Down-regulation Of Receptor Number Research Articles

GABA Induces Activity Dependent Delayed-onset Uncoupling of GABA/Benzodiazepine Site Interactions in Neocortical Neurons

Changes in the function of type A gamma-aminobutyric acid receptors (GABA(A)Rs) are associated with neuronal development and tolerance to the sedative-hypnotic effects of GABA(A)R positive modulators. Persistent activation of GABA(A)Rs by millimolar concentrations of GABA occurs under physiological conditions as GABAergic fast-spiking neurons in neocortex and cerebellum exhibit basal firing rates of 5 to 50 Hz and intermittent rates up to 250 Hz, leaving a substantial fraction of synaptic receptors occupied persistently by GABA. Persistent exposure of neurons to GABA has been shown to cause a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine (BZD) site interactions with a half-life of approximately 24 h. Here, we report that a single brief exposure of neocortical neurons in primary culture to GABA for 5-10 min (t(1/2) = 3.2 +/- 0.2 min) initiates a process that results in uncoupling hours later (t(1/2) = 12.1 +/- 2.2 h). Initiation of delayed-onset uncoupling is blocked by co-incubation with picrotoxin or alpha-amanitin but is insensitive to nifedipine, indicating that uncoupling is contingent upon receptor activation and transcription but is not dependent on voltage-gated Ca2+ influx. Delayed-onset uncoupling occurs without a change in receptor number or a change in the proportion of alpha1 subunit pharmacology, as zolpidem binding affinity is unaltered. Such activity dependent latent modulation of GABA(A)R function that manifests as delayed-onset uncoupling may be relevant to physiological, pathophysiological, and pharmacological conditions where synaptic receptors are transiently exposed to GABA agonists for several minutes.

IgE‐independent atopic dermatitis is associated with a β‐2 adrenergic receptor gene polymorphism

IgE levels are not elevated in about 20% of patients with atopic dermatitis (AD). In this intrinsic AD (IAD), allergic mechanisms are not very important and pathogeny could be mainly neurogenic. β2‐adrenergic receptors are localized on cells involved in AD: Langerhans' cells, keratinocytes and lymphocytes. We wondered whether IAD could be associated with gene polymorphisms 16 and 27 of this receptor. We studied 98 healthy subjects and 83 subjects suffering from DA (UKWP criteria). IgE levels were normal in 12 of them and elevated in 71 (EAD). After DNA extraction, the genotyping was done by PCR and Direct Sequencing of candidate gene. Statistical analysis was performed with EPI‐INFO 6.04 for χ2 test. We found a significant association of Gln27Glu polymorphism with IAD (P = 0.00071 and χ2 = 14.51). There was no difference between healthy subjects and EAD patients. Adrenergic receptor agonists are known to attenuate the proliferative response of human lymphocytes after activation, through the inhibition of interleukin‐2 release. It is known that catecholamines inhibit the antigen‐presenting capability of epidermal Langerhans' cells. Long‐term agonist‐promoted downregulation of receptor number is absent when glu is at position 27. We suggest that the suppression of inhibiting effects of catecholamines could be involved in IAD pathogeny. Dichotomic nature of AD (EAD and IAD) is also associated with polymorphisms (SNP) of the interleukin‐4/interleukin‐13 receptor gene and the differences of cutaneous variables (transepidermal water loss, capacitance and pH). Altogether, these findings indicate that IAD patients exhibit phenotypic and also genotypic features which differ from those patients with EAD. Otherwise, the presence of this polymorphism could provide an explication of rarity of hypertension with AD, because Glu27Gln has been identified as a susceptibility polymorphism for HTA.

Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells.

We have investigated the identity and intracellular cascade of responses resulting from activation of the endogenous 5-hydroxytryptamine receptor in the C6 rat glioma cell line. Sequence analysis of reverse transcription-polymerase chain reaction products derived from C6 glioma cell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine2A receptor. The binding of [3H]ketanserin to cell membranes demonstrated a significant correlation with the 5-hydroxytryptamine2A receptor in rat frontal cortex. On intact cells, 5-hydroxytryptamine stimulated a concentration-dependent increase in phosphatidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydroxytryptamine2A receptors. In whole-cell patch-clamp recordings, 5-hydroxytryptamine induced an outward current mediated predominantly by K+ ions (reversal potential = -80 mV). Using caged molecules containing Ca2+ or inositol 1,4,5-trisphosphate in the patch electrode solution, we found that rapid photolytic release of Ca2+ and particularly inositol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydroxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosphoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+]i and activation of an outward current was observed more frequently in differentiated cells. Prolonged exposure of the cells to 5-hydroxytryptamine led to a decrease in all responses and to the down-regulation of receptor number. We conclude that the rat C6 glioma cell expresses a 5-hydroxytryptamine2A receptor identical to that found in rat brain and that stimulation of the receptor in C6 cells leads to the activation of Ca2+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca2+ ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptamine compared to Ca2+ release and conductance change indicate that a complex relationship exists between the component parts of the receptor-activated cascade.

Receptor-specific functional properties of beta 2-adrenergic receptor autoantibodies in asthma.

beta 2-Adrenergic receptor (beta 2 AR) autoantibodies have been reported in the serum from subjects with asthma but the functional significance of such antibodies is not known. To characterize these antibodies, we developed a Western blot (WB) technique that utilized overexpressed recombinant human beta 2AR as antigen and also developed a control antisera in rabbits directed against the C-terminus of the receptor. beta 2AR autoantibodies were detected in approximately 5% of normal subjects and in approximately 40% of asthmatic subjects. Eighty-four percent of these antibodies were of the IgG class, with the remainder being IgM. Most (73%) of WB-positive sera inhibited [125I]cyanopindolol binding to recombinant solubilized receptors. The mean binding inhibition was 40.4 +/- 5.1% for WB-positive sera versus 7.6 +/- 1.2% for WB-negative sera. Binding of antibody to beta 2AR expressed on intact cells significantly depressed receptor function, with a > 50% attenuation of isoproterenol-stimulated cAMP production. This effect was receptor specific, as forskolin-stimulated cAMP accumulation was not affected by exposure to sera. WB-positive sera that did not inhibit radioligand binding had no effect on receptor function. Thus, some antibodies appear to bind near the ligand binding pocket and act as functional antagonists. In addition, incubation of intact cells expressing beta 2AR with WB-positive sera for 18 h resulted in a 30.3 +/- 0.6% downregulation of receptor number, whereas WB-negative sera induced no downregulation. This downregulation response with WB-positive sera was not affected by coincubation with the antagonist propranolol and was apparently not dependent upon whether the antibody interacted with ligand binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

The palmitoylated cysteine of the cytoplasmic tail of alpha 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation.

Most guanine nucleotide binding protein (G protein)-coupled receptors have a conserved cysteine in the C-terminal cytoplasmic tail near the seventh transmembrane spanning region. This cysteine is known to be palmitoylated in rhodopsin, the beta 2-adrenergic receptor (beta 2AR) and the alpha 2A-adrenergic receptor (alpha 2AAR). For the beta 2AR, this cysteine has been shown to be important for stimulatory G protein (Gs) coupling and agonist-promoted desensitization. For the alpha 2AAR (human alpha 2 C10) palmitoylation occurs at Cys-442, but it is not known what function such fatty acid acylation subserves. The closely related alpha 2CAR subtype denoted alpha 2C4 lacks a cysteine in this region and has different G-protein-coupling characteristics and agonist regulatory properties as compared to alpha 2C10. To assess the role of the palmitoylcysteine in alpha 2AR function, we constructed a mutated alpha 2C10 having a phenylalanine (the analogous amino acid in the alpha 2C4 in this position) substituted for Cys-442, denoted alpha 2C10(Phe-442), and expressed this along with wild-type alpha 2C10 and alpha 2C4 in CHO cells. Functional coupling to inhibitory G protein (Gi) and to Gs was identical between wild-type alpha 2C10 and alpha 2C10(Phe-442). Agonist-promoted desensitization of both the Gi and Gs-mediated pathways was also found to be unaffected by this mutation. Cellular trafficking induced by agonist exposure was evaluated by delineation of intracellular (sequestered) versus cell surface receptors and by determination of net receptor loss. Mutation of Cys-442 did not alter the extent or rate of agonist-promoted sequestration induced by agonists or the recovery from sequestration. However, the downregulation of receptor number after prolonged agonist exposure was completely abolished by this mutation and converted alpha 2C10 to an alpha 2C4 phenotype in regard to this adaptive response. Another mutated alpha 2C10, in which Cys-442 was replaced by alanine, also failed to downregulate. Thus, the function of this cytoplasmic palmitoylcysteine is distinctly different between the alpha 2AR and other G-protein-coupled receptors such as the beta 2AR and rhodopsin, and this suggests that this molecular attribute may subserve diverse roles among members of this family of receptors. For the alpha 2ARs, this may represent an evolved feature that provides for differing needs for regulation of the alpha 2C10 and alpha 2C4 subtypes by agonist.

Metabolism of low‐density lipoprotein in differentiated and undifferentiated HT29 colon cancer cells

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.

Regulation by High Density Lipoproteins of Muscarinic Acetylcholine Receptor Function in Chick Heart Cells Cultured in Defined Medium

Activation of cardiac muscarinic acetylcholine receptors (mAChR) on cultured chick heart cells results in a decrease in cellular cAMP levels and a stimulation of phosphoinositide breakdown. A serum-free culture system has been used to investigate the regulation of mAChR number and function by purified serum high density lipoprotein (HDL). Administration of HDL purified from rooster serum to chick heart cells cultured in defined medium results in an attenuation of the ability of muscarinic agonist to inhibit forskolin-stimulated cAMP accumulation, with no change in its ability to stimulate phosphoinositide hydrolysis or to mediate down-regulation of receptor number. The inclusion of HDL in the culture medium did not result in appreciable changes in mAChR number or affinity, nor were the levels of the inhibitory guanine nucleotide-binding regulatory proteins (G-proteins) altered. However, the ability of guanine nucleotides to inhibit forskolin-stimulated adenylate cyclase activity was reduced by HDL treatment, suggesting that HDL interferes with the capacity of G-proteins to interact with adenylate cyclase. In order to determine which component of native HDL mediates the decreased effectiveness of carbachol, the ability of lipid and apoprotein fractions to mimic the effect of HDL was tested. HDL lipid fractions were able to mimic the effect of native HDL, while protein fractions were not. This result suggests that the ability of HDL to attenuate muscarinic receptor function is mediated by its lipid constituents. The effect of HDL and HDL lipid fractions were not correlated with changes in membrane cholesterol content.

Glucocorticoid receptor regulation: The effects of adrenalectomy, exogenous glucocorticoid, and stress on hepatic receptor number in male and female mice

Although glucocorticoids are known to regulate their own receptor number, the physiologic significance of this process is not known. In order to assess this process in intact animals the effects of adrenalectomy, stress, and exogenous glucocorticoid on the number of hepatic glucocorticoid receptors in Swiss-Webster mice were evaluated. In males 24 hr after adrenalectomy there was a clear 2- to 2.5-fold increase (upregulation) in glucocorticoid receptor number. Conversely, 24 hr after the ip administration of exogenous corticosterone there was a clear downregulation of receptor number. In each case (upregulation and downregulation) female mice were much less responsive than males. Three stressors were used to evaluate the effect of the endogenous secretion of glucocorticoids on downregulation. Male mice were exposed to ether, vibration, and confinement either once or daily for periods up to 3 days. Animals were sacrificed 24 hr after the last stress and hepatic receptor number was compared to an unstressed control. Cytosolic receptor number was not influenced by any of these stimuli. It is concluded that although glucocorticoids clearly regulate glucocorticoid receptor number, as demonstrated by adrenalectomy and the administration of steroids to adrenalectomized animals, the physiologic significance of this process is uncertain as receptor number does not appear to be changed by stimuli of adrenal glucocorticoids in the intact animal.

Muscarinic receptors on cultured cells of rat hippocampus: cholinergic regulation and presence of subtypes

Muscarinic acetylcholine receptors in intact, cultured explants of rat hippocampus were investigated in binding experiments with tritiated quinuclidinyl benzilate ([ 3H]QNB) as ligand. Dissociation constants (K d) were determined to 320–575 pM and maximal binding capacity (B max) to 67–87 fmol/explant. The K d S obtained in kinetic experiments were very similar. Hippocampal explants cultured alone contained more muscarinic receptors than hippocampal explants reinnervated by cholinergic fibers from co-cultured septal explants. Pretreatment of hippocampal explants with carbachol resulted in a down-regulation of receptor number which was counteracted by the simultaneous addition of atropine. Atropine added alone had no effect on receptor number in hippocampal explants cultured alone whereas it occasionally caused an up-regulation in co-cultured hippocampus. Displacement experiments with scopolamine and oxotremorine as competitors, showed that hippocampal explants cultured alone contain multiple types of muscarinic receptors. With atropine, pirenzepine and AF-DX 116, only one class of receptors could be detected.

The carboxy-terminal segment of the yeast α-factor receptor is a regulatory domain

The α-factor receptor is rapidly hyperphosphorylated on Thr and Ser residues in its hydrophilic C-terminal domain after cells are exposed to pheromone. Mutant receptors in which this domain is altered or removed are biologically active and bind α-factor with nearly normal affinity. However, cells expressing the mutant receptors are hypersensitive to pheromone action and appear to be defective in recovery from α-factor-induced growth arrest. Mutant receptors with partial C-terminal truncations undergo ligand-induced endocytosis, suggesting that down-regulation of receptor number is not the sole process for adaptation at the receptor level. A mutant receptor lacking the entire C-terminal domain (134 residues) does not display ligand-induced endocytosis. Genetic experiments indicate that the contribution of SST2 function to adaptation does not require the C-terminal domain of the receptor.

Certain beta-blockers can decrease beta-adrenergic receptor number: II. Down-regulation of receptor number by alprenolol and propranolol in cultured lymphoma and muscle cells.

We have used two different cultured cell lines--S49 lymphoma cells and BC3H-1 muscle cells--to examine the regulation of beta-adrenergic receptors by receptor antagonists. Rather than an increase ("up-regulation") of receptor number that such antagonists often produce, we found that certain beta-blockers elicit a decrease ("down-regulation") of beta-adrenergic receptors. Alprenolol and propranolol, but not sotalol or ICI 118,551, at concentrations of 10-100 nM down-regulated beta-adrenergic receptors 20-70% following 16-20 hours of treatment of S49 or BC3H-1 cells. Several observations suggest that this phenomenon depends upon beta-receptor interaction, including stereoselectivity [(-)-enantiomers more potent than (+)-enantiomers], blockade of the effect by ICI 118,551, absence of down-regulation of alpha-adrenergic receptors in BC3H-1 cells, and lack of a decrease in beta-adrenergic receptor-independent (forskolin-stimulated) cyclic AMP accumulation in S49 cells. The possibility of retained antagonist interfering with receptor measurement was precluded by the fact that the antagonist-induced decrease in receptor number required several hours incubation and occurred without a prominent change in receptor affinity. The ability of the beta-blockers to elicit down-regulation did not correlate with hydrophobicity of the drugs. Antagonist-induced down regulation of beta-adrenergic receptors did not occur in S49 lymphoma cells that lack the alpha-subunit of Gs, the guanine nucleotide-binding regulatory protein, thus implying a requirement for receptor-alpha s interaction in eliciting beta-receptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of glucocorticoid and beta-adrenergic receptors on lectin-stimulated splenocytes.

Activation of porcine splenocytes with the mitogen, concanavalin A, increases the number of glucocorticoid and beta-adrenergic receptors with no change in the apparent dissociation constant. Incubation of splenocytes with concanavalin A in the presence of hydrocortisone 21-sodium succinate prevented this mitogen-induced increase in glucocorticoid receptors. Isoproterenol also prevented the concanavalin A-induced increase in beta-adrenoceptors at 24 hr and reduced the binding affinity of these receptors at 48 hr. Neither agonist had any significant effect on the receptor number of binding affinity of nonstimulated cells. These data demonstrate that the increase in the number of glucocorticoid and beta-adrenergic receptors that occur on lymphoid cells after activation by a T-cell mitogen can be prevented by appropriate hormone agonists. Down-regulation of receptor number by appropriate agonists appears to be a common regulatory system that is shared by both the neuroendocrine and the immune systems.

Altered platelet alpha2-adrenoceptors in patients with angina pectoris

The alpha 2 -adrenoceptor located on noradrenergic neurons regulates the release of norepinephrine by negative feedback. This receptor is also located on human blood platelets, and the number of these receptors is correlated with the plasma norepinephrine content. The purpose of this study was to determine the status of platelet alpha2-adrenoceptors in patients with symptomatic and asymptomatic coronary artery disease. It was hypothesized that patients with symptomatic coronary disease might have a decrease in alpha2-adrenoceptor number that might be related to increased neuronal norepinephrine release. Specific, high-affinity binding of the alpha2-agonist, 3H-clonidine, and the alpha2-antagonist, 3H-yohimbine, to isolated platelet membranes was used to determine the maximal number of binding sites (in fmol/mg protein ± standard error of the mean) and the dissociation constant (in nM) of the alpha2-receptors. In normal subjects, the number of binding sites for 3 H-clonidine was 32 ± 2 and the dissociation constant was 5.5 ± 0.6 (n = 26); the maximal number of binding sites for 3 H-yohimbine was 165 ±12 and the dissociation constant was 4.0 ± 0.5 (n = 16). In patients with symptomatic coronary artery disease, there was a 38% decrease in 3 H-clonidine binding (number of binding sites = 20 ± 3; dissociation constant = 6.6 ± 1.2; n = 9; p < 0.05) and a 44% decrease in 3 H-yohimbine binding (number of binding sites = 93 ± 8; dissociation constant = 4.5 ± 0.3; n = 18; p < 0.005). In patients with asymptomatic coronary artery disease, there were no significant changes from the normal population in binding with either ligand ( 3 H-clonidine: number of binding sites = 30 ± 2; dissociation constant = 6.9 ± 1.9; n = 6 and 3 H-yohimbine: number of binding sites = 137 ±11; dissociation constant = 5.1 ± 0.6; n = 10). Five patients were studied during a symptomatic phase of their disease and then restudied during a quiescent period. In this group, the number of alpha 2 -adrenoceptors increased markedly as shown in 3 H-clonidine from 16 ± 3 to 29 ± 3 (44%, p < 0.005) and in 3H-yohimbine from 69 ± 22 to 127 ± 17 (46%, p < 0.05). The affinity constants and plasma norepinephrine concentrations did not differ among the three groups. If similar changes in receptor number occur on nerve terminals, this may represent a primary abnormality that permits enhanced release of norepinephrine or a secondary down regulation of receptor number in response to increased synaptic levels of norepinephrine.

Lymphocyte beta-adrenergic refractoriness induced by theophylline or metaproterenol in healthy and asthmatic subjects

Beta-adrenergic refractoriness was assessed in human lymphocytes following in vivo administration of the beta-adrenergic agonist, metaproterenol, the phosphodiesterase inhibitor, theophylline, or both concomitantly, to normal and asthmatic subjects. In normal subjects both beta-adrenergic receptor number and isoproterenol stimulated cAMP response decreases during therapy with metaproterenol (59±3; 51±16% of control, respectively), theophylline (76±6; 78±16), or concommitant metaproterenol and theophylline (47±4; 69±13). The asthmatic subjects were of two types; one type responding to metaproterenol or theophylline therapy by down regulation of receptor number to zero or near zero values, and a second group of asthmatics insensitive to down regulation of receptor number. The results suggest that the induction of the refractory state is different between asthmatics and non-asthmatics, and that there may be a role for cAMP in the development of beta-adrenergic refractoriness, in vivo.

Principles of membrane receptor physiology and their application to clinical medicine.

The clinical involvement of membrane receptors for hormones, neurotransmitters, and other molecules of interest is reported with increasing frequency. In this review, the terminology of the hormone-receptor field is defined, and the current concepts of the participation of membrane receptors in the mechanism of hormone action are outlined. Several major developing concepts of receptor regulation and function, including spare receptors and down regulation of receptor number, provide a basis for understanding the physiology and pathophysiology of cellular sensitivity to specific hormones. The relation of these concepts to the pathophysiology of various metabolic disorders is discussed. Clinical disorders in which membrane receptors play a central role are reviewed, including Graves' disease and myasthenia gravis, which involve antireceptor antibodies, and familial hypercholesterolemia and various conditions of hormone resistance that often involve a marked decrease in receptor number as the primary pathogenic factor. The significance of receptor physiology in other selected areas of clinical medicine is discussed.