Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.     

Effect of hydrocortisone on beta-adrenergic receptors in lung membranes.

It has been observed that glucocorticoids potentiate beta-adrenergic stimulation of cardiovascular and airway tissues. In order to investigate the mechanism of this potentiating action, we examined the effect of glucocorticoids on the number and affinity of beta-adrenergic receptors in animal lung tissues, by a direct binding technique using [125]I-Iodohydroxybenzylpindolol ([125]I-HYP), a potent beta-adrenergic receptor antagonist. Specific binding of [125]I-HYP to rat lung membranes was saturable with 386 fmol of [125]I-HYP/mg protein at saturation. The apparent equilibrium dissociation constant of [125]I-HYP for beta-receptors was 221 nM. Chronic administration of hydrocortisone increased the density of beta-adrenergic receptors by 70% from 386 fmol to 657 fmol/mg with some decrease in the affinity of [125]I-HYP for beta-adrenergic receptors. By contrast, adrenalectomy produced a 29% fall in the number of beta-adrenergic receptors without altering the affinity of [125]I-HYP for beta-receptors, and this change was reversed by exogenous adminstration of hydrocortisone. The present study suggests that glucocorticoids may participate in regulating the density of beta-adrenergic receptors, and may potentiate beta-adrenergic receptors stimulation, at least in part by increasing beta-receptor density in tissue membranes.     

beta-Adrenergic receptors in rat brain.

125I-Iodohydroxybenzylpindolol ([125I] IHYP), a potent beta-adrenergic receptor antagonist, has been used to study beta-adrenergic receptors in rat brain. Binding of [125I] IHYP (30 pM) to a membrane fraction min and dissociation took place with a half time of about 16 min. Phentolamine (10(-4) M) decreased non-receptor binding but it had no effect on the binding of [125I] IHYP to beta-adrenergic receptors in cortex, cerebellum or caudate. In the presence of phentolamine specific binding (defined as binding which was blocked by 0.3 muM dl-propranolol) represented 70-85% of total binding. The binding of [125I] IHYP was inhibited by beta-adrenergic agonists and antagonists. d-Stereoisomers were 2-3 orders of magnitude less potent than the corresponding 1-isomers. The denstiy of [125I] IHYP binding sites was studied in membrane fractions from cerebral cortex, cerebellum, and caudate nucleus by means of Scatchard analysis. The K(D) of [125I] IHYP was similar in the three regions studied, and the density of [125I] IHYP binding sites was approximately 50% greater in the cortex and caudate than in the cerebellum. The Hill coefficient for the binding of [125I] IHYP to membranes from cerebral cortex was 1.02. The properties of the binding of [125I] IHYP are similar to those which would be expected of binding to beta-adrenergic receptors in vitro.     

Discrepancies between the affinities of binding and action of the novel beta-adrenergic agonist BRL 37344 in rat brown adipose tissue

The novel brown adipose tissue (BAT) selective beta-adrenergic agonist, BRL 37344, is 31-fold more potent than (-)-isoproterenol in stimulating the respiratory rate of interscapular BAT fragments. BRL 37344 is also more potent (9-fold) than (-)-isoproterenol in stimulating adenylate cyclase activity of IBAT purified plasma membranes whereas, in the same preparation, it is 81-fold less potent than (-)-isoproterenol in competition displacement studies with the beta-adrenergic ligand, [125I]cyanopindolol. We have previously demonstrated that the photoaffinity reagent [125I]cyanopindolol-diazirine selectively labels a 62 kDa protein in IBAT plasma membranes that displays pharmacological properties of a beta 1-adrenergic subtype. Relatively high concentrations of BRL 37344 (10 microM) are required to displace [125I]cyanopindolol-diazirine binding to the 62 kDa protein. Taken together, the results suggest that two different populations of beta-adrenergic receptors may co-exist in BAT plasma membranes: a small population (about 15%) of atypical beta-receptors and a large population of beta 1-receptors that exhibit high and low affinities for BRL 37344, respectively.     

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.     

Comparison of the pharmacological profiles of adrenergic drugs (including BRL-agonists) at [3H]prazosin and [3H]CGP-12177 binding sites in brown adipose tissue

1. In order to determine the selectivity of classical and novel adrenergic agents for alpha 1- and beta-adrenergic receptors in brown adipose tissue, the ability of these agents to compete for binding sites labelled with [3H]prazosin and [3H]CGP-12177, respectively, was investigated. 2. The beta-antagonist propranolol, known to inhibit norepinephrine-induced respiration in micromolar concentrations, bound to the [3H]CGP-12177 site with nanomolar affinity. 3. Among agonists, only isoprenaline showed high selectivity for beta-receptors, and only oxymetazoline for alpha 1-receptors. 4. Unexpectedly, the novel thermogenic agonists (BRL-agonists), shown to be potent and selective stimulators of brown fat thermogenesis, were unselective and bound only with low affinity to the [3H]CGP-12177 binding sites. 5. These results suggest that the beta-adrenergic binding site in brown adipose tissue identified here with [3H]CGP-12177 may not be the one (or not the only one) coupled to thermogenesis.     

Characterization of beta-adrenergic receptors in cultured human and bovine endothelial cells

We used radioligand binding methods to characterize beta-adrenergic receptors on endothelial cells cultured from adult human iliac vein (HIVE) and bovine fetal aorta (BFAE). For comparison, we also studied the well-characterized C6 glioma cell line (C6). Both human and bovine endothelial cells showed specific saturable binding of [125I]iodopindolol. There was no difference in the binding affinity (KD) of iodopindolol to membranes from the three cell types. However, the beta-receptor density (Bmax) was greater on HIVE cells and BFAE cells than on C6 cells. Displacement of ligand from HIVE and BFAE cells by zinterol or from BFAE cells by ICI 89,406 was consistent with binding to the beta 2-subtype. In contrast, displacement of ligand from C6 cells by zinterol or ICI 89,406 was consistent with binding to both beta 1- and beta 2-subtypes. Exposing BFAE cells in culture to 10 microM isoproterenol for 6 h resulted in a 55% decrease in Bmax without a change in KD. We conclude that 1) human and bovine endothelial cells in culture contain a substantial population of beta-adrenergic receptors, which are predominantly of the beta 2-subtype, and 2) endothelial beta-receptors exhibit downregulation by beta-agonists in culture.     

Characteristics of the beta-adrenergic adenylate cyclase system of developing rabbit bone-marrow erythroblasts.

After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.     

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.     

Involvement of microtubules in the isoproterenol-induced 'down'-regulation of myocardial beta-adrenergic receptors

The number of cardiac beta-adrenergic receptors identified by [3H]dihydroalprenolol binding decreases in a concentration-dependent manner during prolonged administration of isoproterenol. Loss of membrane beta-receptors is paralleled by the appearance of [3H]dihydroalprenolol binding activity in the cytosol. This redistribution of receptors is prevented by colchicine and vinblastine but not lumicolchicine. Cardiac receptor desensitization is, therefore, dependent on microtubules and may be influenced by agents interfering with tubulin polymerization.     

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide: a ligand for in vitro and in vivo studies of serotonin receptors

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide ([125I]-EIL) has been evaluated as a ligand for in vitro and in vivo studies of cerebral serotonin 5-HT2 receptors. [125I]-EIL exhibited high affinity (KD = 209 pM) for 5-HT2 receptors with a high degree of specific binding (80-95%) in membranes from rat prefrontal cortex. The regional distribution of [125I]-EIL binding in vivo to seven areas of mouse brain correlated significantly (Rs = 0.93) with known densities of 5-HT2 receptors. In vivo specificity, defined by tissue to cerebellum radioactivity ratios, reached a maximum for frontal cortex at 6 hr (21.2) and persisted through 16 hr (8.8). Ketanserin, a 5-HT2 receptor antagonist, fully inhibited binding in a dose dependent fashion in all brain regions except cerebellum. By contrast, blockers for dopamine D2, alpha- or beta-adrenergic receptors did not significantly inhibit radioligand binding in any region. [125I]-EIL selectively labels 5-HT2 receptors in vivo with the highest specificity of any serotonergic ligand reported to date, indicating that [123I]-EIL should prove applicable to single photon emission computed tomography studies in living brain.     

N-Bromoacetyl-amino-cyanopindolol: a highly potent beta-adrenergic affinity label blocks irreversibly a non-protein component tightly associated with the receptor

A new chemical affinity label for the beta-adrenergic receptor, based on the structure of pindolol, has been synthesized and iodinated with 125I. The compound, N-bromoacetylamino-cyanopindolol (BAM-CYP), has an apparent dissociation constant of 44 +/- 7 pM towards the turkey erythrocyte membranes. This compound blocks irreversibly both the ability of beta-adrenergic receptors to bind 125I-cyanopindolol and the ability of beta-receptors to activate adenylate cyclase in the presence of beta-agonists. Furthermore, the irreversible binding of BAM-CYP to half of the beta-receptor sites abolishes the ligand binding activity of all the sites. These findings suggest that the beta-receptor is oligomeric in its native state. Although 125I-BAM-CYP blocks irreversibly and specifically the beta-adrenergic receptor, it does so by labeling a non-protein component, most probably a water-soluble lipid. The labeling is stereospecific since it is prevented by l-propranolol and not by d-propranolol. It is suggested that this lipid is tightly associated with the receptor in close proximity to the binding site. It is also suggested that this water-soluble lipid fraction may prove crucial for the optimal interaction between the beta-adrenergic receptor and the components of adenylate cyclase.     

Regulation of Brain Nicotinic Receptors by Chronic Agonist Infusion

Several studies have demonstrated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. To determine whether this effect is elicited by other nicotinic agonists found in tobacco, the effects of chronic infusion with nicotine on brain nicotinic receptors were compared with those after anabasine and lobeline. C57BL/6 mice were infused with saline or equimolar doses (18.5 mumol/kg/h) of nicotine, anabasine, or lobeline for 8 days. Nicotinic receptors, quantified by the binding of [3H]nicotine and [125I]iodo-alpha-bungarotoxin (alpha-[125I]BTX), and muscarinic receptors, quantified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB), were then assayed in eight brain regions. An increase in [3H]nicotine binding was observed in all regions except cerebellum following chronic infusion with nicotine and anabasine, whereas lobeline did not alter the number or affinity of these binding sites. This increase was due to changes in Bmax and not in the affinity of the receptor for the ligand (KD). A slight increase in alpha-[125I]BTX binding was observed in cortex following chronic anabasine infusion. [3H]QNB binding sites were largely unaltered following chronic infusion with any of the nicotinic analogs. The levels of the agonists in the brain were also determined after chronic treatment, and the amounts of lobeline and anabasine were found to be higher than that of nicotine. Thus, the failure of lobeline to elicit changes in nicotine binding is not due to reduced brain concentrations.     

Pharmacological Characterization of Cholinergic Receptors in a Human Neuroblastoma Cell Line

A human neuroblastoma cell line, IMR32, has been characterized as far as morphology, membrane receptors for neurotransmitters, and uptake and release of [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine). These cells expressed at their surface both nicotinic and muscarinic cholinergic receptors, revealed by [125I]alpha-bungarotoxin and [3H]quinuclidinylbenzilate ([3H]QNB) binding, respectively. [125I]alpha-Bungarotoxin binding was efficiently inhibited by alpha-bungarotoxin, nicotine, carbachol, and d-tubocurarine. [3H]QNB binding was competitively inhibited by atropine, pirenzepine, and carbachol. Hexamethonium did not affect the binding of either ligand. In competition experiments with [3H]QNB, pirenzepine recognized only one binding site with "low affinity," and carbachol recognized two sites with different affinities. beta-adrenergic receptors were present in a very low amount, whereas alpha-adrenergic and dopaminergic receptors were not detectable. IMR32 cells had an imipramine-sensitive [3H]dopamine uptake, but carbachol, high levels of K+, the calcium ionophore A23187, and alpha-latrotoxin were not able to induce release of [3H]dopamine that had been taken up. The ultrastructural analysis showed that IMR32 cells contained very few dense-core vesicles, suggesting a low storage capacity for neurotransmitter. These cells could be an useful in vitro model for studying neurotransmitter receptors of the human CNS.     

Beta-adrenergic-receptor-mediated suppression of interleukin 2 receptors in human lymphocytes

Adrenergic receptor agonists are known to attenuate the proliferative response of human lymphocytes after activation; however, their mechanism of action is unknown. Since expression of interleukin 2 (IL-2) receptors is a prerequisite for proliferation, the effect of beta-adrenergic receptor agonists on lymphocyte IL-2 receptors was studied on both mitogen-stimulated lymphocytes and IL-2-dependent T lymphocyte cell lines. In both cell types the beta-adrenergic receptor agonist isoproterenol blocked the expression of IL-2 receptors, as determined with the IL-2 receptor anti-TAC antibody. To determine the effect of beta-adrenergic agonists on expression of the high affinity IL-2 receptors, [125I]IL-2 binding studies were performed at concentrations selective for high affinity sites. No significant effect of beta-adrenergic agonists on high affinity IL-2 receptor sites could be detected. The data demonstrate that beta-adrenergic receptor agonists down-regulate IL-2 receptors primarily affecting low affinity sites.     

Dissociation of beta-adrenergic receptors from hormone responsiveness during maturation of the rat reticulocyte.

Intact rat erythrocytes and reticulocytes have been studied in relation to their concentration of beta-adrenergic receptors and their responsiveness to beta-adrenergic catecholamines. Characteristics of the beta-receptor, as determined by binding of 125I-labelled hydroxybenzylpindolol, were compared among control erythrocytes and reticulocytes. The dissociation constant (Kd = 0.1--0.2 nM), association and dissociation kinetics, and stereospecificity for (--)-isomers of agonists and antagonists were similar in both cell types. The reticulocyte population contained four times more receptors per cell than the control erythrocytes. However, reticulocytes were 25 times more responsive than control cells to isoproterenol, as measured by the formation of cyclic AMP. After peak reticulocytosis, cells rapidly lost 95% of their maximum hormone responsiveness, but beta-receptors declined much more slowly. The 4-fold decrease in beta-receptors was associated with a 4-fold decrease in cell volume as the reticulocytes matured. The density of beta-receptors was unchanged. However, responsiveness to isoproterenol in the reticulocytes when expressed on the basis of cell volume was still nine times greater than the control cells. Thus, maturation of reticulocytes is associated with an uncoupling of persistent beta-receptors from catecholamine responsiveness.     

Norepinephrine-induced down regulation of alpha 1 adrenergic receptors in cultured rabbit aorta smooth muscle cells

Drug-induced refractoriness of alpha-adrenergic receptor-mediated vasoconstriction may be a clinically important phenomenon. We have investigated the possible molecular mechanisms underlying this phenomenon in cultured vascular smooth muscle cells derived from the rabbit aorta. alpha 1-Adrenergic receptors were identified in membranes prepared from these cells by [125I]HEAT binding. The radioligand bound to a high affinity site (Kd = 140 pM) in a saturable fashion (202 fmol/mg protein). Adrenergic agonists and antagonists competed for binding of [125I]HEAT with the expected order of potency for an alpha 1-receptor, (-)epinephrine greater than or equal to (-) norepinephrine greater than (+)epinephrine greater than isoproterenol and prazosin greater than phentolamine greater than yohimbine. Exposure of cells for 26 hours to 10 microM norepinephrine resulted in a 70% decrease in the number of alpha 1-receptors as measured by [125I]HEAT binding without any significant change in the affinity of the receptor for the ligand. When the alpha-receptors were blocked with 10 microM phentolamine the loss of receptors induced by norepinephrine was completely prevented. Similar down-regulation of the [125I]HEAT binding sites was observed when the alpha 1-agonist phenylephrine was used instead of norepinephrine. It is concluded that alpha-agonists induce down-regulation of aortic smooth muscle alpha 1-receptors. This reduction of alpha-receptors could be important in the mechanisms by which vascular smooth muscle develops refractoriness to alpha-adrenergic stimulation.     

Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.     

Beta-adrenergic binding is increased by melatonin and alpha-adrenergic compounds

Binding of the beta-adrenergic ligands [3H]dihydroalprenolol and [125I]cyanopindolol to pineal particulate fractions was increased 1- to 3.5-fold by addition of low concentrations of melatonin, alpha-adrenergic agonists, or alpha-adrenergic antagonists. Minimum concentrations of melatonin or alpha-adrenergic compounds which increased beta-adrenergic binding were between 1 pM and 0.1 nM. The increased binding of [3H]dihydroalprenolol caused by melatonin (0.1 muM) was attributed to a major increase in Bmax, which persisted in protein fractions after removal of melatonin. Melatonin enhancement of [3H]dihydroalprenolol binding was apparent after 5 to 7 min (30(0], was was optimal between 20 and 40 min, and decreased at longer times. Alpha-Adrenergic receptors are unchanged during beta-receptor enhancement.     

Effect of the Tricyclic Antidepressant Desipramine on β-Adrenergic Receptors in Cultured Rat Glioma C6 Cells

Rat glioma C6 cells, cultured in the presence of the tricyclic antidepressant desipramine, lost a significant number of beta-adrenergic receptors in a time- and dose-dependent manner. A similar loss was observed whether binding was determined on intact cells with the hydrophilic beta-adrenergic antagonist (+/-)-[3H]4-(3-tert-butylamino-2-hydroxypropoxyl)benzimidazole-2-o n HCl ([3H]CGP-12177) or on cell lysates with the more hydrophobic antagonists [125I]iodocyanopindolol or [3H]dihydroalprenolol. When stimulated with the agonist isoproterenol, desipramine-treated cells accumulated less cyclic AMP than control cells. The affinity of the beta-adrenergic receptors for either antagonist or agonist was unchanged after desipramine treatment. Desipramine interacted only weakly with the receptors and competed for [125I]iodocyanopindolol binding with a Ki of 30 microM. The presence in the culture medium of alprenolol or propranolol, potent beta-adrenergic antagonists, however, did not prevent the reduction in receptors by desipramine. Desipramine also caused a loss of beta-adrenergic receptors from cells maintained in serum-free medium and the cells themselves did not contain or secrete endogenous catecholamines. Although desipramine is a potent inhibitor of catecholamine uptake, it appears unlikely that the observed loss of beta-adrenergic receptors in rat glioma C6 cells exposed to the drug is due to an increase in extracellular catecholamine levels or to a direct interaction with the receptors.