Rodolphe Janssens

Advances in signalling by extracellular nucleotides. the role and transduction mechanisms of P2Y receptors.

Nucleotides are ubiquitous intercellular messengers whose actions are mediated by specific receptors. Since the first clonings in 1993, it is known that nucleotide receptors belong to two families: the ionotropic P2X receptors and the metabotropic P2Y receptors. Five human P2Y receptor subtypes have been cloned so far and a sixth one must still be isolated. In this review we will show that they differ by their preference for adenine versus uracil nucleotides and triphospho versus diphospho nucleotides, as well as by their transduction mechanisms and cell expression.

Effects of extracellular nucleotides and nucleosides on prostate carcinoma cells

The purpose of this work was to characterize the receptors involved in the action of nucleotides on the human prostate carcinoma cell lines LNCaP, PC‐3 and DU145. Northern blotting revealed the presence of P2Y2, P2Y6 and P2Y11 messengers in the three cell lines. P2Y1 mRNA was only observed in the DU145 cells. In both PC‐3 and DU145 cells, ATP and UTP stimulated inositol phosphate accumulation in an equipotent, equiactive and non‐additive way, suggesting the involvement of P2Y2 receptors. ATP also increased cyclic AMP, but this effect is likely to result from degradation into adenosine and activation of A2 receptor. A2 receptor activation led to a synergistic enhancement of prostate‐specific antigen secretion induced by vasoactive intestinal peptide. RT – PCR experiments detected the expression of the P2X4 and P2X5 receptors in the DU145 cells and the P2X4, P2X5 and P2X7 receptors in the PC‐3 cells. The calcium influx induced by BzATP confirmed the functional expression of P2X receptors. ATP inhibited the growth of PC‐3 and DU145 cells. This effect was mimicked neither by UTP nor by adenosine, indicating that it does not result from phospholipase C or adenylyl cyclase activation. On the contrary, in PC‐3 cells, BzATP reproduced the effect of ATP, which was associated to a moderate decrease of proliferation and an increase of apoptosis. In DU145 cells, ATP was more potent than BzATP and growth inhibition was mainly associated with necrosis. We suggest that P2X receptors might be involved in the inhibition by nucleotides of prostate carcinoma cell growth.

Human P2Y2 receptor polymorphism: identification and pharmacological characterization of two allelic variants

In the process of cloning the human P2Y2 receptor in order to establish 1321N1 cell lines expressing this receptor, we detected a gene polymorphism characterized by an arginine 334 to cysteine 334 transition. The frequency distribution of the polymorphism was studied in a European population. We observed that 66% of the tested persons are homozygotes R/R, 29% are heterozygotes R/C and 5% are homozygotes C/C. The frequency of the R allele was 0.8 versus 0.2 for the C allele. We stably expressed each form of the human P2Y2 receptor into 1321N1 cells and isolated clones by limiting dilution. The effects of nucleotides and antagonists on inositol trisphosphate accumulation and cyclic AMP formation were compared between the two cell lines. The time‐courses of inositol trisphosphate accumulation as well as concentration‐response curves characterizing the effects of UTP, ATP, AP4A and ATPγS were mostly similar, except for slight kinetic differences (slower time‐course with the 334C form). The sensitivity to pertussis toxin of inositol trisphosphates accumulation was critically dependent on the agonist concentration and stimulation duration, suggesting the involvement of a Gi,0 protein during the early stimulation by low nucleotide concentrations. No inhibition of cyclic AMP accumulation could be detected. These properties were observed with both polymorphic receptors.

Rapid up-regulation of P2Y messengers during granulocytic differentiation of HL-60 cells

HL‐60 cells are human promyelocytic cells expressing two ATP receptors: the P2Y2 and P2Y11 subtypes. Our Northern blotting experiments have shown that P2Y2 and P2Y11 messengers were up‐regulated in these cells, rapidly and independently of protein synthesis, following treatment with granulocytic differentiating agents such as retinoic acid, dimethylsulfoxide, granulocyte‐colony stimulating factor, dibutyryl cyclic AMP and ATP. AR‐C67085 and adenosine 5′‐O‐(3‐thiotriphosphate), two potent agonists of the recombinant P2Y11 receptor, increased intracellular cAMP concentration in HL‐60 cells more potently than ATP itself. These observations support the conclusion that the effect of ATP on HL‐60 cell differentiation is mediated by the P2Y11 receptor.

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).

Receptors responsive to extracellular uracil nucleotides

The cloning of P2Y4 and P2Y3 (chicken) / P2Y6 (rat, human) receptors has been the ultimate proof that receptors for pyrimidine nucleotides exist. However, on the basis of their structure these receptors do not constitute a family of their own but belong to the P2Y family, which thus encompasses selective purinoceptors (P2Y1, P2Y11), nucleotide receptors responsive to both adenine and uracil nucleotides (P2Y2, P2Y8), and pyrimidinoceptors (P2Y4 responsive to UTP, P2Y3/P2Y6 responsive to UDP). Although the occurrence of uracil nucleotides in extracellular fluids remains poorly documented, the very existence of P2Y receptors selectively responsive to them strongly suggests that UTP and UDP may play a role as intercellular mediators, independently from adenine nucleotides. Northern blotting revealed an expression of the P2Y6 receptor mRNA in human spleen, thymus, and blood leukocytes, as well as in Jurkat and MOLT‐4 T‐cell lines. Other experiments revealed an expression in 1HAEo and 16HBE14o cell lines derived from the airway epithelium. These studies suggest a possible role of the P2Y6 receptor in the immune and respiratory systems. Drug Dev. Res. 45:130–134, 1998.