Receptor Agonist Affinity Research Articles

Adenylate cyclase regulation in intact cultured myocardial cells.

To examine the coupling of cardiac cell-surface beta-adrenergic receptors to adenylate cyclase activation and contractile response, we studied this receptor-effector response system in monolayers of spontaneously contracting chick embryo ventricular cells under physiological conditions. The hydrophilic ligand 3H-CGP12177 identified uniformly high-agonist affinity beta-adrenergic receptors (isoproterenol KD = 15 +/- 9 nM). Isoproterenol-stimulated cyclic AMP (cAMP) accumulation with 50% effective concentration at (EC50) = 12.1 nM and augmented contractile response with EC50 = 6 nM under identical conditions. One micromolar isoproterenol induced receptor loss from the cell surface with t1/2 = 13.2 min; under identical conditions cAMP content declined with t1/2 = 13.5 min and contractile response with t1/2 = 20.7 min. After agonist removal cAMP response recovered with t1/2 = 15.7 min and receptors with t1/2 = 24.7 min. Sixty minutes after agonist removal there was recovery of 52% of maximal cAMP responsiveness and 82% of the initial number of receptors; receptor occupancy was associated with 78% of initial contractile response. Agonist affinity for cell-surface receptors was changed only modestly by agonist exposure. We conclude that for this system there is relatively close coupling between high-affinity receptors, adenylate cyclase stimulation, and contractile response.

Beta-adrenergic agonist-mediated desensitization in senescent rats

The capacity of senescent rats to develop the catecholamine refractory state was investigated in CDF (F-344) rats of 3 and 24 months of age. Beta-adrenergic receptor number, receptor-agonist affinity, and adenylate cyclase activity in heart membranes were assessed, following the chronic in vivo administration of the beta-adrenergic agonist, metaproterenol. Drug treatment leads to marked myocardial hypertrophy, receptor down-regulation, and reduced isoproterenol-stimulated adenylate cyclase activity. The extent of catecholamine-refractoriness was not different in the older rats, indicating the catecholamine desensitization of myocardial beta-adrenergic responsiveness is not impaired in senescence. Receptor agonist affinity and the percent of receptors in the high-affinity state decrease with age. These parameters are further reduced by agonist treatment but to a lesser extent in the older animals. Thus, the effects of age and agonist desensitization are not additive and suggest that aged animals may already be partially desensitized.

Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations.

The affinity of agonists but not antagonists at hepatic membrane alpha 1-adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low-temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo effects of estrogen and 2-hydroxyestradiol on D-2 dopamine receptor agonist affinity states in rat striatum.

The administration of estrogen to ovariectomized rats will result in a dopamine receptor hyposensitive phase at 24 hours followed within 48 hours by a hypersensitive phase. In an attempt to characterize further the molecular mechanism(s) which underlie this biphasic response, we studied the effects of estrogen and one of its metabolites (2-hydroxyestradiol) on striatal D-2 dopamine receptor agonist affinity states. Ovariectomized rats were treated daily with estradiol benzoate (EB; 10 micrograms/kg/day) for 3 days and sacrificed 24 hours after the last dose. Additional groups were treated with 2-hydroxyestradiol (2-OHE 2; 3 micrograms/kg/day), EB (100 micrograms/kg/day) or appropriate vehicle for 3 days and sacrificed 72 hours after the last injection. Animals sacrificed during the hyposensitive phase (i.e. 24 hours) displayed a significant decrease in the ratio of high/low agonist affinity states of the striatal D-2 receptor, while animals sacrificed during the EB- or 2-OHE 2-induced hypersensitive phase (i.e. 72 hours) showed no change in the ratio of high/low agonist affinity states.

Temperature and isoproterenol modulation of beta-adrenergic receptor characteristics

Since agonists and temperature affect receptor affinity, and since these factors may influence the actual determination of receptor affinity, we assessed the in vitro effects of temperature and isoproterenol on the high and low affinity states of beta-adrenergic receptors in rat membrane preparations. There was a temperature-dependent decrease in beta-adrenergic receptor agonist affinity which was further promoted by the presence of isoproterenol. The decrease in receptor agonist affinity was reflected by a decrease in the number of receptors in the high affinity state. These data suggest that receptor desensitization may occur in membrane preparations in vitro and that the present methodology used to assess agonist affinity in vitro may itself alter the very properties being measured.

How does salt raise blood pressure? A hypothesis.

Existing data in the literature indicate that alpha 2-adrenergic receptor agonists have a profound hypotensive action, that sodium attenuates the affinity of alpha 2-adrenergic receptors for agonists, that the location of these receptors in the central nervous system is mainly at the sites of cardiovascular regulation, and that these sites exert a constant tonic inhibition of sympathetic vasoconstrictor tone. This article proposes the theory that sodium exerts its hypertensive action by decreasing the state of affinity of the alpha 2-adrenergic receptors of the central nervous system for locally occurring agonist neurotransmitters, which results in disinhibition of sympathoinhibitory neurons and leads to the hyperadrenergic state characteristic of salt-induced hypertension.

Prenatal exposure of rats to antidepressants enhances agonist affinity of brain dopamine receptors and dopamine-mediated behaviour

The effects of acute treatment or prenatal exposure of rats to antidepressants on locomotor activity and [ 3H]spiroperidol binding to striatal membranes were studied in 25 day old rats. Prenatal exposure or acute treatment with the antidepressants chlorimipramine, iprindole or mianserin reduced locomotor activity on postnatal day 25. On the contrary, prenatal exposure to nomifensine induced locomotor hyperactivity. It was also found that chlorimipramine, iprindole or nomifensine enhanced the locomotor response to apomorphine. Acute treatment with mianserin, markedly decreased locomotion but in utero mianserin did not modify the apomorphine-induced hyperactivity. Scatchard analysis showed no change in the characteristics of binding of [ 3H]spiroperidol to striatal dopamine receptors after antidepressant treatment. However, the ability of dopamine to compete for these sites was significantly enhanced after prenatal exposure to all the antidepressants. This increase in agonist affinity for dopamine receptors may explain the long-lasting behavioural supersensitivity of dopamine receptors observed after chronic treatment with typical or atypical antidepressants.

Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors.

Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose-dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP-binding proteins, IAP-specific substrates, in the brain membranes.

Effects of Na+ on the Muscarinic Cholinergic Receptor of Rat Ventricular Myocytes

The effects of Na+ on alpha 2-adrenergic receptors coupled to inhibition of adenylate cyclase have been well studied. Effects of Na+ on other receptor types also linked to adenylate cyclase inhibition are not clear. We therefore studied the effects of Na+ on the muscarinic cholinergic receptor of rat ventricular myocardium. Intact ventricular myocytes bound the muscarinic receptor antagonist radioligand [3H]quinuclidinyl benzylate with high affinity (Kd 72 +/- 18 pM) to a large number of sites (138,000 +/- 25,000/cell). In ventricular membranes, Na+ (100 mM) increased receptor affinity for radioligand two-fold and decreased receptor affinity for agonist (carbachol) twofold. Sodium was not required for and did not alter the pattern of cholinergic inhibition of adenylate cyclase in ventricular membranes. We conclude that Na+ has minimal effects on both binding and function of cardiac cholinergic receptors in rat. Our results suggest that monovalent cations are not important regulators of agonist binding and function of cardiac muscarinic cholinergic receptors.

Adenylate cyclase modulation by ammonium ion: GTP-like effect on muscarinic and alpha 2-adrenergic receptors.

The stimulatory influence of ammonium sulphate on adenylate cyclase activity has been investigated. By competition binding experiments on the beta-adrenergic stimulatory receptor in rat myocardial membranes, no influence could be detected of ammonium sulphate neither in receptor coupling to the stimulatory guanine nucleotide binding protein nor in the GTP-induced uncoupling. In order to detect an impaired inhibition instead of an increased stimulation of adenylate cyclase activity by ammonium sulphate the investigation was extended to inhibitory receptors. The same type of effect by ammonium sulphate was detected on both the muscarinic cholinergic receptor in rat myocardial membranes as well as on the alpha 2-adrenergic receptor in human platelets. The influence of ammonium sulphate noted in competition binding studies and off-kinetics experiments was GTP-like, i.e. causing a decrease in agonist-receptor affinity leaving all the inhibitory receptors in the low affinity state. In conclusion, this paper indicates that the observed stimulatory effect of ammonium sulphate is exerted by the ammonium ion on the inhibitory guanine nucleotide binding protein, impairing the negative control of adenylate cyclase activity.

Chronic haloperidol does not alter agonist affinity for dopamine receptors in vitro

Agonist competition for [ 3H]spiperone binding to striatal dopamine D 2 receptors was studied in rats rendered supersensitive by chronic treatment with haloperidol. The classical dopamine agonist (-)-N-propylnorapomorphine displaced [ 3H]spiperone biphasically, with IC 50 values of 0.5 and 140 nM for the high and low affinity components, respectively. Neither the relative density nor the affinity of either site for (-)-N-propylnorapomorphine was affected by chronic haloperidol treatment. On the other hand, the novel agonist EMD 23 448 displaced [ 3H]spiperone monophasically. Although this agent only displays potent dopaminergic agonism in supersensitive animals, chronic treatment with haloperidol likewise did not alter the affinity of this drug for [ 3H]spiperone binding sites. The results suggest that the enhanced in vivo potency of certain agonists in supersensitive animals is probably not mediated by changes in D 2 receptor affinity.

Desensitized beta-adrenoceptors of C6-glioma cells have distinct binding properties.

When C6-rat glioma cells were incubated for 20 min with beta-adrenoceptor agonists, a part of the beta-adrenoceptors was localized in light density vesicles. These receptors have the same affinity for dihydroalprenolol as plasma membrane receptors but have a lowered affinity for agonists as well as for the hydrophilic beta-adrenoceptor antagonist CGP-12177. The affinity of these vesicular receptors for a variety of hydrophobic beta-blockers as well as for the hydrophilic beta-blocker timolol is compared with that of plasma membrane receptors. None of the ligands investigated showed any difference in affinity but CGP-12177. Both, introducing a lipophilic side chain into CGP-12177 or altering the benzimidazol-2-one ring system of CGP-12177 led to an increase in the affinity of the vesicular receptors. Since in the presence of the pore-forming agent alamethicin the same affinity was determined for vesicular receptors as for those located on the plasma membrane, it is concluded that the apparent low affinity of the vesicular receptors in the absence of alamethicin is caused by a membrane barrier. The lower affinity of the vesicular receptors for agonists was only slightly increased by alamethicin.

Regulation of myocardial and vascular alpha-adrenergic receptor affinity. Effects of guanine nucleotides, cations, estrogen, and catecholamine depletion.

The threshold sensitivity of cardiovascular tissues to alpha-adrenergic stimulation is determined largely by the affinity of alpha-adrenergic receptors for agonists. To determine whether changes in alpha-adrenergic receptor affinity could contribute to the regulation of cardiac and vascular responsiveness, we used the alpha-adrenergic-selective radioligands, [3H]prazosin, [3H]-WB-4101, and [3H]rauwalscine, to study and contrast the determinants of alpha-adrenergic receptor affinity in myocardium and vascular smooth muscle from the rat. In both tissues, l-epinephrine binding to the alpha 1-adrenergic receptor describes a shallow curve suggesting more than one affinity state. Computer analysis of binding to myocardial alpha 1-receptors indicates that 15% are of high affinity (Kd = 11 nM) and 85% are of low affinity (Kd = 400 nM). Expression of high affinity sites is magnesium dependent (maximum effect, 5-10 mM), and suppressed by the guanosine 5'-triphosphate analogue Gpp(NH)p (maximum effect, 1 mM) and sodium (maximum effect, 100-200 mM). In vascular smooth muscle, agonist-binding curves are also shallow and exhibit a similar response to that of Gpp(NH)p. Basal receptor affinity in myocardium is significantly higher (5.4-fold) than in vascular smooth muscle. Unlike vascular smooth muscle, in which alpha 1-adrenergic receptor affinity is increased by estrogen or reserpine treatment of the animal, the receptor in myocardium is unaffected by these treatments. In vascular smooth muscle, following reserpine-induced increase in alpha-adrenergic receptor affinity, the Gpp(NH)p effect is still present. Thus, alpha 1-adrenergic receptors in both myocardium and vascular smooth muscle exist in two affinity states and are subject to regulation by several factors, including guanine nucleotides, mono- and divalent cations, tissue of origin, sex hormones, and the level of sympathetic stimulation. Potentially, alterations in alpha 1-adrenergic receptor affinity, independent of a change in receptor number, may play an important role in the regulation of cardiovascular tissue responsiveness to catecholamines.

Differences and similarities in muscarinic receptors of rat heart and retina: effects of agonists, guanine nucleotides, and N-ethylmaleimide.

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.

Alterations in leukocyte beta-receptor affinity with aging. A potential explanation for altered beta-adrenergic sensitivity in the elderly.

The elderly have reduced beta-adrenergic sensitivity to agonists, but no change in receptor density. We investigated the relation between beta-receptor affinity for agonists and age in beta-receptors on lymphocytes from 20 healthy men 21 to 74 years old. As an index of beta-receptor affinity for agonists, we determined the IC50 of isoproterenol--the concentration of isoproterenol required to inhibit 50 per cent of iodohydroxybenzylpindolol binding in vitro. We found that receptor affinity for agonists was correlated with age and plasma norepinephrine concentration. Twelve subjects (six 21 to 29 years old and six 55 to 74 years old) were also studied in both the supine and upright positions. In samples obtained in the supine position, the proportion of receptors binding agonist with a high affinity was decreased in the older subjects as compared with the young subjects (22 +/- 1 per cent vs. 38 +/- 3 per cent; P less than 0.05). With upright posture and the associated acute elevation of endogenous plasma catecholamines, the proportion of receptors binding agonist with a high affinity was reduced in the young; no such changes were seen in the older subjects. The data suggest that reduced beta-receptor affinity for agonists may be an explanation for altered beta-adrenergic sensitivity in the elderly.

Inhibition of opiate receptor-mediated signal transmission by rabies virus in persistently infected NG-108-15 mouse neuroblastoma-rat glioma hybrid cells.

Acute and persistent rabies virus infection of mouse neuroblastoma-rat glioma hybrid cells (NG-108-15) results in a loss of the normal inhibiting function of opiates via the opiate receptor on hormone-stimulated adenylate cyclase activity. Previous studies of these persistently infected cells have shown a decrease in the affinity of the opiate receptors for agonists without any change in the number of these receptors. We now demonstrate that persistently infected cells are unable to couple the opiate receptors to the inhibitory regulatory protein Ni of the adenylate cyclase, as measured by the loss of stimulation of the GTPase activity of this protein. However, the unstimulated basal GTPase activities of the regulatory components Ni and Ns are unchanged in the persistently infected cells. These studies also reveal a disorder of the stimulation of the adenylate cyclase by GTP or fluoride via the stimulating regulatory G/F protein (Ns) in persistently infected cells, whereas direct stimulation of the catalytic subunit of the adenylate cyclase by forskolin remains unchanged. Therefore, there are different points of dysfunction caused by the persistent rabies infection in the signal pathway from the opiate receptor to the adenylate cyclase and from the stimulating Ns protein to the enzyme: (i) opiate receptor binding is reduced by a decrease of agonist affinity (previously published data), (ii) the stimulation of GTPase activity of the inhibiting regulatory component Ni of the adenylate cyclase system is inhibited, and (iii) the signal pathway from the stimulating regulatory component of the adenylate cyclase system to the unchanged activity of the catalytic subunit is defective.

Changes in tissue sensitivity to drugs in relation with alteration in receptors by treatment of drugs.

Tissues responses for drugs are often altered by chronic administration of drugs and this alteration is one kind of functional abnormality of the tissues. One mechanism under this functional abnormalities is considered to be in changes in receptors for drugs. In this study, we examined amounts and properties of receptors in sub- and supersensitive states caused by pre-exposure of tissues to drugs and the results obtained were summarized as follows. Long term exposure of tissue to agonists or to conditions which elevate concentration of agonist around the receptor caused reduction in amount of receptor without change in its property. Short term exposure to agonists altered the configuration of the receptor accompanying reduction in sensitivity of the tissue to drugs. But amount of receptor in the tissue was not changed. Heterologous desensitization was observed in some cases by exposure to drugs and no significant change was observed in the receptor of the tissue, qualitatively and quantitatively, indicating dysfunction in the process(es) after activation of the receptor. Denervation and chronic treatments with antagonist and ganglion blocker caused supersensitive state accompanying increase in amount of receptor. But in some cases, qualitative change of receptor, increase in affinity of receptor for agonist, seemed to relate with supersensitive state of tissues.

Pertussis toxin-catalyzed ADP-ribosylation: effects on the coupling of inhibitory receptors to the adenylate cyclase system.

The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (Gi). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of Gi. ADP-ribosylation of Gi has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.

Selective decrease in the affinity of D2 dopamine receptor for agonist induced by islet-activating protein, pertussis toxin, associated with ADP-ribosylation of the specific membrane protein of bovine striatum

When the crude synaptic membrane preparations from bovine striatum were treated with islet-activating protein (IAP), one of the pertussis toxins, a protein with a molecular weight of about 40,000 was ADP-ribosylated. In parallel with this ADP-ribosylation, there was a decrease in D2 dopamine receptor affinity for agonist, while the affinity for antagonist remained unaltered. Addition of GTP to nontreated membranes also resulted in a decrease in the affinity of D2 receptor for agonist, and there was no further reduction of affinity for agonist with addition of GTP to the IAP-treated membranes. As IAP specifically acts on the guanine nucleotide regulatory protein which mediates the inhibition of adenylate cyclase activity (Ni), our findings indicate a possible molecular interaction between the brain D2 dopamine receptor and Ni.

Potentiation of monovalent cation effects on ligand binding to cardiac muscarinic receptors in N-ethylmaleimide treated membranes

Guanine nucleotides and monovalent cations decrease the affinity of cardiac muscarinic receptors for agonists and are required for muscarinic receptor mediated inhibition of adenylate cyclase. N-ethylmaleimide abolished the effects of Gpp(NH)p on the ability of the agonist oxotremorine to inhibit the binding of the antagonist [3H]quinuclidinyl benzilate to purified chick heart membranes. However, the effects of NH4+ to decrease the IC50 for oxotremorine were retained in N-ethylmaleimide treated membranes. The N-ethylmaleimide treatment mimicked the effects of Gpp(NH)p and the oxotremorine inhibition curves obtained with treated membranes in the presence of NH4+ were identical to those obtained in control membranes in the presence of NH4+ and Gpp(NH)p. The results suggest that monovalent cation effects on muscarinic receptors are mediated at a site distinct from effects produced by guanine nucleotides and are greater on free receptors than on receptors coupled to guanine nucleotide binding proteins.