclet Ngezahayo
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ABSTRACT: The expression and physiology of purine receptors of the human blood–brain barrier endothelial cells were characterised by
application of molecular biological, gene-silencing and Ca2+-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled
receptors P2Y2-, P2Y6-, P2Y11- as well as the ionotropic P2X4-, P2X5- and P2X7-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in
the intracellular Ca2+ concentration ([Ca2+]i) from 150 to 300 nM in single cells. The change in [Ca2+]i corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput
experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate
(ADP), BzATP, αβ-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB)
showed a PLC-IP3 cascade-mediated Ca2+ release, indicating that the nucleotide-induced Ca2+ signal was mainly related to P2Y2, 6 and 11 receptors. The gene silencing of the P2Y2 receptor reduced the ATP- or UTP-induced Ca2+ signal and suppressed the Ca2+ signal mediated by P2Y6 and P2Y11 more specific agonists like UDP (P2Y6), BzATP (P2Y11) and ATPγS (P2Y11). This report identifies the P2Y2 receptor subtype as the main purine receptor involved in Ca2+ signalling of the hCMEC/D3 cells.
KeywordsP2 receptors–G-Protein–Neurovascular unit–Gene silencing–siRNA
Purinergic Signalling 04/2012; 8(1):71-80. · 3.16 Impact Factor
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Willem Bintig,
Daniela Begandt,
Barbara Schlingmann,
Linda Gerhard,
Maria Pangalos, Lutz Dreyer,
Natalija Hohnjec,
Pierre-Olivier Couraud,
Ignacio A Romero,
Babette B Weksler,
Anaclet Ngezahayo
[show abstract]
[hide abstract]
ABSTRACT: The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca(2+)-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca(2+) concentration ([Ca(2+)](i)) from 150 to 300 nM in single cells. The change in [Ca(2+)](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTPγS, ATPγS, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, αβ-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP(3) cascade-mediated Ca(2+) release, indicating that the nucleotide-induced Ca(2+) signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca(2+) signal and suppressed the Ca(2+) signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATPγS (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca(2+) signalling of the hCMEC/D3 cells.
Purinergic Signalling 09/2011; 8(1):71-80. · 3.16 Impact Factor
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ABSTRACT: Endothelial cells (ECs) are permanently exposed to the blood flow and the resulting shear stress, its magnitude varying with the EC site in the blood stream. Along with other mechanical stimuli like vessel wall stretching or hydrostatic blood pressure, this shear stress modulates the endothelial cell function, morphology and gene expression. Here, we describe our improved cone-and-plate reactor that applies up to 10 dyn/cm(2) uniform wall shear stress on a defined, ring-shaped region on a culture dish. At the same time, a hydrostatic pressure of up to 195 mmHg can be applied by increasing the atmospheric pressure in the incubator box. Gas composition can be controlled additionally, used for maintaining CO2-homeostasis or inducing hypoxic conditions. For better comparability, six cone-and-plate systems can be used at the same time at different rotational velocities. The effects on cell morphology, cytoskeleton and cell alignment can be monitored during application using a laser scanning microscope. Flow conditions have been studied and a sufficient area of uniform wall shear stress could be shown. To exceed 10 dyn/cm2, we suggest an increase in medium viscosity.
Clinical hemorheology and microcirculation 01/2011; 49(1-4):391-7. · 3.40 Impact Factor
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Dorothee Meyer,
Sebastian Seth,
Jana Albrecht,
Michael K Maier,
Louis du Pasquier,
Inga Ravens, Lutz Dreyer,
Renate Burger,
Martin Gramatzki,
Reinhard Schwinzer,
Elisabeth Kremmer,
Reinhold Foerster,
Günter Bernhardt
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ABSTRACT: The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes.
Journal of Biological Chemistry 01/2009; 284(4):2235-44. · 4.77 Impact Factor
