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Modulation of beta₁-adrenoceptor activity by domain-specific antibodies and heart failure–associated autoantibodies

Roland Jahns, MD^* , Valérie Boivin, PhD, Thorsten Krapf, MD, Gerd Wallukat, MD, Fritz Boege, MD^* and Martin J. Lohse, MD

^* Department of Internal Medicine, Medizinische Poliklinik, University of Wuerzburg, Wuerzburg, Germany
Institute of Pharmacology, University of Wuerzburg, Wuerzburg, Germany
Max Delbrück Center for Molecular Medicine, Berlin, Germany

View larger version (96K): [in a new window]   Figure 1 Detection of anti-beta1-AR by various approaches. (a) Western blots of crude cell lysates, and (b) immunoprecipitates of membrane-biotinylated Sf9 cells expressing recombinant human beta-1-AR (R) or the wildtype vector (C), and (c) fixed and permeabilized, or (d) intact unfixed Sf9-beta1 cells were probed with either (a, c, d) domain-specific rabbit anti-beta1-N/-ECII/-C (Rabbit) or mouse anti-beta1-ECII (Mouse), or (b) with POD-coupled streptavidin (see Methods). Their respective immunoreactivities are compared with those of anti-beta1-ECII-positive or -negative cardiomyopathic patients (Human, Pat. ß1-Pos./-Neg.). Note, that SDS-denatured beta1-AR (lane a) are stained less clearly (Rabbit) or not at all (Mouse, Human) by anti-beta1-ECII antibodies, whereas all of them immunoprecipitate (lane b) or recognize the native beta1-AR (lane d).

View larger version (29K): [in a new window]   Figure 2 Decrease in ligand-binding to human beta1-AR expressed in Sf9 cells (a) in the presence of constant 3 nmol/liter [3H]CGP 12177 and the indicated concentrations of anti-beta1-AR, or (b) in the presence of a fixed concentration of the different anti-beta1-AR (mouse, 7 nmol/liter; rabbit, 70 nmol/liter; human: 2 µmol/liter) and various concentrations of [3H]CGP 12177 (3 x 10–11 to 5 x 10–9 mol/liter). Error bars indicate the mean ± SD of three experiments. Bmax = total ligand binding capacity; KD = receptor-ligand dissociation constant.

View larger version (29K): [in a new window]   Figure 3 Increase in basal or (–)isoprenaline (10 µmol/liter)-stimulated cAMP-levels upon incubation of CHW-beta1 cells with the indicated concentrations of rabbit, mouse or human anti-beta1-ECII (open symbols), or rabbit anti-beta1-C/-N (filled circles/triangles). IgG-preparations from healthy subjects served as a control (filled diamonds). Error bars indicate the mean ± SD of three experiments.

View larger version (28K): [in a new window]   Figure 4 Increases in (–)isoprenaline-stimulated cAMP-levels (10–12 to 10–5 mol/liter) upon incubation of CHW-beta1 cells with a fixed concentration of rabbit (70 nmol/liter) or human anti-beta1-ECII (2 µmol/liter; patient-subgroups A and B). Filled circles or diamonds correspond to the values obtained for rabbit anti-beta1-C or IgG-preparations from healthy subjects, respectively. Error bars indicate the mean ± SD of three experiments. Significance between control values (filled symbols) and those obtained in the presence of anti-beta1-ECII (open symbols) was analyzed by the Scheffé F test. All the values obtained for patient anti-beta1-ECII (subgroups A/B) differed significantly (p < 0.05) from control values in the presence of low (10–12 to 10–10 mol/liter) or high (10–9 to 10–5 mol/liter) isoprenaline concentrations, except those from patient subgroup B at 10–10 mol/liter isoprenaline (p = 0.15, not significant).