arl-67156 and 8-phenyltheophylline

Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood.. Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2).. Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1delta catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells.. The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1delta and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.

Topics: Adenosine Triphosphate; Amides; Blotting, Western; Cells, Cultured; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Marine Toxins; Myosin-Light-Chain Phosphatase; Nucleotidases; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Proteins; Purinergic P1 Receptor Antagonists; Pyridines; rho-Associated Kinases; Theophylline; Thrombin; Transfection

At sites of purinergic neurotransmission, synaptic ecto-ATPase is believed to limit the actions of ATP following its neural release. However, details of the modulation by this enzyme of the ATP-mediated conductance change and the possible mechanisms mediating this modulation remain unelucidated. We have addressed these issues by studying the effect of ARL 67156, a selective ecto-ATPase inhibitor, on ATP-mediated electrical and contractile activity in the sympathetically innervated guinea-pig vas deferens. ARL 67156 at 100 mum significantly potentiated the amplitude of spontaneous excitatory junction potentials (SEJPs) by 81.1% (P < 0.01) and prolonged their time courses (rise time by 49.7%, decay time constant by 38.2%; P < 0.01). Moreover, the frequency of occurrence of SEJPs was strikingly increased (from 0.28 +/- 0.13 to 0.90 +/- 0.26 Hz; P < 0.01), indicating an additional, primarily presynaptic, effect of ecto-ATPase inhibition. The frequency of occurrence of discrete events (DEs), which represent nerve stimulation-evoked quantal release of neurotransmitter, was also increased ( approximately 6-fold; P < 0.01), along with the appearance of DEs at previously 'silent' latencies. Purinergic contractions of the vas deferens were potentiated significantly (P < 0.01) by ARL 67156; these potentiated contractions were suppressed by the A1 agonist adenosine (P < 0.01) but left unaffected by the A1 antagonist 8-phenyltheophylline (8-PT). Our results indicate (i) that ecto-ATPase activity, in addition to modulating the ATP-mediated postjunctional conductance change, may regulate transmitter release prejunctionally under physiological conditions, and (ii) that the prejunctional regulation may be mediated primarily via presynaptic P2X, rather than A1, receptors.

Topics: Action Potentials; Adenosine; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Electrophysiology; Guinea Pigs; Male; Muscle Contraction; Neuromuscular Junction; Receptor, Adenosine A1; Receptors, Purinergic P2; Receptors, Purinergic P2X; Signal Transduction; Synaptic Transmission; Theophylline; Time Factors; Vas Deferens

Ischemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5'-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect.. Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used.. After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP (ARL 67156; 100 microM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 microM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 microM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 microM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT.. ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.

Topics: Adenosine; Adenosine Triphosphate; Animals; Apyrase; Cells, Cultured; Coronary Vessels; Endothelial Cells; Extracellular Fluid; Male; Myocardial Reperfusion Injury; Perfusion; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Theophylline