TY - JOUR A1 - Nolte, D. A1 - Lorenzen, A. A1 - Lehr, H.-A. A1 - Zimmer, F.-J. A1 - Klotz, Karl-Norbert A1 - Messmer, K. T1 - Reduction of postischemic leukocyte-endothelium interaction by adenosine via A\(_2\) receptor N2 - The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. leukocytes were stained in vivo with acridine orange (0.5 mg kg\(^{-1}\) min\(^{-1}\) i.v. ). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. ·Adenosine, the adenosine A1-selective agonist 2-chloro-N\(^6\) -cyclopentyladenosine (CCPA), the Arselective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener {XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused viajugular vein starting 15 min priortorelease of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p< 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces postischemic leukocyte-endothelium interaction via A\(_2\) receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion. KW - Toxikologie KW - Adenosine receptors KW - Ischemia/reperfusion KW - Leukocyte/endothelium interaction KW - Microcirculation Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60424 ER - TY - JOUR A1 - Bommakanti, R. A1 - Bokoch, G. M. A1 - Tolley, J. O. A1 - Schreiber, R. E. A1 - Siemsen, D. W. A1 - Klotz, Karl-Norbert A1 - Jesaitis, A. J. T1 - Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein: Inhibition by pertussis toxin-catalyzed ADP ribosylation N2 - Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent Sedimentation coefficients of approximately 4 and 7 S. Tbe 7 S form can be converted to the 4 S form by guanosine 5' -0- (3-thiotriphosphate) (GTP-yS) with an EC&o of -20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. 0., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP-yS-treated neutrophil plasma membranes, was incubated with purified (>95%) G. protein from bovine brain (containing both G\(_{ia1}\) and G\(_{ia2}\)) or with neutrophil G protein (G\(_a\)), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC\(_{50}\) of 7 S complex formation induced by the two G proteins was 70 \(\pm\) 25 and 170 \(\pm\) 40 DM for G\(_a\) and G\(_1\), respectively. No complexation was measurable when bovine transducin (G\(_t\)) was used up to 30 times the EC\(_{50\) for G\(_a\). The EC\(_{50}\) for G\(_t\) was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 \(\mu\)M GTP-yS to the reconstituted 7 S complex caused a complete reversion of the receptor to the 4 S form, and anti-G\(_1\) peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gt prevented formation of the 7 S form even at 20 times the concentration of unribosylated G. normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a pbysical complex containing N-formyl chemotactic peptide receptor and G protein. KW - Toxikologie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60406 ER - TY - JOUR A1 - Gonzales-Calero, G. A1 - Cubero, A. A1 - Klotz, Karl-Norbert T1 - G protein coupled A\(_1\) adenosine receptors in coated vesicles of mammalian brain. Characterization by radioligand binding and photoaffinity labeling N2 - A\(_1\) adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaflinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1 ,3-[\(^3\)H]dipropyl-xanthine ([\(^3\)H]DPCPX) gave a Kdvalue of 0.7 nM and a Bmax value of 82± 13 fmol/mg protein. For the highly A\(_1\)-selective agonist 2-chloro-N\(^6\)-[\(^3\)H]cyclopentyladenosine ([\(^3\)H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [\(^3\)H]DPCPX binding gave a pharmacological profile with R-N\(^6\)-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCP A to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N\(^6\)- \(^{125}\)I-p-hydroxyphenylisopropyladenosine ([\(^{125}\)I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A\(_1\) receptors labelled in brain membranes. Therefore, coated vesicles contain A\(_1\) adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A\(_1\) receptors in these vesicles are not a processed receptor fonn. These results confirm that A\(_1\) receptors in coated vesicles are coupled to a G-protein, and it appears that the A\(_1\) receptor systems in coated vesicles andin plasma membranes are identical. KW - Toxikologie KW - Adenosine receptors KW - coated vesicles KW - G-protein KW - radioligand KW - photoaffinity labelling KW - brain membranes Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-60435 ER - TY - JOUR A1 - Spielman, William S. A1 - Klotz, Karl-Norbert A1 - Arend, Lois J. A1 - Olson, Barbara A. A1 - LeVier, David G. A1 - Schwabe, Ulrich T1 - Characterization of adenosine A1 receptor in a cell line (28A) derived from the rabbit collecting tubule N2 - We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both uf these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A 1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist f:1H]DPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of [: 1H]DPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent Kd value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of [: 1H]DPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A 1 receptor population in 28A cells has similar agonist binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine ;)' -triphosphate ( 100 ,uM) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic. indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins. In the absence of evidence for subpopulations of the A1 receptor, it appears that in 28A cells. A single A1 receptor population. As defined by ligand binding characteristics, couples via one or more pertussis toxin-sensitive guanine nucleotide binding proteins to two different biological signaling mechanisms. KW - calcium KW - phosphoinositides KW - adenosine 3',5'-cyclic monophosphate KW - receptor binding KW - signal transduction KW - G proteins Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86083 ER - 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 -