Sabine Pirotton

Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells.

The role of ATP and ADP as intercellular mediators is now well established. The presence of the nucleotides in extracellular fluids can result from several mechanisms: cell lysis, selective permeabilization of the plasma membrane and exocytosis of secretory vesicles, such as platelet dense bodies. Extracellular adenine nucleotides are rapidly degraded by ectonucleotidases expressed inter alia on the surface of endothelial cells. They act on cells via the family of P2 receptors which encompasses more than 5 subtypes, some of which have been cloned recently. The P2T, P2U and P2Y receptors belong to the superfamily of receptors coupled to G proteins, whereas the P2X receptor is a cation channel and the P2Z receptor a non-selective pore. ATP and ADP stimulate the endothelial production of prostacyclin (PGI2) and nitric oxide (NO), two vasodilators and inhibitors of platelet aggregation, via an increase in cytosolic Ca2+. This action of adenine nucleotides is believed to limit the extent of intravascular platelet aggregation and to help localize thrombus formation to areas of endothelial damage. The endothelial response to nucleotides is mediated by at least two distinct subtypes of P2 receptors, P2Y and P2U, both coupled to phospholipase C.

Coexpression of P2Y and P2U Receptors on Aortic Endothelial Cells : Comparison of Cell Localization and Signaling Pathways

Depending on the vascular bed considered, the actions of ATP on the endothelium are mediated by either P2Y or P2U receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, where they are both coupled to phospholipase C. In this study, we have investigated whether they are truly coexpressed on the same cells and whether their signaling pathways diverge beyond phospholipase C activation. Measurements of [Ca2+]i in single cells showed that almost all bovine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P2Y receptors, and UTP, an agonist of P2U receptors. UTP stimulated the release of prostacyclin from freshly isolated bovine aortic endothelial cells, even when they were exposed to cycloheximide at the time of their collection: this indicates that P2U receptors must already be expressed on endothelial cells in situ and do not appear during cell culture. The time course of inositol phosphate (InsP) accumulation and the relative proportion of Ins(1,4,5)P3, Ins(1,3,4,5)P4, and Ins(1,3,4)P3 were similar in cells stimulated by 2MeSATP or UTP. UTP and 2MeSATP both stimulated the hydrolysis of phosphatidylcholine by phospholipase D, as reflected by the release of [3H]choline from prelabeled cells. The responses to both agents were blocked after downregulation of protein kinase C, resulting from a prolonged exposure to phorbol 12-myristate 13-acetate: this blockade occurred at a step distal to phospholipase C activation. A single difference between the two pathways has been identified: the effect of 2MeSATP on InsP3 was significantly more inhibited after a short exposure to phorbol 12-myristate 13-acetate than that of UTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual role of GTP-binding proteins in the control of endothelial prostacyclin.

Pretreatment of bovine aortic endothelial cells with pertussis toxin inhibited partially the accumulation of inositol phosphates in response to ATP, whereas cholera toxin had no effect. Both pertussis and cholera toxins enhanced the stimulatory effect of ATP on prostacyclin release from the same cells. This action of cholera toxin was mimicked neither by an increase of cyclic AMP nor by the dissociated subunits of the toxin. Cholera and pertussis toxins, as well as aluminum fluoride, also potentiated the release of prostacyclin induced by ionophore A23187. These results suggest that a pertussis toxin-sensitive GTP-binding protein is involved in the coupling between P2-purinergic receptors and phospholipase C. In addition, another GTP-binding protein would play a crucial role at a further step in the control of PGI2 biosynthesis.

Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells.

1 The response of bovine aortic endothelial cells to adenosine 5′‐triphosphate (ATP) is mediated by both P2y and nucleotide/P2u receptors. In order to determine which form of the nucleotide is the true ligand of these receptors, we have investigated the effects of divalent cations on ATP‐, uridine 5′‐triphosphate (UTP)‐ and 2 methylthioadenosine 5′‐triphosphate (2MeSATP)‐induced inositol phosphate accumulation in these cells. 2 Omisson of Mg2+ from a calcium‐free incubation buffer caused a shift to the left of the ATP concentration‐action curve. 3 In the presence of EDTA (1 mm), the basal level of inositol trisphosphate (InsP3) was markedly increased and the absolute maximal response to ATP was decreased; however, the response to low concentrations of ATP was enhanced. 4 When the results were plotted in terms of calculated ATP4‐ concentrations, the concentration‐response curves obtained in the presence of 1.25 mm Mg2+ lay closer to the respective curves obtained when Mg2+ was omitted from the medium or when Mg2+ was omitted and EDTA (1 mm) was added. The curves became almost superimposable when the baseline value was subtracted. 5 A similar shift to the left of the concentrations‐action curves was also observed with both UTP and 2MeSATP. 6 Our data provide evidence that a form of ATP uncomplexed with divalent cation is the preferential agonist of both the nucleotide/P2u and the P2y receptors expressed on bovine aortic endothelial cells.

P2-purinergic receptors in vascular endothelial cells: from concept to reality.

ATP exerts at least 2 actions on arterial endothelial cells: it stimulates the release of endothelium-derived relaxing factor, a still unidentified vasodilator, and of prostacyclin, a potent inhibitor of platelet aggregation. A study of agonist specificity indicates that these responses are mediated by P2-purinergic receptors. We have now demonstrated that in these cells, the P2-receptors are coupled to a phospholipase C hydrolysing phosphatidylinositol-bisphosphate and that this coupling involves a pertussis toxin-sensitive GTP-binding regulatory protein.

Nucleotide Receptors Coupling to the Phospholipase C Signaling Pathway

The earliest papers reporting a stimulatory effect of extracellular nucleotides on inositol phosphate formation were published in 1985–1987 (Charest et al., 1985; Horstman et al., 1986; Pirotton et al., 1987; Forsberg et al., 1987). Since then the number of organs and cells in which nucleotides have been shown to produce an accumulation of inositol phosphates has been growing. A list which is not claimed to be exhaustive is provided in Table 1. These actions are mediated by at least two distinct receptors, P2Y and P2U, which have been identified according to the rank order of potency of various natural and synthetic agonists and on the basis of cross-desensitization experiments. Basically, the P2Y receptor is characterized by the high potency of 2-Methylthio ATP (2MeSATP) and related agonists (Burnstock et al., 1994), similar potencies of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) (the ratio being somewhat variable from one system to the other) and the lack of activity of UTP. It became apparent in the late eighties that, in many tissues and cells, not only ATP but also UTP is able to induce inositol phosphate formation. At that time it was proposed that the action of UTP is mediated by specific pyrimidinoceptors distinct from the purinoceptors (Seifert and Schultz, 1989). The existence of nucleotide or P2U receptors common to ATP and UTP constituted an alternative possibility, in favor of which experimental evidence started to accumulate: mainly the lack of additivity and the cross-desensitization of the responses to the two nucleotides (Brown et al., 1991; O’Connor et al., 1991; O’Connor, 1992).

P2‐Purinergic Receptors on Vascular Endothelial Cells

ATP and ADP, via P,, receptors, stimulate the release of nitric oxide’ and of prostacyclin *.’ from aortic endothelial cells and increase their rate of proliferation? These actions might have a physiological importance in the interaction between platelets and the vascular endothelium. The release of prostacyclin will limit the extent of platelet aggregation following a lesion of the endothelium, while the mitogenic effect will accelerate the repair of that lesion. Bovine aortic endothelial cells (BAECs) provided a useful model to elucidate the transduction mechanisms associated with PZy receptors. The occupancy of these receptors induces the hydrolysis of several phospholipids by various phospholipases, generating different second messengers. These responses have different time courses and are triggered by distinct mechanisms (FIG. 1). ATP and ADP induce a rapid and transient accumulation of inositol trisphosphate ( lP3), reflecting the hydrolysis of phosphatidylinositol bisphosphate (PIP,) by phospholipase C (FIG. lA).’ The rise of cytoplasmic Ca2+ that results6 is responsible for a burst of prostacyclin release, probably via the activation of a Ca2+-dependent phospholipase A,.’ The coupling of the P,, receptors and the phospholipase C seems to involve a GTP-binding protein (FIG. lC).* ATP and ADP also induce a sustained increase of the choline level inside endothelial cells (FIG. lB), an increase likely to result from the activation of phospholipase D, which hydrolyzes phosphatidylcholine into phosphatidic acid and choline? Phosphatidic acid might play a role in the mitogenic effect of ATP.” Depletion of protein kinase C by a prolonged exposure to phorbol 12-myristate 13-acetate abolished the ATP-stimulated release of choline metabolites (FIG. 1D). This indicates that the activation of protein kinase C by diacylglycerol released from PIP, plays a crucial role in the stimulation of phospholipase D.’

Palmitoyl-l-carnitine increases the release of prostacyclin from vascular endothelial cells

Prostacyclin biosynthesis is dramatically increased in patients with acute myocardial infarction. As palmitoylcarnitine accumulates during myocardial ischemia, the action of this metabolite on the endothelial production of prostacyclin was studied. Palmitoyl-L-carnitine (10-100 microM) enhanced the release of prostacyclin and free arachidonic acid from bovine aortic endothelial cells. This action was mimicked by lysophosphatidylcholine, but by none of the following compounds: acetylcarnitine, carnitine, palmitic acid, sphingosine, dihydrosphingosine and N-stearoyl-dihydrosphingosine. In addition to mobilizing free arachidonate, palmitoylcarnitine induced the release of free choline and phosphorylcholine presumably via the activation of phospholipases C and D. Palmitoyl-L-carnitine had also a cytotoxic effect on the endothelial cells. These data suggest that the increased biosynthesis of prostacyclin in myocardial infarction might be partially explained by the accumulation and release of palmitoyl-L-carnitine.

Transduction mechanisms of p2 purinergic receptors: Role of phospholipase c and calcium

Abstract The transduction mechanisms of P2 receptors have remained uncharacterized until recently. Data accumulated in the last few years demonstrate that, in many cell types, ATP induces a rise of cytoplasmic Ca2+, which often results from the direct coupling between P2 receptors and phospholipase C.

The Role of Nucleotide Receptors in the Cardiovascular System

The first observation of a biological activity for adenine nucleotides on the cardiovascular system was reported by Drury and Szent-Gyorgyi in 1929: when administered iv to guinea pigs, adenosine triphosphate (ATP) induced a decrease in heart rate and arterial blood pressure and a dilation of coronary blood vessels. Since that time, multiple effects of nucleotides have been described and much progress has been made in the understanding of their mechanisms of action on different components of the cardiovascular system, including endothelium, smooth muscle cells (SMC), and cardiomyocytes.