1 In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y(2), P2Y(4) and P2Y(6), but not P2Y(1) receptors.2 None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATP gamma S, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.3 Comparison of the concentration response curves to ATP gamma S with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.4 Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N-6-cyclopentyladenosine nor CGS 21680 were agonists.5 These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors.

The P2Y6 receptor is a uridine nucleotide‐specific G protein‐linked receptor previously reported to stimulate the phosphoinositide (PI) pathway. We have investigated its effect in neurones, by micro‐injecting its cRNA into dissociated rat sympathetic neurones and recording responses of N‐type Ca2+ (ICa(N)) and M‐type K+ (IK(M)) currents. In P2Y6 cRNA‐injected neurones, UDP or UTP produced a voltage‐dependent inhibition of ICa(N) by ∼53% in whole‐cell (disrupted‐patch) mode and by ∼73% in perforated‐patch mode; no inhibition occurred in control cells. Mean IC50 values (whole‐cell) were: UDP, 5.9±0.3 nM; UTP, 20±1 nM. ATP and ADP (1 μM) had no significant effect. Pertussis toxin (PTX) substantially (∼60%) reduced UTP‐mediated inhibition in disrupted patch mode but not in perforated‐patch mode. Uridine nucleotides also inhibited IK(M) in P2Y6 cRNA‐injected cells (by up to 71% at 10 μM UTP; perforated‐patch). Mean IC50 values were: UDP, 30±3 nM; UTP, 115±12 nM. ATP (10 μM) again had no effect. No significant inhibition occurred in control cells. Inhibition was PTX‐resistant. Thus, the P2Y6 receptor, like the P2Y2 subtype studied in this system, couples to both of these two neuronal ion channels through at least two different G proteins. However, the P2Y6 receptor displays a much higher sensitivity to its agonists than the P2Y2 receptor in this expression system and higher than previously reported using other expression methods. The very high sensitivity to both UDP and UTP suggests that it might be preferentially activated by any locally released uridine nucleotides. British Journal of Pharmacology (1999) 126, 1009–1017; doi:10.1038/sj.bjp.0702356

Studies using selective agonists have suggested that the contractile effect of extracellular nucleotides, such as ATP and UTP, in blood vessels is mediated mainly by P2X1 receptors with a smaller contribution of P2Y receptors while the mitogenic effect is mediated by P2Y (P2Y1, P2Y2, P2Y4, and P2Y6) receptors with no effect of P2X1 receptors. This indicates a difference in P2 receptor expression between the contractile and the synthetic phenotype of the SMC. To measure the expression of mRNA for these receptors a competitive RT-PCR assay was developed that utilised synthetic RNA-competitors allowing determination of the number of mRNA copies for each receptor in the samples. In the synthetic phenotype the mitogenic P2Y1 and P2Y2 receptor transcripts were upregulated by 342- and 8-fold, respectively, while the contractile P2X1 receptor is totally downregulated and the P2Y4 and P2Y6 receptors were unchanged. This plasticity of the receptor expression may be important in the transition from the contractile to the synthetic SMC phenotype.

The distribution of the P2Y(1) receptor protein and transcript in the one-day-old chick brain were determined by quantitative in vitro ligand autoradiography and in situ hybridization histochemistry. We have previously used [S-35]2'-deoxy 5'-O-(1-thio) ATP as a radioligand for the recombinant P2Y(1) receptor transiently expressed in COS-7 cells and have also shown that such sites are present al high density (B-max: similar to 37 pmol radioligand bound/mg protein) in chick brain membranes. Here we report the macroscopic localization of these [S-35]2'-deoxy 5'-O-(1-thio) ATP binding sites within the chick brain. They were found to be widely distributed there (within the range of 0.047 +/- 0.012 to 0.309 +/- 0.035 pmol bound/mg wet tissue). The affinities of P-2 agonists and antagonists at these binding sites was comparable to that found previously for the recombinant P2Y(1) receptor. In parallel experiments, the regional and cellular localization of the P2Y(1) receptor messenger RNA was examined by ia situ hybridization. The transcript was also found to be widely distributed throughout the brain. High levels of hybridization were detected in the cortex piriformis, ectostriatum, hippocampus, cerebellum and in a range of discrete nuclei throughout the brain, including the ovoidalis, isthmo-opticus and spiriformis lateralis nuclei. Localization at cellular level indicates that this receptor transcript is expressed in neurons and also at non-neuronal sites. Furthermore, the distribution of the P2Y(1) transcript and the [S-35]2'-deoxy 5'-O-(1-thio) ATP binding sites matched in a number of the regions and structures mentioned above.The present study clarifies the anatomical distribution of the P2Y(1) receptor within the chick brain. Its broad distribution coupled with its neuronal expression suggest an important role for for this type of metabotropic nucleotide receptor within the brain. (C) 1998 IBRO. Published by Elsevier Science Ltd.

Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2‐methythio‐ATP and α,β‐methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y 1 or P2Y 6 receptors. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y 2 receptors. 1321N1 cells transfected with human P2Y 4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. Use of the reverse transcriptase‐polymerase chain reaction confirmed the presence of mRNA encoding P2Y 4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y 2 , P2Y 4 and P2Y 6 receptors, but not P2Y 1 receptors, were detected. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [ 3 H]‐thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y 4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.

Degenerate PCR was used to amplify DNAs encoding members of the P2Y receptor family from rat brain RNA. A full-length sequence obtained for one novel clone (R5) contained an intronless open reading frame that encoded a polypeptide of 361 amino acids, sharing 84% sequence identity with the human P2Y(4) receptor. When R5 was stably expressed in Jurkat cells, calcium fluxes resulting from stimulation of the receptor showed that UDP, ADP, 2-methylthio-ATP, and diadenosine tetraphosphate were inactive, whereas UTP and ATP were both full agonists with similar potency. At the human receptor, ATP has significantly lower potency than UTP. The R5 transcript was not detected in brain by northern hybridization. Therefore, its tissue distribution was assessed by PCR, and the mRNA was found to be widely distributed at a low abundance, being present in brain, spinal cord, and a variety of peripheral organs. Localization of the receptor transcript in adult rat brain sections by in situ hybridization indicated that it is expressed at highest levels in the pineal gland and ventricular system. It is presumed that R5 is a species orthologue of the human P2Y(4) receptor but with this significant difference in agonist pharmacology.

Objectives: The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFE respond to extracellular ATP by inducing expression of c-Sos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFE. Methods: Expression of P2Y(1) and P2Y(2) receptors and induction of c-Sos were examined by Northern blot analysis. CAFB growth was assessed by measuring [H-3]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. Results: Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y(1) and P2Y(2)) an expressed in cultured CAFE. Extracellular ATP induced the expression of c-foss mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca2+ signaling. Extracellular ATP inhibited the NE-stimulated increases in DNA content and in [3H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [H-3]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. Conclusions: These data demonstrate that CAFE express both P2Y(1) and P2Y(2) receptor mRNA and that CAFE respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [H-3]thymidine incorporation into DNA and on expression of c-Sos mRNA are exerted via distinct P2Y receptor subtypes. (C) 1998 Elsevier Science B.V.

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5′‐triphosphate (UTP) or adenosine 5′‐triphosphate (ATP) inhibits the N‐type voltage‐gated calcium current by ∼65%, with an IC50 of 0.5 μM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 ± 6 nM), much less for UTP and ADP (~1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.

We have determined the identity of the orphan G-protein coupled receptor cDNA, 6H1, present in activated chicken T cells, as a subtype of P2Ypurinoceptor. This identification is based first on the degree of sequence identity shared with recently cloned members of the P2Yreceptor family and second on the pharmacological profile. Upon transient expression in COS-7 cells the 6H1 receptor bound the radiolabel [35S]dATPαS specifically and with high affinity (Kd, 10 nM). This specific binding could be competitively displaced by a range of ligands active at P2purinoceptors, with ATP being the most active (Ki, 116 nM). Such competition studies have established the following rank order of activity: ATP ADP 2-methylthioATP α,β-methyleneATP, UTP, thus confirming 6H1 as a member of the growing family of P2Ypurinoceptors. As the fifth receptor of this type to be identified we suggest that it be named P2Y5.

1. Application of molecular biology to the study of P2Y purinoceptors has led to the identification of seven such receptors. Here we briefly review their properties and investigate qualitatively the expression of four rat receptor transcripts in heart. 2. The reverse transcriptase-polymerase chain reaction was used to ascertain whether the rat P2Y1, P2Y2, P2Y4 and P2Y6 receptor transcripts were expressed in whole heart, neonatal cardiac fibroblasts, neonatal cardiac myocytes and adult cardiac myocytes. 3. All receptor sequences could be amplified from neonatal rat whole heart, with P2Y6 appearing the most abundant transcript of the four. P2Y1 is expressed at higher levels in comparison to P2Y2, P2Y4 and P2Y6 in the neonatal myocyte. In the adult myocyte P2Y1, P2Y2 and P2Y6 could be amplified but P2Y4 could not be detected. In the neonatal fibroblast, P2Y1 and P2Y6 appear to be expressed at higher levels than P2Y2 and P2Y4. 4. In summary, it is concluded that multiple P2Y receptor subtypes are expressed in heart and that the expression in myocytes changes from the neonate to the adult.

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

A cDNA encoding a P2Y purinoceptor was originally cloned from chick brain and the bovine and human homologues have recently been obtained. These are seven-transmembrane-domain polypetides, i.e. G protein-coupled receptors. When activated by agonists, this P2Y receptor mobilizes intracellular Ca2+ and has been shown to be coupled to inositol-1,4,5-trisphosphate formation. Its pharmacology has been established in several expression systems, using both ligand binding and functional responses: 2-methylthioATP has the highest potency of nucleotides and derivatives tested, while UTP and alpha, beta-methylene ATP are inactive. This was hence assigned as a new subtype of the pharmacologically defined P2Y receptors, P2Y1. P2Y1 receptors are exceptionally abundant in the brain. A P2U receptor reported by others can be designated P2Y2. Another P2 receptor subtype, P2Y3, now cloned as a cDNA from the brain and expressed in oocytes and in transfected cells, shows a quite different ligand potency profile to the first two. A fourth subtype is expressed primarily in certain haemopoietic cells and in cardiac muscle. A putative fifth subtype is expressed only in T lymphocytes, upon activation. Yet other P2Y subtypes are indicated by recent cloning studies. The amino acid sequences of all of these P2 receptors, while displaying some homology, are strikingly diverse: they form a separate and unusual new family in the G protein-coupled receptor main superfamily.

A partial cDNA was isolated by hybridization screening of an embryonic chick brain library for P2Y purinoceptors. After extension to full length, it revealed an open reading frame that encoded a protein, P2Y3, of 328 amino acids that is nearest in sequence identity to the G protein-coupled P2 purinoceptors obtained by DNA cloning. Expression of P2Y3 in cRNA-injected Xenopus oocytes confirmed that this cDNA encodes a member of the metabotropic purinoceptor family, with a novel order for the relative activities of nucleotides. At 100 microM concentrations, ADP gave the highest activity, and UTP and UDP were also strongly active. When expressed in the human T cell line Jurkat, P2Y3 mediated transient increases in intracellular Ca2+ in response to various nucleotides. Again, an unusual agonist rank order was revealed, with uridine nucleotides being more potent than adenosine nucleotides and UDP being the most potent agonist tested (half-maximal concentration, 0.13 microM) and 10-fold more potent than UTP. 2-Methylthlo-ATP was of relatively low activity in both systems. The receptor transcript is expressed in brain, spinal cord, kidney, and lung and is highly abundant in the spleen but not in other peripheral tissues that we tested. The results indicated that P2Y3 is a previously unknown P2 purinoceptor subtype with a preference for nucleoside diphosphates.

We have previously cloned a cDNA encoding a G-protein-coupled P2 purinoceptor from chick brain and designated this as a P2Y1 purinoceptor (Webb, T.E., J. Simon, B.J. Krishek, A.N. Bateson, T.G. Smart, B.J. King, G. Burnstock and E.A. Barnard, 1993, FEBS Lett. 324, 219). Here, we describe the further characterisation of this recombinant receptor expressed in both simian kidney endothelial (COS-7) cells and Xenopus oocytes. In transfected COS-7 cell membranes, the recombinant receptor showed a high level of expression (Bmax = 7.9 ± 2.2 pmol [35S]dATPαS bound/mg protein) and affinity (Kd = 6.6 ± 0.3 nM). In these COS-7 cells, the activation of the implanted purinoceptor induced a suramin-sensitive formation of inositol 1,4,5-triphosphate (1,4,5InsP3). Upon expression in Xenopus oocytes, ATP was the only natural nucleoside triphosphate to elicit a Ca2+-activated chloride current. The P2 purinoceptor antagonists suramin and Reactive Blue-2 were both able to inhibit this evoked current. Utilizing both expression systems, the binding affinity profile and the functional pharmacological profile of the agonists, the common series found was: 2-methylthioATP (2-MeSATP) ⩾ ATP > ADPβS > ADP. These two agonist series and the lack of activity of adenosine, α,β-methyleneATP (α,β-meATP), 3′-O-(4- benzoyl)benzoyl-ATP (Bz-ATP) and UTP, together confirmed that this receptor is a specific subtype of the P2Y purinoceptors.

Little has been known of the abundance in the brain of any of the G protein coupled P2 purinoceptors nor their pharmacology. Here we show that [35S]dATP alpha S is a suitable radioligand for investigating these receptors and hence that they are exceptionally abundant both in one-day-old chick (Bmax: 37 pmol agonist sites/mg protein) and adult rat brain membranes (Bmax: 39 pmol/mg protein). [35S]dATP alpha S (which is selective for P2Y over the P2X types of purinoceptor) binds with high affinity to these sites in the chick (Kd: 13.3 nM) and in the rat brain membranes (Kd: 9.1 nM). The rank order of potency of purinoceptor-active agonists and antagonists displacing [35S]dATP alpha S binding is: dATP alpha S > (3'-deoxyATP, 2-methylthioATP, ATP alpha S, ATP) > 2'-deoxyATP > 2-methylthioADP > ADP >> suramin, Reactive Blue-2 >> UTP, L-beta,gamma-methyleneATP, adenosine; this defines these binding sites as P2Y subtypes of the P2 purinoceptors. This pharmacological profile of purinergic ligands is in excellent agreement with the potency order established for the recombinant P2Y1 purinoceptor from chick brain, identifying the great majority of the brain P2 purinoceptors as identical or very similar to the native P2Y1 receptor.

Using a chick P2Y1 receptor cDNA probe we have isolated a mammalian P2Y receptor clone from a bovine aortic endothelial cell Library. The sequence has a high degree of similarity to the chick P2Y1 clone. When transfected into the Jurkat cell line, the cDNA conferred sensitivity to purinoceptor agonists. Using fura-2 loaded cells the potency order at the receptor was found to be 2-methlythioadenosine 5′ triphosphate = adenosine 5′ diphosphate > adenosine 5′ triphosphate >> α,β-methyleneadenosine 5′ triphosphate and uridine 5′ triphosphate. This corresponds to the agonist potency order expected for the bovine aortic endothelial cell P2Y receptor.

Extracellular ATP acts at specific cell surface purinoceptors to elicit a wide range of physiological responses. We have recently isolated a cDNA for a G-protein-coupled P2 purinoceptor (P2y1) from chick brain. It has been defined as a P2Y-like purinoceptor by the rank order of potency of P2 purinoceptor ligands, determined electrophysiologically in the Xenopus oocyte expression system. Here, we examine the ligand selectivity of this recombinant receptor, expressed transiently in COS-7 cells. The regional distribution of the P2y1 purinoceptor transcript within the one-day-post-hatch chick brain was also determined. It is widely expressed in the cerebellum and telencephalon and in specific nuclei of the mesencephalon and diencephalon, suggesting a neuronal localization of the P21 purinoceptor.

The etiological agent of coronavirus disease (COVID-19) is the new member of the Coronaviridae family, a severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2), responsible for the pandemic that is plaguing the world. The single-stranded RNA virus is capable of infecting the respiratory tract, by binding the spike (S) protein on its viral surface to receptors for the angiotensin II-converting enzyme (ACE2), highly expressed in the pulmonary tissue, enabling the interaction of the virus with alveolar epithelial cells promoting endocytosis and replication of viral material. The infection triggers the activation of the immune system, increased purinergic signaling, and the release of cytokines as a defense mechanism, but the response can become exaggerated and prompt the so-called "cytokine storm", developing cases such as severe acute respiratory syndrome (SARS). This is characterized by fever, cough, and difficulty breathing, which can progress to pneumonia, failure of different organs and death. Thus, the present review aims to compile and correlate the mechanisms involved between the immune and purinergic systems with COVID-19, since the modulation of purinergic receptors, such as A2A, A2B, and P2X7 expressed by immune cells, seems to be effective as a promising therapy, to reduce the severity of the disease, as well as aid in the treatment of acute lung diseases and other cases of generalized inflammation.