Background-Catecholamines hasten cardiac relaxation through ␤-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of ␤ 1 -and ␤ 2 -adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. Methods and Results-Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (nϭ5) or ischemic cardiomyopathy (nϭ5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 mol/L) or zinterol (10 mol/L), mediated through ␤ 2 -adrenergic receptors, and of norepinephrine (10 mol/L), mediated through ␤ 1 -adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17. Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15Ϯ3%, 5Ϯ2%, and 20Ϯ3%, respectively, and reduced the time to half-relaxation by 26Ϯ3%, 21Ϯ3%, and 37Ϯ3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14Ϯ3, 12Ϯ4, and 12Ϯ3; troponin I 40Ϯ7, 33Ϯ7, and 31Ϯ6; and C-protein 7.2Ϯ1.9, 9.3Ϯ1.4, and 7.5Ϯ2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both ␤ 1 -and ␤ 2 -adrenergic receptors. Conclusions-Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both ␤ 1 -and ␤ 2 -adrenergic receptors, thereby potentially improving diastolic function. (Circulation. 1999;99:65-72.)

Phospholamban is a critical regulator of sarcoplasmic reticulum Ca2+-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca2+ and cAMP pathways, we have investigated the β-adrenergic-induced phosphorylation of Ser16 and Thr17 of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser16 and Thr17 with strikingly different half-maximal values (EC50 = 4.5 ± 1.6 and 28.2 ± 1.4 nmol/l, respectively). The phosphorylation of Ser16 induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation ( r = 0.96), whereas phosphorylation of Thr17 did not. Elevation of extracellular Ca2+did not induce phosphorylation at Thr17; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr17 increased eightfold without additional effects on relaxation rate. Thr17 phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 μmol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser16 by cAMP-dependent protein kinase is the main regulator of β-adrenergic-induced cardiac relaxation definitely preceding Thr17 phosphorylation and 2) the β-adrenergic-mediated phosphorylation of Thr17 by Ca2+-calmodulin-dependent protein kinase required influx of Ca2+through the L-type Ca2+ channel.

Although primary causes of Alzheimer’s and vascular dementia are unknown, the importance of preceding vascular lesions is widely accepted. Furthermore, there is strong evidence for the involvement of autoimmune mechanisms. Here, we report the presence of agonistic autoantibodies directed at adrenergic receptors in the circulation of patients with mild to moderate Alzheimer’s and vascular dementia. In 59% of these patients, agonistic autoantibodies against the α1‐adrenergic receptor and the β2‐adrenergic receptor were identified. The majority of positive patients (66%) contained both types of autoantibodies in combination. In a control group of patients with neurological impairments others than Alzheimer’s and vascular dementia, only 17% were found to harbour these autoantibodies. The autoantibodies to the α1‐adrenergic receptor interacted preferably with the extracellular loop1 of the receptor. They were further studied in IgG preparations from the column regenerate of a patient who underwent immunoadsorption. The α1‐adrenergic receptor autoantibodies specifically bound to the extracellular loop1 peptide of the receptor with an apparent EC50 value of 30 nm. They mobilized intracellular calcium in a clonal cell line expressing the human form of the α1‐adrenergic receptor. Our data support the notion that autoimmune mechanisms play a significant role in the pathogenesis of Alzheimer’s and vascular dementia. We suggest that agonistic autoantibodies to the α1‐adrenergic and the β2‐adrenergic receptor may contribute to vascular lesions and increased plaque formation.

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Dementia has been shown to be associated with agonistic autoantibodies. The deleterious action of autoantibodies on the α1-adrenergic receptor for brain vasculature has been demonstrated in animal studies. In the current study, 169 patients with dementia were screened for the presence of agonistic autoantibodies. 47% of patients suffering from mild to moderate Alzheimer's disease and/or vascular dementia carried these autoantibodies. Eight patients positive for autoantibodies underwent immunoadsorption. Patients treated on four consecutive days were subsequently negative for autoantibodies and displayed stabilization of cognitive and mental condition during 12-18 months' follow-up. In patients treated for 2-3 days, autoantibodies were reduced by only 78%. They suffered a rebound of autoantibodies during follow-up, benefited from immunoadsorption too, but their mental parameters worsened. We provide first data on the clinical relevance of agonistic autoantibodies in dementia and show that immunoadsorption is safe and efficient in removing autoantibodies with overall benefits for patients.