Changes In Receptor Number Research Articles

Down-regulation of parathyroid (PTH)/PTH-related peptide receptor immunoreactivity and PTH binding in opossum kidney cells by PTH and dexamethasone.

Recent data have shown that PTH down-regulation of its receptor on opossum kidney (OK) cells is not associated with any change in the steady state level of the PTH/PTH-related peptide (PTHrP) receptor messenger RNA. For analysis of down-regulation of the PTH/PTHrP receptor in OK cells, the present work uses a specific receptor anti-serum, SR-2, that is useful for detection and quantification of PTH/PTHrP receptor immunoreactivity on intact cells bearing the opossum PTH/PTHrP receptor. SR-2 specifically binds to COS-7 cells transiently expressing the opossum PTH/PTHrP receptor complementary DNA (OK-O), to LLCPK1 cells stably expressing the recombinant opossum PTH/PTHrP receptor (AOK cells), and to OK cells expressing endogenous PTH/PTHrP receptors, but not to mock-transfected COS-7 cells or untransfected LLCPK1 cells. SR-2 binding was also linearly correlated with PTH binding in COS-7 cells transfected with different amounts of OK-O plasmid DNA. Treatment with PTH (100 nM) for 4 and 6 h did not significantly down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased to 51% and 49% of control, respectively, and PTH-stimulated cAMP accumulation was decreased to 27% and 28% of control, respectively. Treatment with PTH (100 nM) for 24 and 48 h significantly decreased PTH binding to 51% and 60% of control and decreased PTH/PTHrP receptor immunoreactivity to 68% and 58% of control, respectively. Incubation of OK cells with 0.1 nM to 1 microM PTH for 4 h did not down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased dramatically. Scatchard blot analysis revealed that the binding affinity was decreased by 7-fold in OK cells treated with PTH for 4 h without change in receptor number. Conversely, treatment of OK cells with PTH for 24 h resulted in a parallel decrease in both receptor number and receptor immunoreactivity without any change in receptor binding affinity. Treatment of OK cells with dexamethasone (0.1 nM to 1 microM) had no effect on PTH binding or PTH/PTHrP receptor immunoreactivity. Incubation of OK cells with both dexamethasone (1 microM) and PTH (0.1 nM to 1 microM), however, caused a significantly greater down-regulation of both PTH binding and PTH/PTHrP receptor immunoreactivity than in cells treated with PTH alone. These data indicate that during the first 4 h of exposure of OK cells to PTH, PTH/PTHrP receptors remain on the cell surface but have lowered affinity to bind the ligand and that dexamethasone potentiates the effect of PTH on PTH/PTHrP receptor down-regulation in OK cells.

Radioligand binding methods: Practical guide and tips

Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.

Parallel changes in dopamine D2 receptor binding in limbic forebrain associated with chronic mild stress-induced anhedonia and its reversal by imipramine.

Chronic sequential exposure to a variety of mild stressors has previously been found to cause an antidepressant-reversible decrease in the consumption of palatable sweet solutions, associated with abnormalities of dopaminergic neurotransmission in the nucleus accumbens. In the present study, 5 weeks of treatment with imipramine (10 mg/kg b.i.d.) reversed the decreased sucrose intake of rats exposed to chronic mild stress. Stress also caused a decrease in D2-receptor binding in the limbic forebrain (but not the striatum), which was completely reversed by imipramine. In nonstressed animals, imipramine decreased D1-receptor binding in both regions. However, in stressed animals, imipramine did not significantly alter D1-receptor binding in either area. Stress alone slightly increased D1-receptor binding, in striatum only. Scatchard analysis showed that all changes in receptor binding resulted from changes in receptor number (Bmax) rather than receptor affinity (KD). The results support the hypothesis that changes in D2-receptor function in the nucleus accumbens are responsible for chronic mild stress-induced anhedonia and its reversal by antidepressant drugs. They do not support the hypothesis that the sensitization of D2-receptors seen following chronic antidepressant treatment is caused by a down-regulation of D1-receptors.

Effects of Butyrate on the Erythropoietin Receptor of Cell Line IW201

The murine erythroleukemic cell line, IW201, normally expresses only low-affinity erythropoietin receptors. Exposure of these cells for 48 hours to sodium butyrate results in a change in receptor kd from about 600 pmol/L to 100 to 200 pmol/L. This change in affinity is accompanied by downregulation of both receptor number and receptor mRNA. Cells exposed to sodium butyrate for 2 hours show a similar change in kd but no change in receptor number. The butyrate effect on kd at 2 hours is abrogated by either cycloheximide or actinomycin D. These data indicate that an accessory protein induced by sodium butyrate is responsible for high-affinity binding of erythropoietin.

Effects of butyrate on the erythropoietin receptor of cell line IW201

The murine erythroleukemic cell line, IW201, normally expresses only low-affinity erythropoietin receptors. Exposure of these cells for 48 hours to sodium butyrate results in a change in receptor kd from about 600 pmol/L to 100 to 200 pmol/L. This change in affinity is accompanied by downregulation of both receptor number and receptor mRNA. Cells exposed to sodium butyrate for 2 hours show a similar change in kd but no change in receptor number. The butyrate effect on kd at 2 hours is abrogated by either cycloheximide or actinomycin D. These data indicate that an accessory protein induced by sodium butyrate is responsible for high-affinity binding of erythropoietin.

Kinetic analysis of the trafficking of muscarinic acetylcholine receptors between the plasma membrane and intracellular compartments.

We have studied the kinetics of muscarinic receptor trafficking between the plasma membrane and intracellular compartments in NG108-15 cells. A model of trafficking is proposed that includes four transport steps, delivery to the plasma membrane from the Golgi complex (linear) and endosomes (exponential) internalization from the plasma membrane into endosomes (exponential), and a degradative route from endosomes into lysosomes (linear). Based upon this model, a general equation has been derived that describes changes in receptor number at the plasma membrane and in endosomes in response to agonist stimulation. By following the movement of receptors into and out of the plasma membrane under various experimental conditions, it has been possible to determine the values of the four rate constants in the general equation and also the size of the endosomal receptor pool created by agonist stimulation. The consequences of changes in these constants for receptor trafficking are demonstrated. The model accounts for the effect of varying the duration of agonist stimulation on the size of the endosomal receptor pool and also permits an estimation of the receptor trafficking that underlies the well-established phenomenon of agonist-induced receptor internalization.

Increased respiratory burst activity is associated with normal expression of IgG-Fc-receptors and complement receptors in peripheral neutrophils from patients with juvenile periodontitis.

The respiratory burst activity in peripheral neutrophils from nine patients with localized juvenile periodontitis (LJP) and age- and sex-matched healthy controls was studied by measuring the intensity of luminol-enhanced chemiluminescence (CL) induced by unopsonized and three differently opsonized zymosan particles, formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). The neutrophils from LJP patients showed in general more intense CL with all activators than did their controls. Particularly, the CL response induced by unopsonized zymosan particles and FMLP were significantly higher (p < 0.05 and 0.001). When comparisons were made between female LJP patients (n = 6) and matched controls, also serum-opsonized and IgG-opsonized zymosan particles produced CL was significantly increased (p < 0.05). In order to determine whether the elevated CL responses to zymosan particles were due to altered levels of the interacting receptors on neutrophil surface, an immunofluorescence analysis of the expression of IgG-Fc-receptors (FcR) and complement receptors (CR) was performed with flow cytometry. No significant difference in the expression of FcRII, FcRIII, CR1 and CR3 was detected in LJP group compared to controls. Since the elevated CL responses can not be explained by changes in receptor numbers it is hypothesized that the increased respiratory burst activity in LJP may be caused by altered post receptor signalling pathway.

Enhanced Negative Inotropic Effect of an Adenosine A 1-Receptor Agonist in Rat Left Atria in Hypothyroidism

Left atria were isolated from rats made hypothyroid by adding propylthiouracil to their drinking water, such rats after saturating doses of thyroid hormones, and from control rats. Isoproterenol (ISO: 1 μM) increased the values of developed tension (DT), maximal rate of tension development (+dT/d t) and tension fall (-dT/d t). The effect was largest in hypothyroid and lowest in hyperthyroid atria. The adenosine A 1-receptor agonist N 6-(phenylisopropyl)-adenosine (PIA) has a powerful negative inotropic effect in ISO-stimulated atria. The effects of PIA on +dT/d t. -dT/d t and DT were enhanced in hypothyroidism. Adenosine receptor number was not decreased. The amount of total G i-like proteins was estimated by pertussis toxin labeling. The amounts of G i2 and G i3 were estimated in Western blots such antisera raised in rabbits against peptides corresponding to parts of their sequences, using purified recombinant α subunits as standards. The amounts of low and high molecular weight forms of G s were estimated by cholera toxin labeling. G i2, G i3 and pertussis toxin substrate concentrations were slightly lower in the hypothyroid animals, while the amounts of both forms of G s per mg of protein were only half of those in euthyroid rat atria. The levels of G i2 and G i3 were greatly elevated as compared to G s as membrane marker. These changes were reversed by treatment of the hypothyroid rats with thyroid hormones. In conclusion, the present results show an enhanced negative inotropic effect of an adenosine A 1-receptor agonist in rat left atria in hypothyroidism, reflected both in sensitivity and responsiveness, and suggest that this is not caused by a change in receptor number but by a relative in G i proteins.

Regulation of TNF-α Receptor Expression on Human Neutrophils by Various Cell Activating Factors

Since tumor necrosis factor-α (TNF-α) has been implicated in a wide range of inflammatory states, we sought to examine the regulation of TNF-α receptors on human neutrophils (PMNs). We were particularly interested in the relationship of TNF-α binding characteristics to various known neutrophil-priming stimuli such as temperature, N -formylmethionyl-leucyl-phenylanine (fMLP), phorbol myristate acetate (PMA), C5a des Arg, and human recombinant C5a (HrC5a). Competition binding studies indicated that there was no significant change in receptor number or affinity due to the temperature used for neutrophil isolation. PMNs isolated at 4 or 25°C had a single class of receptors with a K d of 83.1 ± 3.3 vs 90.0 ±8.3 pmole/liter and a similar number of receptors, 1575 ± 99 vs 1876 ± 279 receptors/cell. However, when either group of isolated PMNs was further incubated in buffer at 37°C for 15 min, a significant increase in K d (4-37°C, 147.9 ± 23.7 pmole/liter; 25-37°C, 165.2 ± 29.2 pmole/liter) and a decrease in receptor number (4-37°C, 1116 ± 86 receptors/cell; 25-37°C, 957 ± 71 receptors/cell) were seen. To determine the effect of other activating substances such as fMLP, PMA, C5a des Arg, HrC5a, 4°C isolated PMNs were used. Pretreatment of PMNs (15 min at 37°C) by all of these agents down-regulated TNF-α receptors by decreasing receptor number and by decreasing receptor affinity. In the course of these studies, we identified a substantial disparity in the chemotactic deactivation and TNF-α receptor down-regulation of ED 50's for native C5a des Arg compared to recombinant C5a. These results would suggest that both loss of receptors and a change in affinity of the TNF-α receptor could be a means of modulating TNF-α response in neutrophils. We speculate that activation and release of the TNF-binding portion of the receptor from a proposed trimer receptor complex explains the affinity loss.

The role of the NMDA receptor in ethanol withdrawal.

The function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is very sensitive to acute inhibition by ethanol. Because of the role of this receptor in processes such as synaptic plasticity and neuronal development, it may contribute to the acute cognitive deficits caused by ethanol, or to the deleterious effects of ethanol during gestation. Excessive stimulation of the NMDA receptor is believed to be involved in the generation of epileptiform seizure activity as well as in excitotoxic cell death. Our studies have demonstrated that there is an adaptive "up-regulation" of NMDA receptor function in brains of chronically ethanol-treated animals and in cultured cells that have been exposed chronically to ethanol. This up-regulation appears to contribute to ethanol withdrawal seizure activity, since withdrawal seizures can be attenuated by specific NMDA receptor antagonists, and the time course of the change in receptor number parallels the time course of withdrawal seizures. In addition, cells exposed chronically to ethanol are significantly more susceptible to glutamate-induced cell death, which is mediated by the NMDA receptor, indicating a key role of the NMDA receptor in the well-characterized neuronal damage that is observed after chronic ethanol exposure and withdrawal in animals and humans. Understanding the basis for withdrawal seizures and withdrawal-induced neurotoxicity provides for the development of specific and selective therapeutic agents to ameliorate these consequences of chronic ethanol exposure and withdrawal.

Possible role of neuropeptide Y in experimental cancer anorexia.

The efficacy of NPY to elicit feeding in TB rats was reduced prior to the onset of overt anorexia, with the feeding response decreasing further as anorexia developed. Hypothalamic concentration of NPY was reduced in TB rats, with the magnitude of the decrease paralleling the degree of anorexia. Binding affinity of NPY to hypothalamic membranes taken from TB rats suggested decreased binding affinity with no change in receptor number. Infusing ammonium salts at a concentration and rate necessary to increase blood ammonia levels to the degree observed in TB rats, produced anorexia and decreased NPY feeding. These results suggest that NPY feeding systems are abnormal in TB rats and that hyperammonemia may be of primary importance in this dysfunction.

Homologous up-regulation of the gonadotropin-releasing hormone receptor in alpha T3-1 cells is associated with unchanged receptor messenger RNA (mRNA) levels and altered mRNA activity.

In primary cultures of rat pituitary, GnRH receptor has been reported to up-regulate when exposed to GnRH. The molecular basis of this regulation could, in theory, involve modulation of gene transcription, RNA processing or stability, translation, and/or receptor degradation. The role of altered biosynthesis was investigated in the mouse gonadotrope cell line, alpha T3-1 cells, by studying the homologous up-regulation of GnRH receptor binding, mRNA levels, and mRNA activity. After GnRH exposure, ligand binding was performed on alpha T3-1 cell membranes. To correlate changes in receptor number and measurements of biosynthetic activity, GnRH receptor mRNA levels were quantitated by solution hybridization/RNase protection assay and Northern blot analysis, and the capacity of RNA from treated cells to direct the synthesis of new receptors (mRNA activity) was evaluated using a Xenopus oocyte-based bioassay. GnRH agonist radioligand binding assay results showed that exposure of alpha T3-1 cells to 10(-10) or 10(-8) M GnRH for 20 min induced an approximately 50% increase in the number of GnRH receptors, similar to previously reported results in rat pituitary primary culture. Despite the increase in receptor number, however, cytosolic and total GnRH receptor mRNA levels assayed by solution hybridization/RNase protection assay with GnRH receptor cRNA probes and by Northern blot analysis were not altered. In contrast to the unchanging mRNA levels, the measurements of mRNA activity paralleled the changes observed at the binding level. alpha T3-1 RNA from treated and control cells were injected into Xenopus oocytes, and the GnRH-induced Cl- current was quantified 48 h later by voltage clamp recording of the response to GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of membrane Ig receptor density and affinity on B cell signaling by antigen. Implications for affinity maturation.

We have initiated a series of experiments to explore the effect of changes in density of the surface Ig receptor (mIg) on Ag:mIg interactions. We transfected into the surface Ig-negative B cell line M12.4 H and L chain constructs known to effect a 10-fold change in antibody binding affinity for the naturally occurring hapten phosphocholine (PC). Two sets of stable transfectants were generated and those expressing levels of mIg comparable to the range normally seen on splenic B cells were studied. One set expressed an unmutated VH and an unmutated VL. The second set expressed the same pair of V regions except for a single somatic change in CDR3 of VH; this substitution increases the affinity of antibody for PC from 3 x 10(4) M-1 to 3 x 10(5) M-1. Ag:mIg interactions were assessed in the transfected cell lines by measuring calcium mobilization induced by stimulation with soluble PC Ag. As expected, the mutation that increased affinity for PC increased the sensitivity of transfectants to PC Ag. Relatively small changes in receptor number had a dramatic effect in the quantity and quality of a calcium response. Significantly, we found that Ag-specific signaling could occur with only a few thousand receptors per cell. Signaling differences were most noticeable with PC protein Ag (T-dependent form) compared with PC polysaccharide Ag (T-independent form). These results suggest that the down-regulation of mIg that follows B cell activation may have evolved to assist in the selection of B cell clones with higher affinity for Ag. Furthermore, the results also provide an explanation for why selection of higher affinity clones can occur with protein Ag but only poorly so with polymeric Ag.

Radioligand binding methods: practical guide and tips.

Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.

Control of receptor sensitivity at the mRNA level.

Neurons are able to adjust the sensitivity of receptor-mediated processes according to the level of receptor activation. Extrapolating from our knowledge of other cellular proteins, regulation of receptor mRNA availability would provide a highly economical means of achieving this objective. Epidermal growth factor is able to induce long-lasting increases in its receptor binding by increasing receptor mRNA levels, and similar effects have been shown for other growth factors. Studies on G-protein-coupled receptors, in particular using adrenoceptor clones transfected into cultured cell lines, have shown that changes in receptor number are generally associated with an alteration in receptor mRNA content. At the neuromuscular junction, dramatic increases in nicotinic acetylcholine receptor number are achieved by activating receptor subunit gene transcription. Less information is available concerning the regulation of ligand-gated ion channels in the brain. Overall, the evidence suggests that receptor mRNA levels are frequently controlled by the degree of receptor stimulation. Receptor mRNA levels are therefore likely to be one of the most important control points for both homologous and heterologous regulation of receptor sensitivity.

High-affinity kainate binding sites in living slices of rat neocortex: Characterization and regulation

We have characterized a high-affinity kainate binding site in in vitro living rat neocortical slices using [ 3H]kainate. [ 3H]Kainate labelled at least two binding sites, the higher affinity site with a K d of 7.1 nM and a B max of 71.2 fmol/mg protein. This high-affinity binding site showed a pharmacology consistent with a kainate receptor with competition by kainate and domoic acid, as well as the (RS)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline. Increases in cellular depolarization induced by 2-h preincubations in veratridine and glutamate led to a significant 55% average decrease in [ 3H]kainate binding in adult cortex. Similarly, preincubation in kainate led to a significant average 26% decrease in binding. In both instances, Eadie-Hofstee analysis of saturation binding data revealed that the decreased binding reflected changes in receptor number. At different postnatal ages, increases in cellular depolarization significantly decreased binding (<20 days postnatal age, −86%; >60 days, −48%). Kainate treatment also significantly decreased binding at all ages (−64% at <20 days; >60 days, −18%), with significant differences noted between ages. These age-dependent effects are unlike those previously described for either N-methyl- d-aspartate [ Lanius and Shaw (1992) Anat. Rec. 232, 54(A)] or (RS)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate high affinity receptors [ Shaw and Lanius (1992) Devl Brain Res. 68, 225–233]. The age-dependent changes noted for kainate receptors in cortex more closely resemble the inhibitory GABA A receptors [ Shaw and Scarth (1992) Molec. Brain Res. 14, 207–212]. These results reveal complex and dynamic interactions of neural activity and receptor regulation for kainate receptors as well as for a number of other amino acid receptors in cortex, especially during postnatal development.

Arginine vasopressin-induced sensitization in brain: facilitated inositol phosphate production without changes in receptor number.

Arginine vasopressin (AVP) has been shown to have a unique sensitization effect whereby repeated injection of AVP into a lateral cerebral ventricle or a mediobasal region of the rat forebrain below the lateral septum and including the anterior hypothalamus referred to as the ventral septal area, causes enhanced motor responses to the ligand. To elucidate possible neuronal mechanisms responsible for AVP sensitization, 1) we determined the dose and the time required for the development and expression of AVP sensitization, and 2) we tested the hypotheses that AVP sensitization may result in a) alteration of septal AVP V1 receptor affinity or number, and/or b) alteration of septal AVP V1 receptor signal transduction (phosphatidylinositol hydrolysis) mechanisms. Our behavioral data show that the magnitude of AVP sensitization varies with dose and time, and the effect is dependent on the time interval between injections, in that an initial intracerebroventricular AVP injection enhances the sensitivity of the animals to the motor effects of similar AVP injections given 6 h to 6 days later but not to injections given hourly or weekly. No changes in septal AVP binding site density and affinity, as measured by [3H]AVP binding to septal synaptic plasma membrane, were found in sensitized animals; [3H]inositol monophosphate stimulation in response to AVP in septal slices, however, was found to be significantly enhanced. This enhanced [3H]inositol monophosphate stimulation appears specific to a V1-type receptor because it was significantly reduced in the presence of the V1 receptor antagonist, d(CH2)5Tyr(Me)AVP, and was not found using oxytocin or the V2 receptor agonist, DDAVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms involved in the adaptations of the adipocyte adrenergic signal-transduction system and their modulation by growth hormone during the lactation cycle in the rat.

The mechanisms responsible for the diminished lipolytic response of adipocytes to catecholamines after litter removal from lactating rats and their modulation by growth hormone have been investigated. Lactation, litter removal and growth-hormone treatment did not alter the ability of noradrenaline to activate protein kinase A (A-kinase), showing that the defect in signal transduction in rats after litter removal is after A-kinase. Litter removal had no effect on hormone-sensitive lipase activity itself, but the proportion of the lipase associated with the fat droplet was decreased; growth-hormone treatment increased hormone-sensitive lipase activity and the proportion associated with the fat droplet. In addition, a number of other adaptations in the beta-adrenergic signal-transduction system occur during the lactation cycle and in response to growth hormone treatment, including changes in receptor number, adenylate cyclase activity and cyclic AMP phosphodiesterase activity, but a defect in the ability of hormone-sensitive lipase to associate with the lipid droplet appears to be the major reason for the diminished response to catecholamines on litter removal.

Comparative pharmacology of selective serotonin re-uptake inhibitors (SSRIs)

Tricyclic antidepressants (TCAs) affect a series of neurotransmitter functions. The uptake of noradrenaline (NA) and serotonin (5-HT) is inhibited to a varying degree. Many of the TCAs potently inhibit a series of neurotransmitter receptors. A series of newer antidepressants preferentially increases 5-HT transmission by inhibiting 5-HT uptake. Selective serotonin re-uptake inhibitors (SSRIs) are those which have a high potency ratio of 5-HT-uptake inhibition as compared to NA-uptake inhibition, together with slight or no effect on other uptake mechanisms, neurotransmitter receptors, enzymes, etc. The marketed SSRIs, citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline all fulfil the above-mentioned criteria. Paroxetine is the most potent 5-HT-uptake inhibitor, whereas citalopram is the most selective. In general, the rank order of selectivity is equal in in vitro studies, in biochemical in vivo studies and in behavioural studies. Potency and selectivity for 5-HT uptake do not coincide. In contrast to TCAs, most of which inhibit receptors for acetylcholine, histamine, NA, 5-HT or DA, most SSRIs neither bind to nor inhibit these receptors. Likewise, monoamine oxidase (MAO) is not inhibited by these drugs. Citalopram, fluoxetine and sertraline are metabolized to compounds possessing similar properties as the parent drugs, whereas this is not the case with the metabolites of fluvoxamine and paroxetine. Upon repeated administration the potency and selectivity of SSRIs are maintained. Although repeated treatment with TCAs and electroconvulsive shocks leads to changes in receptor number and pharmacological responsiveness, e.g. down-regulation of β-adrenoceptors and down-regulation of 5-HT2-mediated function, no consistent changes are seen after prolonged treatment with SSRIs. Since SSRIs show antidepressant effect, this receptor regulation seems not to be a prerequisite for clinical antidepressant activity.

Ligand-mediated internalization of glucagon receptors in intact rat liver.

The ligand-induced internalization of the hepatic glucagon receptor has been studied in rats in vivo using cell fractionation. Injection of glucagon (11 nmol/100 g BW) led to a 2- to 3-fold increase in glucagon-binding activity in Golgi-endosomal (GE) fractions along with a 10-20% decrease in binding activity in plasma membrane (PM) fractions. These changes were time and dose dependent, reaching a maximum by 12-24 min and undergoing reversal in 2 h. Glucagon injection also caused a 20% decrease in glucagon binding to the total particulate fraction, which did not occur when binding was measured in the presence of the detergent octylglucoside. The change in glucagon-binding activity in PM and GE fractions resulted mainly from a change in receptor number; affinity remained unaffected (apparent Kd, 0.5 and 5 nM, respectively). A 5- to 10-fold increase in the glucagon content of GE fractions was observed in glucagon-treated rats. Neither the distribution of PM and Golgi marker enzymes nor that of the asialoglycoprotein receptor was affected by glucagon treatment. Regardless of glucagon treatment, glucagon receptors in GE fractions were less sensitive to GTP than receptors in PM fractions with respect to both inhibition of steady state binding and dissociation of prebound ligand. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, glucagon-receptor complexes formed in PM and GE fractions and subsequently cross-linked showed the same apparent mol wt (57 kilodaltons). In addition, they were identically sensitive to N-glycanase treatment, with two major species of lower mol wt generated. However, only cross-linked complexes associated with PM fractions showed detectable GTP sensitivity. GE fractions displayed a GTP-sensitive adenylate cyclase activity that was about 12 times lower than that of PM fractions. In both fractions, activity was stimulated by the addition of forskolin (8-fold) and, to a lesser extent, glucagon (3-fold). In vivo glucagon treatment led to an increase in activity in GE, but not PM, fractions. These results are consistent with the view that upon acute occupancy, hepatic glucagon receptors are rapidly and specifically internalized along with their ligand. During this process, receptor retained structural integrity and uncouple, albeit partially, from other components of the adenylate cyclase system.