Quantitative autoradiographic analysis of 125I-pindolol binding in Fischer 344 rat brain: changes in beta-adrenergic receptor density with aging

Age-related changes in beta-adrenergic receptor density in Fischer 344 rat brain were examined using in vitro 125I-pindolol (IPIN) binding and quantitative autoradiographic analysis. Localized protein concentrations were determined using a new quantitative histological technique, and these were used to normalize the densities of receptors. Saturation binding studies in brain sections revealed 40-50% decreases in beta-adrenergic receptor density in the thalamus of 23-25-month-old and the cerebellum and brainstem of both 18-19-month-old and 23-25-month-old compared to 4-6-month-old rats. The loss of cerebellar beta-adrenergic receptors may be correlated with reports of deficits in sensitivity to beta-adrenergic-mediated transmission in the cerebellum of aged rats. No changes in specific IPIN binding with age were observed in rat cortex or hippocampus. In all areas examined no age-related differences were observed in receptor affinity. No changes in protein concentration were found in any of the areas examined in the different aged animals. These results demonstrate a region-specific loss of beta-adrenergic receptors with age in the brain of Fischer 344 rats.     

In vivo activation of pineal N-acetyltransferase but not type II thyroxine 5'-deiodinase by phenylephrine in young rats.

The regulation by alpha- and beta-adrenergic agonists of pineal N-acetyltransferase (NAT) and type II thyroxine 5'-deiodinase (5'-D) in rats at either 2 or 6 weeks of age was studied. The pattern of stimulation was different because NAT activity could be clearly activated by an alpha-adrenergic agonist, phenylephrine, only in 2-week-old rats. However, isoproterenol, a beta-adrenergic agonist, was able to stimulate NAT activity in rats at both 2 and 6 weeks of life. On the other hand, phenylephrine was always ineffective in stimulating 5'-D activity, while isoproterenol clearly activated it at both ages. These results strongly suggest a role for alpha-adrenergic receptors, in addition to beta-adrenergic receptors, in regulating rat pineal NAT activity during development.     

Changes in adipocyte beta-adrenergic receptor of cold-acclimated rats.

The changes in the number and affinity of binding sites in the beta-adrenergic receptors of rat white adipocytes after cold exposure were studied with the aid of (p)-[3H]dihydroalprenolol. One day cold exposure did not change the number and affinity of binding sites in beta-adrenergic receptors. Chronic exposure of rats to cold (5 degrees C) for 1 and 4 weeks significantly decreased the affinity of beta-adrenergic receptors without any alteration in the number of binding sites. Such changes in the binding affinity observed in cold-acclimated rats (4 weeks, 5 degrees C) remained for 18 hr after these animals were transferred to a warm environment of 25 degrees C. The decreased affinity of binding sites in beta-adrenergic receptor induced by cold acclimation could not explain the enhanced metabolic response of cold-acclimated animals to noradrenaline.     

Autonomic abnormalities and autoantibodies to beta-adrenergic receptors

We identified autoantibodies to beta 2-adrenergic receptors in the plasma of three apparently normal subjects, four patients with allergic asthma, one subject who was "preallergic" (at risk of allergy), and one patient with cystic fibrosis. Although these antibodies appeared to be heterogeneous, they shared the ability to affect binding of [125]protein A to calf-lung membranes, to inhibit beta-adrenergic ligand binding to calf-lung bet-adrenergic receptors, and to precipitate solubilized calf-lung beta-adrenergic receptors in an indirect immunoprecipitation assay. The presence of autoantibodies to beta-adrenergic receptors in these subjects correlates with abnormal autonomic responsiveness characterized by alpha-adrenergic and cholinergic hypersensitivity and beta-adrenergic hyposensitivity. These findings suggest that autoantibodies to beta-adrenergic receptors may play a part in the development of ment of autonomic abnormalities.     

Does the beta-adrenergic receptor function as a reovirus receptor?

A reovirus (type 3) receptor has previously been identified on the mouse thymoma R1.1 cell line and shown to be structurally similar to the mammalian beta-adrenergic receptor [M. S. Co, G. N. Gaulton, A. Tominaga, C.J. Homcy, B.N. Fields, and M.I. Greene (1985). Proc. Natl. Acad. Sci. USA 82, 5315-5318]. To determine whether beta-adrenergic receptors are universal recognition signals for reovirus binding, we studied the human epidermoid carcinoma cell line A431 which is known to possess large numbers of functional beta-adrenergic receptors and was found in the present study to be susceptible to reovirus infection. It was observed that unlike beta-adrenergic agonists, reovirus binding and internalization did not result in the triggering of cellular adenylate cyclase activity. The presence of reovirus also had no effect on cellular response to the agonist (-)-isoproterenol, nor on the binding of the hydrophilic antagonist [3H]CGP-12,177 to intact A431 cells. Furthermore, sequestration of beta-adrenergic receptors from the cell surface by (-)-isoproterenol had no effect on reovirus binding. Conversely, the binding of [3H]CGP-12,177 to cells with internalized reovirus receptors was found to be normal. These data strongly suggest that reovirus and beta-adrenergic receptors on A431 cells are distinct from each other. Similar observations were also made with the mouse L fibroblasts, with the exception that these cells appear to possess few functional beta-adrenergic receptors.     

Alpha- and beta-adrenergic receptors of the rat cerebral cortex and cerebral microvessels in aging, and their response to denervation.

We studied the effects of aging and norepinephrine depletion 2 weeks after unilateral locus ceruleus lesion on alpha 1-, alpha 2- and beta-adrenergic receptors by ligand binding methods in the ipsilateral and contralateral cerebral cortex of Fischer-344 rats. We also studied the effects of aging and noradrenergic denervation on beta-adrenergic receptors in isolated cerebral microvessels. We found that specific [125I]HEAT binding to alpha 1-adrenergic receptors was not affected by aging or by norepinephrine depletion. Although aging also had no effect on the density or affinity of [3H]UK-14,304 and [125I]pindolol binding to alpha 2- and beta-adrenergic receptors, the density of receptors increased significantly in all age groups after noradrenergic denervation. beta-Adrenergic receptors of cerebral microvessels also were unaffected by aging, but increased their density after noradrenergic denervation at all ages. In all instances, there were no significant effects on the affinity of ligand binding. We conclude that aging does not affect the density or the affinity of adrenergic receptors in the cerebral cortex of Fischer-344 rats, nor does it affect the response of these receptors to norepinephrine depletion.     

Modulation of beta-adrenergic receptors in the pituitary gland following adrenalectomy in rats

The effects of adrenalectomy on beta-adrenergic receptors in the rat pituitary were examined using quantitative in vitro autoradiography with 125I-iodocyanopindolol (125ICYP). 125ICYP binding in the anterior, intermediate and posterior lobes of the pituitary gland was significantly increased in chronically adrenalectomized rats. The increase in 125ICYP binding sites in the rat pituitary following adrenalectomy was not reversed by glucocorticoid replacement with dexamethasone. These data indicate that catecholamines of adrenomedullary origin are capable of modulating beta-adrenergic receptors in the pituitary gland and suggest that peripheral epinephrine may be important in regulating pituitary hormone secretion.     

Huntington's chorea. Changes in neurotransmitter receptors in the brain.

Neurotransmitter-receptor binding sites for apparent muscarinic cholinergic, beta-adrenergic, gamma-aminobutyric acid and serotonin receptors were measured in the caudate nucleus and frontal cerebral cortex from post-mortem brains of 16 patients with Huntington's chorea and 16 controls. In addition, the samples were assayed for the gamma-aminobutyric-acid-synthesizing enzyme, glutamic acid decarboxylase, and for the acetylcholine-synthesizing enzyme, choline acetyltransferase. In the caudate nucleus of choreic brain, both enzyme activities were markedly lower, with significant decreases in muscarinic cholinergic and serotonin receptor binding, whereas enzyme activities and receptor binding were unchanged in the cerebral cortex. By contrast, gamma-aminobutyric acid and beta-adrenergic receptor binding were not significantly different in choreic and control caudate nucleus or cortex, suggesting that, despite the loss of gamma-aminobutyric-acid-synthesizing ability in the corpus striatum, gamma-aminobuytric acid mimetic drugs might alleviate the movement disorders in Huntington's chorea.     

Alpha- and beta-adrenergic receptor function in the brain during senescence

Alpha- and beta-adrenergic receptors and their second messengers play an important role in brain neurotransmission. Changes in receptor function with age may be involved in the age-related changes in arousal, mood and memory. The predominance of data indicates there is decreased beta-adrenergic receptors in all areas of the brain with the exception of the cortex. Evidence suggests a decreased rate of receptor synthesis may be contributing to this loss of receptors with age. Alpha-adrenergic receptor synthesis is also diminished with age. The modulation of receptor concentrations by hormonal factors is impaired with age, especially the time to recover from receptor down-regulation.     

Myocardial beta-adrenergic receptor characteristics in T(3)-induced ascites and in broiler lines differing in ascites susceptibility.

The present study was designed to investigate the myocardial beta-adrenergic receptor characteristics in T3-induced ascites in two genetic lines of broilers, in order to look for possible involvement of myocardial beta-adrenergic receptors in the development of broiler ascites syndrome. Myocardial membrane fractions were prepared from 6 and 8 week old chickens fed a 1.5 parts/10(6) T(3) supplemented diet from day 1 in order to increase ascites incidence. Also, a similar assay was performed on myocardial cells of ascites sensitive and ascites resistant chickens at 5 weeks of age. The binding capacity and binding affinity of the myocardial beta-adrenergic receptor of the two genetic lines of birds did not differ significantly, but the tendency of beta-adrenergic receptor binding capacity and affinity constants in ascites-sensitive birds was slightly higher compared to ascites-resistant birds. In commercial broilers, although dietary T(3) significantly increased ascites mortality and the RV/TV ratio compared to control, it did not affect significantly beta-adrenergic receptor characteristics.     

Endogenous opioid regulation of norepinephrine release in guinea pig hippocampus.

Release of endogenous norepinephrine was detected in guinea pig hippocampal slices using a radioligand displacement assay. Focal electrical stimulation released endogenous norepinephrine and caused a calcium-dependent reduction in specific [3H]propranolol binding at beta-adrenergic receptors in the brain slice. The mu-opioid agonist PL017 decreased norepinephrine release, and the inhibition by PL017 could be blocked by the opioid antagonist naloxone. Endogenous opioid peptides concomitantly released by tissue stimulation also decreased norepinephrine release in a naloxone-sensitive manner. These results support the hypothesis that endogenous opioids can regulate excitability in the hippocampus by presynaptic modulation of norepinephrine release.     

Chronic reserpine administration selectively up-regulates beta 1- and alpha 1b-adrenergic receptors in rat brain: an autoradiographic study.

Rats were treated for 15 days with reserpine or vehicle. One day after the last treatment, animals were killed and frozen brain sections were prepared for in vitro autoradiography. Binding to beta-adrenergic receptors was measured with [125I]iodocyanopindolol, and binding selective for beta 1 and beta 2 subtypes was assessed by including non-radioactive drugs that selectively mask beta receptor subtypes. Total alpha 1-adrenergic receptor binding was measured with [3H]prazosin, while alpha 1a binding was measured with [3H]WB4101 (in the presence of unlabeled serotonin). Quantitative densitometric analysis revealed that chronic reserpine treatment caused an increase in beta binding throughout the brain, including the cortex, thalamus, amygdala, hippocampus, caudate-putamen and hypothalamus. This effect of reserpine was entirely confined to the beta 1 subtype in all regions examined. [3H]Prazosin binding (alpha 1a plus alpha 1b) was also increased after chronic reserpine in several regions of the cortex and thalamus, as well as the ventral hippocampus and caudal amygdala. No effect of chronic reserpine was seen on [3H]WB4101 binding, indicating that the effect of reserpine on alpha 1 receptors is limited to the alpha 1b subtype. The increase in alpha 1b binding after reserpine administration in rats was generally smaller and less widespread than that seen with beta 1 binding. Thus the effect of reserpine upon noradrenergic neurotransmission demonstrates a high degree of receptor specificity and regional selectivity.     

Reduced beta-adrenergic responsiveness in hypothyroid rats.

The beta-adrenergic agonist isoproterenol (100-200 mug/kg body wt sc) induces vasodilation and an increase in skin temperature of the tail of the euthyroid but not the hypothyroid rat. Administration of thyroxine (25 mug/kg body wt per day) to rats made hypothyroid by means of the antithyroid drug aminotriazole (0.5 g/kg of foof) returned responsiveness to control level. Reduced responsiveness to isoproterenol occurred between 1 and 5 wk of treatment with aminotriazole. Increase in tail skin temperature induced in euthyroid rats by isoproterenol was blocked by administration of propranolol (7.5 mg/kg ip). Other beta-adrenergic-induced responses, including increased water intake and increased plasma glucose concentration, also were reduced in hypothyroid rats and returned to control level by administration of thyroxine. Thus, hypothyroidism appears to be accompanied by a reduced beta-adrenergic responsiveness as assessed by changes in tail skin temperature, water intake, and plasma glucose concentration after injection of isoproterenol. Since administration of thyroxine returned the responses of hypothyroid rats to control levels, it appears that thyroxine is important in maintaining beta-adrenergic responsiveness under these conditions.     

Changes in beta-adrenergic receptors in dog livers during endotoxic shock

The effects of endotoxin administration on beta-adrenergic receptors in dog liver plasma membranes were studied using [3H]dihydroalprenolol as a radioactive ligand. The Scatchard analysis revealed a one-component binding characteristic both in control and endotoxin-injected dogs. The Kd (dissociation constant) value was increased by 60% (4.2 +/- 0.5 nM for control vs. 6.7 +/- 0.5 nM for endotoxic; P less than 0.01) and the Bmax (maximum binding capacity) was decreased by 38% (600 +/- 60 and 370 +/- 70 fmol/mg protein for control and endotoxic, respectively; P less than 0.01) 2 h following endotoxin administration. The competitive inhibition studies show that the apparent Kd values for (-)-isoproterenol, (-)-epinephrine, and (-)-norepinephrine were increased by 14, 51, and 5 times, respectively, 2 h postendotoxin. In addition, endotoxin in vitro had a dose-dependent inhibitory effect on the specific binding of [3H]dihydroalprenolol, and it also reduced the number of beta-receptors. These data demonstrate that endotoxin, both in vivo and in vitro, decreased the binding affinity and the number of beta-adrenergic receptors in dog liver plasma membranes. A modification of the beta-adrenergic receptors in dog livers induced by endotoxin administration may play an important role in the development of hepatic glucose dyshomeostasis during shock.     

Alzheimer's disease: changes in hippocampal N-methyl-D-aspartate, quisqualate, neurotensin, adenosine, benzodiazepine, serotonin and opioid receptors--an autoradiographic study

The following receptors were assessed post-mortem in the hippocampi (anterior region) of eight patients with Alzheimer's disease and nine age-matched controls, using autoradiography: N-methyl-D-aspartate (including glutamate, phencyclidine and glycine binding sites), quisqualate, kainic acid, adenosine A1, benzodiazepine, serotonin (1 and 2), muscarinic cholinergic, beta-adrenergic, neurotensin and opioid receptors. In CA1 there were significant parallel losses of binding to the three N-methyl-D-aspartate-linked sites (average reduction 46%) and also losses of quisqualate (38%) and serotonin2 (58%) receptor binding, with a 47% loss of binding to A1 sites. Binding to all of these receptors was also reduced in CA3 (except binding to A1 sites which was normal) but only the serotonin2 receptor binding loss reached significance (52%). A significant reduction in binding was also observed in the entorhinal area to the N-methyl-D-aspartate receptor-linked sites (average reduction = 39%), benzodiazepine (40%) and serotonin2 receptors (45%), and there was a loss of binding to neurotensin (57%) and opioid receptors (42%). Significant reductions in the dentate gyrus molecular layer were seen for serotonin2 receptors (44%), and binding to opioid (44%) and A1 receptors (46%). Levels of ligand binding to muscarinic cholinergic, serotonin1, beta-adrenergic and kainic acid receptors were not significantly different from control values in any of the four areas examined. These results provide support for observations of selective receptor changes in Alzheimer's disease involving a broad range of receptor types which encompass both excitatory amino acid and other receptors (notably serotonin2, A1, benzodiazepine, neurotensin and opioid receptors). The implications of the pattern of receptor changes for the suggestion that excitotoxicity plays a role in the disease are discussed, as is the possible contribution of the receptor changes to the symptomatology of Alzheimer's disease.     

Autoradiographic localization of binding sites for [3H]histamine and H1- and H2-antagonists on cultured neurones and glial cells

By means of autoradiography we have studied the cellular localization of binding of [3H]histamine and H1- and H2-antagonists in explant cultures of rat cerebellum, brain stem and spinal cord. In brain stem and spinal cord cultures, a relatively great number of neurones revealed binding sites for [3H]histamine and to a lesser extent also for the H1-antagonist [3H]pyrilamine and for the H2-antagonist [3H]tiotidine. In contrast, only a small number of labelled neurones was found in cerebellar cultures. The intensity of labelling was usually much stronger for [3H]histamine than for its antagonists, suggesting that binding sites for histamine might reflect both H1- and H2-receptors. Glial cells also showed binding sites for [3H]histamine and the H1- and H2-antagonists, the number of labelled astrocytes by these radioligands was, however, smaller than that observed with [3H]noradrenaline and alpha- and beta-adrenergic antagonists. It is suggested that in addition to alpha- and beta-adrenoceptors, glial cells also possess receptors for histamine.     

Demonstration of beta-adrenergic receptors in rat nasal glands by an in vitro autoradiographic technique

The distribution of beta-adrenergic receptors in rat nasal glands has been investigated with the use of an in vitro autoradiographic technique. Radioligand binding studies indicated that [125I]cyanopindolol binds specifically to beta-adrenergic receptors in cryostat sections of these glands. Autoradiograms generated after incubation with 0.02 nM [125I]cyanopindolol and dipping in nuclear K2 emulsion, showed specific labelling of the striated excretory ducts. These in vitro observations suggest a sympathetic control of the ion and water content of the glandular secretion.     

Distribution of beta-adrenergic receptors on human lymphocyte subpopulations.

A technique is described allowing the quantification and the characterization of specific beta-adrenergic receptors in intact living human lymphocytes. 125I-Iodohydroxybenzylpindolol, a potent beta-adrenergic antagonist was used to label specific binding sites on unfractionated lymphoid cells and on purified subpopulations of T (F1 and F2) and B cells. F1 and F2 were obtained by filtration through nylon wool column as previously described (Delespesse et al., 1976), they differ in their response to mitogens, and in their interactions with adherent cells and B cells. 125I-HYP binding to unfractionated lymphocytes was a saturable, stereospecific and rapid process with a dissociation constant of 2.5 10(-10) M and a binding capacity of 400--600 sites/cell. Bindings on unfractionated lymphocytes, purified B cells and T cells of the F2 fraction were similar. No detectable binding was noted on T cells from the F1 fraction. Enriched T cells obtained by a rosetting technique displayed 200 receptors/cell.     

Decreased density of beta-adrenergic and muscarinic cholinergic receptor sites in the vasa nervorum of aged rats

The pharmacological profile and the anatomical localization of beta-adrenergic and muscarinic cholinergic receptors of the vasa nervorum were studied in sections of sciatic nerve using radioreceptor binding and light microscope autoradiography techniques. Sprague—Dawley rats of 4 and 24 months of age were used. [³H]Dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) were used to label beta-adrenergic and muscarinic cholinergic receptors, respectively. The ligands were bound to sections of rat sciatic nerve in a manner consistent with the labelling of beta-adrenergic or muscarinic cholinergic receptors in the 2 age groups investigated. The dissociation constant (K_d) values (about 1.37 nM for [³H]DHA and 0.75 nM for [³H]QNB) did not significantly change between 4- and 24-month-old rats. The maximum concentration of binding sites (B_max) for [³H]DHA was decreased by about 35% in 24 in comparison with 4-month-old rats. The B_max value autoradiogaphy revealed the development of specific silver grains in the medial layer of epineurial and perineurial arteries in sections of sciatic nerve exposed either to [³H]DHA or [³H]QNB. The number of silver grains developed in epineurial and perineurial arteries of rats of 24 months is significantly lower than in animals of 4 months. The above results suggest the occurrence of an age-dependent loss in the density of beta-adrenergic and muscarinic cholinergic receptors of vasa nervorum.     

Effects of thyroid hormones on cardiac hypertrophy and beta-adrenergic receptors during aging

Administration of thyroxine daily at a dosage of 6.4 mu g/g body weight stimulates cardiac hypertrophy in both 9--13-month-old (mature, n = 34) and 22--24-month-old (senescent, n = 45) Wistar rats. The increase due to thyroxine as well as the time course of the effect do not change with age, although ventricular wet weight to tibial length ratios are higher in senescent animals at 0, 3, and 7 days of treatment (senescent controls = 0.331; senescent hypertrophied = 0.395; mature controls = 0.295; mature hypertrophied = 0.336). Hypertrophy is maximal after 3 days. beta-Adrenergic receptor levels as measured by stereospecific binding of dihydroalprenolol in mature rats are increased about two-fold after 7 days of thyroxine treatment (mature controls = 35 +/- 3 fmol/mg protein, mature treated = 65 +/- 6 fmol/mg protein). Although both stimulated and control values do not differ significantly between mature and senescent groups (senescent controls = 45 +/- 4 fmol/mg protein, senescent treated = 56 +/- 9 fmol/mg protein), the effect of thyroxine on senescent hearts is not statistically significant. In addition, 3-day levels do not increase over 0-day controls at either age. No significant differences in either beta-adrenergic receptor concentrations or affinities between age groups are observed at 0, 3, and 7 days of treatment. However, senescent membrane preparations contain 33% more sialic acid (a rough plasma membrane marker) per unit of protein than mature preparations. Thus, the beta-adrenergic receptor density per unit of sialic acid may be reduced very slightly in the senescent hearts.