Brian F. King
University College London
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FEBS Letters | 1993
Tania E. Webb; Joseph Simon; Belinda J. Krishek; Alan N. Bateson; Trevor G. Smart; Brian F. King; Geoffrey Burnstock; Eric A. Barnard
A cDNA encoding a novel member of the G‐protein‐coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) in Xenopus oocytes a slowly‐developing inward current was observed in response to application of ATP. The pharmacology of this expressed protein defines it as a P2Y purinoceptor.
Trends in Pharmacological Sciences | 2003
Maria P. Abbracchio; Jean-Marie Boeynaems; Eric A. Barnard; José L. Boyer; Charles Kennedy; María Teresa Miras-Portugal; Brian F. King; Christian Gachet; Kenneth A. Jacobson; Gary A. Weisman; Geoffrey Burnstock
The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.
British Journal of Pharmacology | 1998
Yuri Bogdanov; Scott S.P. Wildman; Mp Clements; Brian F. King; Geoffrey Burnstock
An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y4 (hP2Y4) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y4 receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2‐MeSATP=Ap4A>UDP(pure). ADP, ATPγS, 2‐MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y4 is more like the P2U receptor subtype. The rP2Y4 receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y2 receptor (a known P2U receptor). Thus, rP2Y4 and the P2Y2 subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi‐active) but can be distinguished as suramin‐insensitive and suramin‐sensitive P2U receptors, respectively.
Trends in Pharmacological Sciences | 1998
Brian F. King; Andrea Townsend-Nicholson; Geoffrey Burnstock
In the past five years, an extended series (P2Y1-n) of metabotropic nucleotide (P2) receptors has been cloned from vertebrate tissues; these receptors are activated by either ATP or UTP, or both nucleotides. While certain cloned P2Y receptors appear to correspond functionally to particular native P2 receptor phenotypes, such pharmacological phenotypes could be explained by either a combination of several members of the P2Y1-n series being coexpressed in the same tissue or the existence of novel, uncloned P2Y subtypes. Here, Brian King, Andrea Townsend-Nicholson and Geoffrey Burnstock review recent findings on the matter of pharmacological relationships between native P2 and cloned P2Y receptors.
Drug Development Research | 1996
Geoffrey Burnstock; Brian F. King
Over the last 3 years (March, 1993 to June, 1996), DNA sequences have been isolated for some 40 proteins that act as P2 purinoceptors. This explosion in receptor subtypes for nucleotide signalling and rate of advance (1 new receptor per month) have caused problems with the classification of these recombinant purinoceptors. At the request of the research community in this field, a “Purinoceptor Update Newsheet” was compiled at University College London with the view to monitoring and opening for general discussion new findings on P2 purinoceptor cloning and rapidly disseminating this information to investigators in this field. The present paper explains the system of numbering of the two families of receptor subtypes as proposed by Abbracchio and Burnstock [(1994): Pharmacol Ther 64:445–475] (P2X1‐n and P2Y1‐n purinoceptors, as extended families of ligand‐gated and G protein‐coupled receptors), and the order in which these subtypes appear in “Newsheet” tables.
British Journal of Pharmacology | 1997
Brian F. King; Scott S.P. Wildman; Lilia E. Ziganshina; Jesús Pintor; Geoffrey Burnstock
Under voltage‐clamp conditions, the activity of agonists and antagonists at a recombinant P2X2 receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pHe). In normal Ringer (Mg2+ ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pHe (8.0–5.5). ATP‐responses reached a maximum at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.05±0.05. Receptor affinity for ATP was lowered when extracellular Ca2+ ions were replaced with equimolar Mg2+ ions. However, the amplitude of the ATP‐responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.35±0.05. ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X2 receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. The potency order for agonists of P2X2 receptors was: ATP>2‐MeS‐ATPATPγS> ATPαS>>CTPBzATP, while other nucleotides were inactive. EC50 and nH values for full agonists were determined at pH 7.4 and re‐examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2‐MeSATP, ATPγS and ATPαS), without altering the potency order. The potency order for antagonists at P2X2 receptors was: Reactive blue‐2>trinitrophenol‐ATPPalatine fast blackCoomassie brilliant bluePPADS>suramin (at pH 7.4). IC50 values and slopes of the inhibition curves were re‐examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (IC50 values: 10.4±2 μM, at pH 7.4; 78±5 nM, at pH 6.5; 30±6 nM, at pH 5.5). In summary, a lowered pHe enhanced the activity of all agonists at P2X2 receptors but, with the exception of suramin, not antagonists. Since a lowered pHe is also known to enhance agonist activity at P2X receptors on sensory neurones containing P2X2 transcripts, the sensitization by metabolic acidosis of native P2X receptors containing P2X2 subunits may have a significant effect on purinergic cell‐to‐cell signalling.
British Journal of Pharmacology | 1996
Brian F. King; Lilia E. Ziganshina; Jesús Pintor; Geoffrey Burnstock
A full pharmacological characterization was carried out on a recombinant ATP‐gated ion channel (P2times2 purinoceptor) expressed in Xenopus oocytes. This slowly‐desensitizing neuronal P2times2 purinoceptor, activated by ATP (EC50 = 4.6 ± 1 μm at pH 7.4; n 4), showed the agonist potency order: ATP ≥ 2‐ MeSATP = ATPγS ≥ ATPαS < < Bz‐ATP. The receptor affinity for ATP was enhanced 5–10 fold by acidifying the bathing solution (to pH 6.5) but was diminished 4–5 fold in an alkaline solution (pH 8.0). The maximum activity of P2times2 purinoceptors and the activity order of a series of nucleotides were unaltered by changing extracellular pH. Interestingly, ATP sensitivity at a recombinant P2Y1, purinoceptor remained unaltered with changing extracellular pH. These results indicate that acidotic conditions in the synaptic cleft could strengthen purinergic transmission at neuronal P2times2 purinoceptors.
British Journal of Pharmacology | 2003
Scott S.P. Wildman; Robert J. Unwin; Brian F. King
Two molecularly distinct rat P2Y receptors activated equally by adenosine‐5′‐triphosphate (ATP) and uridine‐5′‐triphosphate (UTP) (rP2Y2 and rP2Y4 receptors) were expressed in Xenopus oocytes and studied extensively to find ways to pharmacologically distinguish one from the other. Both P2Y subtypes were activated fully by a number of nucleotides. Tested nucleotides were equipotent at rP2Y4 (ATP=UTP=CTP=GTP=ITP), but not at rP2Y2 (ATP=UTP>CTP>GTP>ITP). For dinucleotides (ApnA, n=2–6), rP2Y4 was only fully activated by Ap4A, which was as potent as ATP. All tested dinucleotides, except for Ap2A, fully activated rP2Y2, but none were as potent as ATP. ATPγS and BzATP fully activated rP2Y2, whereas ATPγS was a weak agonist and BzATP was inactive (as an agonist) at rP2Y4 receptors. Each P2Y subtype showed different sensitivities to known P2 receptor antagonists. For rP2Y2, the potency order was suramin>>PPADS= RB‐2>TNP‐ATP and suramin was a competitive antagonist (pA2, 5.40). For rP2Y4, the order was RB‐2>>suramin>PPADS> TNP‐ATP and RB‐2 was a competitive antagonist (pA2, 6.43). Also, BzATP was an antagonist at rP2Y4 receptors. Extracellular acidification (from pH 8.0 to pH 5.5) enhanced the potency of ATP and UTP by 8–10‐fold at rP2Y4 but did not affect agonist responses at rP2Y2 receptors. Extracellular Zn2+ ions (0.1–300 μM) coapplied with ATP inhibited agonist responses at rP2Y4 but not at rP2Y2 receptors. These two P2Y receptors differ significantly in terms of agonist and antagonist profiles, and the modulatory activities of extracellular H+ and Zn2+ ions. These pharmacological differences will help to distinguish between rP2Y2 and rP2Y4 receptors, in vivo.
The Journal of Physiology | 1998
Matthew S. Morrison; Luca Turin; Brian F. King; Geoffrey Burnstock; Timothy R. Arnett
1 There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non‐selective cation channels and Ca2+‐dependent K+ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation. 2 We found that ATP stimulated resorption pit formation up to 5.6‐fold when osteoclast‐containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0.2‐2 μM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6.9‐7.0). Pit formation by acid‐activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto‐ATPase and by suramin, an antagonist of P2 receptors. 3 Over the same concentration range at which rat osteoclast activation occurred (0.2‐2 μM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3.3‐fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20–200 μM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation. 4 These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts.
Neuroscience | 1996
Brian F. King; Q. Zhu; S. Wang; M.D. Norenberg; Geoffrey Burnstock
Extracellular ATP is known to activate intracellular enzymes in astrocytes via P2 purinoceptors that appear to play important physiological and pathological roles in these supporting brain cells. In this study, major P2 purinoceptor subtypes on astrocytes of neonatal rat cerebral cortices were identified in receptor expression experiments, when astrocytic messenger RNA was injected into Xenopus oocytes and recombinant P2 purinoceptors were characterized pharmacologically. In messenger RNA-injected oocytes, ATP evoked inward chloride currents (ICl,Ca) typical of stimulating metabotropic receptors that release intracellular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hill coefficient was 0.5, which indicated that ATP stimulated two subtypes of P2 purinoceptor. UTP and 2-methylthioATP were the most active (and equipotent) of a series of nucleotides activating recombinant P2 purinoceptors. These results indicated that the two P2 purinoceptors expressed by astrocytic messenger RNA were of P2U and P2Y subtypes. Responses to ATP were antagonized by the P2 purinoceptor antagonist (suramin) but not by the P1 purinoceptor blocker (sulphophenyltheophylline). Findings in expression studies were confirmed in assays of intracellular signalling systems using primary cultures of rat astrocytes. UTP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP or 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as measured by fura-2/AM luminescence) which, in cross-desensitization experiments, indicated the involvement of two subtypes of P2 purinoceptors. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, raise intracellular Ca2+ and also stimulate mitogen-activated protein kinase.