TY - JOUR A1 - Cristalli, G. A1 - Eleuteri, A. A1 - Vittori, S. A1 - Volpini, R. A1 - Lohse, M. J. A1 - Klotz, Karl-Norbert T1 - 2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamides as selective agonists at A\(_2\) adenosine receptors N2 - In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A\(_2\) receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cycla.se, and platelet aggregation studies. Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited by compounds 2a-d with K\(_i\) values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affmity binding at solubilized A\(_2\) receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [\(^3\)H]DPCPX and for the agonist [\(^3\)H]CCPA gave K\(_i\) values in the nanomolar range, and the compounds showed moderate A\(_2\) selectivity. In order to improve this selectivity, the correaponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. A\(_1\) expected, the 5'-N-ethyluronamide derivatives retained the A\(_2\) affinity whereas the A\(_1\) affinity was attenuated, resulting in an up to 10-fold increase in A\(_2\) selectivity. A similar patternwas observed in adenylate cyclase assays andin platelet aggregation studies. A 30- to 45-fold selectivity for platelet A\(_2\) receptors compared to A\(_1\) receptors was found for compounds 8a-c in adenylate cyclase studies. KW - Toxikologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60412 ER - TY - JOUR A1 - Reddington, M. A1 - Klotz, Karl-Norbert A1 - Lohse, M. J. A1 - Hietel, B. T1 - Radiation inactivation analysis of the A\(_1\) adenosine receptor: decrease in radiation inactivation size in the presence of guanine nucleotide N2 - Radiation inactivation analysis of the binding of the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes yielded a radiation inactivation size of 58 kDa. In the presence of GTPyS this was reduced to 33 kDa, in good agreement with the size of the ligand-binding subunit detected after photoaffinity labelling. The data indicate that the structural association of A\(_1\) adenosine receptors with G-protein components is altered in situ in the presence of guanine nucleotides. KW - Toxikologie KW - Adenosine receptor KW - A1 KW - Radiation inactivation KW - Target size KW - G-protein KW - (Rat brain membrane) Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60318 ER - TY - JOUR A1 - Lohse, M. J. A1 - Maurer, K. A1 - Klotz, Karl-Norbert A1 - Schwabe, U. T1 - Synergistic effects of calcium-mobilizing agents and adenosine on histamine release from rat peritoneal mast cells N2 - 1 Adenosine and its metabolically stable analogue N.etbyl-carboxamidoadenosine (NECA) enhance histamine release from rat peritoneal mast cells when tbese are stimulated by calciummobilizing agents. NECA and adenosine shift the concentration-response curve of tbe calcium ionophore A23187 to lower concentrations. 2 The potencies of NECA or adenosinein enhancing A23187-induced histamine release are dependent on the Ievel of stimulated release in tbe absence of adenosine analogues. At high Ievels of release their potencies are up to 20 times higher than at low Ievels. Consequently, averaged concentration-response curves of adenosine and NECA for enhancing bistamine release are shallow. 3 The adenosine transport blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) has no effect by itself at low Ievels of stimulated histamine release, but abolishes the enhancing effect of adenosine. At high Ievels of release, however, NBTI alone enhances the release of histamine. 4 lt is concluded that adenosine and calcium reciprocally enhance the sensitivity of the secretory processes to the effects of the other agent. The Ievels of intracellular adenosine obtained by trapping adenosine inside stimulated mast cells are sufficient to enhance histamine release substantially, suggesting that this effect may play a physiological and pathophysiological role. KW - Toxikologie Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60346 ER - TY - JOUR A1 - Klotz, Karl-Norbert A1 - Lohse, M. J. A1 - Schwabe, U. A1 - Cristalli, G. A1 - Vittori, S. A1 - Grifantini, M. T1 - 2-Chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) - a high affinity agonist radioligand for A\(_1\) adenosine receptors N2 - The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10000-fold selectivity for A1 adenosine receptors, has been examined as a new agonist radioligand. [3H]CCP A was prepared with a specifi.c radioactivity of 1.58 TBqjmmol ( 43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinity K0 -value of 0.2 nmol/1. In the presence of GTP a K0 -value of 13 nmol/1 was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [3H]CCPA binding to rat brain membranes confrrmed binding to an A1 receptor. Solubilized A1 receptors bound [3H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgC12, as has been shown for the agonist [ 3H]N6-phenylisopropyladenosine ([3H]PIA). [3H]CCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A K0 -value of 0.4 nmol/1 and a Bmax-value of 16 fmol/ mg protein was determined in these membranes. In human platelet membranes no specific binding of [3H]CCPA was measured at concentrations up to 400 nmoljl, indicating that A2 receptors did not bind [3H]CCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors [ 3H]CCPA proved to be a useful agonist radioligand for characterization of A 1 adenosine receptors also in tissues with very low receptor density. KW - Toxikologie KW - Adenosine receptors KW - Radioligauds KW - agonists Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60328 ER - TY - JOUR A1 - Lohse, M. J. A1 - Elger, B. A1 - Lindenborn-Fotinos, J. A1 - Klotz, Karl-Norbert A1 - Schwabe, U. T1 - Separation of solubilized A\(_2\) adenosine receptors of human platelets from non-receptor [\(^3\)H]NECA binding sites by gel filtration N2 - Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)dimethylammonio]- 1-propanesulfonate) and the solubilized extract subjected to gel ftltration. Binding of the adenosine receptor agonist [\(^3\)H]NECA (5'-N-ethylcarboxamidoadenosine) was measured to the eluted fractions. Two [\(^3\)H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [\(^3\)H]NECA binding activity eluted from the column. It bound [\(^3\)H]NECA in a reversible, saturable and GTPdependent manner with an affinity of 46 nmol/1 and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [\(^3\)H]NECA to the frrst peak with a pharmacological proftle characteristic for the A\(_2\) adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [\(^3\)H]NECA binding to the second, major peak. These results suggest that a solubilized A\(_2\) receptor-Gs protein complex of human platelets can be separated from other [\(^3\)H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A2 adenosine receptor of human plate1ets. KW - Toxikologie KW - A2 Adenosine receptor KW - Human platelets KW - Radioligand binding KW - Adenylate cyclase Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60309 ER - TY - JOUR A1 - Cristalli, G. A1 - Franchetti, P. A1 - Grifantini, M. A1 - Vittori, S. A1 - Klotz, Karl-Norbert A1 - Lohse, M. J. T1 - Adenosine receptor agonists: Synthesis and biological evaluation of 1-deaza analogues of adenosine N2 - In a search for more selective A\(_1\) adenosine receptor agonists, N\(^6\)-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N\(^6\)-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N\(^6\)-cyclohexyl-l-deazaadenosine (1-deazaCHA, Sc), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-ß-D-ribofuranosyl-3Himidazo[ 4,5-b]pyridine (1). On the other band, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-ß-D-ribofuranuronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-ß-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine (4), in an attempt to find a more selective A\(_2\) agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase andin radioligand binding studies. 1-DeazaNECA (10) proved tobe a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N\(^6\)-substituted 1-deazaadenosines largely retain the A\(_1\) agonist activity of their parent compounds, but lose some of their A\(_2\) agonist activity. This results in A\(_1\)-selective compounds, of which N\(^6\)cyclopentyl- 2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A\(_1\) adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A\(_1\) receptor mediated adenosine actions. KW - Toxikologie Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60262 ER - TY - JOUR A1 - Lohse, M. J. A1 - Klotz, Karl-Norbert A1 - Schwabe, U. A1 - Cristalli, G. A1 - Vittori, S. A1 - Grifantini, M. T1 - 2-Chloro-N\(^6\)-cyclopentyladenosine: a highly selective agonist at A\(_1\) adenosine receptors N2 - 2-Chloro-N\(^6\)-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for At adenosine receptors. Binding of [\(^3\)H]PIA to A1 receptors of rat brain membranes was inhibited by CCP A with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N\(^6\)-cyclopentyladenosine (CPA). Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A\(_1\)-selectivity of CCPA. CCP A inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A\(_1\) receptor, with an IC\(_{50}\)-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC\(_{50}\)-value of 3500 nM. The more than 100-fold A\(_1\)-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A\(_1\) adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard At receptor agonist R-N\(^6\) -phenylisopropyladenosine (R-PIA). CCP A represents the agonist with the highest selectivity for A\(_1\) receptors reported so far. KW - Toxikologie KW - Adenosine receptors KW - Adenylate cyclase Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60279 ER - TY - JOUR A1 - Lohse, M. J. A1 - Klotz, Karl-Norbert A1 - Diekmann, E. A1 - Friedrich, K. A1 - Schwabe, U. T1 - 2',3'-Dideoxy-N\(^6\)-cyclohexyladenosine: an adenosine derivative with antagonist properties at adenosine receptors N2 - Tbe 2',3'-dideoxy analogue of the potent A\(_1\) receptor agonist, N\(^6\)-cyclohexyladenosine (CHA), was synthesized as a potential antagonist for the A\(_1\) adenosine receptor. In sturlies on adenylate cyclase 2',3'-dideoxy-N\(^6\)-cyclohexyladenosine (ddCHA) did not show agonist properties at A\(_1\) or at A\(_2\) receptors. However, it antagonized the inhibition by R-PIA of adenylate cyclase activity of fat cell membranes via A\(_1\) receptors with a K\(_i\) value of 13 \(\mu\)M. ddCHA competed for the binding of the selective A1 receptor antagonist, [\(^3\) HJ8-cyclopentyl-1,3-dipropylxantbine ([\(^3\)H]DPCPX), to rat brain membranes with a K\(_i\) value of 4.8 \(\mu\)M; GTP did not affect the competition curve. In contrast to the marked stereoselectivity of the A\(_1\) receptor for the cx- and the natural ß-anomer of adenosine, the cx-anomer of ddCHA showed a comparable affinity for the A\(_1\) receptor (K\(_i\) value 13.9 \8\mu\)M). These data indicate that the 2'- and 3'-hydroxy groups of adenosine and its derivatives are required foragonist activity at and high affinity binding to A\(_1\) adenosine receptors and for the distinction between the cx- and ß-forms. KW - Toxikologie KW - Adenosine receptors KW - Adenylate cyclase KW - Adenosine receptor antagonists Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60282 ER - TY - JOUR A1 - Klotz, Karl-Norbert A1 - Lohse, M. J. A1 - Schwabe, U. T1 - Chemical modification of A\(_1\) adenosine receptors in rat brain membranes - evidence for histidine in different domains of the ligand binding site N2 - Chemical modification of amino acid residues was used to probe the ligand recognition site of A\(_1\) adenosine receptors from rat brain membranes. The effect of treatment with group·specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N\(^6\)-[\(^3\)H]phenylisopropyladenosine ([\(^3\)H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [\(^3\)H]8- cyclopentyl-1 ,3-dipropylxanthine ([\(^3\)H]DPCPX). Antagonists protected A\(_1\) receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [\(^3\)H]DPCPX, but reduced receptor number. From the selective protection of [\(^3\)H] PIA and [\(^3\)H]DPCPX binding from inactivation, it is concluded that agonists and antagonists oocupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of tbe inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [\(^3\)H]PIA and [\(^3\)H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the Iigand binding site. In addition, it was shown that no further amino acids witb polar side chains are present. The absence of bydrophilic amino acids frout the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A\(_1\) receptors. KW - Toxikologie Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60295 ER - TY - CHAP A1 - Lohse, M. J. A1 - Klotz, K.-N. A1 - Schwabe, U. A1 - Christalli, G. A1 - Vittori, S. A1 - Grifantini, M. T1 - Pharmacology and Biochemistry of Adenosine Receptors N2 - Adenosine modulates a variety of physiological functions via membrane-bound receptors. These receptors couple via G proteins to adenylate cyclase and K+channels. The A1 subtype mediates an inhibition of adenylate cyclase and an opening of K+-channels, and the A2 subtype a Stimulation of adenylate cyclase. Both subtypes have been characterized by radioligand binding. This has facilitated the development of agonists and antagonists with more than 1000-fold A1 selectivity. A1-selective photoaffinity labels have been used for the biochemical characterization of A1 receptors and the study of their coupling to adenylate cyclase. Such selective ligands allow the analysis of the involvement of adenosine receptors in physiological functions. Selective interference with adenosine receptors provides new pharmacological tools and eventually new therapeutic approaches to a number of pathophysiological states. KW - Adenosinrezeptor KW - Pharmakologie KW - Toxikologie Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86251 ER - TY - JOUR A1 - Lohse, M. J. A1 - Böser, S. A1 - Klotz, Karl-Norbert A1 - Schwabe, U. T1 - Affinities of barbiturates for the GABA-receptor complex and A\(_1\) adenosine receptors: A possible explanation of their excitatory effects N2 - The effects of barbiturates on the GABA·receptor complex and the A\(_1\) adenosine receptor were studied. At the GABA-receptor complex the barbiturates inhibited the binding of [\(^{35}\)S]t-butylbicyclophosphorothionate [\(^{35}\)S]TBPT) and enhanced the binding of [\(^3\)H]diazepam. Kinetic and saturation experiments showed that both effects were allosteric. Whereas all barbiturates caused complete inhibition of [\(^{35}\)S]TBPT binding, they showed varying degrees of maximal enhancement of [\(^3\)H]diazepam binding; (±)methohexital was idenafied as the most efficacious compound for this enhancement. At the A\(_1\) adenosine receptor all barbiturates inhibited the binding of [\(^3\)H]N\(^6\)-phenylisopropyladenosine (\(^3\)H]PIA) in a competitive manner. The comparison of the effects on [\(^3\)H]diazepam and [\(^3\)H]PIA binding showed that excitatory barbiturates interact preferentially with the A\(_1\) adenosine receptor, and sedative/anaesthetic barbiturates with the GABA-receptor complex. It is speculated that the interaction with these two receptors might be the basis of the excitatory versus sedative/ anaesthetic properties of barbiturates. KW - Toxikologie KW - GABA-receptor complex KW - Adenosine receptors KW - Barbiturates Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60250 ER - TY - JOUR A1 - Lohse, M. J. A1 - Klotz, Karl-Norbert A1 - Lindenborn Fotinos, J. A1 - Reddington, M. A1 - Schwabe, U. A1 - Olsson, R. A. T1 - 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) - a selective high affinity antagonist radioligand for A\(_1\) adenosine receptors N2 - The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A\(_1\) adenosirre receptors were examined and conipared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A\(_1\) adenosirre receptors and the stimulationvia A\(_2\) adenosirre receptors. The K\(_i\)-values of this antagonism were 0.45 nM at the A\(_1\) receptor of rat fat cells, and 330 nM at the A\(_2\) receptor of human platelets, giving a more than 700-fold A\(_1\)-selectivity. A similar A\(_1\)-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMPphosphodiesterase activity of human platelets. [\(^3\)H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A\(_1\) receptors in membranes of bovine brain and heart, and rat brain and fat cells (K\(_D\) -values 50-190 pM). Its nonspecific binding was about 1% of total at K\(_D\) , except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [\(^3\)H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A\(_1\) receptor. KW - Toxikologie KW - Adenosine receptors KW - Adenylate cyclase KW - Phosphodiesterase KW - Xanthines KW - Radioligands Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60246 ER - TY - JOUR A1 - Klotz, Karl-Norbert A1 - Lohse, M. J. A1 - Schwabe, U. T1 - Characterization of the solubilized A\(_1\) adenosine receptor from rat brain membranes N2 - A\(_1\) adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-( cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A\(_1\) adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-\(N^6\)-[\(^3\)H]phenylisopropyladenosine([\(^3\)H]PJA) with K\(_D\) values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A\(_1\) adenosine receptors could be labelled not only with the agonist [\(^3\)H]PIA but also with the antagonist I ,3-diethyi-8-[\(^3\)H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein N; and that all regulatory functions are retained on solubilization. Key Words: A1 adenosine receptors - Solubilization- Rat brain membranes. Klotz K.-N. et al. Characterization of the solubilized A1 adenosine receptor from rat brain membranes. J. Neurochem. 46, 1528-1534 (1986). KW - Toxikologie KW - A1 adenosine receptors KW - solubilization KW - rat brain membranes Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60222 ER - TY - JOUR A1 - Klotz, Karl-Norbert A1 - Lohse, M. J. T1 - The glycoprotein nature of A\(_1\) adenosine receptors N2 - A\(_1\) adenosine receptors from different tissues and species we~e photoaffinity labelled and then the carbohydrate content was examined by both enzymatic and chemical treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labelled membrane receptors shows that neuraminidase treatment alters the electrophoretic mobility of the receptor band indica ting the presence of terminal neurandnie acids. Neuraminidase digestion does not influence the binding characteristics of the receptor. The totally deglycosylated receptor protein obtained by chemical treatment has an apparent molecular weight Of 32,000. KW - Toxikologie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60231 ER - TY - JOUR A1 - Lohse, M. J. A1 - Klotz, Karl-Norbert A1 - Jakobs, K. H. A1 - Schwabe, U. T1 - Barbiturates are selective antagonists at A\(_1\) adenosine receptors N2 - Barbiturates in pharmacologically relevant . concentrations inhibit binding of (R)-\(N^6\)-phenylisopropyl[\(^3\)H]adenosine ([\(^3\)H]PIA) to solubilized A\(_1\) adenosine receptors in a concentration-dependent, stereospecific, and competitive manner. K\(_i\) values are similar to those obtained for membrane-bound receptors and are 31 \(\mu\)M for ( ± )-5-(1 ,3-dimethyl)-5-ethylbarbituric acid [( ± )DMBB] and 89 \(\mu\)M for ( ± )-pentobarbital. Kinetic experiments demoostrate that barbiturates compete directly for the binding site of the receptor. The inhibition of rat striatal adenylate cyclase by unlabelled (R)-\(N^6\)-phenylisopropyladenosine [(R)-PIA] is antagonized by barbiturates in the same concentrations that inhibit radioligand binding. The Stimulation of adenylate cyclase via A\(_2\) adenosine receptors in membranes from NIE 115 neuroblastoma cells is antagonized only by 10-30 times higher concentrations of barbiturates. lt is concluded that barbiturates are selective antagonists at the A1 receptor subtype. In analogy to the excitatory effects of methylxanthines it is suggested that A\(_1\) adenosine receptor antagonism may convey excitatory properties to barbiturates. Key Words: Adenosine receptors-Barbiturates - Adenylate cyclase-Receptor solubilization-[3H]PIA binding-N1E 115 cells. Lohse M. J. et al. Barbiturates are selective antagonists at A1 adenosine receptors. KW - Toxikologie KW - adenosine receptors KW - barbiturates KW - adenylate cyclase KW - receptor solubilization KW - N1E 115 cells KW - [3H]PIA binding Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60187 ER - TY - JOUR A1 - Klotz, Karl-Norbert A1 - Cristalli, G. A1 - Grifantini, M. A1 - Vittori, S. A1 - Lohse, M. J. T1 - Photoaffinity labeling of A\(_1\) adenosine receptors JF - The journal of biological chemistry N2 - The ligand-binding subunit of the A\(_1\)-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R- \(N^6\)-phenylisopropyladenosine, R-2-azido-\(N^6\)-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific Iigand for A\(_1\)-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R·AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A\(_1\)-subtype. It competes for [\(^3\)H].\(N^6\)- phenylisopropyladenosine binding to Arreceptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [\(^3\)H)\(N^6\)-phenylisopropyladenosine binding afterextensive washing; the K; value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity Iabel of high specific radioactivity (\(^{125}\)I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for \(^{125}\)I-AHPIA binding to rat brain membranes with an order of potency characteristic for A\(_1\)-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of M\(_r\) = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A\(_1\)-subtype. The results indicate that \(^{125}\)I-AHPIA identifies the ligand-binding subunit of the A\(_1\)-adenosine receptor, which is a peptide with M\(_r\) = 35,000. KW - Toxikologie Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60198 VL - 27 IS - 260 ER - TY - JOUR A1 - Lohse, M. J. A1 - Klotz, K.-N. A1 - Ukena, D. A1 - Schwabe, U. T1 - Characterization of \([^3H]\)Phenobarbital Binding to Rat Brain Membranes JF - Neuroscience Letters N2 - The binding of \([^3H]\)phenobarbital to rat brain membranes was studied in order to determine its characteristics and specificity. The binding reaction was rapid and occurred at sites of low affinity. \((K_d = 700 μM)\) and very high density \((B_{max} = 2.7 nmoll/mg protein)\). It was unaffected by temperature changes from O°C to 95°C and was maximal at pH 5. Detergents in low concentrations markedly decreased the binding, apparently without solubilizing the binding sites. It is concluded that the binding of \([^3H]\) phenobarbital is a rather non-specific interaction with the plasma membrane. KW - barbiturates KW - radioligand binding KW - rat brain membranes Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127894 VL - 52 ER -