Diadenosine polyphosphates in cultured vascular smooth-muscle cells and endothelium cells--their interaction with specific receptors and their degradation.
The role of diadenosine polyphosphates (ApnA, where "A" denotes "adenosine" and "n" denotes the number of phosphate groups "p") as vasoconstrictors of smooth-muscle cells and as blood-pressure regulating and insulin-releasing compounds has been described. It was the aim of this study to investigate whether specific receptors for these compounds, mediating the above mentioned effects, occur in cultured vascular smooth-muscle cells (VSMC) and in endothelium cells, and whether these compounds are degraded during incubation. Saturable binding sites for diadenosine polyphosphate [3H]Ap4A with an extremely quick saturation equilibrium, even at low temperature (4 degrees C), are present in vascular smooth-muscle cells. Diadenosine polyphosphates at micromolar concentrations displaced [3H]Ap4A from binding sites; the ranking order was Ap4A > Ap3A > Ap5A approximately Ap6A. Compounds interacting with purinergic P2X receptors such as suramin, alpha,beta-methylene ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), albeit at high concentrations, displaced [3H]Ap4A from its binding sites. Surprisingly, at low concentrations the compounds tested increased the binding of [3H]Ap4A, which might imply the occurrence of positive receptor cooperativity or inhibition of [3H]Ap4A degradation. By use of thin-layer chromatography it was observed that [3H]Ap4A was quickly degraded (half-life approx. 12 min) in the extracellular medium to (mainly) adenosine and inosine. [3H]Ap4A and its degradation products were quickly taken up by the cells. Degradation can be inhibited by Ap6A, alpha,beta-methylene ATP or PPADS. Rather similar degradation and uptake results were also obtained when endothelium cells were used. These data indicate that specific binding sites for [3H]Ap4A are present in vascular smooth-muscle cells and that diadenosine polyphosphates at physiological concentrations displace binding. The receptors involved might be distinct diadenosine polyphosphate receptors, although the involvement of others, such as P2X receptors, is also possible. Ap4A is quickly degraded in the extracellular space and compounds that inhibit degradation result in an increase in [3H]Ap4A binding. It should be remembered that when diadenosine polyphos-phates are being investigated in physiological and pathophysiological studies of their impact on smooth-muscle cell proliferation and on vasoconstriction (blood-pressure regulation), results obtained from long-term incubations might be critical.
Verspohl
E J
EJ
Department of Pharmacology, Institute of Pharmaceutical Chemistry, University of Münster, Germany.
Johannwille
B
B
Kaiserling-Buddemeier
I
I
Schlüter
H
H
Hagemann
J
J
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D015226:Dinucleoside Phosphates / Q000378:metabolism* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011983:Receptors, Purinergic / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000378:metabolism* / Q000494:pharmacology
D015226:Dinucleoside Phosphates; D011983:Receptors, Purinergic; D018048:Receptors, Purinergic P2; D014662:Vasoconstrictor Agents; C080426:adenosine 5',5''',P1,P4-tetraphosphate receptor; C020733:diadenosine tetraphosphate