| 1. |
- Magnusson, Yvonne, 1957, et al.
(författare)
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Functional analysis of rabbit anti-peptide antibodies which mimic autoantibodies against the beta 1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy.
- 1991
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Ingår i: Journal of autoimmunity. - 0896-8411. ; 4:6, s. 893-905
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Tidskriftsartikel (refereegranskat)abstract
- A synthetic peptide corresponding to the second extracellular loop of the beta 1-adrenergic receptor was used as an antigen for antibody production in three rabbits. Antibodies of high titers were obtained in all rabbits. Only one rabbit yielded antibodies which decreased radioligand binding on the receptor in a similar way to that described for autoantibodies in patients with dilated cardiomyopathy. These antibodies recognized the receptor protein in immunoblots. Epitope mapping indicated that the N-terminal sequence of the loop used as antigen was the target of the major antigen fraction. Incubation of antibodies with C6 glioma cell membranes or inner membranes of E. coli, which express the human beta 1-adrenergic receptor, resulted in a decrease in number of radioligand binding sites. This decrease was dependent on the concentration of antibody and of Mg++ ions. It was not affected by the GTP analog GppNHp or the beta 1 subtype-specific antagonist metoprolol. The agonist, isoproterenol, also induced a decrease but the effects of antibody and agonist were not additive. These results suggest that the antibodies induce a Mg(++)-dependent, 'active', labile conformation of the receptor, independent from coupling to the GTP regulatory protein, but similar to that induced by the agonist isoproterenol. This interpretation was corroborated by the beta 1-adrenergic receptor agonist-like effect of the antibodies on cardiomyocytes in culture.
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| 2. |
- Fu, Michael, 1963, et al.
(författare)
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Characterization of anti-peptide antibodies directed against an extracellular immunogenic epitope on the human alpha 1-adrenergic receptor.
- 1994
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Ingår i: Clinical and experimental immunology. - 0009-9104. ; 97:1, s. 146-51
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Tidskriftsartikel (refereegranskat)abstract
- A synthetic peptide corresponding to amino acids 192-218 of the second extracellular loop of the human alpha 1A-adrenergic receptor was used to raise antibodies in rabbits. Affinity-purified antibodies specifically recognized main bands with a molecular weight of about 68, 40 and 37 kD on the electrotransferred membrane proteins of rat ventricle membranes. The incubation of these antibodies with rat myocardial membranes resulted in a decrease in the number of binding sites for the specific radiolabelled alpha 1-antagonist prazosin. These antibodies were also able to mimic the effects of agonist stimulation as demonstrated by a positive chronotropic effect on cultured cardiomyocytes. These results constitute the first immunochemical evidence of the presence of both the A and B subtypes of the alpha 1-adrenergic receptor in the heart. They also confirm that the second extracellular loop of the alpha 1-adrenergic receptors is an immunologically and functionally important domain.
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| 3. |
- Fu, Michael, 1963, et al.
(författare)
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Functional autoimmune epitope on alpha 1-adrenergic receptors in patients with malignant hypertension.
- 1994
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Ingår i: Lancet. - 0140-6736. ; 344:8938, s. 1660-3
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Tidskriftsartikel (refereegranskat)abstract
- Because of the growing evidence that hypertensive disease is accompanied by immunological dysfunction, we have investigated autoimmunity in patients with malignant hypertension. Peptides corresponding to the sequence of the second extracellular loops of the human alpha 1-adrenergic receptor and the M2-muscarinic receptor were used as antigens in an ELISA. Serum from 4 (12%) of 33 healthy controls, 3 (20%) of 15 patients with malignant essential hypertension, and 7 (64%) of 11 with secondary hypertension showed positive responses in the ELISA for the alpha 1-adrenergic receptor peptide. Positive responses were significantly more common among the patients with secondary hypertension than in the other two groups (p < 0.01). By contrast, no autoantibodies against the M2-muscarinic receptor peptide were detected in either hypertensive group. Autoantibodies against the alpha 1-adrenergic receptor, affinity-purified from patients with positive responses, specifically recognised bands with molecular masses of 68, 40, and 37 kDa on immunoblotted membrane proteins of rat ventricles. The patients' autoantibodies caused a decrease in tritiated prazosin binding sites and an increase in heart beating frequency of neonatal cultured rat cardiomyocytes; antibodies purified from the controls had no effect. Circulating autoantibodies against the alpha 1-adrenergic receptor are present in a subgroup of patients with malignant hypertension. These autoantibodies have pharmacological activity in vitro, which suggests that they may be involved in the pathogenesis of malignant hypertension.
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| 4. |
- Magnusson, Yvonne, 1957, et al.
(författare)
-
Autoimmunity in idiopathic dilated cardiomyopathy. Characterization of antibodies against the beta 1-adrenoceptor with positive chronotropic effect.
- 1994
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Ingår i: Circulation. - 0009-7322. ; 89:6, s. 2760-7
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Autoantibodies against the beta 1-adrenoceptor have been detected in the sera of patients with idiopathic dilated cardiomyopathy (DCM). The mechanisms by which these autoantibodies can alter normal receptor function are investigated, and the results are interpreted in the light of the beneficial effects of beta 1-blockade in some of these patients. METHODS AND RESULTS: Autoantibodies against the beta 1-adrenoceptor, affinity purified from sera of patients with idiopathic DCM, were analyzed in a functional test system of spontaneously beating neonatal rat heart myocytes. Antibodies from rabbits immunized with peptides derived from the amino acid sequence of this receptor were also analyzed. Autoantibodies, against the second extracellular loop increased the beating frequency of isolated myocytes in a concentration-dependent manner, to approximately 80% of maximal isoproterenol stimulation. Rabbit anti-peptide antibodies against the second extracellular loop increased the beating frequency correspondingly. Autoantibodies and rabbit anti-peptide antibodies against the second extracellular loop were able to immunoprecipitate the unliganded receptor but not the antagonist-occupied receptor. In contrast, rabbit antibodies against the extracellular N-terminal sequence 34-57 of the beta 1-adrenoceptor were able to immunoprecipitate both the unliganded and the antagonist-occupied receptor although with no effect on the beating frequency of myocytes. The positive chronotropic effect of the antibodies was completely neutralized both by the addition of increasing concentrations of the beta 1-selective antagonist bisoprolol and by preincubation with the peptide corresponding to the second extracellular loop. The antibody-induced increase in beating frequency remained unchanged for more than 6 hours. This should be compared with the isoproterenol-stimulated beating frequency, which undergoes desensitization within 60 minutes. Addition of isoproterenol to autoantibody-stimulated myocytes resulted in only a small increase in beating frequency and did not cause desensitization. Antibodies had only a marginal effect on cyclic AMP production of stimulated cardiomyocytes compared with the 10-fold increase obtained after stimulation with isoproterenol. CONCLUSIONS: The second extracellular loop of the beta 1-adrenoceptor is a specific target for antibodies with stimulatory activity detected in patients with idiopathic DCM. The antibodies have a positive chronotropic effect on isolated rat heart myocytes. Autoantibody stimulation does not cause the normal agonist-induced desensitization phenomena of the effector system. These findings could contribute to our understanding of the pathophysiological mechanisms of the autoantibodies and of the beneficial effect of beta 1-blocking agents in the treatment of patients with idiopathic DCM.
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