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     

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     

Effects of metal cations on [3H]α,β-methylene ATP binding in rat vas deferens

In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [³H],-methylene ATP ([³H]meATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [³H]meATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl₃ and AlCl₃ (0.1 to 100 M), produced the greatest increases in total binding of [³H]meATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 M and 1–10 mM, increased total binding of [³H]meATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC₅₀ values for potentiating binding of the radioligand were obtained: ZnCl₂ (5.44), MnCl₂ (4.52), CaCl₂ (4.17), CoCl₂ (4.06), MgCl₂ (3.67) and BaCl₂ (3.10). Both EDTA and EGTA (0.01–1 mM) inhibited the binding of the radioligand.The effects of ZnCl₂, CaCl₂ and MgCl₂ were examined in saturation studies. In the absence of added divalent cations, [³H]meATP labelled both high (pK_d = 9.15) and low (pK_d = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl₂ (pK_d = 9.56) and by 30 M ZnCl₂ (pK_d = 9.46) but not by 3 mM MgCl₂. The B_max of the low affinity site for [³H]meATP was increased (approximately 4 fold) by both 3 mM MgCl₂ and 30 M ZnCl₂ but not by 3 mM CaCl₂. The selective effect of CaCl₂ on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl₂ using a low concentration of [³H]meATP (1 nM); the sites exhibited the binding characteristics expected of the P_2x purinoceptor. The selective effect of MgCl₂ on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl₂ and using a high concentration of [³H]meATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [³H]meATP revealed several other differences, in addition to their cation selectivity. First, the adenine analogues ADP, meATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for the high affinity [³H]meATP binding sites than for the low affinity binding sites. Secondly, GTP--S and pyrophosphate were selective ligands for the low affinity [³H]meATP binding sites possessing approximately 43 and 1995 fold, respectively, higher pIC₅₀ values at the low affinity sites than at the high affinity sites. Finally, treatment of the membranes with 0.01–1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of the low affinity sites suggest that they do not equate with functional P_2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [³H]meATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity.In conclusion the present study has demonstrated that cations have a marked effect on the binding of [³H]meATP in rat vas deferens. Of particular interest was the ability of CaCl₂ to increase the affinity of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl₂ to increase the B_max of the low affinity binding sites enabled these sites to be selectively labelled. Correspondence to: A. D. Michel at the above address     

Cultured chick sympathetic neurons: modulation of electrically evoked noradrenaline release by P2-purinoceptors

The present study investigates the pharmacological profile of P₂-purinoceptors modulating noradrenaline release from cultured chick sympathetic neurons. ATP (30 M-3 mM) and 2-methylthio-ATP (3–100 M), but not ,-methylene-ATP (up to 100 M), caused a significant facilitation of electrically evoked [³H]-noradrenaline release when added 2 min before depolarization. The facilitation declined with time of exposure suggesting receptor desensitization. The facilitatory effect was markedly diminished by the P₂-purinoceptor antagonists reactive blue 2 (3 M) and suramin (300 M), but not changed by mecamylamine (10 M), a nicotinic receptor antagonist. At 1 mM and higher concentrations, ATP added for 12 min, inhibited noradrenaline release; release was virtually abolished by 6 mM ATP. The inhibitory effect of ATP was slightly diminished by suramin but not affected by reactive blue 2. Electrically evoked [³H]-noradrenaline release remained unaffected in the presence of the adenosine (P₁)-receptor agonists R(–)N⁶-(2-phenylisopropyl)adenosine (R-PIA), 2-[p-(2-carboxyethyl)phenylethylamino]5-N-ethylcarboxamidoadenosine (CGS-21680), 5-N-ethylcarboxamidoadenosine (NECA), and N⁶-2-(4-aminophenyl)ethyladenosine (APNEA), used up to 1 M.The present results confirm the existence of two P₂-purinoceptors affecting noradrenaline release: 1) a facilitatory receptor which is activated by 2-methyl thio-ATP as well as ATP, and blocked by suramin as well as reactive blue 2, and 2) an inhibitory receptor which is activated by ATP, only slightly affected by suramin but not at all by reactive blue 2 and does not belong to the established P₂-purinoceptor subtypes.     

Prejunctional opioid μ-receptors and adenosine A1-receptors on the sympathetic nerve endings of the rat tail artery interact with the α2-adrenoceptors

Summary Experiments were designed to study the interaction between prejunctional ₂-adrenoceptors and both adenosine and opioid receptors at the postganglionic sympathetic nerve endings innervating the tail artery of the rat. Segments of this vessel were preincubated with [³H]-noradrenaline and then perfused/superfused with [³H]-noradrenaline-free medium. Their perivascular nerves were field stimulated with standard stimulation parameters: 24 pulses at 0.4 Hz, 0.3 ms, 200 mA. In some experiments, the stimulation parameters were adjusted in order to obtain similar reference release values despite the presence of a first release-modulating drug.The adenosine agonist 5-N-ethylcarboxamidoadenosine (NECA; 0.3–10 mol/l) and [D-Ala²,MePhe⁴,Glyol⁵]enkephalin (DAGO; 0.3–10 mol/l) depressed the stimulation-evoked overflow of tritium in a concentration dependent manner. The release-inhibiting effect of both NECA and DAGO was enhanced in the presence of the ₂-adrenoceptor antagonist rauwolscine (3 mol/l) while it was attenuated in the presence of the ₂-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2yl-aminol-quinoxaline (UK-14,304; 0.1 mol/l). These changes occurred both at standard and adjusted stimulation parameters.These results demonstrate that the prejunctional adenosine A₁- and opioid -receptors interact with the prejunctional ₂-adrenoceptors. The level at which these interactions take place (receptors themselves or transduction mechanisms) as well as the physiological significance of the phenomenon remain to be determined. Send offprint requests to B. Bucher at the above address     

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     

Subtype specificity of γ-aminobutyric acid type A receptor antagonism by clozapine

Clozapine, an atypical neuroleptic, functionally antagonizes the -aminobutyric acid-induced chloride uptake via the main central inhibitory receptor, -aminobutyric acid type A (GABA_A) receptor, in brain vesicles. GABA_A antagonism by micromolar concentrations of clozapine is more efficient in rat cerebrocortical and hippocampal membranes than in cerebellar membranes, as evidenced by clozapine reversal GABA-inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding. A typical neuroleptic, haloperidol, failed to antagonize GABA in any of these brain regions, while the specific GABA_A antagonist 2-(3-carboxy-2,3-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (SR 95531) was efficient in all three brain regions. Clozapine action on [³⁵S]TBPS binding was unaffected by the benzodiazepine receptor antagonist flumazenil. Clozapine inhibited the binding of [³H]muscimol and [³H]SR 95531 to the GABA recognition site, but this effect only partially correlated with the regional differences in and the potency of clozapine antagonism of GABA-inhibition of [³⁵S]TBPS binding, suggesting that also other than GABA sites may mediate clozapine actions. Autoradiography of [35S]TBPS binding revealed GABA antagonism by clozapine in most brain regions. Main exceptions were cerebellar granule cell and molecular layers, olfactory bulb external plexiform and glomerular layers and primary olfactory cortex, where clozapine antagonized GABA inhibition less than average, and lateral hypothalamic and preoptic areas where its antagonism was greater than average. Recombinant 622 receptors, the predominant 6 subunit-containing receptor subtype in cerebellar granule cells, failed to show GABA antagonism by clozapine up to 100 M. In contrast, recombinant 122 receptors, forming the predominant receptor subtype in the brain, were clozapine sensitive. Recombinant 622 and 632 receptors resulted in clozapine-insensitive receptors, whereas 612 receptors were clozapine sensitive. The efficacy of clozapine to antagonize GABA in 1x2 receptors decreased in the order of 112>122>132. The results indicate that clozapine antagonizes the function of most GABA_A receptor subtypes, and that the interaction is determined by the interaction of the and subunit variants. GABA antagonism is a unique property of clozapine, not shared by haloperidol, which might be involved in the pharmacological mechanism for the increased seizure susceptibility associated with clozapine treatment.     

Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications

We measured in human atrium the density of 5-HT₄ receptors, labelled with [¹²⁵I]-SB 207710 (1-butyl-4-piperidinyl) methyl 8-amino-7-iodo-1, (4-benzodioxan-5-carboxylate), and compared it with the density of ₁- and ₂-adrenoceptors, labelled with (–)-[¹²⁵I]-cyanopindolol. [¹²⁵I]-SB 207710 (5–1200 pmol/l) labelled a small population of saturable binding sites (B _max 4 fmol/mg protein) with a pK_D of 9.7 and with 5-HT₄ receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT₄ receptor characteristics was 10 and 5 times lower, respectively, than the density of ₁- and ₂-adrenoceptors. We suggest that the small 5-HT₄ receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ₁- and ₂-adrenoceptors.     

Inhibition by nucleotides acting at presynaptic P2-receptors of sympathetic neuro-effector transmission in the mouse isolated vas deferens

Summary Effects of nucleotides and nucleosides on smooth muscle tension and the release of previously stored [³H]-noradrenaline were studied in the mouse isolated vas deferens. The tissue was stimulated twice by 20 electrical field pulses delivered at 2 Hz (S₁, S₂)., \-Methylene-ATP, ATPS, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine (up to 100 mol/1) and uridine (up to 1 mmol/1). The electrically evoked overflow of tritium was reduced by the drugs in the following order of potency: ATPS > ATP = adenosine > UTP; ,\-meth-ylene-ATP (up to 10 µmol/l) and uridine (up to 1 mmol/1) did not significantly change the evoked overflow. 8-(p-Sulphophenyl)theophylline did not alter the contractile responses to the nucleotides; it prevented the overflow-inhibiting effect of adenosine and reduced that of UTP; the overflow-inhibiting effects of ATP and ATPS were not significantly attenuated. After prolonged exposure to ,-methylene-ATP, all contractile nucleotide effects were abolished; in contrast, the depression by adenosine and the nucleotides of the evoked overflow of tritium persisted. None of the effects was changed by indometacin, yohimbine or reactive blue 2.It is concluded that ATP, ATPS, ,\-methylene-ATP and UTP produce contraction of the vas deferens by activation of P_2x-receptors. Moreover, the nucleotides inhibit per se the release of [3H]-noradrenaline (and presumably the co-transmitter mixture of noradrenaline and ATP); the effect of ATP is not, or only to a small extent, due to breakdown to adenosine. The presynaptic site of action of the purine nucleotides is a P₂-receptor which differs from the P_2X-receptor and may be a reactive blue 2-resistant P_2y-like receptor.     

Subclassification of presynaptic 5-HT autoreceptors in the human cerebral cortex as 5-HT1Dβ receptors

Human cerebral cortical synaptosomes were used to determine the 5-hydroxytryptamine (5-HT) receptor subtype to which the inhibitory presynaptic 5-HT autoreceptor belongs. The synaptosomes preincubated with [³H]5-HT were superfused and tritium overflow was stimulated by high K⁺. The K⁺-evoked tritium overflow, which was Ca²⁺-dependent but tetrodotoxin-resistant, was concentration-dependently inhibited by the nonselective 5-HTlD_1D/1D receptor agonist, 5-carboxamidotryptamine. Ketanserin at a concentration which should block the 5-HT_1D but not the 5-HT_1D receptor failed to antagonize the inhibitory effect of 5-carboxamidotryptamine. In contrast, the non-selective 5-HT_1D/1D receptor antagonist, methiothepin, at a concentration which should block both the 5-HT_1D and the 5-HT_1D receptor abolished the effect of 5-carboxamidotryptamine. It is concluded that the presynaptic 5-HT autoreceptor, which has previously been classified as 5-HT_1D, belongs to the 5-HT_1D subtype.     

Pharmacokinetics,anticonvulsant efficacy,and adverse effects of trans-2-en-valproate after acute and chronic administration in amygdala-kindled rats

Summary Effects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz.Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADPS, ATPyS, ,-imido-ATP and ,-methyl-ene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine > ATP ATPyS ,-imido-ATP ADP > ,-methylene-ATP. AMP 30 Etmol/l and AMPS 30 mol/l were approximately equieffective with 30 mol/l of adenosine and ATPS, and ADPS, 30 mol/l was approximately equieffective with 30 mol/l of ADP. ,-Methylene-ADP, 2-methylthio-ATP, UTP and GTPS did not change the evoked overflow of tritium. ,-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to ,-methylene-ATP or when the slices were stimulated by 30 pulses, 10 H₂. Neither adenosine deaminase (100 U/l) nor the blocker of 5-nucleotidase, ,-methylene-ADP (10 mol/l), attenuated the inhibition caused by ATP, ATPyS and ,-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATPyS, ,-imido-ATP and ,-methylene-ATP to the right by very similar degrees. 8(p-Sulphophenyl)-theophylline (30 and 300 mol/l) also markedly antagonized the inhibition produced by ATPS. ,-Methylene-ATP (10 and 30 mol/l) and suramin (100 gmol/l) did not modify the effects of adenosine, ATPS and ,-methylene-ATP.It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A₁ subtype of the P₁-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATPS, ,-imido-ATP and ,-methylene-ATP can be potent P₁ agonists. No evidence was found for presynaptic P_2X-, P_2Y- or P₃-purinoceptors. Send offprint requests to I. von Kugelgen at the above address     

Phosphate and thiophosphate group donating adenine and guanine nucleotides inhibit glibenclamide binding to membranes from pancreatic islets

Summary In microsomes obtained from mouse pancreatic islets, the Mg complex of adenosine 5-triphosphate (MgATP) increased the dissociation constant (K _D) for binding of [³H]glibenclamide by sixfold. In the presence of Mg²⁺, not only ATP but also adenosine 5-0-(3-thiotriphosphate) (ATPS), adenosine 5-diphosphate (ADP), guanosine 5-triphosphate (GTP), guanosine 5-diphosphate (GDP), guanosine 5-0-(3-thiotriphosphate) (GTPTS) and guanosine 5-0-(2-thiodiphosphate) (GDP S) inhibited binding of [³H]glibenclamide. These effects were not observed in the absence of Mg²⁺. Half maximally effective concentrations of the Mg complexes of ATP, ADP, ATPS and GDP were 11.6, 19.0, 62.3 and 90.1 mol/l, respectively. The non-hydrolyzable analogues adenosine 5-(,-imidotriphosphate) (AMP-PNP) and guanosine 5-(,-imidotriphosphate) (GMP-PNP) did not alter [³H]glibenclamide binding in the presence of Mg²⁺. MgADP acted much more slowly than MgATP and both MgADP and MgADP did not inhibit [³H]glibenclamide binding when the concentrations of MgATP and MgATP were kept low by the hexokinase reaction. Development of MgATP-induced inhibition of [³H]glibenclamide binding and dissociation of [³H]-glibenclamide binding occurred at similar rates. However, the reversal of MgATP-induced inhibition of [³H]glibenclamide binding was slower than the association of [³H]glibenclamide with its binding site. Exogenous alkaline phosphatase accelerated the reversal of MgATP-induced inhibition of [³H]glibenclamide binding. MgATP enhanced displacement of [³H]glibenclamide binding by diazoxide. The data suggest that sulfonylureas and diazoxide exert their effects by interaction with the same binding site at the sulfonylurea receptor and that protein phosphorylation modulates the affinity of the receptor.Some of the results described here are part of the medical theses of S. Löser and I. Rietze Send offprint requests to M. Schwanstecher at the above address     

Functional evidence for the presence of adenosine A2-receptors in cultured coronary endothelial cells

Summary Adenosine and the adenosine receptor agonists, R- and S-N⁶-phenylisopropyladenosine (R- and S-PIA) and 5-N-ethylcarboxamidoadenosine (NECA), enhanced [³H]cAMP accumulation in [³H]adenine-labelled cultured endothelial cells isolated from the microvasculature of guinea pig hearts. As shown by their concentration-response curves, NECA was a more potent agonist than R-PIA or adenosine. Their respective concentrations at half-maximal stimulation of [³H]cAMP accumulation were 0.7 M, 10.5 M and 12.6 M, indicating a 15- to 18-fold potency difference between NECA and the other agonists. The increased [³H]cAMP accumulation elicited by 10^–5 M NECA was inhibited by the xanthine derivative 8-phenyltheophylline, 3-isobutyl-l-methylxanthine, theophylline or caffeine. These findings provide functional evidence for the presence of adenosine receptors of the A₂-type in microvascular coronary endothelial cells in culture. The functional significance of these receptors remains to be established, but they may be involved in the regulation of vascular permeability.Abbreviations NECA 5-N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopropyladenosine - 8-PT 8-phenyltheophylline - IBMX 3-isobutyl-l-methylxanthine - IBMX shirlyDL,-4-(3-butoxy-4methoxybenzyl)-2-imidazolidinone Postdoctoral fellow of the Medical Research Council of Canada Send offprint requests to S. Nees at the above address     

Adenosine but not an adenine nucleotide mediates tonic purinergic inhibition,as well as inhibition by glutamate,of noradrenaline release in rabbit brain cortex slices

Summary A possible contribution of adenine nucleotides to the endogenous purinergic, A₁-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [³H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A₁-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1–100 nmol/l) as well as the enzyme adenosine deaminase (0.1–10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The ₁-adrenoceptor-selective agonist methoxamine (10 but not 1 mol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mol/l but then not attenuated further by DPCPX100 nmol/l.L-Glutamate (300 mol/l–2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mol/l but greatly, and to the same extent, attenuated by DPCPX 100 mol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg⁺⁺ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mol/l and 1 mol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg⁺⁺). It is concluded that adenosine but not an adenine nucleotide mediates the tonic purinergic presynaptic inhibition of noradrenaline release in rabbit brain cortex. The marked degree of disinhibition by DPCPX and adenosine deaminase underscores the potential physiological role of this inhibition. The purinergic inhibitory tone is reinforced by glutamate, indicating that glutamate releases adenyl compounds in rabbit brain cortex. Again adenosine but not an adenine nucleotide mediates the indirect inhibition by glutamate of the release of noradrenaline. The noradrenaline-releasing effect that glutamate exerts in rat occipito-parietal cortex does not occur in rabbit occipito-parietal cortex. Methoxamine depresses the release of noradrenaline in rabbit brain cortex directly at presynaptic ₂-adrenoceptors rather than by release of purines.Correspondence to I. von Kügelgen at the above address     

Inhibitory adenosine A1-receptors on rat locus coeruleus neurones

Summary Intracellular recordings were performed in ₁-pontine slice preparation of the rat brain containing the locus coeruleus (LC). Adenosine (100, 300 mol/l) and its structural analogues, namely (–)-N⁶-(R-phenyliso-propyl)-adenosine (R-PIA; 3 – 30 mol/l) and S-PIA (10, 30 mol/l), as well as 5-N-ethylcarboxamido-adenosine (NECA; 3–30 mol/l) inhibited the firing rate of spontaneous action potentials and produced hyperpolarization; their rank order of potency was RPIA - NECA > S-PIA > adenosine. When applied by superfusion, all agonists strongly desensitized the LC cells; the hyperpolarization never surmounted 6 mV. Upon pressure ejection of adenosine 10 mmol/l from ₁- micropipette positioned close to an LC neurone, the membrane potential was raised by 14 mV and the apparent input resistance decreased by 20%. When the membrane potential was hyperpolarized by current injection to ₁- similar extent as adenosine did, the fall in input resistance was only 7%. The adenosine uptake inhibitor S-(p-nitrobenzyl)-6-thioguanosine (NBTG) 30 mol/l decreased the frequency of action potentials alone; on simultaneous bath-application with adenosine 300 mol/l it potentiated the hyperpolarization caused by the purine derivative. 8-Cyclopentyl-1,3-dipropylxanthine (CPDPX) 0.1 mol/l had no effect on its own, but it antagonized both R-PIA 30 mol/l and NBTG 30 mol/l. A higher concentration of CPDPX (1 mol/l) facilitated the spontaneous firing. In conclusion, both exogenous and endogenous adenosine activates somatic and/or dendritic A₁-receptors of LC neurones leading to an enhancement of potassium conductance and thereby to ₁- decreased firing rate and ₁- hyperpolarization. Send offprint requests to P. Illes 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     

Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [³H]-noradrenaline was investigated.Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline (0.05 M), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 LM-3 mM), 2-methylthio-ATP (3–100 M), as well as DMPP (10 and 100 M) induced a significant overflow of tritium. The EC₅₀-value of ATP was 20 M. Both the ATP-induced and the DMPP-induced tritium overflow was Ca²⁺-dependent and sensitive to tetrodotoxin (0.3 M) and -conotoxin (0.1 M); in addition, it was inhibited by the ₂-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 M). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P₂-purinoceptor antagonists suramin (300 M) and reactive blue 2 (3 M); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 M) and markedly inhibited by hexamethonium (100 M). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [³H]-choline.The results suggest that ATP at molar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.     

Cultured chick sympathetic neurons: prostanoid EP1 receptor-mediated facilitation of noradrenaline release

Prostanoid EP receptor-mediated modulation of noradrenaline release from cultured chick sympathetic neurons was investigated. Transmitter release from dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline, was elicited either by electrical field stimulation (36 pulses/3 Hz) or by elevating the extracellular concentration of K⁺ (to 30 mM; for 2 min).Prostaglandin E₂ (PGE₂; 0.01–3 M) enhanced electrically evolved [³H]-noradrenaline release in a concentration-dependent manner with a maximal increase by about 50% at 1 M. Also iloprost (0.1–3 M) increased transmitter release concentration-dependently, whereas misoprostol (0.1–3 M) had no effect. Indometacin (10 M) influenced neither evoked release per se nor the enhancement caused by PGE₂. AH6809 (3 M), a selective EP₁ receptor antagonist, blocked the enhancement caused by both PGE₂ and iloprost. K⁺-evoked noradrenaline release, which was virtually insensitive to tetrodotoxin (0.3 M), was increased by PGE₂ to an extent comparable to that observed after electrical stimulation.In summary, the present data indicate that PGE₂ facilitates noradrenaline release from cultured chick sympathetic neurons by a receptor which shows the pharmacological profile of the EP₁ subtype and is probably located at the processes of the neuron.     

Enhancement of adenosine A1 receptor functions by benzoylthiophenes in guinea pig tissues in vitro

Previous reports on a series of benzoylthiophenes, including PD 81,723 {2-amino-4,5-dimethyl-3-(3-trifluoromethyl-benzoyl)thiophene}, have shown specific enhancement of agonist binding at the adenosine A₁ receptor. We have studied the effects of two substituted benzoylthiophenes, PD 78,416 {thieno[2,3-c]pyridine-6(5H)-carboxylic acid, 2-amino-3-benzoyl-4,7-dihydro-ethyl ester} and RS-74513-000 {2-amino-4-ethyl-5-methyl-3-(3-trifluoro-methyl-benzoyl) thiophene} on response elicited by adenosine A₁ receptors in isolated guinea pig left atrium and ileum.In the electrically paced left atrium, PD 78,416 antagonized negative inotropic effect elicited by the agonist CPA {N⁶-cyclopentyladenosine} with a pK_B value of 6.2 ± 0.2 (n = 4) . At a low concentration which had no antagonistic effect (0.1 M), PD 78,416 enhanced the effect of CPA. The concentration-response curve to CPA was shifted leftward by 5.1 fold (95% confidence limits 2.4–11.2). In field stimulated isolated ileum, PD 78,416 (0.1, 0.3, 1 M) did not enhance or antagonize effects of CPA. At concentrations above 1 M, PD 78,416 decreased electrically induced contraction. This effect was not sensitive to adenosine deaminase and was not antagonized by the A₁ antagonist CPX {8-cyclopentyl-1,3-dipropyl-xanthine} (1 M).Unlike PD 78,416, RS-74513-000 (0.01, 0.1, 1, 3, 10 M) did not antagonize or enhance effects of CPA in the left atrium. However, effects of CPA in ileum were enhanced by RS-74513-000 (1 and 3 M). Maximum enhancement was observed at 3 M; the concentration-response curve to CPA was shifted leftward by 3.2 fold (95% confidence limits 2.4–4.2). Higher concentrations of RS-74513-000 (10 and 30 M) decreased electrically induced contraction, this effect was not reversed by CPX. These findings confirmed that functional effects of A₁ adenosine receptor may be enhanced by substituted benzoylthiophenes in vitro. The differential effect of PD 78,416 and RS-74513-000 on cardiac and ileal A₁ receptors suggests that it may be possible to design selective enhancers for cardiac and neural functions.     

Modulation of 5-hydroxytryptamine release by presynaptic inhibitory α2-adrenoceptors in the human cerebral cortex

Summary Slices and synaptosomes from human cerebral cortex (which had to be removed to reach deeply located tumours) and, for comparison, synaptosomes from guinea-pig and rat cerebral cortex were preincubated with [³H]5-hydroxytryptamine and superfused with physiological salt solution containing an inhibitor of 5-hydroxytryptamine uptake. The effects of -adrenoceptor agaonists and antagonists on the electrically (slices) or potassium-evoked (synaptosomes) tritium overflow were studied.In human cerebral cortical slices, the electrically-evoked [³H] overflow was inhibited by noradrenaline (pIC₂₅ value: 6.35); the non-selective -adrenoceptor antagonist phentolamine, at a concentration of 0.32 mol/l, strongly antagonized the inhibitory effect of noradrenaline (apparent pA₂ value: 8.19) but did not affect the evoked overflow by itself. In synaptosomes from humans, guinea-pigs and rats, noradrenaline also inhibited the K⁺-evoked[³H] overflow in a concentration dependent manner; the ₂-adrenoceptor clonidine (1 mol/l), but not the ₁-adrenoceptor agonist methoxamine (1 mol/l), mimicked the effects of noradrenaline; the effect of noradrenaline (0.3 mol/l) was abolished by the ₂-but not by the ₁-adrenoceptor antagonist prazosin (1 mol/l).It is concluded that release-inhibiting adrenoceptors of the ₂-subtype exist on 5-hydrpxytryptamine terminals innervating the cerebral cortex in human and guinea-pig brain.Send offprint requests to M. Raiteri at the above address