Rank Order Of Agonist Potency Research Articles

Functional characterization of the adenosine receptor mediating inhibition of peristalsis in the rat jejunum.

1. The non-selective adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), is a potent inhibitor of morphine withdrawal diarrhoea in rats. More recently we found that NECA exerts its antidiarrhoeal effect by inhibiting secretion in both the jejunum and ileum and also by inhibiting peristalsis in the ileum. The specific aim of this study was to characterize the receptor in the rat jejunum mediating inhibition of peristalsis via functional studies using a range of metabolically stable adenosine analogues based on the pharmacological criteria of relative agonist and antagonist potencies. 2. Peristalsis in the rat isolated jejunum was achieved by raising the pressure to between 7-11 cmH2O for 3 min followed by a 3 min rest period (pressure at zero). The mean rate of peristalsis during inflation was 7.3 +/- 0.1 peristaltic waves per 3 min and this rate remained consistent for up to 30 min, in 5 separate tissues. The inhibitory effects of the adenosine analogues were quantified by expressing their effects as a % reduction in the mean number of peristaltic contractions derived from the control tissues. 3. The rank order of agonist potency to reduce the rate of peristalsis was: N6-cyclopentyladenosine (CPA) > NECA > R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) > chloroadenosine (2-CADO) > S-PIA > 2-phenylaminoadenosine (CV-1808). This order complies well with the rank order of agonist potency that represents the activation of the A1 receptor subtype (CPA > R-PIA = CHA = > NECA > 2-CADO > S-PIA > CV-1808). 4. The selective A1 adenosine antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and the nonselective adenosine antagonist 8-phenyltheophylline (8-PT) at their respective concentrations of 10 nM and 2 microM caused parallel rightward shifts in the concentration-response curve to the non-selective Al/A2 agonist NECA. DPCPX was significantly more potent at inhibiting NECA than 8-PT as revealed by their apparent pA2 values; DPCPX (9.5) and 8-PT (7.26). The high affinity of DPCPX relative to that of 8-PT suggests the presence of an Al and not an A2B receptor. In addition, the high affinity of DPCPX(pA2:9.37) against the selective Al agonist CPA, further confirms the presence of the Al receptor subtype.5. In this study we found that the Al adenosine receptor is involved in regulating in vitro peristalsis which is different from the adenosine receptor regulating inhibition of secretion (A2B) in the same region of intestine of the same species. We propose that A2B adenosine agonists could be of clinical value in the management of diarrhoea that is due to microbiological organisms where antimotility effects are not desired.

ATP-induced increase in intracellular Ca 2+ concentration in the cultured marginal cell of the stria vascularis of guinea-pigs

The primary culture of marginal cells from the explant of the stria vascularis of guinea-pigs was established and confirmed by morphological, immunohistochemical and physiological characters of the cultivated cells. The positive stain of cytokeratin 18 without vimentin and desmin expressions indicated the epithelial origin of the cultured polygonal cells. Electron microscopical findings of cultured cells resembled the morphological characteristics of marginal cells. The addition of 100 μM ouabain to the cultured cells significantly increased the intracellular Na + concentration, providing the evidence for the presence of Na + pump. Using the cultured marginal cells identified by the above-mentioned findings, the effects of neurotransmitters and hormones in regulating intracellular Ca 2+ concentration ([Ca 2+] i) were investigated. The addition of 10 −4 M ATP caused an increase in [Ca 2+] i, which was independent of the presence of extracellular Ca 2+. The rank order of agonist potency was ATP>α,β-methylene ATP>ADP. Adenosine, however, evoked no response. Cultured marginal cells may possessP 2-purinergic receptors which mobilize Ca 2+ from the intracellular Ca 2+ pool. [Marginal cell, Cell culture, Na + K + ATPase, Intracellular Ca 2+ concentration, ATP]

Differences in agonist potency ratios at human m1 muscarinic receptors expressed in A9L and CHO cells

A9L mouse fibroblast and Chinese hamster ovary (CHO) cells appear to differ in their complement of guanine-nucleotide binding proteins (G proteins) and/or isoform of effectors that lead to inositol-monophosphate formation. The influence of these cellular components on receptor activation was examined by comparing agonist-induced inositol monophosphate formation via human muscarinic ml receptors expressed in the two cell lines. The rank order of agonist potencies of five full agonists differed in the two cell lines. In addition, differences in agonist potency ratios for two of the five agonists (carbachol and methacholine) suggest that the agonists differ in their activation of ml receptors and this is reflected in differences in G protein coupling The results provide biological evidence that muscarinic agonists differentially activate ml receptors and that, at least for the systems examined in this study, receptor-effector coupling in a given system may depend on the structure of the agonist.

A study on P2x purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens

1. We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine-5'-triphosphate (ATP), as well as a number of its structural analogues as agonists at P2X purinoceptors in the rat vas deferens in vitro. 2. Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential-70 mV) with fast flow concentration-clamp applications of agonists in single isolated smooth muscle cells. ATP, 2-methylthio adenosine-5'-triphosphate (2-MeSATP) and alpha,beta methylene adenosine-5'-triphosphate (alpha,beta-meATP) all evoked inward currents over a similar concentration range (0.3-10 microM), being approximately equipotent with similar concentrations for threshold effects (0.3 microM). ADP (10 microM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 microM). 3. alpha,beta-meATP was the most potent agonist in producing concentrations of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 microM). However, ATP and 2-MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration-effect curves were shallow with smaller maximal responses than could be achieved with alpha,beta-meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was alpha,beta-meATP >> ATP = 2-MeSATP. 4. Measurement of inorganic phosphate (iP), as a marker of purine nucleotide metabolism in the vasdeferens whole tissue preparation, indicated that ATP and 2-MeSATP were rapidly metabolized,whereas alpha,beta-meATP was stable for up to 2 h. Removal of divalent cations prevented breakdown of ATP and 2-MeSATP, suggesting that metabolism involved a Ca2+/Mg2+-dependent enzyme.5. It appears that in isolated preparations of rat vas deferens, the low potency of ATP and 2-MeSATP can be explained by rapid agonist breakdown by ectonucleotidases. However, this is not the case in the single cell studies where the use of rapid concentration-clamp applications revealed the true potency of the agonists. Under such conditions the three agonists were all equal in potency indicating that the rank order of agonist potencies of alpha,beta-meATP>> ATP = 2-MeSATP is not in fact characteristic of smooth muscle P2x-purinoceptors as commonly believed.

Bradykinin receptors in mouse and rat isolated superior cervical ganglia.

1. The ability of bradykinin and its analogues to depolarize rat and mouse superior cervical ganglia was studied by use of in vitro grease-gap recording techniques, and the ability of antagonists selective for bradykinin receptor subtypes to block their effects was examined. 2. Bradykinin (3 microM) depolarized ganglia from both species, although the magnitude of the maximal response was less in mouse (15 +/- 5%, n = 7) than rat tissue (33 +/- 6%, n = 7), relative to muscarine (1 microM). 3. Interleukin 1 beta (30 u ml-1 for 18 h at 37 degrees C) increased the depolarization caused by bradykinin (3 microM) in mouse ganglia from 15% to 54% (P < 0.001, n = 12). Responses to the B1 receptor agonist, [des-Arg10]-kallidin (3 microM) were similarly potentiated but this was only detected after inhibition of peptidase activity with 10 microM captopril (4% to 35%, n = 5). 4. In ganglia from both species the rank order of agonist potency was bradykinin = [Lys0]-bradykinin >> [des-Arg10]-kallidin. However, like responses to [des-Arg10]-kallidin in mouse tissue, both the potency of bradykinin and the maximal depolarization achieved (EC50 = 912 nM; 80%, n = 11) was enhanced following inhibition of angiotensin converting enzyme with 10 microM captopril (EC50 = 50 nM; 135%, n = 4). 5. Responses to bradykinin were selectively antagonized by the B2 receptor antagonist, Hoe 140 but not by the B1 antagonist, [Leu8]-bradykinin1-8. From Schild analysis the pA2 value for Hoe 140 in mouse tissue was 9.65, although the slope of the regression line was significantly greater than unity, indicating non-competitive kinetics (slope = 1.88 +/- 0.18, n = 9). The depolarization caused by [Lys0]-bradykinin was also antagonized by Hoe 140 (3 nM).6. Thus the predominant bradykinin receptor in mouse superior cervical ganglia is compatible with a B2 subtype. Furthermore the depolarizations caused by B1 and B2 agonists in this tissue can be increased following exposure to interleukin l beta, and by blocking peptide degradation with captopril.

Binding of [ 35S]adenosine 5′- O-(2-thiodiphosphate) to endothelial cells in culture

We have investigated the binding of [ 35S]adenosine 5′- O-(2-thiodiphosphate) ([ 35S]ADPβS) to intact cultured bovine aortic endothelial cells which have been previously shown to co-express P 2y and P 2u purinoceptors and to bovine adrenal medulla endothelial cells which solely possess P 2u purinoceptors. ADPβS has been shown to stimulate phospholipase C activity in these cells via the P 2y purinoceptor and does not interact with the P 2u purinoceptor. We describe a simple equilibrium binding procedure designed for the study of low affinity agonists and compare these results with those obtained by separation of bound and free by filtration. Saturation analysis of equilibrium binding data revealed two sites for ADPβS binding; one with K D = 3.3 × 10 −8, M, B max = 32 pmol/mg protein; and the other with K D = 4.3 × 10 −6 and B max = 2155 pmol/mg protein. Use of filtration did not significantly alter the K D of either of these sites, nor the B max of the high affinity site, but reduced the B max of the low affinity site by more than 95%. The rank order of agonist potency for competing for [ 35S]ADPβS binding indicated that most of this was to non-P 2y purinoceptor sites as β,γ-methylene ATP, a P 2x purinoceptor agonist, was more potent than 2-methylthio ATP, a P 2y purinoceptor agonist. Binding was also carried out in the presence of β,γ-methylene ATP, in an attempt to reduce non-P 2y purinoceptor binding and produced similar results. Specific [ 35S]ADPβS binding sites were also found in bovine adrenal medulla endothelial cells which do not possess P 2y purinoceptors. These results indicate that [ 35S]ADPβS was able to bind to endothelial cells from different parts of the vasculature but that the ligand can only be considered suitable for investigation of P 2y purinoceptors on mammalian cells when specific conditions are designed to reduce the large amount of non-receptor binding.

Site-directed Mutagenesis of P2U Purinoceptors: POSITIVELY CHARGED AMINO ACIDS IN TRANSMEMBRANE HELICES 6 AND 7 AFFECT AGONIST POTENCY AND SPECIFICITY

Two subtypes of G protein-coupled receptors for nucleotides (P2U and P2Y purinoreceptors) contain several conserved positively charged amino acids in the third, sixth, and seventh putative transmembrane helices (TMHs). Since the fully ionized form of nucleotides has been shown to be an activating ligand for both P2U and P2Y purinoceptors (P2UR and P2YR), we postulated that some of these positively charged amino acids are involved in binding of the negatively charged phosphate groups of nucleotides. To investigate the role of the conserved positively charged amino acids in purinoceptor function, a series of mutant P2UR cDNAs were constructed so that lysine 107 and arginine 110 in TMH 3, histidine 262 and arginine 265 in TMH 6, and arginine 292 in TMH 7 were changed to the neutral amino acid leucine or isoleucine. The mutated P2UR cDNAs were stably expressed in 1321N1 astrocytoma cells and receptor activity was monitored by quantitating changes in the concentration of intracellular Ca2+ upon stimulation with full (ATP, UTP) or partial (ADP, UDP) P2UR agonists. Neutralization of His262, Arg265, or Arg292 caused a 100-850-fold decrease in the potency of ATP and UTP relative to the unmutated P2UR and rendered ADP and UDP ineffective. In contrast, neutralization of Lys107 or Arg110 did not alter the agonist potency or specificity of the P2UR. Neutralization of Lys289 in the P2UR, which is expressed as a glutamine residue in the P2Y subtype, did not alter receptor activity; however, a conservative change from lysine to arginine at this position altered the rank order of agonist potency so that ADP and UDP were approximately 100-fold more potent than ATP and UTP. A three-dimensional model of the P2UR indicates the feasibility of His262, Arg265, and Arg292 interactions with the phosphate groups of nucleotides.

Evidence for two different imidazoline sites on pancreatic B cells and vascular bed in rat

The relative potencies of imidazoline compounds to induce insulin secretion and vascular resistance were compared in the isolated perfused rat pancreas. On insulin secretion, only the two imidazolines, antazoline and efaroxan, induced a concentration-dependent response, antazoline being 10 times more potent than efaroxan. In contrast, idazoxan, a blocker of imidazoline I 1 sites, at concentrations up to 30 μM, antagonized the insulin response to 10 μM efaroxan (IC 50 ≅ 14 ± 2 μM) without affecting that to 3 μM tolbutamide. On pancreatic vessels, not only antazoline and efaroxan but also idazoxan induced a concentration-dependent vasoconstriction; the rank order of agonist potency was antazoline > efaroxan > idazoxan. In addition, cimetidine, an imidazole known to bind imidazoline I 1 sites, ineffective per se, partially reversed the insulin stimulatory effect of efaroxan without affecting its vasoconstrictor effect. This study demonstrates that the insulin secretory and vasoconstrictor actions of imidazolines involve different imidazoline sites in rat pancreas. The results provide evidence for an I 1 type mediating insulin secretion on B cells and an I 2 type mediating vasoconstriction in vessels.

Molecular cloning, functional expression and pharmacological characterization of the human metabotropic glutamate receptor type 4

A cDNA encoding the human metabotropic glutamate receptor type 4 (hmGluR4) was isolated from human brain cDNA libraries by cross-hybridization with rat mGluR4 probes. The deduced amino acid sequence of human mGluR4 consists of 912 residues and shows a sequence identity of 96% to the amino acid sequence of rat mGluR4. Northern blot analyses indicate that hmGluR4 is strongly expressed in the cerebellum of the adult human brain but also at low levels in hippocampus, hypothalamus and thalamus. Stimulation of hmGluR4 with L-2-amino-4-phosphonobutyrate ( l-AP4), l-serine- O-phosphate ( l-SOP), l-glutamate or (1 S,3 R)-1-aminocyclo-pentane-1,3-dicarboxylic acid ((1 S,3 R)-ACPD) in stably transfected Chinese hamster ovary (CHO) cells depressed forskolin-induced cAMP accumulation, whereas quisqualate (0.5 mM) was ineffective. The rank order of agonist potencies is: l-AP4> l-SOP> l-glutamate > (1S,3R)-ACPD> > quisqualate. ( A, S)- α-methyl-4-carboxyphenylglycine (1 mM), a reported antagonist at some mGluR subtypes, did not reduce the depression of forskolin-induced cAMP accumulation by L-AP4.

Functional characterization of the adenosine receptor mediating inhibition of intestinal secretion

1. Previous studies have shown that the mixed A1/A2 adenosine agonist 5'-N-ethylcarboxamido-adenosine (NECA) inhibits intestinal fluid secretion which is thought to contribute to its antidiarrhoeal effect in the rat. The aim of this study was to characterize the adenosine receptor mediating this antisecretory effect via functional studies using a range of selective agonists and antagonists and by applying the pharmacological criteria of relative agonist and antagonist potencies. 2. Adenosine agonists and antagonists were administered i.v. to anaesthetized rats. Intestinal secretion was then stimulated by i.a. infusion of vasoactive intestinal peptide (VIP, 0.8 microgram min-1) and the net fluid transport across the wall of the jejunum was measured by a recirculation technique. 3. The rank order of agonist potency to reduce the response to VIP was: NECA > N6-cyclopentyladenosine (CPA) > R-N6-(2-phenylisopropyladenosine) (R-PIA) > S-PIA > chloroadenosine (2-CADO) > 2-phenylaminoadenosine (CV-1808). This order best complies with the rank order of agonist potency that represents activation of the recently described A2B receptor: NECA > 2-CADO > R-PIA = CHA > S-PIA > = CV-1808 > = CGS-21680. The most potent agonists (NECA, CPA and RPIA) had ED50 values in the low microgram range. 4. The anitsecretory action of NECA (submaximal dose of 40 micrograms kg-1) was antagonized equally (approximately 50%) by the selective adenosine antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 mg kg-1) and 8-phenyltheophylline (8-PT, 0.1 mg kg-1). This equipotent activity indicates the presence of an A2 and not an A1 receptor. 5. It is suggested that adenosine A2B receptor agonists could be evaluated for potential use as antidiarrhoeal drugs.

5-Hydroxytryptamine 3 receptor antagonists inhibit enterotoxin B-induced emesis in the ferret

We report on the pharmacological profile of the 5-HT receptor which induces contraction of the bovine isolated cerebral arteries. Several 5-HT receptor agonists were tested for their ability to induce vasoconstriction in bovine pial arteries and their potencies were compared to that of 5-HT. The rank order of agonist potency can be summarized as 5-carboxamidotryptamine (5-CT) = RU 24969 ≧ 5-HT > sumatriptan > α-methyl-5-HT > methysergide > 2-methyl-5-HT > ((±)-2- dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene (8-OH-DPAT). Only methysergide induced a contraction which was smaller than that elicited by 5-HT. Antagonists with selective affinity at 5-HT1A/1B (propranolol), 5-HT1C (mesulergine), 5-HT2 (ketanserin, mianserin) and 5-HT3 (MDL 72222) sites were inactive to block the 5-HT-induced contraction. In contrast, the 5-HT1/5-HT2 receptor antagonists methiothepin and metergoline inhibited the 5-HT-induced response with relatively high affinity (pA2 = 8.16 ± 0.26 and 6.73 ± 0.05, respectively). Overall, this pharmacological profile indicated clearly that a 5-HT1 receptor, must closely related to the 5-HT1D subtype, is responsible for the 5-HT-induced contraction of bovine cerebral arteries. Correlation analysis of the potencies of a series of 5-HT receptor agonists and antagonists in bovine and human cerebrovascular preparations showed a highly significant positive correlation (r=0.94, P=0.0051). Analyses of the correlation between the agonist and antagonist potencies at bovine cerebrovascular receptors and their published affinities at the cloned human 5-HT1Dα and 5-HT1Dβ (or human 5-HT1B) receptors showed a highly significant correlstion only with the 5-HT1Dβ (r=0.82; P=0.006) subtype. We conclude that cerebral vasoconstriction in bovine cerebral arteries is mediated by a receptor homologous to the human cerebrovascular 5-HT1D receptor and that bovine pial arteries appear to be the best available pharmacological model for the human cerebrovascular 5-HT1D receptor. Further, the results suggest that bovine cerebrovascular 5-HT1D receptors resemble the cloned human 5-HT1Dβ (or human 5-HT1B) receptor subtype.

D-1 dopamine receptors mediate dopamine-induced pancreatic exocrine secretion in anesthetized dogs.

Characterization of dopamine (DA) receptor subtypes was examined on the canine exocrine pancreas using selective DA receptor agonists and antagonists in anesthetized dogs. Each drug was injected i.a. in a single bolus fashion. Graded doses of DA (0.01-3 mumol) produced dose-dependent increases in the secretory rate of pancreatic juice, with a maximum effect at approximately 1 mumol. SCH23390 (3-30 nmol), a selective D-1 DA receptor antagonist, caused a progressive parallel shift to the right in the dose-response curve for DA-induced pancreatic secretion without changes in the maximal response. However, domperidone (3 mumol), a selective D-2 DA receptor antagonist, did not antagonize the DA-induced pancreatic exocrine secretion. A Schild analysis of the data indicates that the inhibitory constant value for SCH23390 to inhibit DA-stimulated secretion was 6.9 nmol. In addition, the stimulatory effects of SKF38393 (0.1-10 mumol) and YM435 (0.3-30 nmol), selective D-1 DA receptor agonists, and LY171555 (1-10 mumol), a selective D-2 DA receptor agonist, on pancreatic secretion were demonstrated. The rank order of agonist potency was YM435 > DA > SKF38393 >> LY171555. These results suggest that DA-induced pancreatic exocrine secretion is mediated by activation of D-1 DA receptors.

Cloning and characterization of a dog 5-HT 1Dβ serotonin gene

We report on the pharmacological profile of the 5-HT receptor which induces contraction of the bovine isolated cerebral arteries. Several 5-HT receptor agonists were tested for their ability to induce vasoconstriction in bovine pial arteries and their potencies were compared to that of 5-HT. The rank order of agonist potency can be summarized as 5-carboxamidotryptamine (5-CT) = RU 24969 ≧ 5-HT > sumatriptan > α-methyl-5-HT > methysergide > 2-methyl-5-HT > ((±)-2- dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene (8-OH-DPAT). Only methysergide induced a contraction which was smaller than that elicited by 5-HT. Antagonists with selective affinity at 5-HT1A/1B (propranolol), 5-HT1C (mesulergine), 5-HT2 (ketanserin, mianserin) and 5-HT3 (MDL 72222) sites were inactive to block the 5-HT-induced contraction. In contrast, the 5-HT1/5-HT2 receptor antagonists methiothepin and metergoline inhibited the 5-HT-induced response with relatively high affinity (pA2 = 8.16 ± 0.26 and 6.73 ± 0.05, respectively). Overall, this pharmacological profile indicated clearly that a 5-HT1 receptor, must closely related to the 5-HT1D subtype, is responsible for the 5-HT-induced contraction of bovine cerebral arteries. Correlation analysis of the potencies of a series of 5-HT receptor agonists and antagonists in bovine and human cerebrovascular preparations showed a highly significant positive correlation (r=0.94, P=0.0051). Analyses of the correlation between the agonist and antagonist potencies at bovine cerebrovascular receptors and their published affinities at the cloned human 5-HT1Dα and 5-HT1Dβ (or human 5-HT1B) receptors showed a highly significant correlstion only with the 5-HT1Dβ (r=0.82; P=0.006) subtype. We conclude that cerebral vasoconstriction in bovine cerebral arteries is mediated by a receptor homologous to the human cerebrovascular 5-HT1D receptor and that bovine pial arteries appear to be the best available pharmacological model for the human cerebrovascular 5-HT1D receptor. Further, the results suggest that bovine cerebrovascular 5-HT1D receptors resemble the cloned human 5-HT1Dβ (or human 5-HT1B) receptor subtype.

Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP

1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells.

Characterization of the prejunctional adenosine receptors in the rat anococcygeus muscle.

Adenosine receptor agonists inhibited electrically-evoked contractions of the rat isolated anococcygeus muscle. The compounds tested were: N6-cyclopentyladenosine (CPA), N((S, trans)-2-hydroxycyclopentyl)adenosine (GR79236), the R- and S-isomers of phenylisopropyladenosine (PIA), 5'-N-ethylcarboxamidoadenosine (NECA), ((2-(4-(2-carboxyethyl)phenyl)ethyl)amino)-N-ethylcarbox-amidoa denosine (CGS 21680) and N-((2-methylphenyl)methyl)adenosine (metrifudil). The rank order of agonist potency was: CPA = R-PIA = GR79236 = NECA >> S-PIA > metrifudil > CGS 21680, which is consistent with an effect mediated by adenosine A1 receptors. A similar rank order of potency was obtained for inhibition of electrically-evoked contractions of the guinea-pig ileum. However, there may be a lower receptor reserve in rat anococcygeus compared with the guinea-pig ileum, since higher concentrations of agonists were necessary to produce effects in the anococcygeus than in the guinea-pig ileum and S-PIA behaved as a partial agonist. The effect of NECA was antagonized in rat anococcygeus and guinea-pig ileum by the mixed A1/A2 receptor antagonist, 8-phenyltheophylline (pA2 values of 6.8 and 6.9, respectively). The selective A1-receptor antagonist, 8-cyclopentyl-1,3- dipropylxanthine (DPCPX), also blocked the inhibitory response to NECA in both tissues. Here, however, the pA2 values (9.6 and 8.6, respectively) were slightly but significantly different. These values confirm that the prejunctional adenosine receptors of the rat anococcygeus are of the A1 type, and suggest that they are similar but not necessarily identical to those of the guinea-pig ileum. The differing potencies of DPCPX as an antagonist of NECA between the preparations may reflect a tissue-dependent variation in sensitivity to this antagonist.

Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification

1. By use of a 'grease-gap' technique, the depolarizing effects of adenosine 5'-triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg(2+)-free (+ 1 x 10(-3) M ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2. In normal PSS, ATP produced concentration-dependent depolarization responses but the concentration-effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 x 10(-5) M and at the highest concentration tested (1 x 10(-3) M) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5-hydroxytryptamine (5-HT, 1 x 10(-5) M). 3. In Ca2+/Mg(2+)-free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1-3 x 10(-7) M and the maximal response to ATP amounted to 526% of the response to 5-HT (1 x 10(-5) M) in normal PSS. The concentration-effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 x 10(-6) M. 4. In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either alpha, beta-methylene ATP or 5-HT. However, the maximum responses to both alpha, beta-methylene ATP and 5-HT were significantly increased in Ca2+/Mg(2+)-free PSS. 5. The depolarizing effects of several analogues of ATP were determined in Ca2+/Mg2+-free PSS.ATP-gamma-S and 2-methylthioATP were of similar potency to ATP (respective equi-effective molar ratios(EMRs) of 1.9 and 1.3, where ATP = 1) and similar maximum responses were obtained. Alpha, beta-MethyleneATP, beta, gamma-methylene ATP and ,beta, gamma-imido ATP were considerably less potent than ATP, analysis yielding mean EMRs of 48.9, 85.0 and 60.0, respectively. Maximum responses to these latter three agonists were not obtained at the highest concentrations tested (1 x 10-4-3 X 10- M). Benzoyl ATP, adenosine 5'-0-(2-thiodiphosphate) and adenosine diphosphate produced only small depolarizing responses at high concentrations (>1 x 10-4 M). Adenosine monophosphate, adenosine and uridine S'-triphosphate each had little or no depolarizing effect in Ca2+/Mg2+-free PSS.6. These data demonstrate that in the absence of divalent cations the excitatory actions of some, but not all, purine nucleotides in the rat vagus nerve are markedly potentiated. In Ca2+/Mg2+-free PSS, the rank order of agonist potencies was ATP = 2-methylthioATP = ATP-gamma-S>> alpha,beta-methylene ATP = beta, gamma imido ATP = P,y-methylene ATP. These findings are in stark contrast to our previous observations in normal PSS where the rank order of agonist potencies for these nucleotides was alpha,beta-methyleneATP> ATP-gamma-S > beta,gamma-imido ATP = beta,gamma-methylene ATP> 2-methylthioATP> ATP.7. We suggest that the two different rank orders of potency can be explained by differential metabolism involving Ca2+/Mg2+-dependent ectonucleotidases. If so, these data indicate that ATP and 2-methylthioATP are inherently more potent than alpha,beta-methylene ATP as agonists at neuronal P2X purinoceptors in the rat vagus nerve. The possible implications of these findings to the present system for subclassifying P2 purinoceptors are profound.

The 5-HT1-like receptor mediating the increase in canine external carotid blood flow: close resemblance to the 5-HT1D subtype.

1. It has recently been shown that the increase in external carotid blood flow induced by 5-hydroxy-tryptamine (5-HT) in the anaesthetized dog, being mimicked by 5-carboxamidotryptamine (5-CT), inhibited by methiothepin, vagosympathectomy and sympatho-inhibitory drugs, and resistant to blockade by ritanserin and MDL 72222, is mediated by stimulation of prejunctional 5-HT1-like receptors leading to an inhibitory action on carotid sympathetic nerves; these 5-HT1-like receptors are unrelated to either the 5-HT1A, 5-HT1B or 5-HT1C (now 5-HT2C) receptor subtypes. Inasmuch as 5-CT, 5-methoxytryptamine, sumatriptan and metergoline display high affinity, amongst other 5-HT binding sites, for the 5-HT1D subtype, in the present study we have used these drugs in an attempt to determine whether the above inhibitory prejunctional 5-HT1-like receptors correlate with the 5-HT1D subtype. 2. One-minute intracarotid (i.c.) infusions of 5-HT (0.3, 1, 3 and 10 micrograms), 5-CT (0.01, 0.03, 0.1 and 0.3 micrograms), 5-methoxytryptamine (1, 3, 10 and 30 micrograms) and sumatriptan (1, 3, 10, 30 and 100 micrograms) resulted in dose-dependent increases in external carotid blood flow (without changes in mean arterial blood pressure or heart rate) with the following rank order of agonist potency: 5-CT >> 5-HT > 5-methoxytryptamine > or = sumatriptan. Interestingly, sumatriptan-induced vasodilatation was followed by a more pronounced vasoconstriction. 3. The external carotid vasodilator effects of 5-HT, 5-CT, 5-methoxytryptamine and sumatriptan were dose-dependently and specifically antagonized by metergoline (10, 30 and/or 100 micrograms kg-1, i.v.). In addition, 5-methoxytryptamine- and sumatriptan-induced vasodilator effects were, respectively, markedly inhibited or abolished after vagosympathectomy, as previously shown for 5-CT and 5-HT.4. Sumatriptan showed tachyphylaxis in its vasodilator component and antagonized 5-HT-induced external carotid vasodilatation in a specific manner, suggesting that a common site of action may be involved.5. Taken together, the above results support our contention that 5-HT, 5-CT, 5-methoxytryptamine and sumatriptan produce external carotid vasodilatation in the dog by an action that might primarily involve a prejunctional inhibition on carotid sympathetic nerves; a secondary component of this vasodilator response may be postsynaptic (endothelium-dependent and/or even directly on the vasculature).Based on the rank order of agonist potency, inhibition by vagosympathectomy and blockade by metergoline, we suggest that the inhibitory prejunctional 5-HT1-like receptors mediating external carotid vasodilatation in the dog closely resemble the 5-HTID receptor subtype. The pharmacological profile of these receptors is similar (sympathetic nerves of the rat kidney and human saphenous vein, as well as porcine coronary endothelium) to other putative 5-HTID receptors mediating vascular responses.

Pituitary ATP receptors: characterization and functional localization to gonadotropes.

We have used real-time dynamic video imaging of Fura-2 fluorescence to study the acute effects of extracellular ATP on [Ca2+]i in pituitary cells and found that ATP produced a rapid biphasic cytosolic Ca2+ increase in a dose-dependent manner in both rat pituitary gonadotropes and gonadotrope-derived alpha T3-1 cells. Removal of extracellular Ca2+ only attenuated the plateau phase of ATP-induced intracellular Ca2+ response and suramin (100 microM), a P2-purinoceptor antagonist, reduced the ATP effect by 40-60%. Further studies revealed the following rank-order of agonist potency in the Ca2+ response: -ATP = ADP = UTP > 2-methylthioATP >> beta, gamma-methylene ATP/alpha,beta-methylene ATP. Both homologous and heterologous desensitization occurred with ATP and UTP and these did not have additive effects. Together the data suggest that the intracellular Ca2+ responses of gonadotropes and alpha T3-1 cells to these nucleotides are mediated by a single class of receptor pharmacologically characterized as the P2U subtype. This is the first demonstration of functional ATP receptors in pituitary gonadotropes.

Pituitary ATP receptors: characterization and functional localization to gonadotropes

We have used real-time dynamic video imaging of Fura-2 fluorescence to study the acute effects of extracellular ATP on [Ca2+]i in pituitary cells and found that ATP produced a rapid biphasic cytosolic Ca2+ increase in a dose-dependent manner in both rat pituitary gonadotropes and gonadotrope-derived alpha T3-1 cells. Removal of extracellular Ca2+ only attenuated the plateau phase of ATP-induced intracellular Ca2+ response and suramin (100 microM), a P2-purinoceptor antagonist, reduced the ATP effect by 40-60%. Further studies revealed the following rank-order of agonist potency in the Ca2+ response: -ATP = ADP = UTP > 2-methylthioATP >> beta, gamma-methylene ATP/alpha,beta-methylene ATP. Both homologous and heterologous desensitization occurred with ATP and UTP and these did not have additive effects. Together the data suggest that the intracellular Ca2+ responses of gonadotropes and alpha T3-1 cells to these nucleotides are mediated by a single class of receptor pharmacologically characterized as the P2U subtype. This is the first demonstration of functional ATP receptors in pituitary gonadotropes.

The P2Z-purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP.

1. Extracellular adenosine triphosphate (ATP) is known to open a receptor-operated ion channel (P2Z class) in human lymphocytes which conducts a range of cationic permeants. The activity of a range of different agonists and inhibitors towards the P2Z-purinoceptor was investigated by measuring the agonist-induced influx of Ba2+ into fura-2 loaded lymphocytes. 2. The most potent agonist was 2' & 3'-0-(4-benzoylbenzoyl)-ATP (benzoylbenzoic ATP) which gave 2 fold greater maximum Ba2+ influx and had a 10 fold lower EC50 than for ATP. The rank order of agonist potency in K(+)-media was benzoylbenzoic ATP >> ATP = 2-methylthio ATP = 2-chloro ATP > ATP-gamma-S. ADP, UTP and alpha,beta-methylene ATP were unable to stimulate Ba2+ influx. 3. Extracellular Na+ inhibited the increment of Ba2+ influx induced by all concentrations of ATP, 2-methylthio ATP, 2-chloroATP and ATP-gamma-S. This inhibitory effect of extracellular Na+ is also reflected in the different EC50s for benzoylbenzoic ATP (8 microM in K(+)-media, 18 microM in Na(+)-media) but the maximal response to this agonist was the same in the presence or absence of Na+. 4. Treatment of lymphocytes with 2,3 dialdehyde ATP (oxidized ATP0 at 300 microM for 60 min gave total and irreversible inhibition of ATP-induced Ba2+ influx. 5'-p-Fluorosulphonyl benzoyladenosine (FSBA) also was an irreversible inhibitor but the maximal inhibition achieved was 90%. 5. It is concluded that the P2z-purinoceptor of human lymphocytes has a rank order of agonist potency which clearly distinguishes it from other P2-receptors and that oxidized ATP is a convenient irreversible inhibitor for the P2Z-purinoceptor.