Maddalena Fratelli

Certain beta-blockers can decrease beta-adrenergic receptor number: I. Acute reduction in receptor number by tertatolol and bopindolol.

We have previously reported that a potent new beta-blocker, tertatolol, when given at therapeutic doses to healthy volunteers, rapidly reduced the number of human mononuclear leukocyte beta-receptors. In the present study, the mechanism of receptor regulation by beta-antagonists incubated with target cells in vitro was investigated. Two different cell types (human mononuclear leukocytes and S49 murine lymphoma cells) were used, and beta-adrenergic receptors were measured using either the hydrophilic ligand 3H-CGP 12177 (specific for surface receptors) or lipophilic 125I-pindolol (which measures total receptors). In a comparison between beta-blockers, tertatolol and bopindolol, but not propranolol and pindolol, were found to rapidly (1 hour at 37 degrees C) reduce the number of beta-adrenergic receptors. This was paralleled by a reduction in isoproterenol-stimulated cyclic AMP accumulation. The reduction in receptors was the same whether surface or total receptors were measured; thus, it was not due to receptor sequestration. This effect was not caused by partial agonist activity (bopindolol is a weak partial agonist); in parallel experiments, tertatolol and bopindolol, but not pindolol (potent partial agonist) and isoproterenol (full agonist), reduced beta-adrenergic receptors. Finally, this effect was not due to irreversible binding: the receptor reduction induced by the irreversible blocker bromo-acetyl-alprenolol-methane (BAAM) was stable for several hours, while the effect of tertatolol and bopindolol was slowly reversed over the same time course. We suggest that tertatolol and bopindolol have two effects on beta-adrenergic receptors: they bind competitively, and then they modify the receptors so that they are no longer available for binding by ligands or catecholamines.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress induced desensitization of lymphocyte β-adrenoceptors in young and aged rats

The effects of different times of immobilization stress on intact lymphocyte beta-adrenoceptors and plasma corticosterone were compared in 3-month and 24-month-old rats. In young animals after 30 min restraint 3H-dihydroalprenolol specific binding was significantly reduced (61% of control value) and plasma corticosterone significantly raised (186% of control). The effect on beta-adrenoceptors was due changes in receptor number (Bmax) without any effect on affinity (KD). In aged rats both effects were only seen after 180 min restraint and were less pronounced. Isoproterenol treatment in vitro reduced beta-adrenoceptors on lymphocytes. This effect was less pronounced in lymphocytes from aged rats. Corticosterone in vitro increased 3H-dihydroalprenolol specific binding. We therefore suggest that the decrease of beta-adrenoceptors reflects an adaptive response to the stress-induced catecholamine release and that corticosterone could play a role in reversing this effect. This adaptive response to stress seems to be impaired in aged animals.

Agonist-induced α1-adrenergic receptor changes: Evidence for receptor sequestration

Short‐term receptor regulation by agonists is a well‐known phenomenon for a number of receptors but little is known about the regulation of α1‐adrenergic receptors. In the present study we provide evidence of α1‐adrenergic receptor changes induced by agonists on DDT1 MF‐2 smooth muscle cells. The cells were preincubated with the agonist and receptor changes were investigated in the cells washed free of the agonist. On cells pretreated with norepinephrine the number of receptors recognized by [3H]prazosin at 4°C was reduced by 38%. The receptors were not degraded as the number of sites was the same in control and norepinephrine‐treated cells when binding was measured at 37°C. When binding was measured on fragmented membranes (at 4°C), the number of receptors was the same in control and norepinephrine‐treated cells, suggesting that the disruption of cellular integrity might expose receptors which are probably sequestered after agonist treatment. We conclude that agonists induced rapid sequestration of receptors on intact DDT cells.Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors but little is known about the regulation of α1-adrenergic receptors. In the present study we provide evidence of α1-adrenergic receptor changes induced by agonists on DDT1 MF-2 smooth muscle cells. The cells were preincubated with the agonist and receptor changes were investigated in the cells washed free of the agonist. On cells pretreated with norepinephrine the number of receptors recognized by [3H]prazosin at 4°C was reduced by 38%. The receptors were not degraded as the number of sites was the same in control and norepinephrine-treated cells when binding was measured at 37°C. When binding was measured on fragmented membranes (at 4°C), the number of receptors was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose receptors which are probably sequestered after agonist treatment. We conclude that agonists induced rapid sequestration of receptors on intact DDT cells.

Low affinity of β-adrenergic receptors for agonists on intact cells is not due to receptor sequestration

The low affinity of beta-adrenergic receptors for agonists described on intact cells at 37 degrees C has usually been interpreted in terms of reduced accessibility of agonists (which are usually hydrophilic) for sequestered receptors. We challenged this hypothesis by eliminating the plasma membrane barrier with low doses of the detergent digitonin. In human mononuclear leukocytes (MNL) permeabilized with digitonin, sequestered receptors became accessible to hydrophilic ligands such as agonists, but the affinity was still low. Then we investigated the relationship between low affinity agonist binding and sequestration using concanavalin A, which blocks sequestration. Even when sequestration was blocked, the affinity of the beta-adrenergic receptors for agonists was low. We conclude that: (a) low affinity agonist binding is independent of receptor sequestration; (b) the receptors which undergo conformational change are those that are sequestered; (c) the low affinity appears before sequestration occurs. This receptor conformational change could be the first step in agonist-induced desensitization.

Two subpopulations of α1-adrenergic receptors with high and low affinity for agonists: Short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including beta-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in alpha 1-adrenergic receptors induced by agonists. alpha 1-receptors were studied on DDT1 MF-2 smooth muscle cells (DDT1-MF-2 cells) by specific [3H]prazosin binding. In competition binding on membranes and on intact cells at 4 degrees C or at 37 degrees C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37 degrees C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [3H]prazosin binding inhibited by 20 microM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [3H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4 degrees C, but only 30% at 37 degrees C. On DDT1-MF-2 cells preequilibrated with [3H]prazosin at 4 degrees C, and then shifted to 37 degrees C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4 degrees C it is the native form of alpha 1-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37 degrees C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 microM norepinephrine. The nature of this low-affinity form was further investigated. On DDT1-MF-2 cells preincubated with the agonist and then extensively washed at 4 degrees C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [3H]prazosin at 4 degrees C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4 degrees C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37 degrees C may be due to the agonist-induced sequestration of alpha 1-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.

Catecholamines Do Not Induce Fibrinolytic Activity Increase in Cultured Bovine Endothelial Cells

This study has investigated the catecholamine involvement in the fibrinolytic modulation of cultured bovine aortic endothelial cells (BAEC). Adrenaline and isoproterenol, at concentrations ranging from 10 to 100 microM, were unable to modulate the fibrinolytic response of these cells. beta-Adrenergic binding studies using 3H-CGP 12177 as radioligand evidenced the presence of about 23,113 +/- 2,065 sites/cell, with a KD of 1.23 +/- 0.29 nM. Isoproterenol stimulated cAMP accumulation at concentrations ranging from 1 to 100 microM, with a maximal accumulation of 30 pmol/10(6) cell. Hence, in our experimental conditions BAEC, although possessing functional beta-adrenergic receptors, were unable to increase any fibrinolytic activity in response to catecholamines.