Agonist Competition Curves Research Articles

Exercise function and biochemical properties of β-adrenergic receptors of canine heart

We tested the hypothesis that the biochemical properties of cardiac beta-adrenergic receptors (βAR), as determined by standard in vitro methods, limit physiological responsiveness of dogs to PAR stimulation during peak exercise. Accordingly, we measured oxygen consumption, left ventricular dP/dt at 40 mmHg developed pressure, heart rate, and cardiac output during maximal dynamic exercise in 13 adult mongrel dogs. These studies were followed by biochemical analysis of PAR in membrane homogenates of left ventricle from the same animals using saturation binding and agonist competition curves for [125I] cyanopindolol. There was a substantial range in both biochemical and physiological variables within this population of normal dogs. However, there were no significant correlations between biochemical descriptors of cardiac PAR and any physiological variables measured during exercise. These results suggest that receptor-effector coupling and maximal cardiac performance during intense sympathetic stimulation of peak exercise are limited by factors other than the biochemical properties of cardiac PAR.

Site-directed mutagenesis studies of human A 2A adenosine receptors : Involvement of glu 13 and his 278 in ligand binding and sodium modulation

To provide insights into interactions between ligands and A 2A adenosine receptors, site-directed mutagenesis was used to test the roles of a glutamic acid residue in the first transmembrane domain (Glu13) and a histidine residue in the seventh transmembrane domain (His278). The two residues, which have been suggested to be closely linked in molecular modeling studies, were mutated to glutamine (E13Q) and tyrosine (H278Y), respectively. Saturation experiments revealed that [ 3H]ZM241385 (4-{2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5- a][1,3,5]triazin-5-yl-amino]ethyl}phenol) bound wild-type and mutant receptors in membranes from COS-7 cells expressing human A 2A adenosine receptors with high affinity and low non-specific binding. It was found from the competition experiments that the affinity of the A 2A adenosine receptor agonists for the mutant receptors was 3- to 200-fold lower than for the wild-type receptor. Among antagonist competitors of binding at E13Q and H278Y mutant receptors, there was variation in the affinity depending on their different structures, although changes were relatively minor (<3-fold) except in the case of theophylline, whose affinity was decreased approximately 20 times on the H278Y mutant. The possible involvement of the two residues in sodium ion regulation was also tested. The agonist competition curves for [ 3H]ZM241385 were shifted to the right in both wild-type and mutant receptors in the presence of 1 M sodium ions, but the extent of shift (2- to 27-fold) in wild-type receptor was generally larger than for the mutant receptors. Sodium ions also decreased [ 3H]ZM241385 dissociation from both wild-type and mutant receptors, being more influential on the former than the latter. The results suggest that the two closely linked residues Glu13 and His278 in A 2A adenosine receptor are most important for agonist recognition and are partly responsible for the allosteric regulation by sodium ions.

Negative regulation of α2-adrenergic receptor-mediated Gi signalling by a novel pathway

Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.

Negative regulation of α2-adrenergic receptor-mediated Gi signalling by a novel pathway

Chinese hamster ovary (CHO) cells stably expressing α2 adrenergic receptor (α2AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The α2AAR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of α2AAR-mediated inhibition was not observed when α2AAR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to Gi-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates Gi signalling in a protein-kinase-C-dependent manner.

Alternations in beta-Adrenoceptor Mechanisms in Hearts Perfused With Xanthine Plus Xanthine Oxidase.

BACKGROUND: Although beta-adrenoceptors and adenylyl cyclase are known to be affected upon exposing cardiac membranes to some oxyradical generating systems, the results are conflicting. Furthermore, functional significance of alterations in the beta-adrenoceptor-adenylyl cyclase systems in terms of changes in the inotropic responses to catecholamines is not clear. METHODS AND RESULTS: The positive inotropic effect of isoproterenol was augmented on perfusing the isolated rat hearts with xanthine (X) plus xanthine oxidase (XO) for 5 minutes but was attenuated by perfusion for 15 minutes. The isoproterenol-stimulated adenylyl cyclase activity in cardiac membranes showed an increase at 10 minutes and a decrease at 30 minutes perfusion of hearts with X plus XO. The density of beta-adrenoceptors in cardiac membraners was reduced after 10 minutes and 30 minutes of perfusion with X plus XO, whereas the affinity of beta-adrenoceptors was increased after 10 minutes and reduced after 30 minutes. Although beta-adrenoceptors was increased after 10 minutes and reduced after 30 minutes. Although beta-adrenoceptors were unaltered by 10 minutes of perfusion with X plus XO, their affinity was increased and density was decreased by 30 minutes of perfusion. The agonist competition curves using isoproterenol indicated an increase in the number of coupled receptors in the high affinity state on 10 minutes of perfusion and an increase in the low affinity state of coupled receptor due to 30 minutes of perfusion with X plus XO. The basal as well as forskolin-, NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities in cardiac membranes exhibited an increase after 10 minutes and decrease after 30 minutes of perfusion with X plus XO. Although the presence of superoxide dismutase plus catalase in the perfusion medium prevented most of the alterations due to X plus XO, it did not alter the increased affinity of the beta-adrenoceptor upon perfusing hearts for 10 minutes with X plus XO. CONCLUSIONS: The results in this study suggest the biphasic nature of the oxyradical-induced alterations in both the inotropic responses to catecholamines and the beta-adrenoceptor-mediated signal transduction mechanism in the heart.

Heparin inhibits melatonin binding and signal transduction in chick brain.

The high-affinity receptor for melatonin is coupled to a pertussis toxin-sensitive, inhibitory guanine nucleotide regulatory protein, Gi, which mediates inhibition of adenylate cyclase activity in the chick and other species. Heparin has been found to uncouple alpha 2-adrenoceptors from a similar Gi; therefore it was of interest to examine the effect of this agent on melatonin binding and signal transduction in chick brain. In competition studies, melatonin inhibited the binding of 2-[125I]iodo-melatonin with high- and low-affinities of KH = 20 pM and KL = 15 nM, respectively. In the presence of heparin (100 units/ml), a single site with an affinity of about 6 nM was detected. The monovalent cations, Na+ and Li+, produced a greater rightward shift of the agonist competition curve than heparin and converted all high-affinity sites to a low affinity of approximately 15-18 nM. In saturation studies, heparin reduced the affinity of high-affinity sites and caused a significant decrease in the density of low-affinity sites. In keeping with its ability to reduce high-affinity binding, heparin blocked melatonin's inhibitory effect on forskolin-stimulated adenylate cyclase activity in chick brain synaptosomal membranes. These findings indicate that heparin impairs the coupling between the melatonin receptor and Gi, with a consequent decrease in binding affinity and a loss of receptor-mediated signalling.

Characterization of Cardiac β-Adrenergic Receptors in the Guinea Pig Heart: Application to Study of β-Adrenergic Receptors in Shock Models

The myocardial response to catecholamines is significantly diminished in many types of shock or heart failure. The guinea pig heart is an ideal model for the study of shock, as it is relatively inexpensive, and the cardiovascular system of the guinea pig most closely resembles that of the human. Using this model, we have developed techniques to characterize and quantitate changes in β-adrenergic receptors (βAR) in the guinea pig heart after burn injury. Preliminary experiments were performed to determine the optimum binding conditions, e.g., incubation time and conditions, protein concentrations, rinsing, etc. Additional experintents were conducted using agonists and antagonists to characterize the rank order of potency and stereospecificity of the βAR. Crude membrane preparations (50 μg/250μl) from sham-burned and burned hearts were incubated with 8-10 concentrations of 125 I-cyanopindolol (10-450 pM) at 37°C for 1 hr. Under these conditions, binding assays were linear with respect to protein concentration and time. Alprenolol (10 μM) was used to determine nonspecific binding. The membrane preparations used in this study bound both agonists and antagonists with a rank order of potency and stereospecificity characteristic of a β-adrenergic receptor. Finally, agonist competition curves were performed with isoproterenol in the presence and absence of Gpp(NH)p to determine receptor regulation by the Gs protein. Analysis using computer-assisted techniques suggests that the fraction of high-affinity β-receptors is significantly reduced after burn injury (41.2 ± 4.7%) compared to sham-burned controls (54 ± 2%, P ⩽ 0.023). The methods described here can be easily adapted to correlate cardiac dysfunction with changes in β-adrenergic receptors during many types of shock or trauma.

Characterization of metabotropic glutamate receptors coupled to a pertussis toxin sensitive G-protein in bovine brain coated vesicles

Glutamate metabotropic receptors (mGluRs) in bovine brain coated vesicles have been characterized by pharmacological and kinetic binding experiments. Saturation experiments revealed a single binding site with a K d = 607.9 ± 78.5 nM and a B max = 6.45 ± 0.88 pmol mg protein. The specific binding of l-[ 3H]glutamate to mGluRs is regulated by guanine nucleotides. Guanosine-5'-triphosphate (GTP; 100 μM) shifts the agonist competition curves to the right, increasing the IC 50 values. Pertussis toxin treatment produces a pharmacological binding profile for quisqualate similar to that obtained in the presence of 100μM GTP. These results indicate the presence of metabotropic glutamate receptors in coated vesicles and its coupling to a pertussis toxin sensitive G-protein.

Burn-induced alterations in cardiac beta-adrenergic receptors.

Previous studies in our laboratory have demonstrated that burn injury (45% total body surface area, 3rd-degree scald burn) diminishes contractile and relaxation function in the isolated perfused guinea pig heart. The mechanisms responsible for the burn-mediated dysfunction are not well understood. Therefore the purpose of this study was to examine the inotropic response to isoproterenol, a beta-adrenergic agonist, and burn-induced alterations in beta-adrenergic receptors (beta-AR) in adult guinea pig hearts. Isoproterenol dose-response curves were generated in isolated perfused hearts from sham-burned and burned guinea pigs. In addition, binding studies were performed using [125I]iodocyanopindolol on hearts from sham-burned and burned guinea pigs. Both the functional response and sensitivity to isoproterenol were significantly diminished 24 h after burn injury. beta-AR density (binding capacity, Bmax) and affinity were determined by Scatchard analysis. Agonist competition curves were performed in the presence or absence of 0.1 mM 5'-guanylyl imidodiphosphate. There was no difference in Bmax in membranes from sham-burned and burned hearts; however, the affinity of beta-AR was significantly decreased after burn injury compared with sham burn [dissociation constant = 32.5 +/- 1.9 (mean +/- SE), n = 10, vs. 26.7 +/- 1.7 pM, n = 10, P = 0.039]. Furthermore, the fraction of receptors in a high-affinity state (those functionally coupled to Gs protein) was significantly decreased after burn injury compared with sham burn (41.2 +/- 4.7%, n = 9, vs. 54 +/- 2%, n = 9, P = 0.023).(ABSTRACT TRUNCATED AT 250 WORDS)

Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. I. Coupling of alpha 2-adrenergic receptor subtypes to distinct G-proteins.

alpha 2-Adrenergic receptor (alpha 2-AR) subtypes couple to pertussis toxin (PT)-sensitive G-proteins to elicit both stimulatory and inhibitory cell responses. Signal specificity may be generated by the ability of the receptor subtypes to "recognize" distinct G-proteins with different affinity. To address this issue we stably expressed three alpha 2-AR subtypes, RNG alpha 2 (alpha 2B-AR), RG10 (alpha 2C-AR), and RG20 (alpha 2D-AR), in NIH-3T3 fibroblasts, which express two PT-sensitive G-proteins (Gi alpha 2, Gi alpha 3), and analyzed receptor/G-protein interactions by determining: 1) functional coupling to adenylylcyclase and 2) the ability of the receptors to exist in a high affinity state for agonist. In alpha 2D-AR transfectants expressing 200 or 2,200 fmol of receptor/mg of protein, epinephrine (10 microM) inhibited forskolin-induced elevation of cellular cAMP by 26 +/- 4.8% and 72 +/- 6.2%, respectively. Similar results were obtained in alpha 2B-AR transfectants. However, in alpha 2C-AR transfectants (200 fmol/mg) the forskolin-induced elevation of cellular cAMP was not altered by agonist treatment. In alpha 2C-AR transfectants expressing higher receptor densities (650-1,200 fmol/mg), epinephrine inhibited the effect of forskolin by 30 +/- 3.2%. This difference in functional coupling among the alpha 2-AR subtypes is reflected at the receptor/G-protein interface. In membrane preparations of alpha 2B and alpha 2D-AR but not alpha 2C-AR transfectants, agonist competition curves were biphasic, indicating high and low affinity states of the receptor for agonist. The high affinity state was guanyl-5'-yl imidodiphosphate- and PT-sensitive, indicative of receptor/G-protein coupling. These data suggest that the alpha 2C-AR differs from the alpha 2B and alpha 2D-AR subtypes in its ability to recognize PT-sensitive G-proteins expressed in NIH-3T3 fibroblasts. The alpha 2C-AR may couple preferentially to PT-sensitive G-proteins (Gi1, Go1,2) not expressed in NIH-3T3 fibroblasts and thereby elicit different cellular responses.

Proximal Tubular α2-Adrenoceptor Density in the Spontaneously Hypertensive Rat

In the kidney of the spontaneously hypertensive rat (SHR), alpha 2-adrenoceptors are increased and may be related to hypertension. We measured alpha 2-adrenoceptor density and characteristics in a proximal tubule suspension with the alpha 2-adrenoceptor antagonist, 3H-rauwolscine. SHR had a significantly greater density of alpha 2-adrenoceptors when compared to their normotensive controls, the Wistar-Kyoto (WKY) rats (146 +/- 13 v 58 +/- 7 fmol/mg protein; P less than .001). Competition experiments determined the alpha 2-adrenoceptor to be an alpha 2B-subtype. Agonist competition curves were shallow and exhibited pseudo-Hill slopes of less than 1, indicating that they bind to both high and low affinity receptor sites. In summary, alpha 2-adrenoceptors are increased in the proximal tubule of SHR and appear to be of the alpha 2B-subtype. An increase in alpha 2-adrenoceptors may be related to abnormalities in hypertension.

Different effects of N-ethylmaleimide on the binding characteristics of 5-hydroxytryptamine receptor subtypes in rat brain membranes

To characterize 5-hydroxytryptamine (5-HT) receptor subtypes in the CNS in terms of their binding and coupling properties with GTP-binding proteins (G proteins), we examined the effects of serotonergic agents GTP?S and N-ethylmaleimide (NEM), on the binding of [(3)H]8-OH-DPAT, [(125)I]iodo-cyanopindolol (ICYP), [(3)H]mesulergine, [(3)H]DOB and [(3)H]ketanserin in rat brain membranes. [(3)H]8-OH-DPAT, [(125)I]ICYP, [(3)H]mesulergine and [(3)H]ketanserin are radiolabeled ligands for 5-HT(1A), 5-HT(1B), 5-HT(1C) and 5-HT(2) sites, respectively. Correlation of the values of the dissociation constants (K(D)) of the serotonergic agonists or antagonists was evaluated in two categories of 5-HT receptor subtypes. Highly significant correlations were found among 5-HT(1A), 5-HT(1B) and 5-HT(1D) sites, and among 5-HT(1C) and 5-HT(2) sites. A significantly reversed correlation was observed between 5-HT(1A) and 5-HT(2). GTP?S caused a shift to the right on the 5-HT competition curve for the binding of [(125)I]ICYP, [(3)H]mesulergine or [(3)H]ketanserin. Although NEM-treatment partially induced a right shift on the agonist competition curve for [(125)I]ICYP binding, this treatment induced a left shift on the curve for [(3)H]mesulergine and [(3)H]ketanserin binding. In addition, the binding of [(3)H]DOB, a 5-HT(2) agonist, was increased by NEM-treatment. NEM seems to characterize agonist binding to the 5-HT receptor subtypes and the coupling of these subtypes to G proteins. These results suggest that 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors belong to one category, and 5-HT(1C) and 5-HT(2) receptors belong to another category.

Purification and characterization of bovine cerebral cortex A 1 adenosine receptor 1

A 1 adenosine receptors (A 1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A 1AR from bovine cerebral cortex. This A 1AR is distinct from other A 1ARs in that it displays an agonist potency series of N 6-R-phenylisopropyladenosine ( R-PIA)>N 6- S-phenylisopropyladenosine > ( S-PIA)>5′- N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA>NECA> S-PIA. The A 1AR was solubilized in 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of M r 36,000 by silver staining, Na 125I iodination with chloramine T and photoaffinity labeling with [ 125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy-phenyl]-1,3-dipropylxanthine. This single protein displayed all the characteristics of the A 1AR, including binding an antagonist radioligand ([ 3H]XAC) with high affinity ( K d = 0.7 nM) and in a saturable manner ( B max> 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A 1AR pharmacology: R-PIA> S-PIA>NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A 1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A 1AR from M r 36,000 to M r, 32,000 in a single step. The A 1AR was sensitive to neuraminidase but resistant to α-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A 1AR similar to rat brain and fat A 1AR in terms of its carbohydrate chains yet the purified A 1AR retains its unique agonist potency series observed in membranes.

Human M1-, M2- and M3-muscarinic cholinergic receptors: Binding characteristics of agonists and antagonists

The muscarinic acetylcholine receptors were identified in membrane preparations from human tissues by the specific binding of 1-[ benzilic-4,4′- 3H] quinuclidinyl benzilate. Saturation binding isotherms of this radioligand yielded a total amount of receptors of 435 ± 208, 159 ± 65 and 913 ± 89 fmol/mg protein, respectively, in the hippocampus, pons and submandibular gland. Non linear least squares analysis of competition binding studies with the antagonists pirenzepine and AF-DX 116 indicates that the majority of receptors are of the M1-type in the hippocampus (83%, high affinity for pirenzepine, intermediate affinity for AF-DX 116), the M2-type in the pons (low affinity for pirenzepine and high affinity for AF-DX 116), and the M3-type in the submandibular gland (low affinity for pirenzepine and AF-DX 116). Competition binding parameters of the agonists carbachol, arecoline, oxotremorine, pilocarpine and MCN-A-343 were compared for M1, M2 and M3 receptors in the human hippocampus, pons and submandibular gland. GTP caused a shift to the right and a steepening of the shallow agonist competition curves in the 3 tissues but did not affect the initially steep ones. This effect is explained by a GTP-mediated conversion of high- to low-agonist affinity sites. The extent of the nucleotide shift was much greater for M2 receptors as compared with M1 and M3 receptors. The GTP effect was impaired by the sulphydryl reagent N-ethylmaleimide, probably due to alkylation of GTP-binding proteins. Moreover, the reagent provoked also an increase of the agonist affinity for the uncoupled muscarinic receptors. For all agonists, this increase was more pronounced for the M2 receptors than for the M1 and M3 receptors. These findings suggest structural differences between the agonist binding sites of M1 and M3 receptors versus the M2 receptors.

Developmental changes in guanine nucleotide regulatory proteins in the rat myocardial alpha 1-adrenergic receptor complex.

During development, the cardiac alpha 1-adrenergic chronotropic response changes from positive in the neonate to negative in the adult. The negative chronotropic effect of alpha 1-adrenergic stimulation in the adult depends on maturation of sympathetic innervation and the presence of a pertussis toxin (PT)-sensitive guanine nucleotide-binding (G) protein. To examine the possibility of a developmental change in coupling of a PT-sensitive G protein to the alpha 1-adrenergic receptor, radioligand binding experiments with the iodinated alpha 1-selective radioligand [125I]-I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([ 125I]-IBE 2254) were performed on membranes prepared from control and PT-treated neonatal and adult rat hearts. Scatchard analysis showed fewer alpha 1-adrenergic receptors in the adult than in the neonate (168 +/- 10 fmol/mg protein in the neonate vs. 124 +/- 13 fmol/mg protein in the adult), but similar affinities (equilibrium dissociation constant 124 +/- 29 pM in the neonate vs. 140 +/- 34 pM in the adult). PT treatment did not alter the results. In both the neonate and adult, 5'-guanylylimidodiphosphate [Gpp(NH)p, 500 microM] shifted the l-epinephrine competition curve to the right and increased the slope factor toward unity. PT had no effect on the l-epinephrine competition curve in the neonate. However, in the adult PT itself caused a partial shift in the agonist competition curve, reducing but not eliminating the effect of Gpp(NH)p. Consistent with the results from the binding experiments, PT did not have any effect on the alpha 1-adrenergic-mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1-adrenergic-mediated negative chronotropic response was completely converted to a positive one after PT treatment. These results indicate the presence of a PT-insensitive G protein in the neonatal and adult rat heart and the acquisition of a PT-sensitive G protein linked to the negative chronotropic response during development.

Coupling of adenosine receptors to a pertussis toxin-sensitive G protein in the human heart

The adenosine receptors in myocardial membranes of human explanted hearts were labeled with the radiolabeled ligand [ 3H]DPCPX (1,3-dipropyl-8-cyclopentylxanthine). Agonist competition curves revealed high- and low-affinity states. The addition of guanylyl imidodiphosphate (Gpp (NH)p) converted all receptors to a low-affinity state. The portion of high-affinity states and the influence of guanine nucleotides were most pronounced at 22°C. Only low-affinity states could be detected in pertussis toxin-treated membranes. It is concluded that adenosine receptors in the human ventricle couple via a G protein sensitive to pertussis-toxin. Alterations of the coupling of adenosine receptors might have a pathophysiological role in dilated cardiomyopathy in which pertussis toxin substrates are increased.

Chronic Estrogen Treatment Promotes a Functional Uncoupling of the D2Dopamine Receptor in Rat Anterior Pituitary Gland

The effect of chronic estrogen treatment on the anterior pituitary D2 dopamine receptor was studied by treating rats with diethylstilbestrol (DES) over a 6-week period. DES treatment resulted in an increase in anterior pituitary weight and PRL content and serum PRL levels compared to those in sham-treated controls. The status of the anterior pituitary D2 dopamine receptor was evaluated using both radioligand binding and adenylate cyclase assays. [125I]N-(p-aminophenethyl)spiroperidol [( 125I]NAPS), a derivative of the D2-selective antagonist spiperone, was used to quantitate D2 receptors. Saturation analysis of [125I]NAPS binding indicated that DES treatment had no effect on the affinity or maximum binding capacity of the radioligand for the D2 receptor. Competition analysis with unlabeled D2 antagonists for [125I]NAPS binding also indicated that DES treatment did not affect antagonist interactions with the receptor. In contrast, the interactions of agonists with the D2 receptors from DES-treated rats were modified, as assessed through [125I]NAPS competition analysis. Using control tissue, agonist competition curves revealed both high and low affinity agonist binding states of the receptor. In the presence of guanine nucleotides, the high affinity agonist binding state is abolished, reflecting coupling of the receptor with a guanine nucleotide regulatory (G) protein. In DES-treated tissue, agonist competition curves indicated the presence of only low affinity agonist binding, with minimal effects of guanine nucleotides, suggesting uncoupling of receptor-G-protein interactions. The functionality of the D2 receptor was further assessed by examining dopaminergic inhibition of vasoactive intestinal peptide-stimulated adenylate cyclase activity. Although DES treatment resulted in a reduction of vasoactive intestinal peptide-stimulated enzyme activity itself, the ability of dopaminergic agonists to inhibit this activity was reduced by about 50%. These results suggest that estrogen is capable of attenuating the functional coupling of the D2 receptor with its biochemical effector system in the anterior pituitary gland.

A1 adenosine receptor-G protein coupling in bovine brain membranes: effects of guanine nucleotides, salt, and solubilization.

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of cardiac alpha- and beta-adrenoceptors in spontaneously hypertensive rats

The effects of isoprenaline, Ca2+ and phenylephrine (in the presence of propranolol) on force of contraction were studied in isolated electrically driven papillary muscles of spontaneously hypertensive rats (SHR) and age-matched (14-18 weeks) Wistar Kyoto control rats (WK). Cardiac alpha- and beta-adrenoceptors were characterized by radioligand binding studies. The positive inotropic effect of isoprenaline in SHR was less effective than in control rats. The EC50 values did not differ in both groups. In SHR, isoprenaline was less effective than Ca2+ to increase force of contraction whereas in WK it had the same effectiveness as Ca2+. The positive inotropic effect of phenylephrine in the presence of propranolol was similar in SHR and WK. In SHR, both the densities of cardiac alpha- and beta-adrenoceptors were reduced. In beta-adrenoceptor binding experiments, the nonhydrolysable GTP analog Gpp(NH)p caused a rightward shift of agonist competition curves of isoprenaline. Biphasic competition curves revealed a similar percentage of low and high affinity sites in SHR and WK, respectively. In alpha-adrenoceptor binding experiments, Gpp(NH)p caused no detectable shift of agonist competition curves with norepinephrine. It is suggested that cardiac beta-adrenoceptor down-regulation is involved in the reduced positive inotropic effect of isoprenaline in SHR. Functional uncoupling of beta-adrenoceptors does not appear to be involved in the reduced beta-adrenoceptor-mediated positive inotropism in SHR. Binding studies do not show evidence for a large number of alpha-adrenoceptors coupling to a guanine-nucleotide binding protein in the rat heart. Finally, in ventricular myocardium of SHR, cardiac alpha-adrenoceptors do not serve as a reserve mechanism during impaired beta-adrenergic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of L-prolyl-L-leucyl glycinamide with dopamine D2 receptor: evidence for modulation of agonist affinity states in bovine striatal membranes.

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)