Interactions of agonists and antagonists with beta-adrenergic receptors on intact L6 muscle cells

A binding assay has been developed to characterize beta-adrenergic receptors on intact L6 muscle cells. The affinity of beta-adrenergic receptors for the radioligand iodohydroxybenzylpindolol (IHYP) was the same in membrane preparations and in intact cells when determined by either equilibrium binding or kinetic analysis. The number of specific IHYP binding sites per cell was approximately the same on intact cells as on membranes. The pharmacological properties of antagonists indicated that the receptors on intact cells were identical to those on membranes. However, the beta-adrenergic receptors on intact cells had a 100-400 fold lower affinity at equilibrium for the agonist isoproterenol than did beta-adrenergic receptors on membranes. This low affinity of the receptor for agonists as measured by inhibition of radioligand binding in intact cells has also been observed in C6 (2) and S49 (3) cells. Our results suggest that beta receptors on intact cells after a 1 minute incubation was similar to the KD value for isoproterenol measured in membranes at equilibrium in the presence of GTP. After 1-2 minutes of exposure to a low concentration of agonist, binding of IHYP was no longer inhibited. These results suggest that agonists rapidly convert the beta receptors on intact cells to a state which has a low affinity for agonists. The affinity of the receptor for antagonists did not change during the incubation.     

Effects of serotonergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami

Experimental evidence suggests that serotonin (5HT) is excitatory to the hypothalamic-pituitary-adrenal axis and that this effect involves activation of both hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion. The present study was undertaken to examine the mechanism by which 5HT stimulates the central component of the HPA axis. To accomplish this we employed an in vitro rat hypothalamic organ culture system in which CRH secretion from single explanted hypothalami was measured by specific radioimmunoassay (IR-rCRH). All experiments were performed after an overnight (15-18 hr) preincubation. Serotonin stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped and the peak effect was observed at the concentration of 10(-9) M. The stimulatory effect of 10(-9) M 5HT was antagonized by the 5HT1 and 5HT2 receptor metergoline and by the selective 5HT2 receptor antagonists ketanserin and ritanserin. The muscarinic antagonist atropine, the nicotinic antagonist hexamethonium and the alpha-adrenergic receptor antagonist phentolamine, on the other hand, did not inhibit 5HT-induced IR-rCRH secretion. The specific 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped with peak of effect reached at the concentration of 10(-9) M. We also tested the ability of the 5HT agonist meta-chlorophenylpiperazine (m-CPP) and of the selective 5HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) to cause CRH secretion. Although both m-CPP and 8-OH-DPAT stimulated IR-rCRH secretion in a dose-dependent fashion, several differences were observed when their effect was compared to that of 5HT. These included a different shape of the dose-response curve, a lower maximal stimulatory effect and a different maximal stimulatory concentration. These findings suggest that serotonin stimulates CRH secretion by explanted rat hypothalami and that this effect appears to be mediated mainly through a 5HT2 receptor mechanism.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

Functional activation of beta-adrenergic receptors by thiols in the presence or absence of agonists

Treatment of beta-adrenergic receptor with dithiothreitol (DTT) or other thiol compounds caused its functional activation in the presence or absence of agonist ligands. Such activation was observed in reconstituted unilamellar phospholipid vesicles that contained beta-adrenergic receptors, purified to greater than or equal to 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (Gs) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0 degrees C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of Gs by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding, an effect about equivalent to the addition of beta-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTP gamma S binding, increasing the initial rate about 10-fold. DTT treatment was as effective as agonist in stimulating GTPase activity, and maximal stimulation was obtained when DTT-treated vesicles were assayed in the presence of agonist. Other thiol compounds produced effects similar to those of DTT but were at least 10-fold less potent. Stimulation of GTP gamma S binding or GTPase activity required active receptor, and treatment of the receptor with DTT prior to reconstitution also increased its efficacy. There was no effect of DTT on Gs alone. Thus, the site of action of DTT appears to be on the beta-adrenergic receptor itself, and the reduction of disulfides and the binding of agonist act synergistically to activate the receptor. DTT treatment made the receptor more labile to thermal denaturation. Inclusion of cholesterol or cholesteryl-hemisuccinate (5-25%) in the vesicles protected the reduced receptor against such denaturation and enhanced its recovery during reconstitution. No effect of cholesterol or cholesteryl-hemisuccinate was observed on the stability of the nonreduced receptor, which was comparable to that observed in native membranes.     

Binding of agonists and antagonists to muscarinic receptors

The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [³H] acetylcholine. Mechanisms that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.     

Novel antagonists of serotonin-4 receptors: Synthesis and biological evaluation of pyrrolothienopyrazines

Based on the definition of a 5-HT₄ receptor antagonist pharmacophore, a series of pyrrolo[1,2-a]thieno[3,2-e] and pyrrolo[1,2-a]thieno[2,3-e] pyrazine derivatives were designed, prepared, and evaluated to determine the properties necessary for high-affinity binding to 5-HT₄ receptors. The compounds were synthesized by substituting the chlorine atom of the pyrazine ring with various N-alkyl-4-piperidinylmethanolates. They were evaluated in binding assays with [³H]GR113808 (1) as the 5-HT₄ receptor radioligand. The affinity values (K_i or inhibition percentages) were affected by both the substituent on the aromatic ring and the substituent on the lateral piperidine chain. A methyl group on the tricyclic ring produced a marked increase in affinity while an N-propyl or N-butyl group gave compounds with nanomolar affinities. Among the most potent ligands, 34d was selected for further pharmacological studies and evaluated in vivo. This compound acts as an antagonist/weak partial agonist in COS-7 cells stably expressing the 5-HT_4(a) receptor and is of great interest as a peripheral antinociceptive agent.     

The agonist paradox: agonists and antagonists of acetylcholine receptors and opioid receptors

In contrast to antagonists, agonists tend to induce considerable conformational changes in their receptors, resulting in opening of ion channels, either directly or via secondary messengers. These conformational transformations require great energy expenses. However, the experimentally determined free energies of complexation between agonists and receptors are often relatively smaller than those for the corresponding antagonists. To rationalize this so-called 'agonist paradox', which has not been clarified in the literature, we have developed an alternative model. Our model may help to discriminate between agonists and antagonists of the acetylcholine (ACh) and mu-opioid receptors. For this purpose, a series of ligands (1-18) have been analyzed both in structural terms and with respect to complexation geometry within the anionic binding sites of these two receptor types.     

Guanine nucleotides modulate the affinity of antagonists at beta-adrenergic receptors

Investigation of the properties of the binding of the radiolabelled antagonists (125I)-iodohydroxybenzylpindolol, (125I)-iodopindolol, and (125I)-iodocyanopindolol to beta-adrenergic receptors of L6 myoblast membranes revealed that guanine nucleotides caused a 2 to 4.5 fold increase in the apparent affinity of these antagonists. No significant effects of GTP were observed on the density of binding sites determined with each radioligand. GTP, GDP, and GMPPNP were of similar high affinity in producing this effect, while GMP was much less potent, and ATP was without effect. Under similar assay conditions GTP reduced the apparent binding affinity of the agonist isoproterenol for the beta-adrenergic receptors of L6 cells. The results indicate that, contrary to previous observations, guanine nucleotides affect not only the interactions of agonists with beta-adrenergic receptors, but also the interaction of antagonists with these adenylate cyclase-linked receptors.     

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.     

Effects of adrenergic agonists and antagonists on muscle O2 uptake and lactate metabolism

To investigate adrenergic receptor-mediated responses in dog gastrocnemius-plantaris muscle, several catecholamine agonists, isoproterenol, epinephrine, norepinephrine, and phenylephrine, and two antagonists, propranolol and phenoxybenzamine, were given during repetitive, isotonic, tetanic contractions. The response variables that were measured were muscle blood flow, shortening during constant load contractions, and arterial and venous O2 and lactate concentrations. The calculated variables were O2 uptake (VO2), net lactic acid output (L), and power output. In the control experiments, the contractions increased VO2 to approximately 50 times rest by 2 min. Thereafter, shortening, work, and VO2 declined together by 17% at 30 min, indicating muscle fatigue. L increased rapidly to nearly 0.8 mumol X g-1 X min-1 by 2 min, declined to 0.3-0.4 mumol X g-1 X min-1 by 7 min, and was like rest at 15, 22.5, and 30 min. The arterial lactate concentration rose steadily from rest to 30 min of contractions. Epinephrine infusion stopped the decline of VO2 during the contractions, but this effect was not observed with the other agonists. Propranolol decreased VO2 compared with controls at 22.5 and 30 min of contractions. Phenoxybenzamine decreased VO2 compared with controls at all times during contraction, and the decline with time was present. Coinfusion of epinephrine with propranolol reduced the decline in VO2 observed with propranolol alone. Both epinephrine and isoproterenol increased L compared with controls. This epinephrine response was antagonized by propranolol but enhanced by phenoxybenzamine. Both isoproterenol and epinephrine infusions increased arterial lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian rhythms in rat brain alpha- and beta-adrenergic receptors are modified by chronic imipramine

Circadian rhythms of α- and ß-adrenergic receptor number, with different wave forms, as well as differences in timing of maximal binding, are present in rat brain. Chronic treatment with the tricyclic antidepressant drug imipramine modifies these rhythms: peak binding of both receptors occurs 4–12 hours later than in controls, the 24-hour mean is decreased by 15–30%, and the amplitude is increased by 20–30%. Delaying of the phase position of neurotransmitter receptor rhythms by a tricyclic antidepressant may be relevant to its clinical mode of action, since depressive patients appear to have abnormally phase-advanced circadian rhythms.     

Action of agonists and antagonists on adrenergic receptors in isolated porcine coronary arteries

The adrenergic receptors of porcine coronary arteries were investigated in helically cut strips of small (less than or equal to 0.5 mm outer-diameter (od), medium (0.8-1.2 mm od), large (1.5-2.5 mm od), and very large (greater than 4 mm od) coronary arteries. Both the beta1 agonist dobutamine and the beta2 agonist terbutaline relaxed coronary arteries partially contracted by 25 mM of KCl. Dobutamine contracted small coronary arteries at 10(-5) M concentrations, then relaxed them at 10(-4) M. The beta1-adrenoceptor antagonist metoprolol contracted coronary arteries relaxed by either dobutamine or terbutaline, but the beta2 antagonist H35/25 did so only in high and probably nonselective concentrations. Alpha1-adrenoreceptor stimulating concentrations of phenylephrine did not contract any of the arteries. Metoprolol and high concentrations of H35/25 further contracted large coronary arteries partially contracted by 25 mM potassium. These contractions were blocked by verapamil and papaverine but not by atropine, phentolamine, yohimbine, mepyramine, or methysergide. This seems to indicate that beta-adrenergic receptors in porcine coronary arteries are beta1-receptors, or closely resemble beta1-receptors. They differ from many other beta1-receptors, however, in that they are stimulated by terbutaline. Alpha1 adrenoreceptors seem not to be present in these porcine coronary arteries to a significant extent. Metoprolol and high concentrations of H35/25 have a direct contractile effect in large porcine coronary artery that is not mediated by alpha-adrenergic, muscarinic, histaminergic, or serotonergic receptors but requires verapamil-sensitive calcium.     

Effects of agonists and antagonists of rhyanodine receptors on potassium contractures in twitch and tonic frog skeletal muscle fibers

A comparative analysis of the effects of the concentrations of Ca2+ in external medium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of rhyanodine-sensitive Ca2+ channels of carcoplasmic reticulum on the characteristics of potassium contracture in frog twitch and tonic skeletal muscles has been performed. It was shown that the duration of contracture in tonic muscles is not restricted by the presence of Ca2+, as distinct from twitch muscles. Dandrolene does not practically affect the contractile responses of tonic fibres, and the concentration of cresol eliciting the contracture for tonic fibres is substantially higher (1 mM) than for twitch fibers (0.25 mM). In twitch fibers, the potassium contracture activated in the presence of cresol is comparable in amplitude and dynamics with the contracture under control conditions, and in tonic fibers a summing of responses without relaxation after the washing of excessive potassium is observed. This suggests that, in twitch fibers, the influx of Ca2+ can directly create the concentration sufficient for the maintenance of contraction, and in tonic fibers its involvement is mediated through the Ca(2+)-dependent activation of the beta-isoform of rhyanodine-sensitive channels.     

Effects of beta-adrenergic agonists on bone-resorbing activity in human osteoclast-like cells

In the present study, we demonstrate for the first time that beta-adrenergic agonists stimulate bone-resorbing activity in human osteoclast-like multinucleated cells (MNCs). Osteoclast-like MNCs constitutively expressed mRNA for alpha1B-, alpha2B- and beta2-adrenergic receptor (AR) in addition to characteristic markers of mature osteoclast, such as calcitonin receptor (CT-R), tartrate-resistant acid phosphatase (TRAP), alphaV-chain of integrin (Int alphaV), carbonic anhydrase II (CA-II) and cathepsin K (Cathe K). Epinephrine (1 microM; alpha,beta-adrenergic agonist) up-regulated expression of Int alphaV, CA-II and Cathe K in the osteoclast-like MNCs. Osteoclastic resorbing activity was markedly increased by isoprenaline (1 microM; beta-adrenergic agonist), moderately by epinephrine, but poorly by phenylephrine (1 microM; alpha1-adrenergic agonist). The actin ring, which was suggested to be correlated with bone-resorbing activity, was clearly observed in osteoclast-like MNCs treated with isoprenaline and epinephrine, but faintly in those treated with phenylephrine. These findings suggest that beta-adrenergic agonists directly stimulate bone-resorbing activity in matured osteoclasts.     

Ruminant placental lactogens act as antagonists to homologous growth hormone receptors and as agonists to human or rabbit growth hormone receptors

Growth hormone receptor (GHR)-mediated activity of ruminant placental lactogens (PLs) and ovine (o) GH was compared, using cells transfected with full size human (h), rabbit (rb), and oGHRs. All three PLs acted as agonists in heterologous bioassays, whereas in homologous bioassays in cells transfected with oGHRs they antagonized the oGH activity. Despite these differences, oGH and PLs bound with similar affinity to the oGHR extracellular domain (oGHR-ECD), indicating that the binding occurs through hormone site I. Gel filtration of complexes between oPL and oGHR-ECD showed a 1:1 stoichiometry, confirming this conclusion. The oPL T185D and bPL T188D, which exhibited weak biological activity mediated through GHRs, behaved as site I antagonists, whereas oPL G130R and bPL G133R formed a 1:1 complex with GHR-ECDs and bound to h/rb/oGHR-ECDs with affinity similar to that of wild-type oPL. They had no agonistic activity in all models transfected with h/rb and oGHRs, but were antagonistic to all of them. In conclusion, ruminant PLs antagonize the activity of oGH in homologous systems, because they cannot homodimerize oGHRs, whereas in heterologous systems they act as agonists. The structural analysis hints that minor differences in the sequence of the GHR-ECDs may account for this difference. Since the initial step in the activity transduced through cytokine/hemapoietic receptors family is receptor homodimerization or heterodimerization, we suggest that the question of homologous versus heterologous interactions should be reexamined.     

Effects of alpha- and beta-adrenergic agonists on Trypanosoma cruzi interaction with host cells

We studied the effects of adrenergic agonists on the capacity of blood trypomastigote forms of Trypanosoma cruzi to associate with (i.e., bind and/or penetrate) host cells in vitro. The extent of T. cruzi association with mouse macrophages in the presence of the beta-adrenergic agonist L-isoproterenol was significantly decreased with respect to mock-treated controls. Similar results were obtained when the parasite was pretreated with L-isoproterenol and was then allowed to interact with untreated macrophages. In contrast, pretreatment of trypomastigotes with either L-phenylephrine or methoxamine-alpha-adrenergic agonists--enhanced their reactivity with macrophages. Interaction with a nonphagocytic host cell was also decreased and increased by parasite pretreatment with beta- and alpha-adrenergic agonists, respectively. The L-isoproterenol and L-phenylephrine effects were no longer detectable 2 and 3 hr after their removal, respectively, and were therefore reversible. Atenolol, a specific beta 1 adrenoreceptor blocker inhibited the L-isoproterenol effect, whereas butoxamine, a specific beta 2 blocker, did not. Thus, beta 1-like but not beta 2-like binding sites appeared to be expressed on T. cruzi. Both prazosin and yohimbine, preferential alpha 1- and alpha 2-receptor blockers, respectively, abolished the L-phenylephrine effect. The opposite effects of alpha- and beta-adrenergic agonists suggested that the infectivity of T. cruzi may be regulated by activation of surface components comparable to the adreno-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)