Diadenosine Polyphosphates in Cultured Vascular Smoothmuscle Cells and Endothelium Cells—Their Interaction with Specific Receptors and their Degradation

Abstract

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.

Effects of diadenosine polyphosphates on glomerular volume

“…The effects of dinucleotides on glomeruli can be induced, at least in part, through their vasoactive metabolites, such as mononucleotides and adenosine. It has been reported for several cell types that dinucleotides are degraded by cell surface ecto‐nucleotides that may be of importance for biological effects of extracellular dinucleotides (Verspohl et al ., 1999). We analysed the ability of isolated glomeruli to hydrolyse Ap n A and we observed significant release of adenosine (from Ap 3 A, Ap 4 A and Ap 5 A) and ATP (from Ap 4 A and Ap 5 A) during incubation (Table 1).…”

“…Ap n As are present in all living cells and certain amounts of these are released into extracellular space during platelet aggregation, metabolic stress and neurotransmission (Luthje & Ogilvie, 1988; Schluter et al ., 1994; Miras‐Portugal et al ., 1998). The effects of Ap n As are mediated via P1 and P2 receptors or dinucleotide receptors (Hoyle et al ., 1996; Vahlensieck et al ., 1996; Pintor et al ., 1997; Ralevic & Burnstock, 1998; Verspohl et al ., 1999). P1 receptors (G proteins coupled receptors) are classical receptors for adenosine and are further subdivided, according to convergent molecular, biochemical and pharmacological evidence into the following subtypes: A 1 , A 2A , A 2B and A 3 .…”

Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications

“…Extracellular AP n As bind to and activate or block endogenous P2X and P2Y receptors (e.g., P2Y AP4A =P2 D receptor, most sensitive to AP 4 A and ADPβS) in a variety of tissues and cells and with a diversity of effects (Pintor and MirasPortugal 1995;Miras-Portugal et al 1996;Hourani et al 1998;Verspohl et al 1999). Furthermore, a novel receptor for AP n As, distinct from the P2 D receptor, has been proposed (P4) which is not activated by ATP and synthetic analogues (Pintor and Miras-Portugal 1995;Miras-Portugal et al 1999).…”

“…However, it has to be taken into consideration that the extracellular actions of AP n As are rapidly terminated by the enzymatic degradation by ecto-diadenosine polyphosphate hydrolases (Miras-Portugal et al 1996, 1999Zimmermann 1996). This factor can greatly decrease the apparent potency and selectivity of AP n As (Westfall et al 1997;Hourani et al 1998;Verspohl et al 1999;Lewis et al 2000).…”

Ecto‐diadenosine polyphosphates hydrolase activity on human prostasomes

“…Extracellular diadenosine polyphosphates have been identi®ed as neurotransmitters and vasomodulators [4]. Binding sites for diadenosine polyphosphates have already been shown in different cells: heart [11], brain [12,13], liver [14], insulin-secreting cells [15], and cultured vascular smooth muscle and endothelium cells [16].…”