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The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14748. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Adenosine 5'-triphosphate (ATP) is a cotransmitter with classical transmitters in most nerves in the peripheral and central nervous systems, although the proportions vary between tissues and species and in different developmental and pathophysiological circumstances. There was early evidence that ATP was released together with acetylcholine (ACh) from motor nerves supplying skeletal muscle, although it was considered at the time as a molecule involved in the vesicular uptake and storage of ACh. Later it was shown that in the developing neuromuscular junction, released ATP acted on P2X receptor ion channels as a genuine cotransmitter with ACh. Adenosine triphosphate was shown to be released from sympathetic nerves supplying the guinea-pig taenia coli in 1971. Soon after, the possibility was raised that ATP was coreleased with noradrenaline from sympathetic nerves to guinea-pig seminal vesicle, cat nictitating membrane and guinea-pig vas deferens. Sympathetic purinergic cotransmission has also been demonstrated in many blood vessels. Parasympathetic nerves supplying the urinary bladder use ACh and ATP as cotransmitters; ATP acts through P2X ionotropic receptors, whereas the slower component of the response is mediated by the metabotropic muscarinic receptor. Adenosine triphosphate and glutamate appear to be cotransmitters in primary afferent sensory neurons. Adenosine triphosphate, calcitonin gene-related peptide and substance P coexist in some sensory-motor nerves. A subpopulation of intramural enteric nerves provides non-adrenergic, non-cholinergic inhibitory innervation of gut smooth muscle. Three cotransmitters are involved, namely ATP, nitric oxide and vasoactive intestinal polypeptide. In recent years, studies have shown that ATP is released with ACh, noradrenaline, glutamate, gamma-aminobutyric acid, 5-hyroxytryptamine and dopamine in different subpopulations of neurons in the central nervous system.

Vascular endothelial cells have been identified as a source of substance P (SP) which may act in an autocrine/paracrine fashion to bring about nitric oxide (NO)-dependent vasodilatation and mitogen-induced cell division or immunologic and inflammatory responses. Whilst SP is localised in and released from endothelial cells, an endothelial mRNA expression of SP has not previously been shown. In the present study, mRNA expression of SP in human dermal microvascular endothelial cells is demonstrated using in situ hybridisation techniques with enhancement procedures. Incubation of microvascular endothelial cells with nerve growth factor (NGF) under conditions of increased shear stress increases the mRNA expression and release of SP Endothelin (ET) release is also enhanced. These changes are pertinent to circulatory, events that may occur in Raynaud's phenomenon in systemic sclerosis.

1. Application of ATP and alpha,beta-methylene ATP (alpha beta meATP) to voltage-clamped guinea-pig pelvic neurons produced three types of inward currents. A fast-desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly-desensitizing currents, and the remainder had biphasic responses. 2. Slowly-desensitizing responses were characterized pharmacologically. The response to alpha beta meATP 100 microM was 46+/-27% (range 0--100%) of that evoked by ATP 100 microM in the same cell. Cross-desensitization indicated the presence of alpha beta meATP-sensitive and -insensitive receptors. 3. The concentration-response curve for alpha beta meATP had an EC(50) of 55 microM, and a Hill coefficient of 0.99, while at the alpha beta meATP-insensitive receptor, ATP had an EC(50) of 73 microM, with a Hill coefficient of 1.78. 4. The response to alpha beta meATP was blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and Cibacron blue. However, the alpha beta meATP-insensitive receptor was inhibited by PPADS, but not by the other two antagonists. 5. 2'- (or 3'-) O-trinitrophenyl-ATP was 10 times more potent in inhibiting responses to alpha beta meATP than to ATP (at the alpha beta meATP-insensitive receptor). 6. Lowering extracellular pH potentiated responses to alpha beta meATP and ATP, while raising pH attenuated them. 7. Co-application of Zn(2+) (3--300 microM) inhibited the responses to alpha beta meATP and ATP, with IC(50) values of 286 and 60 microM, respectively. 8. In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X(2) homomers, at least three distinct P2X receptors are present in guinea-pig pelvic neurons, probably homomeric P2X(2), P2X(3) and heteromeric P2X(2/3) receptors. However, some of the novel pharmacological properties observed suggest that the guinea-pig P2X receptor subtypes may differ from their rat orthologues.

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5'-nucleotidases). The activity of soluble ATPase was progressively increased from 1.62+/-0.27 to 12.7+/-1.0 pmoles ml(-1) h(-1) after 60 min of stimulation by shear stress. The rate of [(3)H]-ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid, and remained insensitive to the classic inhibitors of ion-pumping and intracellular ATPases. Shear stress also increased the activity of 5'-nucleotidase in the medium from 2.0+/-0.5 to 27.2+/-2.8 pmoles ml(-1) h(-1). When shear stress was applied after removal of ecto-5'-nucleotidase by phosphatidylinositol-specific phospholipase C, the release of 5'-nucleotidase was drastically reduced. These results show that soluble ATPase and 5'-nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.

1. The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X(1-4) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5'-adenylic deaminase, and purified using reverse-phase chromatography. 3. Against ATP-responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)Ip4I (6.3)Ip3I (4.5). Ip5I (10-100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP effect. However, the Schild plot was non-linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non-surmountable antagonist. 4. Against ATP-responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6. 0)Ip5I (5.6)Ip3I (4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. 5. Diinosine polyphosphates failed to inhibit ATP-responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1 - 4 receptors. 6. Thus, Ip5I acted as a potent and relatively-selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high-affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (/=100 nM) concentrations but has more complex actions at higher (100 nM) concentrations.

1 The modulatory activity of extracellular H+ and Zn2+ was examined on ATP-responses at rat P2X1 (rP2X1) and rat P2X3 (rP2X3) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2 Superfused ATP (0.03-30 microM, at pH 7.5) evoked inward currents at rP2X1 receptors (EC50 value, 300+/-7 nM). ATP potency was reduced 2 fold at pH 6.5, and 6 fold at pH 5.5, without altering the maximum ATP effect. Alkaline conditions (pH 8.0) did not alter ATP activity. 3 Superfused ATP (0.01 - 300 microM, at pH 7. 5) evoked inward currents at rP2X3 receptors (EC50 value, 1.8+/-0.3 microM). ATP activity was affected only at pH 5.5, reducing agonist potency 15 fold without altering the maximum ATP effect. 4 Extracellular Zn2+ inhibited ATP-responses at rP2X1 receptors in a time-dependent manner, a 20 min pre-incubation being optimal (IC50 value, 1.0+/-0.2 microM). However, the Zn2+ effect was pH-independent, suggesting Zn2+- and H+-inhibition of ATP-responses occur through independent processes. 5 Extracellular Zn2+ weakly potentiated ATP-responses at rP2X3 receptors (EC50 value, 11+/-1 microM). The Zn2+ effect was dependent on pre-incubation time and, with 20 min pre-incubation periods, Zn2+ potentiated then inhibited ATP-responses in a concentration-dependent, but pH-independent, manner. 6 In summary, ATP activity at rP2X1 receptors was decreased by both extracellular H+ and Zn2+ and their effects were additive. ATP activity at rP2X3 receptors was less sensitive to H+-inhibition and, in contrast, was potentiated by Zn2+ in a pH-independent manner. These differential effects may help distinguish P2X1 and P2X3 receptors in whole tissues.

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 = ATPgammaS = 2-MeSATP = Ap4AUDP(pure). ADP, ATPgammaS, 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.

1. Purinergic and cholinergic components of parasympathetic neurotransmission and contractile responses to exogenous alpha,beta-methylene ATP, acetylcholine, substance K, substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and capsaicin have been investigated in the urinary bladder of hibernating hamsters (4 weeks), cold exposed (4 weeks) and age-matched controls. 2. Electrical field stimulation (EFS) evoked increased frequency-dependent contractions in the detrusor strips from hibernating hamsters compared with those obtained from cold-exposed and age-matched animals. Tetrodotoxin (10(-6) M) completely blocked the frequency-dependent contractions in all groups. 3. The purinergic component of the parasympathetic neurotransmission was not affected in hibernating and cold-exposed animals while the cholinergic component was increased with respect to age-matched animals. The neurogenic response to EFS, still present after incubation with atropine (10(-6) M) and suramin (10(-4) M), was attenuated by indomethacin (10(-6) M) and blocked by tetrodotoxin (10(-6) M). 4. Exogenous administration of alpha,beta-methylene ATP elicited a significantly reduced contraction in strips from hibernating and cold-exposed hamsters relative to age-matched animals. The contractile response to exogenous acetylcholine was greater in the detrusors from hibernating hamsters than in cold-exposed and age-matched animals. Substance K elicited reduced contractions in preparations from hibernating animals compared with cold-exposed and control animals. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, substance P and capsaicin did not elicit any relaxant or contractile response either at resting tone or in carbachol (5 x 10(-7) M)-precontracted tissues. 5. In summary, our findings indicate that 4 weeks of hibernation can significantly increase neurogenic responses in the hamster urinary bladder. This appears to be due to an increase in postjunctional responses to acetylcholine. In contrast, there was a decrease of the postjunctional responses to the parasympathetic cotransmitter ATP and also to the sensory-motor neurotransmitter substance K.

1. Sympathetic neurotransmission and noradrenaline content of the tail artery of Donryu rats fed for 2 months with a cholesterol-supplemented diet enriched with 4% cholesterol, 1% cholic acid, 0.5% thiouracil (CCT), were examined. 2. Total serum cholesterol level of CCT fed rats (7.05 +/- 1.77 mg ml(-1), n = 8) was significantly greater than lab-chow fed controls (2.58 +/- 0.32 mg ml(-1), n = 8). Low density lipoprotein level was also significantly increased in CCT-fed (1.79 +/- 0.26 mg ml(-1), n = 8) compared with control fed rats (1.35 +/- 0.25 mg ml(-1), n = 8) but plasma levels of triglyceride and high density lipoproteins did not differ significantly between the two groups. 3. Contractile responses of the arterial rings to transmural nerve stimulation (65 V, 0.1 ms, 4-64 Hz, 1 s), were markedly attenuated in the CCT fed animals compared with the controls. This reduction involved the noradrenergic rather than purinergic component of sympathetic transmission. 4. Vasoconstrictor responses to exogenous noradrenaline (0.01-300 microM) and adenosine 5'-triphosphate (0.3-1000 microM) were unaffected by CCT diet, indicating prejunctional alteration of sympathetic neurotransmission during CCT-induced hyperlipidaemia. 5. The noradrenaline content of the tail arteries of CCT fed animals (2.64 +/- 0.36 ng mg(-1), n = 6) was significantly lower than that of controls (3.82 +/- 0.32 ng mg(-1), n = 6). 6. These findings show that chronic treatment of Donryu rats with a cholesterol-supplemented diet led to altered levels of circulating lipid fractions accompanied by attenuated sympathetic noradrenergic neurotransmission and reduced noradrenaline content of the rat tail artery.

1. 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). 2. 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. 3. 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. 4. 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. 5. The potency order for agonists of P2X2 receptors was: ATP2-MeS-ATP ATPgammaSATPalphaSCTPBzATP, 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, ATPyS and ATP alpha S), without altering the potency order. 6. The potency order for antagonists at P2X2 receptors was: Reactive blue-2trinitrophenol-ATPor = Palatine fast blackor = Coomassie brilliant blueor = PPADSsuramin (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 microM, at pH 7.4; 78 +/- 5 nM, at pH 6.5; 30 +/-6 nM, at pH 5.5). 7 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.

1. The aim of this study was to characterize P2 receptors in the arterial vascular bed of human perfused placental cotyledons. Vasoconstrictor responses to bolus injections of purine and pyrimidine nucleotides were tested at basal tone, and vasodilator responses in preparations with tone raised by perfusion with prostaglandin F2alpha (PGF2alpha; 10-50 nM). 2. At basal tone, bolus injections of the P2X-selective agonist alpha,beta-methylene ATP (alpha,beta-meATP; 0.5-500 nmol) elicited dose-dependent vasoconstriction. ATP (0.005-5 micromol) also elicited dose-dependent vasoconstriction, but was less potent than alpha,beta-meATP. Vasoconstriction was also elicited by other nucleotides, but only at the highest dose tested (5 micromol): UTPCTP = ITP (n = 6). GTP and TTP did not cause vasoconstriction. 3. Constrictor responses to bolus injections of alpha,beta-meATP were resistant to desensitization and were not significantly affected when carried out in the presence of 1 microM alpha,beta-meATP added to the perfusate. However, responses to bolus injections of alpha,beta-meATP were partially blocked by perfusion with 10 microM alpha,beta-meATP. In contrast, responses to ATP and UTP were unaffected by 10 microM alpha,beta-meATP. The P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 and 100 microM) had no significant effect on vasoconstriction mediated by alpha,beta-meATP and ATP. 4. Removal of the endothelium had no significant effect on constrictor responses to alpha,beta-meATP, ATP and UTP. Inhibition of nitric oxide (NO) synthesis with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 microM) had no significant effect on vasoconstriction to ATP and alpha,beta-meATP. 5. In preparations with tone raised with PGF2alpha (10-50 nM) vasodilatation was elicited by nucleotides with the following order of potency: 2MeSATP = ADPATPUTPCTP = GTP = ITP = TTP. pD2 values were: 2MeSATP, 10.03+/-0.26 (n=7); ADP, 9.97+/-0.40 (n=5); ATP, 8.89+/-0.18 (n=7); UTP, 7.79+/-0.35 (n=7). Maximal responses to 2MeSATP and ADP were similar and were approximately 40% greater than maximal responses to ATP and UTP. 6. Vasodilator responses to nucleotides were abolished by L-NAME (100 microM) and by removal of the endothelium. 7. In conclusion, contractile responses mediated by alpha,beta-meATP and ATP in human placental smooth muscle are resistant to desensitization and insensitive to PPADS and, thus, show a dissimilar pharmacological profile to the classic smooth muscle P2X1 receptor. There may be two subtypes of smooth muscle P2 receptor based on differential antagonism of alpha,beta-meATP and ATP with alpha,beta-meATP. A smooth muscle P2 receptor mediates vasoconstriction to UTP, and may indicate a further subtype. Endothelium-dependent, NO-dependent, vasodilatation to 2MeSATP and ADP may be mediated by P2Y1 receptors, while endothelial P2Y2 receptors are likely to mediate NO-dependent relaxation to ATP and UTP.

1. The effects of NF023, the symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid), and its parent compound suramin were investigated on vasoconstrictor responses to alpha, beta-methylene ATP in rabbit isolated saphenous artery and vasodilator responses to ATP in noradrenaline-precontracted rabbit isolated thoracic aorta. 2. In rabbit isolated saphenous artery, alpha, beta-methylene ATP-induced vasoconstrictor responses via P2X-receptors were concentration-dependently and competitively antagonised by NF023 (30-300 microM; pA2 = 5.69 +/- 0.04). Suramin (100-1000 microM) also competitively blocked vasoconstrictor responses to alpha, beta-methylene ATP, albeit with lower potency (pA2 = 4.79 +/- 0.05). In contrast, NF023 (100 microM) did not significantly affect contractile responses to noradrenaline or histamine in the saphenous artery. 3. In noradrenaline-precontracted rabbit isolated thoracic aorta preparations, ATP (3-3000 microM) concentration-dependently induced relaxations via endothelium-dependent or smooth muscle P2Y-receptor subtypes. NF023 (30-300 microM) failed to block relaxant responses to ATP at endothelium-dependent P2Y-receptors, whereas suramin (100-1000 microM) did antagonise endothelium-dependent vasodilator responses to ATP. Neither NF023 (100 microM) nor suramin (300 microM) influenced vasorelaxant responses to ATP via endothelium-independent P2Y-receptors. 4. In conclusion, this study outlines the selectivity of NF023 as an effective P2X-receptor antagonist in rabbit isolated blood vessels without affecting endothelium-dependent or endothelium-independent P2Y-receptor subtypes, adrenoceptors or histamine receptors.

Stimulation of the vagal non-adrenergic inhibitory innervation caused the release of adenosine and inosine into vascular perfusates from the stomachs of guinea-pigs and toads.Stimulation of portions of Auerbach's plexus isolated from turkey gizzard caused the release of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP).ATP, added to solutions perfused through the toad stomach vasculature, was broken down to adenosine, inosine and adenine.Of a series of purine and pyrimidine derivatives tested for inhibitory activity on the guinea-pig isolated taenia coli, ATP and ADP were the most potent.ATP caused inhibition of twelve other gut preparations previously shown to contain non-adrenergic inhibitory nerves. The inhibitory action of ATP was not prevented by tetrodotoxin.Quinidine antagonized relaxations of the guinea-pig taenia coli caused by catecholamines or adrenergic nerve stimulation. Higher concentrations of quinidine antagonized relaxations caused by ATP or non-adrenergic inhibitory nerve stimulation.When tachyphylaxis to ATP had been produced in the rabbit ileum, there was a consistent depression of the responses to non-adrenergic inhibitory nerve stimulation but not of responses to adrenergic nerve stimulation.It is suggested that ATP or a related nucleotide is the transmitter substance released by the non-adrenergic inhibitory innervation of the gut.

1. This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca2+]i, in a population of rat striatal astrocytes loaded with the fluorescent Ca2+ probe Fura2, by means of fluorescence spectrometry. 2. ATP triggered a fast and transient elevation of [Ca2+]i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), as well as uridine-5'-triphosphate (UTP) resembled that of ATP, while alpha, beta-methylene-ATP (alpha, beta-meATP) and beta, gamma-methylene-ATP (beta, gamma-meATP) were totally ineffective. 3. Suramin (50 microM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (5 microM) significantly depressed the maximum response. 4. Extracellular Ca2+ did not contribute to the observed [Ca2+]i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni2+ (1 mM) or Mn2+ (1 mM) did not modify the nucleotide responses. 5. Furthermore, after preincubation with 10 microM thapsigargin, the nucleotide-evoked [Ca2+]i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved. 6. Both application of the G-protein blocker guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml-1) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 microM), significantly reduced the ATP-evoked [Ca2+]i rise. 7. In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca2+]i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.

1. The modulator effects of a series of neurotransmitters and related substances were tested on responses to adenosine 5'-triphosphate (ATP) at a recombinant P2x2 receptor expressed in defolliculated Xenopus oocytes. 2. Nicotine, 5'-hydroxytryptamine (5-HT), noradrenaline, adenosine, bradykinin and histamine (all 100 microM) potentiated the responses to ATP (3 microM). an effect found due to acidification of the bathing solution by these drugs. 3. Arachidonic acid, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP) (all 100 microM) and nerve growth factor (NGF; 50 ng ml-1) potentiated the responses to ATP (3 microM) through a largely or wholly pH-independent effect. 4. Small acidic and alkaline shifts, as little as 0.03 pH-units, enhanced or diminished the responses to ATP, respectively. A linear relationship existed between the degree of potentiation of the ATP-induced responses caused by nicotine, 5-HT, noradrenaline, adenosine, bradykinin and histamine and the potentiation of these responses induced by the addition of acid to the superfusate. 5. Since P2x receptors on sensory neurones include P2x2 subunits, the attendant acidosis and ATP-release associated with tissue injury may play a role in sensitizing sensory nerve fibres.

1. A brief challenge of rat astrocytes with either alpha, beta-methyleneATP (alpha, beta-meATP) or basic fibroblast growth factor (bFGF) resulted, three days later, in morphological differentiation of cells, as shown by marked elongation of astrocytic processes. The P2 receptor antagonist suramin prevented alpha, beta-meATP- but not bFGF-induced astrocytic elongation. Similar effects on astrocytic elongation were also observed with ATP and other P2 receptor agonists (beta, gamma meATP, ADP beta S, 2meSATP and, to a lesser extent, UTP). 2. Pertussis toxin completely abolished alpha, beta-meATP- but not bFGF-induced effects. No effects were exerted by alpha, beta-meATP on cyclic AMP production; similarly, neomycin had no effects on elogation of processes induced by the purine analogue, suggesting that adenylyl cyclase and phospholipase C are probably not involved in alpha, beta-meATP-induced effects (see also the accompanying paper by Centemeri et al., 1997). The tyrosine-kinase inhibitor genistein greatly reduced bFGF- but not alpha, beta-meATP-induced astrocytic elongation. 3. Challenge of cultures with alpha, beta-meATP rapidly and concentration-dependently increased [3H]-arachidonic acid (AA) release from cells, suggesting that activation of phospholipase A2 (PLA2) may be involved in the long-term functional effects evoked by purine analogues. Consistently, exogenously added AA markedly elongated astrocytic processes. Moreover, various PLA2 inhibitors (e.g. mepacrine and dexamethasone) prevented both the early alpha, beta-meATP-induced [3H]-AA release and/or the associated long-term morphological changes, without affecting the astrocytic elongation induced by bFGF. Finally, the protein kinase C (PKC) inhibitor H7 fully abolished alpha, beta-meATP- but not bFGF-induced effects. 4. Both alpha, beta-meATP and bFGF rapidly and transiently induced the nuclear accumulation of Fos and Jun. Both c-fos and c-jun induction by the purine analogue could be fully prevented by pretreatment with suramin. In contrast, the effects of bFGF were unaffected by this P2 receptor antagonist. 5. It was concluded that alpha, beta-meATP- and bFGF-morphological differentiation of astrocytes occurs via independent transductional pathways. For the purine analogue, signalling involves a Gi/G(o) protein-coupled P2Y-receptor which may be linked to activation of PLA2 (involvement of an arachidonate-sensitive PKC is speculated); for bFGF, a tyrosine kinase receptor is involved. Both pathways merge on some common intracellular target, as suggested by induction of primary response genes, which in turn may regulate late response genes mediating long-term phenotypic changes of astroglial cells. 6. These findings implicate P2 receptors as novel targets for the pharmacological regulation of reactive astrogliosis, which has intriguing implications in nervous system diseases characterized by degenerative events.

This pre-embedding electron-immunocytochemical study investigated the localisation of endothelial (type III) and neuronal (type I) isoforms of nitric oxide synthase, substance P, endothelin-1 and 5-hydroxytryptamine in the human middle cerebral artery taken up to 40 h postmortem. To ¿recover' from the anoxic period some of the vessels were incubated in oxygenated Krebs solution prior to the immunoprocedure. At this long postmortem time, immunoreactivity to type III and type I nitric oxide synthase, substance P, endothelin-1 and 5-hydroxytryptamine was found in a subpopulation of intact cells present in the vessel intima; immunoreactivity to type I nitric oxide synthase was also observed in a subpopulation of adventitial perivascular nerve fibres. Cultures of the cells from the intima of the postmortem vessels showed that the cells were proliferating and positive immunoreactivity to factor VII identified them as endothelial cells. The results therefore indicate that even after up to 40 h postmortem, endothelium of human middle cerebral artery is immunoreactive for a number of vasoactive agents and perivascular nerve fibres show nitric oxide synthase immunoreactivity.

Adenosine 5'-triphosphate (ATP) is a cotransmitter with noradrenaline (NA) in sympathetic nerves supplying the vas deferens and a number of blood vessels. ATP is responsible for the excitatory junctional potentials (EJPs) in response to single nerve impulses and the initial twitch responses of the smooth muscle, while NA produces the longer-lasting tonic contractions. The proportions of ATP to NA vary between different sympathetic nerves; they also change during development and in some pathological conditions, including hypertension. Prejunctional neuromodulation of release of the two cotransmitters appears to involve independent mechanisms and is frequency dependent; this raises the question of whether ATP and NA are stored in separate vesicles or whether there are subpopulations of sympathetic nerves with a predominance of ATP or NA. ATP and NA have synergistic postjunctional actions, whether excitatory (as in the vas deferens and most blood vessels) or inhibitory (as in rabbit coronary vessels). It is suggested that use of the term 'adrenergic nerves' as a synonym for sympathetic nerves is no longer appropriate, although 'adrenergic transmission' or 'purinergic transmission' are still useful terms.

Endothelin-1 is thought to play a role in the regulation of gastrointestinal function including its secretory role. This study investigated the localisation of endothelin-1 in the mucosa of the large intestine of normoxic and chronically hypoxic rats. Chronic hypoxia was produced by maintaining the animals in a hypoxic chamber. After animal perfusion/fixation, colonic segments from normoxic and hypoxic rats were investigated by the postembedding immunogold labelling procedure. In the microvasculature from normoxic (control) animals immunolabelling visualised as single particles was low and confined to the cytoplasm of endothelial cells. In the epithelium, immunolabelling was found mainly at the basal and apical regions of the cells. Chronic hypoxia for 10 d greatly enhanced the level of immunolabelling both in endothelial and epithelial cells of the mucosa. In endothelial cells clusters of gold particles were found in the cytoplasmic matrix and the subendothelium. Epithelial cells showed intense labelling, located mainly in the luminal/apical region within vacuole-like spaces and in the brush-border. In conclusion, hypoxia induces increase in immunoreactivity to endothelin-1 in gut endothelial and especially epithelial cells in the colonic mucosa.

Numerous NADPH-diaphorase positive neurons were localised in the pancreatic ganglia cultured from newborn guinea pigs. Most of these ganglia were found in the head region of the pancreas. A majority of the labelled neurons occurred in groups, although solitary neurons were also present. NADPH-diaphorase labelling was localised mainly in the cell cytoplasm and its processes, but not within the nucleus. There were more nitric oxide synthase (NOS)-immunoreactive neurons in culture as compared with those positive for NADPH-diaphorase. NADPH-diaphorase activity has been colocalised with NOS and vasoactive intestinal polypeptide (VIP) in a subset of neurons in culture. Colocalisation of NADPH-diaphorase with NOS and VIP in cultured neurons was not restricted to the cell body but also to the proximal and distal processes. Some of these labelled processes made contacts with exocrine acinar and endocrine cells. It is concluded that neuronal nitric oxide (NO) may be influencing the secretory roles of the pancreas, thereby participating in homeostasis.

Cells in dissociated culture preparations of the superior cervical ganglion of the adult rat were examined for the presence of insulin receptors. This was assessed immunocytochemically by the demonstration of binding by a mouse monoclonal anti-insulin receptor antibody. A large subpopulation (> or = 90%) of neuronal cell bodies and associated neurites exhibited positive immunostaining. The apparent absence of staining over nuclear regions suggested that the majority of neuronal receptors had an intracytoplasmic localisation. In contrast, a subpopulation of fibroblasts showed punctate immunostaining, which appeared to be confined to the cell surface. Glial (satellite) cells did not appear to be immunostained. The possible effects of insulin on neurons in the peripheral nervous system are discussed.

Sympathetic ganglia from newborn rats and guinea-pigs were grown in modified Rose chambers and examined with scanning electron microscopy after 5–7 days. The cell types seen were macrophages, neurons, glial cells and connective tissue cells. They presented a wide range of surface morphologies and 3-dimensional configurations, from spheroid with an irregular surface to flattened with a smooth surface. The arrangement of the nerve fibres and cells in the outgrowth was essentially 2-layered with connective tissue cells nearest the substrate and nerve fibres, glial cells and macrophages lying over them. The relationships of sympathetic nerve fibres to the different cell types were also investigated. In all cases nerve fibres closely followed the cellular surface contours although the nature of the relationships varied. Fine finger-like cytoplasmic projections were sometimes seen from connective tissue cells and macrophages. The possible role of these structures in adhesion and motility is discussed.

A histological study of the structure of the urinary bladder of the ringtail possum has been made. The innervation of the bladder has been studied in vitro, using the technique of analytical pharmacology. The bladder has well-defined inner longitudinal and outer circular muscle layers. Nerves supplying the bladder are found both in the pelvic nerves and in the vesical nerves which run with the vascular supply of the bladder fundus. Ganglia have been demonstrated along the trunks of the vesical nerves and also aggregated at the bladder neck. The response of the bladder to stimulation of either nerve supply in situ or in vitro is always a simultaneous contraction of both longitudinal and circular muscles. Inhibitory responses to nerve stimulation have never been observed. The optimal frequency for stimulation of these nerves at 30° C. is 50 pulses/sec. The bladder is contracted by ACh and 5-hydroxytryptamine, but is relaxed by adrenaline, noradrenaline and histamine. The response to nerve stimulation is reduced by atropine and potentiated by eserine. Adrenergic blocking agents do not affect the nerve-mediated response unless they also affect the response to applied ACh in a similar manner. Ganglionic blocking agents, in concentrations which do not reduce the response to ACh, cause up to a 40% reduction of the response to stimulation of either the vesical or the pelvic nerves. It is concluded that the nerve fibres supplying the possum bladder are cholinergic, perhaps 40 % of them being stimulated pre-ganglionically. The evolutionary significance of these observations is discussed. Some points of pharmacological interest have been discussed in relation to drug actions on placental mammal preparations.

The distribution of galanin was investigated at the light microscopic level in the human distal colon using immunocytochemical techniques. Galanin-like immunoreactivity was seen in nerve cell bodies in ganglia of the myenteric and submucous plexuses and in nerve fibres innervating all the muscle layers of the colon, the lamina propria, and epithelial cells at the base of crypts or lining the colonic lumen. Immunoreactivity was more intense in the circular muscle than in the longitudinal muscle or the muscularis mucosae. Immunoreactive nerve cell bodies were much scarcer in the myenteric than in the submucous plexus. Within subdivisions of the submucous plexus, galanin-like immunoreactivity was heterogeneously distributed. In Henle's plexus and Meissner's plexus 82-83% of galanin-positive cell bodies were up to 360 microns 2 in profile-area, but in the intermediate plexus nearly all (99.8%) were below 360 microns 2. The frequency-distribution of cell body area of galanin-containing nerve cell bodies was similar for Henle's plexus and Meissner's plexus but these two plexuses contain different size-populations of neurons when stained for NADH-diaphorase activity. Galanin-like immunoreactive nerve fibres were found in the plexus entericus (submucosus) extremus, and this is the first report of neuropeptide in this location.

The distribution of 5-hydroxytryptamine-like immunoreactivity was studied in whole-mount preparations of intestine from human fetuses. Immunoreactive nerve cell bodies were located in the myenteric plexus and were occasionally found in the submucous plexus; they were often seen to have long processes. Varicose fibers were found in the ganglia and internodal strands of the myenteric and submucous plexuses, in the deep muscular plexus of the circular muscle, and in the walls of some small mesenteric blood vessels immediately outside the intestine. This study provides evidence for the presence of serotoninergic nerves in the human intestine.

Adrenergic, cholinergic, and serotoninergic nerves were studied in the myenteric plexus of ileum and colon from streptozotocin-treated rats, an animal model of juvenile-onset diabetes. In view of clinical reports implicating diabetic autonomic neuropathy as the cause of gastrointestinal dysfunction in diabetes mellitus, neurochemical and histochemical techniques were used to study changes in the innervation of the gut. In the myenteric plexus of the ileum from diabetic animals, adrenergic nerves displayed signs of degeneration and the brightness of fluorescence in serotoninlike immunoreactive nerves was lower. Cholinergic nerves, however, did not display any signs of reduction in the ileum, and both choline acetyltransferase and acetylcholinesterase activities per centimeter were increased. In contrast, in the proximal colon 8 wk after induction of diabetes, neurochemical assays revealed significant increases in noradrenaline and serotonin levels as well as choline acetyltransferase activity, although no obvious changes in the pattern of innervation could be detected histochemically. The results indicate that changes do occur in the innervation of the gut of the streptozotocin-diabetic model shortly after the induction of diabetes, although they differ significantly in the ileum and colon; these may be of relevance to the types of gastrointestinal dysfunction displayed in human diabetes.

Fluorescent histochemical localization of monoamines in whole mounts of the bladder of the toad ( Bufo marinus) revealed bundles of fluorescent nerves containing predominantly adrenaline. These formed a wide meshed net work about arteries but not veins. A small proportion of muscle bundles was supplied by fine varicose fluorescent nerves. Loading with adrenaline or noradrenaline increased the intensity of the fluorescence in the nerves, but produced no appreciable increase in the number of muscle bundles supplied by fluorescent nerves. Large, brilliantly fluorescent cells containing a primary monoamine were located along nerve bundles. Chronic reserpine treatment abolished the fluorescence in nerves but not in these cells. Ganglion cells showed no specific fluorescence, nor did fluorescent terminals impinge on them, but many ganglion cells contained autofluorescent granules. The significance of the presence and localization of catecholamines in the toad bladder is discussed in relation to the pharmacology of the organ and to histochemical investigations of the innervation in other vertebrate bladders.