Multisite contacts involved in coupling of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein. Structural and functional studies by beta-receptor-site-specific synthetic peptides.

Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

Lysosomotropic behavior of adrenergic antagonists in interactions with human neutrophils.

Autonomic neurohormones affect the secretory activity of neutrophils by modulating release of lysosomal enzymes in response to immunologic stimuli. Autonomic agents are also weak bases which might modify cell function by accumulating in the acidic interior of the lysosome, in addition to their receptor-mediated activity. We examined the association of the beta-adrenergic antagonist [3H]dihydroalprenolol with human neutrophils and lysosome and membrane fractions derived from neutrophils, and the subcellular distribution of the photoaffinity-labeled beta-adrenergic ligand [3H]azidobenzylcarazolol after incubation with intact cells. Isolated neutrophil lysosomes accumulated significantly more [3H]dihydroalprenolol than isolated membrane preparations. Decreasing the transmembrane pH gradient markedly reduced [3H]dihydroalprenolol accumulation by intact cells or lysosomes but not by membranes. Since [3H]dihydroalprenolol accumulated by intact cells remained rapidly exchangeable, the photoaffinity ligand [3H]azidobenzylcarazolol was used to assess ligand distribution after association with whole cells. After cell disruption, 18.5 +/- 1.3% of this ligand appeared in the lysosome fraction as compared to 2.2 +/- 0.6% in the membrane fraction. The secretagogue phorbol myristate acetate caused release of the ligand as well as lysosomal enzymes from cells. These findings suggest that there is significant pH-dependent lysosomal accumulation of beta-antagonists. This lysosomotropic interaction may be important both as it affects the sequestration and redistribution of the drugs, and as it might in some circumstances affect host defense functions of the neutrophil.

[Primary orthostatic hypotension]. / Primäre orthostatische Hypotonie. Ein Fallbericht.

We studied a 25-year-old man suffering from primary orthostatic hypotension whose blood pressure decreased to 65/45 mm Hg during orthostasis and to 95/70 mm Hg during ergometric exercise (50 and 100 watt), and whose heart rate responses were inadequate. Resting catecholamine levels were within the normal range and did not show any significant increase related to orthostasis or to ergometric exercise. Hypersensitivity was observed to low doses of intravenous noradrenaline and isoproterenol. Specific binding of 3H-Yohimbine to intact platelets revealed a normal number of alpha-2-adrenoreceptors in agreement with the adrenaline-induced platelet aggregation in vitro, which was, however, in contrast to hypersensitivity to noradrenaline. Specific 3H-Dihydroalprenolol binding to intact polymorphonuclear leucocytes revealed an increased beta-2-adrenoreceptor density in agreement with hypersensitivity to Isoproterenol. Prescription of Fludrocortison improved orthostatic hypotension.

Agonist-induced redistribution of beta-adrenergic receptors on intact human mononuclear leukocytes: redistributed receptors are nonfunctional.

Incubation of human mononuclear leukocytes (MLN) with isoproterenol rapidly desensitizes beta-adrenergic receptors, i.e. isoproterenol-stimulated cAMP accumulation decreases. This desensitization is accompanied by a redistribution of the receptor into a cellular environment to which hydrophilic compounds have limited access. We found that the total number of beta-receptors [defined as binding of [3H]dihydroalprenolol (DHA) inhibited by 1 microM propranolol] was unchanged in the desensitized MNL. In control MNL, virtually all DHA binding was inhibited by 1 microM CGP-12177, suggesting that all of these receptors are on the cell surface, whereas in desensitized cells, only 33 +/- 2% (mean +/- SEM) of the DHA binding was inhibited by CGP-12177. We quantitated the sequestered receptors by subtracting the number of surface receptors from the total number of receptors. The sequestered receptors were homogeneous, with an affinity for DHA identical to that of surface receptors (Kd, 0.66 +/- 0.12 vs. 0.62 +/- 0.08 nM). The time courses of desensitization and sequestration were identical. The functional status of the sequestered receptors was assessed using the agonist zinterol, which (unlike catecholamines) is quite hydrophobic. Zinterol competed for DHA binding to both sequestered and surface receptors, whereas isoproterenol only competed for binding to the surface receptors. However, cAMP accumulation in desensitized MNL was reduced to the same extent regardless of whether isoproterenol or zinterol was used as the agonist. These results demonstrate that desensitization of intact cells to beta-agonists cannot be attributed to limited accessibility of the sequestered receptors to catecholamines, but, rather, that the sequestered receptors are not functionally coupled to adenylate cyclase.

Characterization of human platelet beta-adrenoceptors.

The widespread use of beta-adrenoceptor antagonists against hypertension, angina pectoris and migraine or as a preventive treatment after myocardial infarction has encouraged us to investigate the effects of these drugs on platelet function. The aim of this study was to examine whether beta-blocking drugs interfere with platelet beta- adrenoceptors and whether this dependency is related to their selectivity for beta-adrenoceptor subtypes. Beta-adrenoceptor stimulation of human platelets with isoprenaline increased cyclic AMP (cAMP), which is known to inhibit platelet aggregation. Furthermore, our studies showed that cAMP formation in vitro was stimulated by non-selective and beta 2-selective agonists, but not by the predominant beta 1-agonist prenalterol. Isoprenaline- stimulated cAMP formation was blocked by the non- selective beta-adrenoceptor antagonists propranolol, timolol, and alprenolol, while the beta 1-selective antagonists atenolol and metoprolol had no influence on an isoprenaline-induced cAMP formation. Receptor binding studies using (3H)-dihydroalprenolol revealed an IC50 value for propranolol of 85 nM, while metoprolol only displaced the bound (3H)-dihydroalprenolol at far higher concentrations (IC50, 20 microM). We conclude that the human platelet beta-adrenoceptors are mainly of the beta 2- subtype and that beta-adrenoceptor antagonists, especially the non-selective antagonists interfere with platelet function assessed as platelet cAMP formation.

Characterization of monoclonal antibodies to the beta-adrenergic antagonist alprenolol as models of the receptor binding site.

Antibodies to receptor ligands have been valuable in understanding the nature of receptor-ligand interactions. We have developed four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol by immunizing A/J mice with (-)-alprenolol coupled to keyhole limpet hemocyanin. The antisera from these mice displayed specific [3H]dihydroalprenolol ([3H]DHA) binding that was inhibited by alprenolol, propranolol, and isoproterenol. Somatic cell fusion of spleen cells from the immunized mice to SP2/0 myeloma cells, followed by limited dilution subcloning, resulted in the isolation of four hybridomas (1B7, 5B7, 5D9, and 2G9) demonstrating three different classes of ligand binding characteristics. 1B7 had the highest binding affinity for antagonists based on Scatchard analysis (Kd [125I]- CYP = 1.4 X 10(-10) M; Kd [3H]DHA = 6.5 X 10(-9) M), and was the only antibody to demonstrate agonist-inhibition of [3H]DHA binding. Ki values computed from competitive inhibition curves of [3H]DHA binding to 1B7 resulted in a rank order of potency similar to that of beta-2-adrenergic receptors: (-)-propranolol greater than acebutolol amine greater than isoproterenol greater than (+)-propranolol greater than epinephrine greater than norepinephrine. 5B7 and 5D9 exemplified a second class of antibody. This pair had lower antagonist binding affinities (Kd [3H]DHA = 2 X 10(-8) M and 2.5 X 10(-7) M, respectively) and was stereoselective in binding receptor antagonists: (-)-propranolol greater than (+)-propranolol greater than acebutolol amine. Agonist inhibition of [3H]DHA binding to these antibodies could only be observed at very high concentrations (greater than 10(-4) M agonist), and was not dose-dependent. Finally, the class of anti-alprenolol monoclonal antibodies represented by 2G9 had the lowest antagonist binding affinity of all (IC50 alprenolol = 1 X 10(-5) M), did not demonstrate ligand stereoselectivity, and did not recognize agonists. We propose that antibodies raised against beta-adrenergic receptor ligands demonstrating stereoselective agonist binding will also demonstrate high affinity antagonist binding, and that they will closely parallel the binding characteristics of the receptor. According to this "agonist best-fit hypothesis," anti-idiotypic antibodies raised against the binding site of these idiotypes might contain true mirror images of the beta-adrenergic receptor binding site.

Altered plasma catecholamines and numbers of alpha- and beta-adrenergic receptors in platelets and leucocytes in hyperthyroid patients normalized under antithyroid treatment.

We measured plasma catecholamines, alpha- and beta-adrenoreceptor numbers and the accumulation of cyclic adenosine monophosphate (cAMP) in the unstimulated state and in response to 10 mumol/l (-) isoproterenol in blood cells from 29 euthyroid controls and from 18 patients with spontaneous hyperthyroidism. In the thyrotoxic patients plasma norepinephrine (1.14 +/- 0.5 nmol/l) and epinephrine (0.3 +/- 0.14 nmol/l) were significantly decreased compared with plasma norepinephrine (1.87 +/- 0.7 nmol) and epinephrine (0.41 +/- 0.19 nmol/l) in the controls (P less than 0.01 and P less than 0.05, respectively) and the values obtained in subjects rendered euthyroid by antithyroid treatment (P less than 0.001, respectively). alpha-adrenoceptor density in platelet membranes obtained from patients in the hyperthyroid state (114 +/- 38 sites per cell) was significantly decreased when compared with controls (159 +/- 48 sites per cell, P less than 0.01) and the values from patients under effective antithyroid treatment (136 +/- 35 sites per cell, P less than 0.01). On the contrary, a significant increase in beta-adrenoceptor density in mononuclear leucocyte (MNL) membranes was found in hyperthyroid patients (1751 +/- 237 sites/cell) when compared with controls (1510 +/- 351 sites/cell, P less than 0.05) and the same patients following antithyroid treatment (1455 +/- 260 sites/cell, P less than 0.001). The equilibrium dissociation constants (KD) did not change in hyperthyroidism. Basal cAMP concentrations in MNL were higher in untreated thyrotoxicosis (45 +/- 18 pmol/10(6) cells/10 min) than in patients in the euthyroid state (35 +/- 9 pmol/10(6) cells/10 min, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The oligosaccharide moiety of the beta 1-adrenergic receptor from turkey erythrocytes has a biantennary, N-acetyllactosamine-containing structure.

The turkey erythrocyte beta 1-adrenergic receptor can be purified by affinity chromatography on alprenolol-Sepharose and characterized by photoaffinity labeling with N-(p-azido-m-[125I]iodobenzyl)-carazolol. Through the use of the specific glycosidases neuraminidase and endo-beta-N-acetylglucosaminidase H and affinity chromatography on lectin-Sepharose gels, we show here that the receptor is an N-glycosyl protein that contains complex carbohydrate chains. No high-mannose carbohydrate chains appear to be present. The binding of the radiolabeled antagonist dihydroalprenolol to the receptor is affected neither by the enzymic treatments nor by the presence of lectins, suggesting that the carbohydrate moiety is not involved in the catecholamine binding site.

Baboon erythrocyte ghosts contain beta-adrenergic receptors.

We have used the beta-adrenergic antagonist [3H]dihydroalprenolol [( 3H]DHA) to identify binding sites on the erythrocyte membrane of the primate Papio ursinus. Analysis of the saturation isotherm revealed binding to be saturable with a maximal number of binding sites of 499 fmol/mg protein. [3H]DHA binds specifically to the erythrocyte ghosts with an apparent dissociation constant (Kd) of 0.57 +/- 0.06 nM. A similar value for Kd (0.46 +/- 0.07 nM) was evaluated from the rate constants of association (0.013 +/- 0.003 X nM-1 X min-1) and dissociation (0.006 +/- 0.001 X min-1). beta-adrenergic agonists compete for the binding sites with an order of potency (dl-isoproterenol greater than l-epinephrine greater than l-norepinephrine) typical of a beta 2-adrenergic receptor. Binding was shown to be stereospecific with l-stereoisomers being more potent than their corresponding d-stereoisomers in causing half-maximal inhibition. Isoproterenol stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in a concentration-dependent manner, maximal levels (1.130 +/- 0.358 pmol cAMP/10(8) cells) being four times the basal levels. The results demonstrate the existence of a large number of beta-adrenergic receptors on baboon erythrocyte ghosts.

Proteolysis-associated deglycosylation of beta 1-adrenergic receptor in turkey erythrocytes and membranes.

A protease that can be inhibited by glutathione, dithiothreitol, and o-phenanthroline but not by ethylenediaminetetraacetic acid converts the 50-kilodalton beta-adrenergic receptor in turkey erythrocyte membranes to a 40-kDa polypeptide which retains the specific ligand binding site. This conversion is attenuated in intact erythrocytes. The large 50-kDa peptide contains N-linked, complex carbohydrates and is retained on wheat germ agglutinin-Sepharose. The 40-kDa product of proteolysis does not bind to the wheat germ agglutinin and can thus be separated from the 50-kDa polypeptide by lectin chromatography. However, the large difference in molecular weights of the two receptor peptides cannot be accounted for solely by the different extent of glycosylation.

Identification of beta-adrenergic receptors in teleost red blood cells.

Beta-adrenoceptors have been identified in eel erythrocyte membranes using [3H]dihydroalprenolol binding. These receptors exhibit a mean dissociation constant (KD) of 1.36 nM and a mean maximum number of binding sites (Bmax) of 315 fmol/mg protein. These receptors do not appear to belong to the beta 1-subtype.

[Changes in the density of human lymphocyte beta-adrenergic receptors in hypertension]. / Izmenenie plotnosti beta-adrenergicheskikh retseptorov limfotsitov cheloveka pri gipertonicheskoi bolezni.

The beta-adrenoreceptor density in intact lymphocytes of peripheral blood of normotensive and hypertensive adults was measured by the radioligand binding technique with the use of 3H-dihydroalprenolol. The density of beta-adrenoreceptors was found to be higher in lymphocytes of hypertensive subjects while the number of the receptors was equal to 2076 +/- 208 and 1461 +/- 175 receptors per cell, respectively, in hypertensive and normotensive subjects. The dissociation constants in both cases varied from 0.4 to 1.8 nM. Probably, the decreased responses to the adrenergic agents in hypertensive subjects are not connected with the decreased number of beta-adrenoreceptors and may be due to alterations in postreceptor events.

[Determination of beta-receptors on intact polymorphonuclear leukocytes in autologous plasma]. / Bestimmung von beta-Rezeptoren an polymorphkernigen intakten Leukocyten im autologen Plasma.

The binding of tritium labelled radioligand dihydroalprenolol was investigated on live polymorphonuclear leukocytes of 6 endurance trained (VO2 max. 65.7 +/- 2.0 ml/kg . min), and 9 non-endurance trained subjects (VO2 max. 52.0 +/- 4.0 ml/kg . min). The specific binding of dihydroalprenolol is seen as an indicator of the beta-receptor density. The specific binding of dihydroalprenolol is defined as the difference between the total binding and that amount of dihydroalprenolol that could not be displaced. The leukocytes were reincubated for the binding studies in their autologous plasma. The specific binding of dihydroalprenolol on live polymorphonuclear leukocytes shows a levelling off behaviour at a concentration of approximately 2 nmol/l dihydroalprenolol in trained as well as in untrained subjects. The specific binding amounts to about 85% (0.1 nmol/l dihydroalprenolol) to 51% (2.0 nmol/l dihydroalprenolol) of the total binding. Based on Scatchard analysis, Bmax was determined as 41.2 fmol/10(7) cells (trained subjects) and 21.6 fmol/10(7) cells (untrained subjects). KD is 0.44 nmol/l dihydroalprenolol (untrained subjects), and 0.49 nmol/l dihydroalprenolol (trained subjects). The beta-adrenergic binding sites are approximately 1300 (untrained subjects), and 2500 binding sites/cell (trained subjects). The specific binding of dihydroalprenolol on live polymorphonuclear leukocytes is significantly higher in trained than in untrained subjects. This training dependent change in beta-receptor density may be an indicator of an increased sensitivity to catecholamines.

The response of lymphocyte beta-adrenergic receptors to chronic propranolol treatment in depressed patients, schizophrenic patients, and normal controls.

The ability of beta-adrenergic receptors to adapt to reductions in stimulation might be impaired in depressed or schizophrenic patients. To test this hypothesis 12 normal controls, 12 depressed patients resistant to tricyclic antidepressants, and 8 chronic schizophrenic patients were treated with propranolol 160 mg/day for 10 days. A previous study had reported that this dose regimen led to a rise in lymphocyte beta-adrenergic receptors in normal volunteers. Blood was sampled before propranolol treatment and 60 hr after the last propranolol dose. There were no significant differences between any of the groups in lymphocyte beta-adrenergic receptors after 10 days of propranolol treatment.

Decreased beta-adrenergic receptor density in mononuclear leukocytes from thyroidectomized patients.

Beta-adrenergic receptor characteristics were investigated in peripheral blood mononuclear leukocytes taken from patients before and after partial thyroidectomy. In order to discriminate the effect of surgical stress per se from that of thyroidectomy, the analysis was also performed on patients before and after cholecystectomy. Receptor characteristics were determined by using dihydroalprenolol as ligand in direct equilibrium binding experiments. The binding affinity showed no changes either when two different surgical treatments were compared or when the same patient was analysed before and after the operation. On the contrary, a significant decrease in receptor density was found in thyroidectomized patients when compared pre- and post-operatively. This fall in receptor number seems to be linked with thyroid function since no statistically significant changes were observed in cholecystectomized patients in relation to surgical operation. This view is further supported by data on T3 serum levels, which show a significant fall after thyroidectomy but no statistically significant modifications after cholecystectomy. It is concluded that beta-adrenoceptor modulation plays an important role in the relationship between thyroid and beta-adrenergic system.

Lymphocyte beta-adrenergic receptors are not altered in hyperthyroidism.

Binding of (-)3H]-dihydroalprenolol (3H-DHA) to lymphocytes from five thyrotoxic patients and five age- and sex-matched contents were examined to ascertain whether beta-adrenergic receptor number or binding affinity were altered in thyrotoxicosis. Whereas an increase in beta-adrenoceptor density has been reported in triiodothyronine-induced hyperthyroidism, we did not find changes in beta-adrenergic receptor density or binding affinity. In one patient studied sequentially, we did not find alteration in 3H-DHA bindings before or after the subject was rendered euthyroid. We conclude that lymphocyte beta-adrenergic receptor density and affinity are not altered in spontaneous hyperthyroidism.