Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding.
(49/142)
Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system. (+info)
Antibodies to beta 1 and beta 2 adrenoreceptors in Chagas' disease.
(50/142)
Evidence accumulated over the last decade concerning human and experimental models suggests that an immunopathological mechanism may be involved in the pathogenesis of chronic Chagas' disease. In this paper we demonstrate the existence of two different circulating IgG in chagasic patients which bind with myocardial beta 1 and spleen cell beta 2 adrenoceptors, acting as non-competitive inhibitors. Both chagasic IgG against beta 1 and beta 2 adrenoceptor increased intracellular levels of cAMP that could be blocked by specific beta 1 and beta 2 adrenoceptor antagonists. The specificity for beta 1 and beta 2 adrenoceptors and the independence of other tissue reactive antibodies was demonstrated by IgG absorption with turkey red blood cell (TRBC), human lymphocytes (HL) or guinea pig red blood cells (GPRBC). The F(ab')2 fraction acted similarly. This supports the specificity of beta 1 and beta 2 adrenoceptors to the chagasic IgG and the independence of the other tissue reactive antibodies, such as EVI system. The probable pathogenic role of both beta 1 and beta 2 adrenergic chagasic antibody is discussed. (+info)
A possible mechanism in interaction of a partial agonist with beta-adrenoceptor in guinea-pig taenia caecum: effects of Gpp(NH)p on its two different binding sites.
(51/142)
The mechanisms of the actions of the beta-adrenergic partial agonist (befunolol) were studied in isolated guinea-pig taenia caecum. Befunolol, 2-acetyl-7-(2-hydroxy-3-isopropylaminopropoxy)benzofuran hydrochloride was found to be a typical partial agonist in guinea-pig taenia caecum. The pD2-value of befunolol was in agreement with its pKA-value obtained with photoaffinity labeling, but was different from its pA2-value against isoprenaline and pKI-value obtained from the inhibition of specific [3H]-dihydroalprenolol binding. The Scatchard plot of the specific [3H]-befunolol binding showed two affinity sites of the receptor in the absence of Gpp(NH)p, but the low affinity site was reduced while the high affinity site was not affected in the presence of Gpp(NH)p. The pKD-value of the high affinity site of befunolol was in agreement with its pA2-value, and the pKD-value of the low affinity site was in agreement with its pD2-value or pKA-value. These results suggest that the beta-adrenergic partial agonist may interact with two different sites: an agonist binding site and an antagonist binding site. (+info)
Acute change in the cyclic AMP content of rat mammary acini in vitro. Influence of physiological and pharmacological agents.
(52/142)
The cyclic AMP content of acini, freshly prepared from mammary tissue of lactating rats, was measured during incubation in vitro. Neither adrenergic agonists nor cyclic AMP phosphodiesterase inhibitors alone caused a change of more than 2-fold in the basal cyclic AMP content of acini. Together, however, these agents provoked increases of around 20-fold in acini cyclic AMP content. Forskolin caused similar effects. The relative potency of adrenergic agonists in increasing cyclic AMP in acini, together with the ability of selective antagonists to oppose such rises, indicated that beta 2-adrenergic receptors were involved in mediating the effects. Receptor-binding experiments using [3H]dihydroalprenolol and selective beta-antagonists confirmed the predominant presence of beta 2-adrenergic receptors on acini membranes and on membranes prepared from purified mammary secretory epithelial cells. These results elucidate some previous findings [Robson, Clegg & Zammit (1984) Biochem. J. 217, 743-749; Williamson, Munday, Jones, Roberts & Ramsey (1983) Adv. Enzyme Regul. 21, 135-145], questioning the role of cyclic AMP in the regulation of lipogenesis in mammary acini. (+info)
Changes in 3H-spiperone, 3H-WB 4101 and 3H-dihydroalprenolol bindings to brain membranes produced by postnatal pretreatment with chlorpromazine in adult rats.
(53/142)
In order to elucidate possible mechanisms of the learning deficit produced by postnatal pretreatment with chlorpromazine (CPZ), changes in catecholamine receptors in the rat brain were investigated. Male neonates of Wistar strain rats were given s.c. 2 mg/kg/day of CPZ for 7 successive days from days 6 to 12 after birth. Effect of the postnatal pretreatment with CPZ on saturation constants for specific bindings of 3H-spiperone, 3H-WB 4101 and 3H-dihydroalprenolol, respectively, in 8 brain regions was investigated at 60 days after birth. Significant decreases in Bmax values of 3H-WB 4101 binding sites in the cortex, thalamus, hypothalamus, mid brain and medulla oblongata/pons and decreases in Kd values of the binding sites in thalamus, hypothalamus and mid brain were observed in CPZ-pretreated rats when compared with corresponding Bmax and Kd values obtained in saline-pretreated rats. Furthermore, significant decreases in both Bmax and Kd values of 3H-DHA binding sites in the thalamus were detected in CPZ-pretreated rats when compared with those obtained in saline-pretreated rats. However, no alterations in 3H-spiperone binding sites in all brain regions were found between CPZ- and saline-pretreated rats. These results suggest that the learning deficit observed in CPZ-pretreated rats may be produced by a functional disorder of catecholaminergic, in particular alpha 1-noradrenergic neurons in the brain. (+info)
MY336-a, a novel beta-adrenergic receptor antagonist produced by Streptomyces gabonae.
(54/142)
Streptomyces gabonae KY2234 was found to produce a new compound, MY336-a, which bound to beta-adrenergic receptor. The compound was isolated from the fermentation broth of KY2234. MY336-a showed a high affinity for the beta-receptor, labeled with [3H]dihydroalprenolol in the membrane fractions of rat heart (beta 1-adrenergic receptor) or lung (beta 2-adrenergic receptor), whereas the compound bound very weakly to alpha-adrenergic receptor, labeled with [3H]dihydroergokryptine in rat brain. the inhibition constants (Ki) of the compound were 0.73 and 0.14 microM for the beta-receptors of heart and lung, respectively. 5'-Guanylylimidodiphosphate (Gpp(NH)p) did not alter the affinity of the beta-receptors for MY336-a. In isolated guinea-pig atria, MY336-a produced an inhibition of the positive chronotropic and inotropic effects of isoproterenol. MY336-a also antagonized the relaxation of tone induced by isoproterenol in isolated guinea-pig trachea. No partial agonistic activity was detected in MY336-a in the isolated atria and trachea. In anaesthetized dogs, MY336-a (1 mg/kg, iv) exerted negative inotropic action (left ventricular dp/dt max, -32.6%). (+info)
N-Bromoacetyl-amino-cyanopindolol: a highly potent beta-adrenergic affinity label blocks irreversibly a non-protein component tightly associated with the receptor.
(55/142)
A new chemical affinity label for the beta-adrenergic receptor, based on the structure of pindolol, has been synthesized and iodinated with 125I. The compound, N-bromoacetylamino-cyanopindolol (BAM-CYP), has an apparent dissociation constant of 44 +/- 7 pM towards the turkey erythrocyte membranes. This compound blocks irreversibly both the ability of beta-adrenergic receptors to bind 125I-cyanopindolol and the ability of beta-receptors to activate adenylate cyclase in the presence of beta-agonists. Furthermore, the irreversible binding of BAM-CYP to half of the beta-receptor sites abolishes the ligand binding activity of all the sites. These findings suggest that the beta-receptor is oligomeric in its native state. Although 125I-BAM-CYP blocks irreversibly and specifically the beta-adrenergic receptor, it does so by labeling a non-protein component, most probably a water-soluble lipid. The labeling is stereospecific since it is prevented by l-propranolol and not by d-propranolol. It is suggested that this lipid is tightly associated with the receptor in close proximity to the binding site. It is also suggested that this water-soluble lipid fraction may prove crucial for the optimal interaction between the beta-adrenergic receptor and the components of adenylate cyclase. (+info)
Purified rat hepatic beta 2-adrenergic receptor. Structural similarities to the rat fat cell beta 1-adrenergic receptor.
(56/142)
The mammalian beta 2-adrenergic receptor from rat liver has been purified by sequential cycles of affinity chromatography followed by steric exclusion high performance liquid chromatography. In purified preparations, the overall yield of receptor approaches 10%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly purified receptor preparations reveals a single peptide, Mr = 67,000, as judged by silver staining. Purified beta 2-adrenergic receptor migrates on steric-exclusion high performance liquid chromatography in two peaks, Mr = 140,000 and 67,000. Specific binding of (-)-[3H]dihydroalprenolol and (-)-[125I]iodocyanopindolol to purified rat liver beta-adrenergic receptor preparations is stereoselective and displays a rank order of potencies characteristic of a beta 2-adrenergic receptor. The mammalian beta 1-adrenergic receptor of rat fat cells has also been purified (Cubero, A., and Malbon, C.C. (1984) J. Biol. Chem. 259, 1344-1350). When purified in the presence of protease inhibitors, radioiodinated beta 1-adrenergic receptors from rat fat cells and beta 2-adrenergic receptors from rat liver comigrate on sodium dodecyl sulfate-polyacrylamide gels as 67,000 Mr peptides. Autoradiograms of two-dimensional partial proteolytic digests of the purified, radioiodinated rat liver beta-adrenergic receptor, as generated by alpha-chymotrypsin, Staphylococcus aureus V8 protease, and elastase reveal a pattern of peptide fragments essentially identical to those generated by partial proteolytic digests of the purified radioiodinated beta 1-receptor from rat fat cells. This data suggests that a high degree of homology exists between these two pharmacologically distinct mammalian beta-adrenergic receptor proteins. (+info)