guanosine-triphosphate and 3-iodopindolol

Studies using isolated cell membranes have shown that beta-adrenergic receptors exist in two interconvertible conformations, one with high affinity for agonists and the other with low affinity. Guanine nucleotides and sodium shift high-affinity receptor sites into low-affinity sites. We sought to demonstrate affinity states of the beta-adrenergic receptor in slide-mounted sections of rat and human postmortem brain, and to determine using quantitative receptor autoradiography the regional distribution in the brain of high-affinity receptors relative to the total population of beta-adrenergic receptors. The beta-adrenergic agonist isoproterenol inhibited the binding of 125I-iodopindolol to slide-mounted sections of rat forebrain and human premotor cortex in a biphasic manner. Approximately 80% of the beta-receptor sites in both rat and human brain showed high-affinity for isoproterenol (7-14 nM). Treatment with 0.1 mM non-hydrolyzable guanine nucleotide 5'-guanylylimidodiphosphate and 25 mM NaCl abolished high-affinity binding in rat brain sections and reduced it in human brain sections. These findings were confirmed by membrane studies using similar tissue samples. For autoradiographic studies, 17 nM isoproterenol displaced 71% of the high-affinity sites without affecting low-affinity beta-adrenergic receptors. Digital subtraction was used to selectively visualize beta-receptors in the high-affinity conformation. The proportion of beta-adrenergic receptors in the high-affinity conformation differed significantly across rat brain regions and across human cortical layers. We conclude that beta-adrenergic affinity states, presumably reflecting the interaction of the receptor with a G-protein, occur in slide-mounted sections of rat brain and persist in human postmortem material.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Brain; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Isoproterenol; Pindolol; Rats; Receptors, Adrenergic, beta

SOM-1122 was found to be a high-affinity, partial agonist for beta adrenergic receptors. SOM-1122 inhibited the binding of [125I]iodopindolol to membranes prepared from rat cerebral cortex and cerebellum. GTP regulated the binding of SOM-1122 by increasing the Hill coefficient in both tissues and reducing the affinity of the receptor for SOM-1122 in the cerebellum. SOM-1122 increased the concentration of cyclic AMP in slices of rat cerebral cortex in a dose-dependent manner; this effect was antagonized by propranolol. Two lines of evidence suggested that SOM-1122 was centrally active after peripheral administration. First, SOM-1122 inhibited the binding of [125I]iodopindolol in vivo in a dose-dependent manner. Second, after chronic infusion with SOM-1122 for 7 days, the density of beta adrenergic receptors in the cerebellum was reduced; receptor density also was reduced 18 hr after acute administration of SOM-1122, although to a lesser extent. SOM-1122 was found to be behaviorally active. It reduced locomotor activity and reduced response rate under a multiple fixed-interval, fixed-ratio schedule in a dose-dependent manner. SOM-1122 also reduced response rate and increased reinforcement rate under a differential-reinforcement-of-low-rate schedule. These behavioral actions of SOM-1122 appeared to be due to an interaction of the agonist with beta adrenergic receptors, as they were antagonized by propranolol. The behavioral changes produced by stimulation of beta adrenergic receptors with SOM-1122 were generally similar to those caused by other centrally acting beta adrenergic agonists and by antidepressant drugs.

Topics: Adrenergic beta-Antagonists; Animals; Benzimidazoles; Cerebral Cortex; Conditioning, Operant; Cyclic AMP; Guanosine Triphosphate; Male; Motor Activity; Pindolol; Propranolol; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta

Age-related differences in the binding properties of beta-adrenergic receptors on lymphocyte membranes isolated from healthy individuals have been reported. The purpose of the present studies was to determine whether or not beta receptors on polymorphonuclear leukocyte (PMN) membranes showed similar age-related changes. Plasma Percoll gradients were used to isolate PMN and mononuclear leukocyte (MN) cells from blood drawn in the supine position from young (25-34 years) and elderly (60-76 years) healthy volunteers. Both blood pressures and plasma norepinephrine levels were significantly elevated in the elderly subjects. Saturation analysis with [125I]-(-) iodopindolol (IPIN) showed a 2.5-fold higher density of beta 2 receptors on the MN, but not PMN, membranes from the elderly. Neither the affinities of the receptor for IPIN or the agonist isoproterenol, nor the GTP-induced shift in agonist affinity, differed with subject age in either cell type. These results suggest that subpopulations of MN cells on which beta-adrenergic receptors are localized may increase in the elderly and that it is important to measure receptor properties on a more homogeneous leukocyte population such as PMN cells or subpopulations of MN cells as a function of aging.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Adult; Aged; Aging; Drug Interactions; Female; Guanosine Triphosphate; Humans; Isoproterenol; Leukocytes, Mononuclear; Male; Middle Aged; Neutrophils; Pindolol; Receptors, Adrenergic, beta

The beta-adrenergic receptor in membranes from cat, rat, and guinea pig hearts appears to undergo spontaneous coupling to the stimulatory nucleotide-regulatory protein, Ns. This was demonstrated by incubating cardiac membranes from acutely reserpinized animals with the alkylating reagent N-ethylmaleimide (NEM), which is known to "freeze" the beta-receptor Ns-complex. The concentration of norepinephrine in these membrane preparations was less than 0.1 nM. Cat heart membranes were preincubated for 10 min at 30 degrees with NEM (10(-7)-10(-3) M) and subsequently incubated with (-)-[125I]iodopindolol (IPIN) (18 min, 30 degrees). NEM caused a concentration-dependent decrease in specific IPIN binding with a maximal reduction of about 20% at 0.1 mM. This decrease occurred even in the absence of MgCl2 (0.5 mM EDTA added as well). A comparable reduction was also observed in myocardial membranes from rat and guinea pig. This fall reflects a decrease in the number of beta-adrenergic receptor sites, as demonstrated by saturation binding experiments with IPIN. The equilibrium dissociation constant of the radioligand for the remaining receptors was not affected. When increasing concentrations of GTP were included in the preincubation mixture, it resulted in a dose-dependent protection of NEM-induced decrease in IPIN binding. The protection was complete at 0.1 mM GTP. In addition, GTP reversed the NEM effect when added to the incubation mixture 10 min after NEM. The apparent reduction in cardiac beta-adrenergic receptor number by NEM (in the absence of beta-receptor agonist) is compatible with a model in which part of the receptor population is able to undergo spontaneous coupling to Ns.

Topics: Animals; Cats; Cell Membrane; Dose-Response Relationship, Drug; Erythrocyte Membrane; Ethylmaleimide; Female; GTP-Binding Proteins; Guanosine Triphosphate; Guinea Pigs; Heart; Male; Myocardium; Norepinephrine; Pindolol; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta; Reserpine; Turkeys

beta-Adrenergic agonist-sensitive adenylate cyclase activity and binding of the beta-adrenergic antagonist(-)-[125I]iodopindolol were studied in rat liver during development of male Fischer 344 rats ages 6-60 days. In liver homogenates maximum adenylate cyclase response to beta-adrenergic agonist (10(-5) M isoproterenol or epinephrine) decreased by 73% (P less than 0.01) between 6 and 60 days, with most of the decrease (56%; P less than 0.01) occurring by 20 days. beta-adrenergic receptor density (Bmax) showed a corresponding decrease of 66% (P less than 0.01) by 20 days without subsequent change. Binding characteristics of stereospecificity, pharmacological specificity, saturability with time, and reversibility were unchanged with age. GTP-, fluoride-, forskolin-, and Mn2+-stimulated adenylate cyclase activities also decreased during development, suggesting a decrease of activity of the catalytic component and/or guanine nucleotide regulatory component of adenylate cyclase. These results indicate that the developmental decrease of beta-adrenergic agonist-sensitive adenylate cyclase activity may result from decreased numbers of beta-adrenergic receptors. Developmental alterations of nonreceptor components of the enzyme may also contribute to changes of catecholamine-sensitive adenylate cyclase.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Binding Sites; Colforsin; Diterpenes; Female; Fluorides; Growth Inhibitors; Guanosine Triphosphate; Iodine Radioisotopes; Liver; Male; Pindolol; Rats; Rats, Inbred F344; Receptors, Adrenergic, beta; Stimulation, Chemical

The tight binding of noradrenaline (NA) to beta-adrenergic receptors was studied in membranes from the left ventricular myocardium of the rat. Addition of GTP (0.1 mM) to membrane preparations from control rats (NA concentration 8.5 +/- 2.1 nM) caused a 4-35% (N = 8) increase (P less than 0.01) in the number of specific binding sites of [125I](-)pindolol. In contrast, addition of GTP did not cause any changes in the number of beta-adrenergic receptors in heart membranes from reserpinized animals (NA concentration less than 0.1 nM). In heart membranes from reserpinized animals, preincubation with NA (followed by washing) revealed a time- and concentration-dependent decrease with a maximum of 35-40% in the [125I](-)pindolol-binding sites. This agonist-mediated decrease in the number of receptors was prevented if GTP was also present in the NA-preincubation medium. It is concluded that NA can undergo tight binding to beta-adrenergic receptors in rat heart membranes. The heart-membrane preparations contain endogenous NA which, via tight agonist binding, is responsible for masking part of the beta-adrenergic receptor population.

Topics: Adrenergic beta-Agonists; Animals; Cell Membrane; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Myocardium; Norepinephrine; Pindolol; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta; Reserpine

The interactions of the atypical agonists pindolol and celiprolol with beta adrenergic receptors were compared with those of the full agonist, isoproterenol. Studies were carried out using intact cells as well as membranes prepared from C6 glioma cells. Computer-assisted analysis of dose-response curves resulting from the inhibition of the binding of [125I]iodopindolol by the beta-1 and beta-2 selective compounds ICI 89,406 and ICI 118,551 revealed that approximately one-third of the beta adrenergic receptors on these cells were beta-1 receptors. Addition of GTP to the binding assay simplified the dose-response curve for inhibition of the binding of [125I]iodopindolol by isoproterenol and diminished the potency of the agonist. GTP had no effect on the binding of pindolol or celiprolol, suggesting that these drugs do not induce the formation of a ternary complex with the receptor and the guanine nucleotide-binding protein for stimulation of adenylate cyclase activity. When added to the growth medium of intact C6 cells, isoproterenol induced a 40-fold increase in cyclic AMP accumulation. Pindolol and celiprolol, however, caused no elevation of enzyme activity. Addition of isoproterenol to the growth medium of intact cells resulted in an 80% decrease in the density of both beta-1 and beta-2 adrenergic receptors within 8 hr. Growing cells in the presence of pindolol or celiprolol induced a 50% decrease in the density of beta-2 receptors, which was inhibited by beta adrenergic antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Celiprolol; Cells, Cultured; Glioma; Guanosine Triphosphate; Iodine Radioisotopes; Isoproterenol; Pindolol; Propanolamines; Rats; Receptors, Adrenergic, beta

Repeated administration of the centrally acting beta adrenergic agonist clenbuterol to rats reduced the ability of isoproterenol to increase levels of cyclic AMP in slices of cerebral cortex. This lessened response to isoproterenol was not due to a reduction in beta receptor density but appeared to be due to diminished receptor/N-protein coupling. This was determined by measuring the ability of isoproterenol to inhibit the binding of the beta adrenergic antagonist [125I]iodopindolol to membranes prepared from cerebral cortex. Using membranes prepared from vehicle-treated rats, isoproterenol, in the absence of GTP, inhibited the binding of [125I]iodopindolol with an IC50 value of 85 nM and a Hill coefficient of 0.65. GTP (250 microM) increased the IC50 value to 290 nM and the Hill coefficient to 0.98. After repeated administration of 10mg/kg of clenbuterol to rats for 8 days, isoproterenol inhibited the binding of [125I]iodopindolol with an IC50 value of 125 nM and a Hill coefficient of 0.90; GTP increased the IC50 value to 170 nM and the Hill coefficient to 0.98. It was inferred from the results of modeling of the isoproterenol competition curves that the repeated administration of clenbuterol reduced or eliminated the high-affinity component of isoproterenol binding. These effects of clenbuterol were found to depend on dose and duration of treatment and were reversible. Repeated administration of the antidepressant drugs desipramine, imipramine, phenelzine, zimelidine and mianserin twice daily for 21 days, by contrast, did not affect receptor/N-protein coupling.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antidepressive Agents; Cerebral Cortex; Clenbuterol; Ethanolamines; GTP-Binding Proteins; Guanosine Triphosphate; In Vitro Techniques; Isoproterenol; Male; Pindolol; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta

Neurotransmitters and hormones mediate their effects through interaction with specific receptors. A complete understanding of the effects of these chemical signals requires detailed knowledge, at the molecular level, of agonist/receptor interactions. It is likely that agonists and antagonists interact with the same site on a receptor. Agonists, however, are by definition different from antagonists in that agonists are responsible for transducing information across the cell membrane, ultimately resulting in a biological response, while antagonists appear to act through passive occupancy of receptors. Implicit in this concept is the idea that these fundamental differences between agonists and antagonists arise from the sequelae induced by agonist-specific interactions with receptors.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Epinephrine; Guanosine Triphosphate; Humans; Isoproterenol; Magnesium; Models, Biological; Pindolol; Radioligand Assay; Receptors, Adrenergic, beta; Sulfhydryl Reagents; Thermodynamics

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.

Topics: Adrenergic beta-Antagonists; Binding, Competitive; Cells, Cultured; Cyclic AMP; Guanine Nucleotides; Guanosine Triphosphate; Iodocyanopindolol; Kinetics; Muscles; Pindolol; Radioligand Assay