guanosine-triphosphate and cyanopindolol

Beta-adrenergic agonists are widely used for preterm labor treatment, but their effectiveness may be limited by desensitization. We thus investigated the effects of a beta-agonist, isoproterenol, on the myometrial beta-adrenergic receptor (beta-AR)/adenylyl cyclase pathway after administration in vivo to late-pregnant rats (8 mg/kg, twice-daily injections). One hour after the first injection, isoproterenol-stimulated adenylyl cyclase activity was reduced by 37%. This was associated with a rapid and transient uncoupling of the beta2-ARs (53% reduction of high-affinity receptors). After prolonged isoproterenol treatment (76 h), adenylyl cyclase activity was desensitized not only to isoproterenol but also to guanosine triphosphate and forskolin. Such treatment induced 1) a selective decrease of beta2-ARs as assessed by 125I-cyanopindolol binding, which was reversed by 5'-guanylylimidodiphosphate and thus probably did not involve irreversible loss of receptors, and 2) a rapid alteration of their transcript levels. Prolonged isoproterenol treatment also led to myometrial Gi2alpha and Gi3alpha increase (44% and 70%) as assessed by Western blotting. Furthermore, pertussis toxin pretreatment of membranes abolished the decrease in isoproterenol-stimulated adenylyl cyclase activity. Thus, we demonstrated that myometrial adenylyl cyclase desensitization after beta-agonist treatment results mainly from beta2-AR uncoupling and increase in Gi activity.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Colforsin; Drug Tolerance; Female; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Myometrium; Pindolol; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; RNA, Messenger

The coupling efficiencies of beta-1 and beta-2-adrenergic receptors (ARs) in rat C6 glioma cells and the effect of changes in subtype density and ratio caused by dexamethasone (DEX) treatment were studied. Radioligand binding studies in membranes suggested that beta-2-ARs showed slightly larger GTP-induced decreases in agonist affinity than beta-1-ARs, which suggests more ternary complex formation with Gs. Treatment with DEX increased the proportion of beta-2-ARs from 20% to 60% without affecting GTP-induced decreases in agonist affinity. Coupling efficiency was determined directly by progressive inactivation of beta-ARs with the irreversible alkylating agent pindobind, which caused similar reductions in beta-1 and beta-2-AR binding sites. Studies on cyclic AMP accumulation showed that DEX-induced decreases in beta-1/beta-2 ratio reduced the receptor reserve for the beta-1-selective agonist norepinephrine but increased the maximal response to the beta-2-selective agonist zinterol without creating a receptor reserve. DEX treatment did not alter occupancy-response curves or maximal responses to the nonselective agonists isoproterenol and epinephrine. It was concluded that changing beta-2-AR density alters the maximal response to beta-2-AR activation; changing beta-1-AR density alters the apparent beta-1-AR reserve. Both subtypes contribute to the responses to nonselective agonists in a nonadditive manner. Beta-1- and beta-2-ARs appear to couple with different efficiencies (beta-1 > beta-2) to cyclic AMP accumulation in C6 glioma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cyclohexane Monoterpenes; Dexamethasone; Glioma; Guanosine Triphosphate; In Vitro Techniques; Pindolol; Rats; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Tumor Cells, Cultured

We measured ligand binding to the beta-adrenergic receptor from porcine adipocytes using tritiated radioligands, dihydroalprenolol (DHA) and CGP-12177 (CGP), and an iodinated radioligand, cyanopindolol (ICP). Binding was measured in a crude plasma membrane preparation. Equilibrium saturation binding was regular for all three ligands; the Kd were approximately 4,000 pM for DHA, 600 pM for CGP, and 100 pM for ICP. Binding was stereospecific with each radioligand. Association of each radioligand was relatively rapid; dissociation was rapid and complete for DHA, initially rapid but ultimately incomplete for CGP, and minimal for ICP. The Kd estimated from kinetic data were approximately 1,000 pM for DHA and 100 pM for CGP. The receptor did not bind phentolamine, an alpha-adrenergic antagonist, except at concentrations greater than 10(-5) M. Propranolol was bound to the receptor with a Ki of approximately 8 nM regardless of the radioligand used. Metoprolol, a purported beta 1-adrenergic specific antagonist, was bound to the receptor with a Ki of approximately 300 nM when the radioligands were CGP or ICP but with a Ki of approximately 1,000 nM when the radioligand was DHA. The Ki for ICI 118,551, a purported beta 2-adrenergic specific antagonist, were approximately 500 nM when the radioligands were DHA or CGP but 125 nM when the radioligand was ICP. Thus, the choice of radioligand can influence the characterization of the beta-adrenergic receptor being studied.

Topics: Adipose Tissue; Adrenergic beta-Antagonists; Animals; Binding, Competitive; Dihydroalprenolol; Guanosine Triphosphate; Kinetics; Ligands; Molecular Conformation; Pindolol; Propanolamines; Receptors, Adrenergic, beta; Swine; Temperature; Time Factors

Several manipulations that affect G protein/receptor coupling also alter the binding of [125I]iodocyanopindolol ([125I]ICYP) and [corrected] +/- cyanopindolol (+/- CYP) to rat brain 5-HT1B binding sites in radioligand binding assays. Inclusion of 5 mM MgSO4 in these assays results in a small but significant increase in the affinity of [125I]ICYP (from KD = 0.046 nM to KD = 0.037 nM). In contrast, 100 microM Gpp(NH)p, GTP, or GDP reduce [125I]ICYP affinity (KD = 0.056 nM with GTP) while ATP and GMP are less effective. +/- CYP affinity for 5-HT1B sites labeled by [3H]dihydroergotamine [( 3H]DE) also displays a small but significant reduction (from Ki = 1.4 nM to Ki = 3.5nM) by the inclusion of 100 microM GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT1B specific binding of [125I]ICYP. The NEM induced reduction in [125I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [125I]ICYP to 5-HT1B sites is dependent on G proteins. The effects of Mg2+ ion, guanine nucleotides, NEM and G protein reconstitution on [125I]ICYP and +/- CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT1B sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Brain; Cell Membrane; Dihydroergotamine; Ethylmaleimide; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Iodocyanopindolol; Magnesium Sulfate; Pindolol; Rats; Receptors, Serotonin

The goal of this study was to determine the mechanism of beta-adrenergic receptor desensitization after chronic elevation of circulating NE levels. Osmotic minipumps containing either NE or saline were implanted subcutaneously in dogs for 3-4 wk. Physiologic desensitization to isoproterenol was confirmed in conscious dogs, i.e., left ventricular dP/dt increased in response to isoproterenol (0.4 micrograms/kg per min) by 5,625 +/- 731 mmHg/s in control dogs with saline pumps, and significantly less, P less than 0.01, by 2,093 +/- 263 mmHg/s in dogs with NE pumps. Myocardial beta-adrenergic receptor density as determined with 125I-cyanopindolol binding was 49% higher (p less than 0.05) in the NE pump group. However, beta-adrenergic receptor agonist binding with isoproterenol demonstrated a significant shift into the low affinity state for the animals with NE pumps. Basal, GTP plus isoproterenol, 5'-guanylylimidodiphosphate, sodium fluoride, and forskolin-stimulated adenylate cyclase activity in the NE pump group were significantly depressed (P less than 0.05) by amounts ranging from 20 to 40%. The functional activity of the guanine nucleotide binding protein Gs was also reduced (P less than 0.05) in animals with NE pumps. Thus, the process of desensitization in response to chronic elevation of NE levels in intact, normal dogs does not involve a decrease in beta-adrenergic receptor density. Rather, it is characterized by reduced adenylate cyclase activation and uncoupling of the beta-adrenergic receptor in association with decreased activity of the GTP-coupling protein Gs.

Topics: Adenylyl Cyclases; Animals; Colforsin; Dogs; Drug Tolerance; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Heart Ventricles; Hemodynamics; Isoproterenol; Norepinephrine; Organ Size; Pindolol; Receptors, Adrenergic, beta; Sodium Fluoride; Sodium-Potassium-Exchanging ATPase; Ventricular Function

Intrauterine 17 beta-estradiol pellets can induce an up-regulation of guinea pig myometrial beta-adrenergic receptor density and l-isoproterenol-dependent adenylate cyclase activity. Does 17 beta-estradiol influence the ability of beta-adrenergic receptors to form a "high affinity" state with l-isoproterenol, which is a necessary step for adenylate cyclase activation? Nonpregnant, oophorectomized guinea pigs received intrauterine pellets of either placebo, 17 beta-estradiol, progesterone, or 17 beta-estradiol plus progesterone for 1 week. 17 beta-Estradiol resulted in pharmacologic, whereas progesterone resulted in physiologic plasma 17 beta-estradiol and progesterone concentrations, respectively. The affinity of myometrial beta-adrenergic receptors for l-isoproterenol was measured by percentage of inhibition of -[125I]cyanopindolol binding. In all groups, the competition curves in the presence of magnesium chloride could be resolved into two affinity states of the beta-adrenergic receptor, "high" and "low," respectively. The ratio of their dissociation constants was not influenced by hormonal treatment. However, the relative concentration of beta-adrenergic receptors in the high affinity state was significantly higher in the 17 beta-estradiol-treated group than that in the control group. This correlates with the up-regulation in myometrial adenylate cyclase activity and suggests that myometrial beta-adrenergic receptor-adenylate cyclase function may be modulated by 17 beta-estradiol.

Topics: Adrenergic beta-Antagonists; Animals; Estradiol; Female; Guanosine Triphosphate; Guinea Pigs; Isoproterenol; Myometrium; Ovariectomy; Pindolol; Progesterone; Receptors, Adrenergic, beta

The ability of myometrial beta-adrenergic receptors to form a "'high-affinity state" with beta-adrenergic receptor agonists might be greater in pregnant guinea pigs at greater than or equal to 0.9 of gestation than in nonpregnant animals. To determine whether this difference is due to pregnancy in general or is associated only with late pregnancy and to determine whether it persists in the postpartum period, we studied the interaction of l-isoproterenol with beta-adrenergic receptors in myometrial membranes obtained from nonpregnant (nulligravid) animals, pregnant (primigravid) animals at 0.3, 0.7, and 0.9 to 1.0 of gestation (term 65 days), and postpartum guinea pigs (2 to 3 days). The affinity of myometrial beta-adrenergic receptors for l-isoproterenol was measured by percent inhibition of -[125I]cyanopindolol binding. In the presence of magnesium chloride, the competition curves could be resolved into two affinity state of the beta-adrenergic receptor, "high" and "low," respectively, in all groups. The ratio of the dissociation constant of the "low"-affinity state to that of the "high"-affinity state was significantly higher in pregnant guinea pigs at greater than or equal to 0.9 of gestation than in nonpregnant or postpartum animals and in pregnant animals of earlier gestations. In the presence of guanosine triphosphate only one (low-affinity) state of the receptor was detectable. Thus it is only in pregnant guinea pigs at greater than or equal to 0.9 of gestation that the ability of myometrial beta-adrenergic receptors to form a high-affinity state is enhanced.

Topics: Adrenergic beta-Antagonists; Animals; Female; Guanosine Triphosphate; Guinea Pigs; Isoproterenol; Myometrium; Pindolol; Pregnancy; Pregnancy, Animal; Receptors, Adrenergic, beta

The effects of guanosine triphosphate (GTP) and magnesium on the interaction of 1-isoproterenol with beta-adrenoreceptors were studied in myometrial membranes from nonpregnant and late-pregnant (0.9 gestation, term 65 days) guinea pigs. The affinity of the beta-adrenoreceptor for 1-isoproterenol, as measured by inhibition of (-)125I-cyanopindolol binding, was increased by 10 mM MgCl2. The addition of 250 microM GTP reversed this process. In the presence of MgCl2, the competition curves could be resolved into two affinity states of the beta-adrenoreceptor, high and low, respectively. The ratio of the dissociation constant of the high-affinity state to that of the low-affinity state was significantly higher in late-pregnant than in the nonpregnant animals. In the presence of GTP, there was only one (low-affinity) state of the receptor detectable. In both groups of animals, the interaction between beta-adrenoreceptor agonists and myometrial beta-adrenoreceptors was positively modulated by MgCl2. This process was reversed by GTP. However, there appeared to be a differential regulation of the ability of the myometrial beta-adrenoreceptor to form a high-affinity state, depending on the reproductive state of the animal.

Topics: Animals; Female; Guanosine Triphosphate; Guinea Pigs; Iodine Radioisotopes; Isoproterenol; Magnesium; Myometrium; Pindolol; Pregnancy; Propranolol; Radioimmunoassay; Receptors, Adrenergic, beta