ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

Retrospective study on the reliability of an “approximate LD50” determined with a small number of animals

Based on 364 LD₅₀ determinations in mice and rats after intravenous and oral administration of drugs, the reliability of an approximate LD₅₀ was retrospectively tested.The difference between approximate LD₅₀ and LD₅₀ is — independent of species and route of administration — not greater than ± 20% of the LD₅₀ in 90% of the cases.Four to five doses — uniformly distributed over the dose-mortality range can suffice in reliably determining the approximate LD₅₀.The probability is 10% that an approximate LD₅₀ and LD₅₀ are significantly different from each other.152 parallel studies on male and female animals show that the LD₅₀ or approximate LD₅₀ must not be determined for both sexes. It is sufficient to test a dose near the LD₅₀ in the opposite sex. A 50–75% reduction of expenditure in animal material is possible in most of LD₅₀ determinations.     

A comparison of the effects of TMB-8 and nifedipine on pressor responses to α1- and α2-adrenoceptor agonists in pithed rats

TMB-8 has been characterized as an inhibitor of the release of Ca⁺ from intracellular pools. We have studied the modification of the pressor responses to selective _l-adrenoceptor agonists (methoxamine and phenylephrine), and to selective ₂-adrenoceptor agonists (B-HT 920 and B-HT 933) in pithed rats, produced by TMB-8. We have compared this modification with that produced by the calcium antagonist nifedipine. Nifedipine (100 g/kg, 300 g/kg, and 1000 g/kg) inhibited in a dose-dependent manner the pressor responses to the ₁- and ₂-adrenoceptor agonists, the dose-response curves to the ₂-adrenoceptor agonists being shifted further to the right. TMB-8 at a dose of 3000 g/kg did not modify the pressor effects of the _l-adrenoceptor agonists, and neither did it reinforce the inhibition of such responses produced by nifedipine. By contrast, TMB-8 pretreatment (0.03 g/kg, 0.3 g/kg, 3 g/kg, 30 g/kg, 300 g/kg and 3000 g/kg) inhibited the responses to both ₂-adrenoceptor agonists, the inhibition being more pronounced with B-HT 920. A similar effect was obtained with 0.03 g/kg TMB-8 and 0.3 g/kg TMB-8, particularly in the case of B-HT 920. It was stronger with higher doses, but similar for all doses over 3 g/kg. The inhibition of the pressor responses mediated by the stimulation of ₂-adrenoceptors by TMB-8 was less in rats treated with the Ca²⁺ entry promoter BAY K 8644 (300 g/kg), and could also be reduced by the continuous infusion of CaCl₂ (0.25 g/min). These results suggest that in pithed rats TMB-8 may also behave as an inhibitor of the Ca⁺ influx into vascular smooth muscle.     

P2-purinoceptor-mediated autoinhibition of sympathetic transmitter release in mouse and rat vas deferens

Effects of drugs acting at P₂-purinoceptors on the release of newly taken up [³H]-noradrenaline were studied in slices of mouse and rat vas deferens. The slices were superfused and stimulated electrically, in most experiments by trains of 60 pulses/8 Hz.In mouse vas deferens, the P₂-purinoceptor antagonists reactive blue 2 (1.8–100 M) and brilliant blue G (10–300 M) increased the stimulation-evoked overflow of tritium in a concentration-dependent manner as shown previously for suramin. Reactive blue 2, which preferentially blocks the P_2Y-subtype, was the most potent compound and the compound with highest maximal effect, an increase by 104%. Pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), in contrast, caused a small increase only at a single concentration (30 M). The effects of reactive blue 2, brilliant blue G and suramin were not additive. The P₂ agonist adenosine 5-O-(3-thio)-triphosphate (ATPS) reduced the evoked overflow of tritium. As shown previously for suramin, reactive blue 2 30 M and brilliant blue G 100 M antagonized the effect of ATPS. From the shift of the ATPS concentration-response curve to the right, an apparent pK_B value of 5.3 was estimated for reactive blue 2 and an apparent pKB of 4.5 for brilliant blue G. In rat vas deferens, reactive blue 2 (3–30 M), brilliant blue G (10 M) and suramin (30–300 M) also increased the evoked overflow of tritium. As in the mouse, reactive blue 2 was the most potent compound and the compound with highest maximal effect, an increase by 9001o. As previously demonstrated in the mouse, suramin (300 M) increased the evoked overflow of tritium only when rat vas deferens slices were stimulated by trains of 60 pulses at 1 or 8 Hz, but not when they were stimulated by trains of 6 pulses/100 Hz.The results confirm the operation of a P₂-purinoceptor-mediated prejunctional negative feedback controlling the release of noradrenaline in mouse vas deferens and demonstrate the same mechanism in rat vas deferens. The prejunctional P₂-purinoceptors are P_2Y-like in both species. They are a novel kind of autoreceptors, operating in parallel to prejunctional ₂-autoreceptors. Correspondence to: I. von Kügelgen at the above address     

β2-Adrenoceptor-mediated positive inotropic effect of adrenaline in human ventricular myocardium

Summary Experiments were designed to unravel the relative contribution of ₁- and ₂-adrenoceptors to the positive inotropic effects of adrenaline and noradrenaline in isolated tissues of left ventricular myocardium of man. We also analyzed relationships between the fractions of human left ventricular ₁- and ₂-adrenoceptors, estimated from binding assays, and stimulation of adenylate cyclase and contractile force by adrenaline and noradrenaline. 1) Selective blockade of ₂-adrenoceptors by erythro(±)-(-methyl-indan-4-yloxy)-3-isopropylaminobutan-2-ol (ICI 118,551) attenuated the increase of contractile force caused by adrenaline but not by noradrenaline, suggesting some involvement of ₂-adrenoceptors. Selective blockade of ₂-adrenoceptors without affecting ₁-adrenoceptors still enabled both adrenaline and noradrenaline to cause maximum possible increases of contractile force through ₁-adrenoceptors. 2) A direct involvement of ₂-adrenoceptors became manifest by selectively antagonizing ₁-adrenoceptors by 1-[2((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]3-[4(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712 A) without affecting ₂-adrenoceptor. ₂-adrenoceptors can mediate half of the maximum increase of contractile force elicited by low concentrations of adrenaline and also contribute to the increase of contractile force caused by high concentrations of noradrenaline. 3) -adrenoceptors were labelled in membrane particles with ³H-(–)-bupranolol in the absence (₁ & ₂) and presence of 500 nmol/l CGP 20712 A (₂). 71 % of the -adrenoceptors Were ₁ and 29% ₂. Binding inhibition experiments with CGP 20712 A and ICI 118,551 yielded 74% ₁ and 26% ₂-4) With the help of ICI 118,551 and CGP 20712 A it was found that, in membrane particles, 33–36% and 64–67% of maximum stimulation of the adenylate cyclase by noradrenaline was mediated through ₁- and ₂-adrenoceptors, respectively. Adrenaline caused 11–25% and 75–89% of maximum cyclase stimulation through ₁- and ₂-adrenoceptors, respectively. 5) The contribution of ₁- and ₂-adrenoceptors to the positive inotropic effects of adrenaline and noradrenaline cannot be inferred straightforwardly from biochemical estimates of receptor fractions and fractional adenylate cyclase stimulation. Send offprint requests to A. J. Kaumann, ICI Pharmaceutical Division, Mereside Alderley Park, Macclesfield, Cheshire SK10 4TG, UK     

O-Acylated lysergol and dihydrolysergol-I derivatives as competitive antagonists of 5-HT at 5-HT2 receptors of rat tail artery

Summary Twelve ergolines (O-Ayated lysergol and dihydrolysergol-I derivatives) were synthesized to study their antagonism of 5-HT responses in comparison with methysergide and LY 53857 [6-methyl-l-(1-methylethyl)-8-ergoline carboxylic acid 2-hydroxy-l-methyl-propylester hydrogen maleate] in cylindrical segments of the isolated rat tail artery. With regard to (9.10-didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate, the most potent new ergoline derivative, we examined the phenomenon of insurmountable antagonism to 5-HT by methysergide.O-Acylated lysergol and dihydrolysergol-I derivatives competitively antagonized 5-HT-induced contractions with calculated pA₂. values of 7.30 ± 0.42 for the weakest and 8.42 ± 0.35 for the most potent ergoline derivative in this series. N¹-isopropyl substitution did not generally enhance 5-HT₂ receptor affinities but lowered affinities for ₁ adrenoceptors in rat aorta. Methysergide and LY 53857 were insurmountable, antagonists of 5-HT in rat tail artery.Preincubation with (9.10-didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate (1 mol/l) partially prevented the depression of 5-HT-induced contractions caused by methysergide (1–10 nmol/l). Methysergide (100 nmol/l) abolished the protective effect of (9.10-didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate. (9.10-Didehydro-6-methyl-8-ergoline)methyl R,S-2-methylbutyrate (1 mol/l), concomitantly incubated with methysergide (30 nmol/l), partially restored the maximum response to 5-HT that had been depressed by methysergide (30 nmol/l). Partial restoration could not be mimicked by washout of methysergide. In view of this result, it is suggested that insurmountable antagonism by methysergide of 5-HT responses in rat tail artery is due to allosteric modulation of 5-HT₂ receptors rather than pseudoirreversible inhibition. Send offprint requests to H. Pertz at the above address     

Existence of functional M<Subscript>3</Subscript>-muscarinic receptors in the human heart

It has been recently shown that, in adult rat ventricular cardiomyocytes, functional muscarinic receptors (M-receptors) of the M₃-subtype exist that mediate inositol phosphate (IP) formation. The aim of this study was to characterize the M-receptor subtype mediating IP formation in the human heart. For this purpose in [³H]-myo-inositol labeled slices of human right atria, carbachol-induced [³H]-IP formation and its inhibition by several M-receptor antagonists was assessed. Carbachol (0.1 M–100 M) increased [³H]-IP formation; maximal increase at 100 M was 93±16% above basal (n=20); the pEC₅₀-value for carbachol was 5.56. Atropine (1 M) completely suppressed 100 M carbachol-induced [³H]-IP formation. Among the M-receptor subtype selective antagonists himbacine (1 M) and pirenzepine (1 M) only marginally affected carbachol-induced [³H]-IP formation whereas the M₃-receptor antagonist darifenacin (1 nM–1 M) concentration-dependently inhibited carbachol-induced [³H]-IP formation with a pK_i-value of 8.49. We conclude that in human right atrium there exist functional M₃-receptors that couple to IP formation.Abbreviations DAG Diacylglycerol - IP Inositol phosphates - M Muscarinic - PLC Phospholipase C     

Effects of intracerebroventricular administration of prostaglandin D2 on behaviour,blood pressure and body temperature as compared to prostaglandins E2 and F2α

The present work examined some central nervous actions of prostaglandin D₂ (PGD₂), which is the most prevalent prostaglandin in rodentorain. The effects of PGD₂ were compared with those of PGE₂ and PGF₂. The prostaglandins were administered intracerebroventricularly (ICV) to conscious rats using the method of Herman (1970). All three prostaglandins studied produced depressive behavioral effects, causing obvious sedation at doses of 2.0 g and 20.0 g ICV. PGD₂ and PGE₂ significantly reduced spontaneous motor activity at doses of 2.0 g and 20.0 g ICV. PGF₂ was less effective; only 20.0 g significantly inhibited motor activity. At a dose of 20.0 g ICV all three compounds were shown to block convulsions induced by pentylenetetrazol. PGD₂, the most effective prostaglandin in this respect, was still slightly anticonvulsive at a dose of 2.0 g ICV. PGF₂ hat the weakest anticonvulsive potency. PGE₂ and PGF₂ (2.0 g and 20.0 g ICV) caused a marked hypertensive effect, whereas PGD₂ at the same dose levels only produced a small increase in blood pressure. PGE₂ and PGF₂ (2.0 g and 20.0 g) also exerted marked pyrogenic actions. The effects of PGD₂ on body temperature were variable. When given at a dose of 20.0 g ICV, it caused slight hyperthermia whereas a lower dose (2.0 g ICV) induced a moderate fall in body temperature. These findings suggest a relationship between the actions of the different prostaglandins on blood pressure and body temperature.A preliminary report was given at the Spring Meeting of the Deutsche Pharmakologische Gesellschaft, March 1983 (Förstermann and Heldt, 1983)     

Effect of captopril on myocardial β-adrenoceptor density and Giα-proteins in patients with mild to moderate heart failure due to dilated cardiomyopathy

In end-stage heart failure due to idiopathic dilated cardiomyopathy ₁-adrenoceptors are downregulated and G₁-proteins are upregulated. The aim of the present study was to investigate the influence of the angiotensin-converting enzyme inhibitor captopril on -adrenoceptor density and G_i-proteins in sequential endomyocardial biopsies. Nineteen patients with mild to moderate congestive heart failure due to idiopathic dilated cardiomyopathy (NYHA Class II–III) were studied before and after 8–11 weeks of therapy. Patients were randomised into a captopril and a control group; 9 patients received captopril 12.5–50 mg per day, (divided in 2–3 doses) p.o. in addition to conventional therapy with digoxin and diuretics, and 10 controls received conventional therapy only. Echocardiography, spiroergometry, right heart catheterisation and endomyocardial biopsies were performed before (baseline) and after treatment. Compared to baseline, captopril increased total -adrenoceptor density by selectively increasing ₁-adrenoceptors (31.6 vs 41.2 fmol·mg^–1; p<0.05) but had no significant effect on G_i-proteins. The results indicate that treatment with angiotensin-converting enzyme inhibitors partly restores myocardial ₁-adrenoceptor density, and this action effect may contribute to the clinical improvement of patients with idiopathic dilated cardiomyopathy treated in this way.     

Evans blue blocks P2X-purinoceptors in rat vas deferens

Summary In rat vas deferens, Evans blue 100 M increased contractions elicited by high K⁺ and by noradrenaline but markedly reduced contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was shifted to the right by Evans blue 30 M and the maximal contraction was increased. In tissues incubated with nifedipine 10 M, Evans blue 100 M tended to increase the residual contraction elicited by noradrenaline and abolished the residual response to ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was progressively shifted to the right by increasing concentrations of Evans blue in the presence of nifedipine; maximal contractions were increased by Evans blue 10 and 30 but not 100 M. From the shifts to the right caused by Evans blue 30 M, apparent pK_B values of 5.9 (no nifedipine) and 6.0 (nifedipine present) were calculated. It is concluded that Evans blue blocks P_2X-purinoceptors in rat vas deferens and in addition causes a non-receptor-specific enhancement of contractions.Correspondence to: R. Bültmann at the above address     

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

Effect of prednisolone and ketotifen onβ 2-adrenoceptors in asthmatic patients receivingβ 2-bronchodilators

Summary In 13 patients with bronchial asthma, who were on ₂-adrenergic bronchodilator therapy, the effects of prednisolone and ketotifen on lymphocyte ₂-adrenoceptor density and -responsiveness were investigated. The mean lymphocyte ₂-adrenoceptor density and -responsiveness was significantly lower than in healthy controls, presumably due to the long-term ₂-adrenergic bronchodilator treatment. Both prednisolone 100 mg i.v. and ketotifen 1 mg b.d.p.o. for 6 days rapidly improved lymphocyte ₂-adrenoceptor function. 16 h after prednisolone and about 6 days after the first dose of ketotifen lymphocyte ₂-adrenoceptor density and-responsiveness had risen to values within the range in normal volunteers.The improvement of lymphocyte ₂-adrenoceptor function was accompanied by a significant increase in peak expiratory flow rate before and after inhalation of salbutamol.It is concluded that prednisolone and ketotifen may act beneficially on the recovery of ₂-adrenoceptor responsiveness to ₂-adrenergic bronchodilators in tolerant asthmatic patients.     

The selective P2X purinoceptor agonist, β,γ-methylene-L-adenosine 5′-triphosphate,discriminates between smooth muscle and neuronal P2X purinoceptors

The effects of the putative selective P_2X purinoceptor agonist, ,-methylene-l-adenosine 5-triphosphate (me-l-ATP), were determined at rat neuronal and smooth muscle P_2X purinoceptors.Me-l-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 M. In contrast, the archetypal P_2X purinoceptor agonist, , methylene ATP (meATP;1–100 M), produced concentration-related depolarisation responses with a mean EC₅₀ value of 10.8 M. The depolarising effects of meATP were not attenuated by me-l-ATP (100 M). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 M), but not me-l.-ATP (1–300 M), evoked rapid ( < 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, me-d-ATP and meATP competed with high affinity for [³H]Lx meATP binding sites, with mean pIC₅₀ values of 7.7 and 8.3, respectively. However, me-l-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 M (mean pIC₅₀ value 4.1).In prostatic segments of the rat vas deferens, me-l-ATP (1–100 M) and meATP (0.3–100 M) each produced concentration-related contractile responses with mean EC₅₀ values of 17.1 and 3.6 M, respectively. Me-l-ATP (1–10 M) evoked fast inward currents in freshly dispersed vas deferens smooth muscle cells, indicative of an action at ligand-gated ion channels. Binding sites in vas deferens membranes labelled using 1 nM [³H]meATP exhibited high affinity for me-l-ATP, meATP and me-d-ATP with mean PIC₅₀ values of 7.7, 8.4 and 7.3, respectively.These results indicate that me-l-ATP exhibits neither agonist nor antagonist properties at P_2X purinoceptors on rat vagal neurones and possesses only very low affinity for [³H]meATP binding sites in rat brain. In contrast, me-l-ATP is a potent, high affinity agonist at smooth muscle P_2X purinoceptors of the rat vas deferens. This selective agonist action of me-l-ATP suggests that P_2X purinoceptors in smooth muscle and neurones are different and represent distinct P_2X purinoceptor subtypes.     

Reactive red 2: a P2y-selective purinoceptor antagonist and an inhibitor of ecto-nucleotidase

Effects of reactive red 2 and its parent compound acid red 33 were studied in rat vas deferens and guinea-pig taenia coli. In rat vas deferens, reactive red 2 (1 to 10 M) shifted the concentration-response curve for the P_Zx-purinoceptor-mediated contraction effect of , \-methylene ATP slightly to the right and progressively decreased the maximum (apparent antagonist K_d value 0.42 M). Acid red 33 (1000 M) shifted the curve to the right without changing the maximum (apparent K_d 386 M). The concentration-contraction curve of noradrenaline was not altered by reactive red 2. In the carbachol-precontracted guinea-pig taenia coli, reactive red 2 (0.1 to 10 M) shifted the concentration-response curve for the P_2Y-purinoceptor-mediated relaxation effect of adenosine 5-O-(2-thiodiphosphate) (ADP&S) progressively to the right; only at the highest concentration of antagonist (10 M) was the maximum slightly depressed; a pA₂ value of 7.55 (K_d 0.028 M) was derived from the shift. Acid red 33 (1000 M) shifted the concentration-relaxation curve of ADP\S to the right without changing the maximum (apparent K_d 171 M). Reactive red 2 (1 to 10 LM) also shifted the concentration-response curve for the relaxation effect of , \-methylene ATP, which is mediated by an unclassified P₂-purinoceptor, progressively to the right but simultaneously decreased the maximum (apparent K_d1.6 M). The concentration-relaxation curve of 2-chloroadenosine was not altered by reactive red 2. Pieces of vas deferens and taenia coli degraded 76 and 66 % of added ATP (10 M) within 30 min, respectively. Reactive red 2 (0.1 to 100 M) progressively reduced this degradation by up to 95%, with IC₅₀values of 3.9 ± 0.6 and 3.9 ± 2.3 M, respectively. Acid red 33 (1000 M) reduced the degradation by 30 and 20%, respectively.The results indicate that reactive red 2 is a relatively potent antagonist at both P_Zx-purinoceptors in rat vas deferens and P_2Y-purinoceptors in guinea-pig taenia coli, with a 15 fold selectivity for the P_2Y-purinoceptor. It inhibits ecto-nucleotidase in both tissues. The dichloro-triazine residue that distinguishes the compound from acid red 33 greatly enhances the potency at both receptor subtypes as well as at the nucleotidase. As regards P₂-purinoceptor subtypes, the results confirm the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli.     

P2-purinoceptor induced prostaglandin synthesis in primary rat astrocyte cultures

Summary Adenosine triphosphate (ATP) is one of the cotransmitters that are commonly released at catecholaminergic and cholinergic nerve terminals. The glial cell type most closely associated with the synapse is the astrocyte and, thus, is the next cellular element beside the postsynaptic neuron to face the transmitters released. This report gives evidence of P₂-purinoceptors on cultured astroglial cells. Upon stimulation with nucleoside triphosphates and nucleoside diphosphates, the cells respond with synthesis of prostaglandins of the D₂ type, which is the predominant prostaglandin made in rat brain. Nucleoside triphosphate analogues, such as 5-adenylyl-imido diphosphate, ,-methylene, or ,-methylene ATP were less effective than ATP or its non-hydrolysable analogue ATP [ S]. The receptor was desensitized by ATP [ S] within 15 min, whereas desensitization by ,-methylene ATP was significantly delayed. 8-phenyl-theophylline (10^–4 M) had no influence on ATP-stimulated prostaglandin synthesis. Adenosine 5-monophosphate (AMP) and adenosine were unable to stimulate prostaglandin D₂ formation. According to the common nomenclature for purinoceptors, the described astroglial receptor would fulfill the characteristics of a P₂-purinoceptor. Furthermore, it is shown that pertussis toxin sensitive G-proteins influence some early step in prostaglandin synthesis. The inactivation of these proteins results in reduced prostaglandin formation. It is assumed that ATP serves as an important mediator in the cross-talk between neurons and astroglial cells at the synaptic cleft. Send offprint requests to P. J. Gebicke-Haerter at the above address     

Identification of a novel high affinity adenosine binding protein from bovine striatum

Summary In solubilized extracts from bovine striatal membranes three different binding sites for 5-N-ethylcarboxamidoadenosine ([³H]NECA) were observed after separation of the extract by gel filtration on Sepharose CL-6B. The first peak was eluted in the void volume and contained the AZ adenosine receptor. In the second peak, [³H]NECA binding sites were eluted with a pharmacological profile characteristic of adenotin, a low affinity non-receptor adenosine binding protein. The third peak represented approximately 50% of the [³H]NECA binding activity. This site bound [³H]NECA in a reversible and saturable manner with K _D of 17 nmol/l and a binding capacity of 11.3 pmol/mg protein. In competition experiments, adenosine, NECA, NAD, nnosine, 5-AMP and S-adenosyl-L-homocysteine were the most potent ligands. In contrast to adenosine receptors, this site did neither bind adenosine receptor antagonists nor the A₂ selective agonist CGS 21,680 (2-[p-(2-carboxyethyl)phenethylamino]5-N-ethylcarboxamidoadenosine). These results suggest the existence of a novel high affinity binding site for adenosine of unknown function in bovine striatum.Abbreviations AMPPCP ,-methyleneadenosine-5-triphosphonate - CCPA 2-chloro-N⁶-cyclopentyladenosine - CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - CGS 21,680 2-[p-(2-carboxyethyl)phenethylamino]-5-N-ethylcarbox-amidoadenosine - CPA N⁶-cyclopentyladenosine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - GppNHp guanosine-5-[,-imido]triphosphate - GTP[S] guanosine-5-O-(3-thiotriphosphate) - NBTI S-4-nitrobenzyl-6-thioinosine - NECA 5-N-ethylcarbox-amidoadenosine - NECG 5-N-ethylcarboxamidoguanosine - PIA N⁶-phenylisopropyladenosine - SAH S-adenosyl-L-homocysteine - XAC 8-{4-[([{(2-aminoethyl)-amino}carbonyl]-methyl)oxy]-phenyl}-1,3-dipropylxanthine Send offprint requests to A. Lorenzen at the above address     

Differentiation of cardiac chronotropic and inotropic effects of β-adrenoceptor agonists

Summary The relative inotropic and chronotropic activity of -adrenoceptor agonists was studied in the noradrenaline-depleted, anaesthetized cat. Terbutaline, a selective ₂-adrenoceptor agonist, gave at a certain dose a more pronounced chronotropic than inotropic response, while a new ₁-selective adrenoceptor agonist (–)-H 80/62 produced the same degree of chronotropic and inotropic stimulation. The results indicate that there is some difference between the -adrenoceptors in the sinus node mediating chronotropic stimulation and -adrenoceptors in the ventricular myocardium mediating stimulation of the contractile force. It has been shown that there are both ₁- and ₂-adrenoceptors in the heart (Carlsson et al., 1972). In the light of this finding it is hypothetized that there are differences in the relative distribution of ₁- and ₂-adrenoceptors in the sinus node and in the myocardium. Although ₁ is the predominant type of -adrenoceptor in both regions, the ₁: ₂ concentration ratio seems to be higher in the myo-cardium, than in the sinus node.     

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 ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue.All compounds shifted the concentration-response curve of ,-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 K _d values were between 0.8 and 385 M. In the guinea-pig taenia coli, only the members of the trypan blue group were relatively potent, shifting the concentration-response curve of ADPS 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 K _d values in the trypan blue group were between 5.2 and 324 M. The removal of ATP from the medium by vas deferens tissue was decreased mainly by the members of the Evans blue group, with IC_25% values between 13 and 158 (in 1 case >1000) M.The results indicate that the position of the sulphonate residues at the terminal naphthalene rings of these compounds hardly influences P_2X purinoceptor affinity but greatly influences P_2Y 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 P_2X- versus P_2Y-selectivity presently available.