Vera Ralevic

Roles of P2-purinoceptors in the cardiovascular system.

Characterization of P2-purinoceptor subtypes has facilitated understanding of the many diverse effects produced by purine nucleotides. P2X-Purinoceptors are located on vascular smooth muscle where they mediate vasoconstriction resulting from ATP released as a cotransmitter with noradrenaline from sympathetic nerves. P2Y-Purinoceptors are usually located on the vascular endothelium where they have a role as mediators of vascular relaxation by locally produced ATP. In some vessels, P2Y-purinoceptors are also located on the smooth muscle, perhaps in association with purinergic or sensory nerves, where they can elicit direct relaxation to neuronally released ATP. The net effect of ATP and its analogues on isolated vessels or on vascular beds will be the results of actions mediated by P2X- and P2Y-purinoceptor subtypes, although changes in vascular tone and in integrity of nerves and endothelial cells may alter the balance of the response. Such changes have been observed in diseased states (e.g., atherosclerosis) and may have important implications for the involvement of P2-purinoceptors in, for example, vasospasm. The development of selective and potent antagonists to P2X- and P2Y-purinoceptors has so far remained elusive, and their therapeutic potential can only be guessed.

Endothelial Cells Cultured from Human Umbilical Vein Release ATP, Substance P and Acetylcholine in Response to Increased Flow

Cultured human umbilical vein endothelial cells superfused with Krebs’ solution were used to investigate release of ATP, substance P and acetycholine with shear stress. ATP was consistently released when the cells were exposed to increased flow rate; release was rapid, had declined by 1 min and occurred upon a second exposure. Release of substance P and acetylcholine was more varied; increased shear stress led to release of substance P from 4 out of 16 endothelial-cell columns, whereas acetylcholine was released in 4 out of 12 columns. This is the first time that unequivocal evidence has been presented for release of these neurotransmitter substances from vascular endothelial cells. These findings have important implications about the mechanisms of local regulation of vascular tone.

Ultrastructural localisation of substance P and choline acetyltransferase in endothelial cells of rat coronary artery and release of substance P and acetylcholine during hypoxia.

Substance P and choline acetyltransferase have been localised in a small proportion of endothelial cells of rat coronary arteries using electron microscopic immunocytochemistry. During a hypoxic period of 1 min, coronary vasodilatation was produced in the Langendorff heart preparation and increased levels of substance P and acetylcholine were released into the perfusate. The possibility that these substances are released from endothelial cells during hypoxia and contribute to the hyperaemic response is discussed.

Actions mediated by P2-purinoceptorsubtypes in the isolated perfused mesenteric bed of the rat.

1 The effects of adenosine 5′‐triphosphate (ATP) and its analogues on the perfusion pressure of the isolated mesenteric bed of the rat were examined in preparations at resting tone, and with tone raised by noradrenaline. 2 In the preparations at resting tone, the effect of the analogues was to produce vasoconstriction, their rank order of potency being α,β‐methylene ATP > 2‐methylthio ATP > ATP. 3 In raised tone preparations, dose‐dependent vasodilatations were produced by ATP and 2‐methylthio ATP although, at the highest doses tested, responses decreased in magnitude. The rank order of potency of the analogues in eliciting this vasodilator response was 2‐methylthio ATP > ATP, while α,β‐methylene ATP was without effect. 4 Following desensitization of contractile responses to α,β‐methylene ATP, contractile responses to ATP and 2‐methylthio ATP were abolished while their relaxant responses were potentiated. 5 Removal of the endothelium with sodium deoxycholate totally abolished the vasodilator responses and enhanced the contractile responses. 6 It is concluded that, in the rat mesentery, ATP and its analogues cause vasoconstriction via P2x‐purinoceptors and vasodilatation via P2y‐purinoceptors and that these are located on the smooth muscle and on the endothelium, respectively.

Vasoconstrictor and vasodilator responses to various agonists in the rat perfused mesenteric arterial bed: selective inhibition by PPADS of contractions mediated via P2x‐purinoceptors

1 The effect of pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on vasoconstrictor and/or vasodilator responses to various agonists and electrical field stimulation was investigated in the rat mesenteric arterial bed at basal tone and at tone raised by methoxamine (15–50 μm). 2 At basal tone, nucleotides produced vasoconstriction with the following rank order of potency: α,β‐methylene ATP > > 2‐methylthio ATP ≥ ATP = UTP. PPADS (0.3–10 μm) concentration‐dependently antagonized α,β‐methylene ATP‐, 2‐methylthio ATP‐ and ATP‐induced responses. UTP‐, noradrenaline‐ and nerve‐mediated (4–32 Hz) increases in perfusion pressure remained unaffected by 10 μm PPADS. 3 In raised tone preparations, nucleotides produced vasodilations, their rank order of potency being 2‐methylthio ATP > ATP > UTP. Responses to 2‐methylthio ATP were slightly antagonized, whereas ATP‐ and UTP‐induced responses remained unaffected by 10μm PPADS. In addition, acetylcholine‐and adenosine‐elicited relaxations were not influenced by 10 μm PPADS. 4 The present results confirm the previously described selective P2x antagonism by PPADS, this compound being ineffective at muscarinic M3‐ and adenosine P1‐receptors as well as at α1‐adrenoceptors. There was some inhibition of P2y‐purinoceptors but at a much higher concentration than required for inhibition of P2x‐purinoceptors. 5 In addition, this study provides evidence for the ineffectiveness of PPADS at both vasoconstriction‐and vasodilatation‐mediating P2u‐purinoceptors.

Peptides and vasomotor mechanisms.

The multiple and diverse roles played by neuropeptide Y, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide and other biologically active peptides in the cardiovascular system are considered. A model of the vascular neuroeffector junction is described, which illustrates the interactions of peptidergic and nonpeptidergic transmitters that are possible at pre- and postjunctional sites. The effects of peptides on specific endothelial receptors are also described, which highlights the ability of these agents to act as dual regulators of vascular tone at both adventitial and intimal surfaces, following local release from nerves, or from endothelial cells themselves. Changes in expression of vascular neuropeptides that occur during development and aging in some disease situations and following nerve lesion are discussed.

Substance P is released from the endothelium of normal and capsaicin-treated rat hind-limb vasculature, in vivo, by increased flow.

The rat hind-limb vasculature releases substance P when subjected to a rapid increase in flow through the vascular bed. This release also occurs during high flow after rats have been capsaicinized, when loss of substance P-containing nerve fibers was verified by immunohistochemistry. Air treatment, a procedure shown by transmission electron microscopy to have removed endothelial cells from the arteries but not arterioles or capillaries of the hind-limb preparations, eliminated this release. Thus, the substance P released is unlikely to arise from perivascular nerves but rather from arterial endothelial cells.

Nitric oxide is the mediator of ATP‐induced dilatation of the rabbit hepatic arterial vascular bed

1 Livers of 10 New Zealand White rabbits were perfused in vitro with Krebs‐Bülbring buffer via the hepatic artery (HA) and portal vein (PV) at constant flows of 23 ± 1 and 77 + 1 ml min−1 100 g−1 respectively. The tone of the preparation was raised with noradrenaline (concentration: 10 μm). 2 Dose‐response curves for the vasodilatation produced by adenosine 5′‐triphosphate (ATP), acetylcholine (ACh), adenosine, and sodium nitroprusside (SNP) were obtained following injection into the HA supply. Injections were then repeated in the presence of the l‐arginine to nitric oxide pathway inhibitors N‐monomethyl‐l‐arginine (l‐NMMA, n = 6) and N‐nitro‐l‐arginine methyl ester (l‐NAME, n = 4) at concentrations of 30 μm and 100 μm for each inhibitor. 3 Both l‐NMMA and l‐NAME antagonized the responses to ATP and ACh; l‐NAME was 2–3 times more potent than l‐NMMA as an inhibitor of these endothelium‐dependent vasodilatations. Neither l‐NMMA nor l‐NAME attenuated responses of the endothelium‐independent vasodilators, adenosine and SNP. 4 These results indicate that nitric oxide is the mediator of ATP‐induced vasodilatation in the HA vascular bed of the rabbit and that the receptor responsible for the release of nitric oxide, the P2y‐purinoceptor, is located predominantly on the endothelium.

Contribution of P1-(A2b subtype) and P2-purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed.

1 The direct vascular effects of adenosine and ATP were compared in the isolated and perfused mesenteric arterial bed of the rat. The actions of analogues of adenosine and ATP were also examined. 2 In preparations at basal tone, adenosine lacked vasoconstrictor actions, while ATP elicited dose‐dependent vasoconstrictor responses. When the tone of preparations was raised by adding methoxamine to the perfusate, adenosine and its stable analogue, 2‐chloroadenosine (2‐CADO) elicited dose‐dependent vasodilatation. The A2 adenosine receptor agonist, 5′‐N‐ethylcarboxamidoadenosine (NECA) was active at lower doses than adenosine, while the A2a‐selective agonist, CGS 21680 and the selective A1 agonist, N6‐cyclopentyladenosine (CPA) failed to induce vasodilatation. ATP and its analogue, 2‐methylthio ATP, elicited dose‐dependent vasodilatation at doses 400 fold lower than adenosine. 3 Vasodilator responses to adenosine and 2‐CADO were sensitive to antagonism by 1 μm 8‐sulphophenyltheophylline (8‐SPT) and were unaffected by inhibition of nitric oxide synthase by Nω‐nitro‐L‐arginine methyl ester (L‐NAME). In contrast, vasodilator responses to ATP were not sensitive to antagonism by 8‐SPT and were almost abolished by L‐NAME treatment. 4 These results indicate that in the rat mesenteric arterial bed, while both adenosine and ATP participate in the purinergic control of vascular tone, adenosine appears to be a weaker vasodilator than ATP and lacks vasoconstrictor action. A2b adenosine receptors account for the adenosine‐induced vasodilatation which is independent of the production of nitric oxide.

Discrimination by PPADS between endothelial P2Y‐ and P2U‐ purinoceptors in the rat isolated mesenteric arterial bed

1 The main aim of this study was to characterize the antagonistic effects of pyridoxalphosphate‐6‐ azophenyl‐2′,4′‐disulphonic acid (PPADS) at coexisting endothelial P2Y‐ and P2U‐purinoceptors. Studies were conducted in Krebs‐perfused mesenteric arterial preparations isolated from the rat, with tone raised by methoxamine (5–50 μm). 2 Purine and pyrimidine compounds elicited vasodilatation with a rank order of potency of 2‐methylthio ATP (2‐MeSATP) = ADP>ATP = UTP>P1, P3‐diadenosine triphosphate (Ap3A)>P1, P2‐ diadenosine pyrophosphate (Ap2A)>NADP> adenosine. 8‐para‐Sulphophenyltheophylline (8‐PSPT; 3 μm) had no effect on vasodilator responses to 2MeSATP, ADP, ATP, UTP, Ap3A or NADP, but blocked responses to adenosine and the maximal response to Ap2A. 3 PPADS (3–100 μm) attenuated vasodilator responses to the P2Y‐selective agonists 2MeSATP and ADP, shifting the dose‐response curves to the right. The pA2 values for PPADS at 2MeSATP and ADP were 5.97 ± 0.69 and 5.98 ± 0.86 respectively. In contrast, PPADS had no effect on vasodilator responses mediated by the P2U‐selective agonist, UTP, or on vasodilator responses mediated by ATP. 4 PPADS (10 μm) was used to characterize responses mediated by the adenine dinucleotides; dose‐response curves for vasodilator responses to Ap3A and NADP, but not those to Ap2A, were shifted to the right by PPADS. The estimated pA2 values for the effect of PPADS on Ap3A and NADP were 6.38 and 6.26 respectively. 5 Indomethacin (10 μm) had no effect on vasodilator responses to 2MeSATP, ADP, ATP or UTP. 6 In conclusion, these results show that PPADS is an antagonist at endothelial P2Y‐ but not P2U‐ purinoceptors in rat mesenteric arteries. These receptors cannot be discriminated by inhibition of prostaglandin synthesis; P2Y‐purinoceptors are, however, sensitive to ADP. Selective antagonism by use of PPADS showed that ATP acts at P2U‐ and not P2Y‐purinoceptors. Ap3A and NADP mediate vasodilatation via P2Y‐purinoceptors, whereas vasodilatation to Ap2A is mediated partly via P1 and possibly via P2U‐purinoceptors.