Corelease of noradrenaline and adenosine triphosphate from sympathetic neurones.
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Adenosine Triphosphate↗
Biomedical subjects
K Starke
Publications and source records attributed to K Starke.
Presynaptic alpha 2A/D-autoreceptors in the brain cortex of Cercopithecus aethiops.
The existence of presynaptic alpha 2-autoreceptors, and the subtype to which the receptors belong, were studied in the brain cortex from two African green monkeys (Cercopithecus aethiops). Slices of the brain cortex were preincubated with 3H-noradrenaline. The slices were then superfused with medium containing desipramine (1 microM) and stimulated electrically with trains of 72 pulses. 3 Hz. Concentration-response curves of nine alpha-adrenoceptor antagonists were determined. All antagonists enhanced the stimulation-evoked overflow of tritium, i.e. the release of noradrenaline, indicating the existence of presynaptic alpha 2-autoreceptors. The EC30%, values of the antagonists (concentrations that increased the evoked overflow of tritium by 30%) were taken as a measure of affinity for the auto-receptors. The subclassification was based on a comparison of these affinity estimates with affinities for alpha 2A, alpha 2B, alpha 2C and alpha 2D radioligand binding sites and for previously classified alpha 2A- and alpha 2D-autoreceptors. The comparison indicates that the alpha 2-autoreceptors in the brain cortex of Cercopithecus aethiops belong to the genetic alpha 2A/D and not the alpha 2B or alpha 2C subtype. In their pharmacological properties, the autoreceptors are closest to the pharmacological alpha 2D subtype. The results support the notion that alpha 2-autoreceptors belong at least predominantly to the alpha 2A/D subtype of alpha 2-adrenoceptors.
Adrenergic alpha-2 Receptor Antagonists↗
P2-receptor-mediated inhibition of noradrenaline release in the rat hippocampus.
Experiments on hippocampal slices were carried out in order to find out whether the release of noradrenaline in the hippocampus can be modulated through P2-receptors. The slices were preincubated with [3H]-noradrenaline, superfused with medium containing desipramine (1 microM), and stimulated electrically, in most experiments by 4 pulses/100 Hz. The adenosine A1-receptor agonist N6-cyclopentyl-adenosine (CPA) and the nucleotides ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) decreased the evoked overflow of tritium by up to 55%. The adenosine A2a-agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamido-adenosin e (CGS 21680; 0.003-0.3 microM) caused no change. The concentration-response curve of CPA was shifted to the right by the A1-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3 nM) but not by the P2-receptor antagonists cibacron blue 3GA (30 microM) and reactive blue 2 (30 microM); the apparent pKB value of DPCPX against CPA was 9.0. In contrast, the concentration-response curve of ATP was shifted to the right by DPCPX (3 nM), apparent pKB 8.7, as well as by cibacron blue 3GA (30 microM), apparent pKB 5.2, and reactive blue 2 (30 microM), apparent pKB 5.6; the antagonist effects of DPCPX and cibacron blue 3GA were additive in a manner compatible with the blockade of two separate receptors for ATP. The same pattern was obtained with ATP gamma S: its concentration-response curve was shifted to the right by DPCPX as well as by cibacron blue 3GA and reactive blue 2. Suramin (300 microM) antagonized neither the effect of ATP nor that of ATP gamma S. The 5'-nucleotidase inhibitor alpha, beta-methylene-ADP (100 microM) did not change the effect of ATP. Only cibacron blue 3GA (30 microM) but not reactive blue 2 (30 microM), given alone, consistently caused a small increase of the evoked overflow of tritium. Hippocampal slices degraded exogenous ATP, and this degradation was reduced by cibacron blue 3GA (30 microM), reactive blue 2 (30 microM) and suramin (300 microM). The results indicate that the noradrenergic terminal axons of the rat hippocampus possess P2-receptors in addition to the known A1-adenosine receptors. The presynaptic P2-receptors mediate an inhibition of noradrenaline release, are activated by nucleotides but not nucleosides, and are blocked by cibacron blue 3GA and reactive blue 2. ATP and ATP gamma S act at both the A1- and the P2-receptors. An autoreceptor function of cerebral presynaptic P2-receptors remains doubtful.
Adenosine↗
Subtype determination of soma-dendritic alpha2-autoreceptors in slices of rat locus coeruleus.
The aim of the study was to subclassify the soma-dendritic alpha2-autoreceptors in the locus coeruleus (LC) of the rat by means of antagonists. To this end, the frequency of spontaneous action potentials was recorded extracellularly from single LC neurones in brain slices. The neurones fired spontaneously at an average rate of 1 Hz. The selective alpha2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and noradrenaline decreased the action potential discharge with IC50 values of 5 and 510 nM, respectively. The concentration-inhibition curves of UK 14,304 and noradrenaline were shifted to the right by phentolamine (0.15 microM) and rauwolscine (0.15 microM) but not by prazosin (1 microM). Apparent Kd values of phentolamine were 17 nM (against UK 14,304) and 20 nM (against noradrenaline). Apparent Kd values of rauwolscine were 47 nM (against UK 14,304) and 70 nM (against noradrenaline). (+)-Oxaprotiline (1 microM) suppressed the firing of the neurones within 10 to 33 min. In the continued presence of oxaprotiline, phentolamine and rauwolscine restored firing with EC50 values of 120 and 250 nM, respectively. Prazosin (1 microM) again was ineffective. All three antagonist affinity estimates - against UK 14,304, exogenous noradrenaline and endogenous noradrenaline (that accumulates in the extracellular space in the presence of oxaprotiline) - yield an affinity order phentolamine > rauwolscine >> prazosin, prazosin being ineffective even at a concentration of 1 microM. These findings identify the soma-dendritic alpha2-autoreceptors of the LC as the rat variant of the alpha2A/D-adrenoceptor, i.e. alpha2D. Not only presynaptic but also soma-dendritic alpha2-autoreceptors may at least predominantly be alpha2A/D throughout the nervous system.
Action Potentials↗
Characterization by antagonists of P2-receptors mediating endothelium-dependent relaxation in the rat aorta.
The receptors through which 2-methylthio ATP (MeSATP), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), UTP and ATP elicit endothelium-dependent relaxation of noradrenaline-precontracted rings of the rat aorta were characterized by means of a series of antagonists. The acetylcholine-induced relaxation and the degradation of MeSATP, UTP and ATP were also studied. The potency of the nucleotides at producing relaxation decreased in the order MeSATP (EC50 0.24 microM) > ADP beta S (0.43 microM) > UTP (1.09 microM) > ATP (3.53 microM). MeSATP, ADP beta S and UTP did not cause relaxation when the endothelium had been destroyed; high concentrations of ATP still caused some relaxation. The relaxation by MeSATP, ADP beta S and UTP became very small after treatment of the rings with NG-nitro-L-arginine methyl ester; the relaxation by ATP was less affected. Pre-exposure to MeSATP (100 microM) abolished or almost abolished the relaxation normally elicited by MeSATP and ADP beta S, did not change that elicited by UTP and slightly enhanced the relaxation elicited by ATP. Of nine compounds examined as antagonists, six attenuated selectively the effect of some or all of the nucleotides (as compared to acetylcholine): suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), reactive red 2 and 5,5'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-7-amino -6-hydroxy-naphthalene-1,4-disulphonate (NH05). Decreases of maximal relaxations and slopes different from unity in Schild plots often indicated non-competitive kinetics of the antagonism. For each of the six 'selective' antagonist, the apparent Kd values against MeSATP and against ADP beta S were similar: none of the six differentiated between MeSATP and ADP beta S. Also, for each of four 'selective' antagonists, the apparent Kd values against UTP and against ATP were similar: none of the four differentiated between these two nucleotides (two antagonists did not act against UTP and ATP in the 'selective' concentration range). On the other hand, for five of the six 'selective' antagonists (the exception being NH05), the apparent Kd values against MeSATP and ADP beta S were considerably lower than those against UTP and ATP. At the highest concentrations tested against agonist-evoked relaxations, the antagonists did not alter the removal from the incubation medium, by pieces of rat aorta, of MeSATP, UTP and ATP. It is concluded that nucleotides cause endothelium-dependent relaxation of the rat aorta through two sites: a P2Y-receptor and a P2U-receptor. The receptors may be pharmacologically similar to a bovine endothelial P2Y (P2Y1) and a cloned rat P2U (P2Y2) receptor, respectively. ATP acts mainly through the P2U-receptor. Suramin, reactive blue 2, iso-PPADS, PPADS and reactive red 2 are more potent at the P2Y- than the P2U-receptor. NH05 does not discriminate between the two receptors but is the most potent P2U antagonist so far described.
Acetylcholine↗
Vasoconstrictor and vasodilator effects of guanine nucleotides in the rat aorta.
Although GTP, like ATP and UTP, is stored in platelet dense granules, little is known about its vascular effects. The present study was carried out in order to characterize the effects of GTP and related compounds in the rat aorta. Contractions were examined in aortic rings at resting tension. In rings with intact endothelium, GTP, GDP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) caused small contractions. In endothelium-denuded rings, the contractions were unchanged or increased and persisted after desensitization of P2X-receptors by alpha,beta-methylene ATP. Relaxations were examined in aortic rings precontracted with noradrenaline. In rings with intact endothelium, GTP (EC50 131 microM), GDP (no maximal effect obtained), GTP gamma S (EC50 6.8 microM) and guanosine (EC50 822 microM) caused prominent relaxation, whereas GDP beta S caused further contraction. In endothelium-denuded rings, the relaxant effect of GTP was greatly reduced, that of GDP and guanosine was unchanged, and that of GTP gamma S was abolished. Relaxations by GTP and GTP gamma S in endothelium-intact rings were studied in more detail. The relaxation by GTP was slightly and the relaxation by GTP gamma S greatly reduced after treatment with NG-nitro-L-arginine methyl ester. Pre-exposure to a high concentration of the P2Y-receptor agonist 2-methylthio ATP (MeSATP) did not attenuate the effects of GTP and GTP gamma S. Four compounds previously identified as antagonists at the P2Y- and P2U-receptors of rat aortic endothelium--suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS) and 5,5'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-7-amino-6- hydroxynaphthalene-1,4-disulphonate (NH05)--were tested against GTP and GTP gamma S. Suramin, reactive blue 2 and iso-PPADS were much less potent against GTP and GTP gamma S than previously found against (the P2Y effect of) MeSATP. Suramin, iso-PPADS and NH05 were about as potent against GTP and GTP gamma S as previously found against (the P2U effect of) UTP and in particular ATP. It is concluded that guanine nucleotides can cause both contraction and relaxation of the rat aorta. The high concentrations of GTP and GDP required, and in the case of contraction the small size of the response, make a physiological role of the vascular effects of these nucleotides unlikely. GTP and GTP gamma S elicit endothelium-dependent relaxation through P2U-receptors. GTP in addition relaxes the aorta through smooth muscle receptors, possibly by way of its degradation product guanosine. The stable analog GTP gamma S is a relatively potent and selective agonist for the endothelial P2U-receptor.
Animals↗
A re-investigation of questionable subclassifications of presynaptic alpha2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra.
It has been suggested that at least the majority of mammalian presynaptic alpha2-autoreceptors belong to the genetic alpha2A/D-adrenoceptor subtype. The aim of the present study was to re-examine the alpha2-autoreceptors in tissues in which previous assignments conflicted with this alpha2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as alpha2B or similar to, but not identical with, alpha2D, and in the human kidney, where they were classified as alpha2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be alpha2A, in agreement with the rule, but in contrast to indications that the alpha2A/D-adrenoceptor of the guinea pig possesses alpha2D pharmacological properties. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The Kd values of up to 14 antagonists (including the partial agonist oxymetazoline) against the release-inhibiting effect of the alpha2 agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. UK 14,304 reduced the evoked overflow of tritium with an EC50 between 6.3 and 13.2 nM. All antagonists (except prazosin in one case) shifted the concentration-inhibition curve of UK 14,304 to the right. Comparison of the Kd values thus obtained with Kd values at known alpha2 subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were alpha2D and those in the human kidney alpha2A. For example, the pKd values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pKd values at the prototypic alpha2D radioligand binding sites in the bovine pineal gland (r = 0.96, P < 0.001; r = 0.92, P < 0.01; and r = 0.95; P < 0.001) and with the pKd values at the alpha2D-autoreceptors of guinea-pig atria (r = 0.99, P < 0.001; r = 0.95, P < 0.001; and r = 0.98, P < 0.001). The pKd values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pKd values at alpha2A, alpha2B and alpha2C binding sites and alpha2A-autoreceptors. On the other hand, the pKd values of the antagonists in the human kidney were closely correlated with pKd values at the prototypic alpha2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pKd values at the alpha2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pKd values at alpha2B, alpha2C and alpha2D binding sites and alpha2D-autoreceptors. In contrast to previous suggestions, the autoreceptors in rat vena cava and atria are alpha2D, those in the human kidney alpha2A, and those in the guinea-pig urethra equally alpha2D. All, therefore, conform to the rule that alpha2-autoreceptors belong at least predominantly to the genetic alpha2A/D subtype of the alpha2-adrenoceptor. The apparent paradox of an alpha2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic alpha2A/D-adrenoceptor otherwise has alpha2D pharmacological properties, is removed.
Adrenergic Antagonists↗
P2-receptor-mediated inhibition of serotonin release in the rat brain cortex.
The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.
2-Chloroadenosine↗
Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation.
The release of ATP was studied in cultures of astrocytes derived from the brain hemispheres of newborn rats. There was a basal efflux of ATP, which was increased up to 19-fold by glutamate (300-1000 microM). N-methyl-D-aspartate (20-500 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 30-100 microM) and kainate (20 microM). The N-methyl-D-aspartate receptor-selective antagonist 2-amino-5-phosphonopentanoate (100 microM) blocked the effect of N-methyl-D-aspartate but not the effects of AMPA, kainate and glutamate. The AMPA receptor-selective antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (30 microM) blocked the effect of AMPA and also of glutamate and N-methyl-D-aspartate, but not the effect of kainate. The kainate receptor-selective antagonist D-glutamyl-amino-methanesulfonate (30 microM) blocked the effect of kainate but not of glutamate. Glutamate (1000 microM) did not increase the release of lactate dehydrogenase from astrocytes. Excitatory amino acids are known to release adenyl compounds in the brain. The present results identify one adenyl compound thus released, namely ATP, and identify astrocytes as one source. The release is brought about by activation of any of the three ionotropic glutamate receptor types-N-methyl-D-aspartate, AMPA and kainate receptors. AMPA receptors seem to mediate at least a part of the effect of glutamate itself, but the involvement of other receptors cannot be ruled out. ATP and its degradation products, such as adenosine, once released, may exert acute as well as trophic effects on neurons and glial cells.
Adenosine Triphosphate↗
Differences in the mode of stimulation of cultured rat sympathetic neurons between ATP and UDP.
Postganglionic sympathetic neurons possess at least two excitatory receptors for nucleotides: P2X-purinoceptors and separate receptors for uracil nucleotides. In cultured neurons from rat superior cervical ganglia (SCG), both receptors, when activated, induce release of noradrenaline. Here we describe marked differences between the responses of cultured neurons from rat thoracolumbal paravertebral ganglia to ATP and UDP. ATP elicited release of previously taken up [3H]noradrenaline, induced an inward current, and increased the intra-axonal free calcium level, over the same range of micromolar concentrations. UDP was more potent than ATP in releasing [3H]noradrenaline but induced an inward current only at a concentration of 1 mM and caused much smaller increases in intraaxonal free calcium. The mechanism of action of ATP presumably consists of P2X-purinoceptor activation followed by depolarization, calcium entry through voltage-sensitive channels and exocytosis. The mode of action of UDP is different. It probably activates a G-protein-coupled pyrimidinoceptor. The pyrimidinoceptor then possibly mediates mobilization of intracellular calcium close to the sites of transmitter release and in addition an increase in the calcium sensitivity of the exocytotic apparatus.
Adenosine Triphosphate↗
The Porto meetings on adrenergic mechanisms.
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Adrenergic Agents↗
Temperature Dependence of the Exchange Splitting of the Surface State on Gd(0001): Evidence against Spin-Mixing Behavior.
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Journal Article↗
Spin-polarized photoelectrons excited by circularly polarized radiation from a nonmagnetic solid.
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Journal Article↗
ATP release and its prejunctional modulation.
We studied some properties of the release of noradrenaline and ATP in isolated sympathetically innervated tissues. Release was elicited by electric stimulation and assessed as overflow of tritiated compounds (after labelling with [3H]noradrenaline) and enzymically measured ATP, respectively. Evans blue, which inhibits ectonucleotidases, greatly increased the evoked overflow of ATP, indicating that a major part of the ATP was metabolized after release. Much of the ATP was postjunctional in origin. The neural fraction was isolated when postjunctional release was suppressed by prazosin (alpha 1-adrenoceptor antagonist) and suramin (P2 purinoceptor antagonist). Comparison of neural ATP and [3H]-noradrenaline release showed that prostaglandin E2 reduced the release of both co-transmitters to a similar extent. Activation of prejunctional alpha 2-adrenoceptors, however, preferentially reduced the release of [3H]noradrenaline, and activation of prejunctional A1 purinoceptors reduced preferentially the release of ATP. Nucleotides such as ATP depressed the release of [3H]noradrenaline through two receptors: the well-known prejunctional A1 receptors and a separate group of prejunctional P2 purinoceptors. P2 antagonists increased the release of [3H]-noradrenaline. Overall, the results indicate differential storage, release and modulation of release of the two sympathetic co-transmitters. They also indicate that postganglionic sympathetic axons possess receptors for both co-transmitters: alpha 2 and P2 autoreceptors.
Adenosine Triphosphate↗
P2-purinoceptor antagonists: I. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by small aromatic isothiocyanato-sulphonates.
Effects of eight small aromatic isothiocyanato-sulphonates, of the aliphatic 2-isothiocyanatoethene-1-sulphonate (IES), and of the parent amines were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. The aromatic isothiocyanato-sulphonates all reduced contractions of the rat vas deferens elicited by alpha, beta-methylene ATP. The antagonism was non-competitive, with depression of the maximum of the concentration-response curve of alpha, beta-MeATP and incomplete reversibility. The IC50 values were between 11 and 54 microM. In the guinea pig taenia coli, the aromatic compounds shifted the concentration-response curve of ADP beta S to the right in a surmountable manner (one exception), and where three concentrations were tested, the Arunlakshana-Schild regression was linear and its slope did not differ from 1. The apparent Kd values were between 10 and 214 microM. The removal of ATP from the medium by vas deferens tissue was decreased by the aromatic isothiocyanates with IC25% values between 25 and 464 microM. IES and the parent amines were inactive or almost inactive (parent amines not tested on ATP breakdown). The results indicate that the isothiocyanato residue as well as the aromatic core are essential for P2-purinoceptor blockade. At the P2X-purinoceptor, potency increases with the size of the molecules but is independent of the position of the isothiocyanato and sulphonate substituents. No simple structure-activity relationship for the P2Y-purinoceptor and the ATP-degrading ecto-nucleotidases can be derived beyond the apparent lack of a major influence of the position of the substituents. 2-Isothiocyanatonaphthalene-1-sulphonate (beta-INS) seems to be interesting because of relatively high P2X-selectivity versus both the P2Y-purinoceptor and ecto-nucleotidases.
Adenosine Diphosphate↗
P2-purinoceptor antagonists: II. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to Evans blue and trypan blue.
Effects of Evans blue and four derivatives as well as of trypan blue and four derivatives, mostly smaller fragments but two compounds with an additional ethylene bridge in the center of the molecule, were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. All compounds shifted the concentration-response curve of alpha, beta-MeATP in the rat vas deferens to the right, and most compounds increased the maximum of the curve. Each member of the Evans blue series was similar in potency to the corresponding member of the trypan blue series. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent Kd values were between 0.8 and 385 microM. In the guinea-pig taenia coli, only the members of the trypan blue group were relatively potent, shifting the concentration-response curve of ADP beta S to the right in a surmountable manner. In 2 of 3 cases where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent Kd values in the trypan blue group were between 5.2 and 324 microM. The removal of ATP from the medium by vas deferens tissue was decreased mainly by the members of the Evans blue group, with IC25% values between 13 and 158 (in 1 case > 1000) microM. The results indicate that the position of the sulphonate residues at the terminal naphthalene rings of these compounds hardly influences P2X purinoceptor affinity but greatly influences P2Y affinity and ecto-nucleotidase blockade. Among active compounds, apparent purinoceptor affinity and ecto-nucleotidase blockade increase with the size of the molecules up to Evans blue and trypan blue themselves; introduction of a central ethylene bridge does not result in a further gain in potency. NH01, the desmethyl derivative of Evans blue, seems to be interesting because it is the compound with the highest P2X- versus P2Y-selectivity presently available.
Adenosine Diphosphate↗
P2-purinoceptor antagonists: III. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to suramin.
Effects of suramin and five analogs or fragments of suramin were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. One compound, NF023, differed from suramin by removal of two p-methylbenzamido groups, whereas another, BSt101, differed from NF023 by additional removal of the three sulphonate residues from one of the terminal naphthalene rings. The compounds all shifted the concentration-response curve of alpha, beta-MeATP in the rat vas deferens to the right and simultaneously increased the maximum of the curve. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent Kd values were between 1 and 3672 microM. In the guinea-pig taenia coli, the compounds shifted the concentration-response curve of ADP beta S to the right in a parallel manner, but in the one case where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent Kd values were between 10 and 786 microM. The removal of ATP from the medium by vas deferens tissue was decreased only by suramin, NF023 and BSt101, with IC25% values between 170 and 590 microM.
Adenosine Diphosphate↗
Antagonists that differentiate between alpha 2A-and alpha 2D-adrenoceptors.
Four antagonists were examined for their ability to differentiate alpha 2A-from the orthologous alpha 2D-adrenoceptors. The antagonists were (2S,12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12b-octah ydro-2H- benzo[b]furo[2,3-a]quinolizine)-2,4'-pyrimidin-2'-one (MK912), 2-[2-(methoxy-1,4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The alpha 2-autoreceptors in rabbit brain cortex were chosen as alpha 2A-and the alpha 2-autoreceptors in guinea-pig brain cortex as alpha 2D-adrenoceptors. Slices of the brain cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, alpha 2-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) was used as an alpha 2-adrenoceptor agonist. UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) alpha 2D-adrenoceptors (pKd values 10.0, 9.7 and 9.1, respectively) than for (rabbit) alpha 2A-adrenoceptors (pKd 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for alpha 2A-(pKd 7.4) than for alpha 2D-adrenoceptors (pKd 6.9). Ratios calculated from the Kd values of the four compounds differentiated between alpha 2A and alpha 2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate alpha 2A-from alpha 2D-adrenoceptors.
Adrenergic alpha-Agonists↗