guanosine-triphosphate and iodohydroxybenzylpindolol

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Chemical Phenomena; Chemistry; Erythrocyte Membrane; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hormones; In Vitro Techniques; Isoproterenol; Kinetics; Liposomes; Models, Biological; Phospholipids; Pindolol; Propranolol; Rats; Receptors, Adrenergic, beta; Synaptic Transmission; Thermodynamics; Thionucleotides

Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Albuterol; Animals; Binding, Competitive; Cell Line; Cell Membrane; Guanosine Triphosphate; Isoproterenol; Lymphoma; Mice; Pindolol; Receptors, Adrenergic, beta; Stereoisomerism; Structure-Activity Relationship

To determine whether the beta-blocking drug propranolol had any physiologic effect on normal (n = 14) and adenomatous (n = 15) human thyroid tissues, experiments were performed to study the binding of the beta-blockers 125I-iodocyanopindolol (125I-ICYP) and 125I-iodohydroxybenzylpindolol (125I-IHYP) and the stimulation of adenyl cyclase (AC) by isoproterenol. 125I-ICYP and 125I-IHYP failed to show high-affinity binding in 27 of 29 specimens, whereas two (one normal and one adenomatous) thyroid tissues demonstrated high-affinity binding (Kd 5.5 +/- 1 X 10(-9) M) for 125I-ICYP. Thyroid-stimulating hormone (0.3 IU/ml), guanosine triphosphate (10(-4) M), and Gpp (NH)p(10(-4) M) stimulated AC in all thyroid tissues, although in two tissues (normal) Gpp (NH)p failed to cause a significant increase. Isoproterenol (10(-4) M), in contrast, had no effect on basal AC activity or on guanosine triphosphate, and Gpp (NH) p stimulated AC activity in 26 of the 29 thyroid tissues. In one of the two tissues that increased AC in response to isoproterenol, the beta-blocking drugs propranolol hydrochloride, bunitrolol hydrochloride, and tolilprolol hydrochloride decreased AC stimulation to isoproterenol at concentrations of 10(-6) M (p less than 0.05). Higher concentrations of propranolol (10(-4) - 10(-2) M) decreased AC stimulation to thyroid-stimulating hormone (p less than 0.01), not only in this responsive tissue but also in tissues that failed to demonstrate high-affinity binding for 125I-ICYP and AC stimulation to isoproterenol (p less than 0.01). Thus most normal and adenomatous human thyroid tissues lack beta-receptors and a functioning beta-receptor AC system. High concentrations of propranolol in vitro decreased AC response by thyroid-stimulating hormone, but this is probably a nonreceptor-mediated effect.

Topics: Adenoma; Adenylyl Cyclases; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; In Vitro Techniques; Iodocyanopindolol; Isoproterenol; Pindolol; Propanolamines; Propranolol; Receptors, Adrenergic, beta; Thyroid Gland; Thyroid Neoplasms; Thyrotropin

After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.

Topics: Adenylyl Cyclases; Animals; Bone Marrow; Cell Differentiation; Dose-Response Relationship, Drug; Erythroblasts; Erythrocytes; Guanosine Triphosphate; Guanylyl Imidodiphosphate; In Vitro Techniques; Isoproterenol; Kinetics; Male; Pindolol; Rabbits

The interactions of agonists and antagonists with beta-adrenergic receptors appear to be fundamentally different. Antagonists only occupy the receptor while agonists bind to the receptor and induce a specific conformational change. The excellent correlation between the efficacy of full or partial agonists and the magnitude of the changes in the thermodynamic parameters associated with the binding of these compounds suggests that this conformational change is related to the ability of various drugs to stimulate adenylate cyclase. In most instances it is difficult to distinguish an agonist from an antagonist using the in vitro binding of radiolabeled ligands. The thermodynamic analysis described here represents one example of a situation in which agonists and antagonists can be distinguished using in vitro binding techniques. The complexities of agonist interactions with beta-adrenergic receptors are also clearly seen in studies of binding to intact cells and in direct studies using radiolabeled agonists. In the former studies it appeared that there were marked changes in the properties of the receptor that occurred during the 60 min that are required for the binding of IHYP to beta-adrenergic receptors on L6 myoblasts to reach equilibrium. These changes make it difficult to analyze the affinity of the receptor for agonists using intact cells. Finally, the complexities of agonist interactions with the beta-adrenergic receptor are also seen in direct binding studies with radiolabeled agonists. The curvilinear Scatchard plots and the complicated dissociation kinetics attest to this complexity. The importance of the formation of a hypothetical ternary complex between hormone, receptor, and guanine nucleotide-binding regulatory protein is now becoming appreciated. The existence of other components of this system will remain an important subject for future investigation. In any case, it is clear that a full understanding of this system will require the use of not only radiolabeled agonists and antagonists but also of both broken and intact cells.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Cell Membrane; Epinephrine; Erythrocyte Membrane; Guanosine Triphosphate; Kinetics; Lung; Myocardium; Pindolol; Propranolol; Rats; Receptors, Adrenergic; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Temperature; Thermodynamics; Turkeys

The effects of a nonselective (isoproterenol), a beta-1 selective (prenalterol) and a beta-2-selective (procaterol) agonist on beta adrenoceptor occupancy, adenylate cyclase activity and muscle contractions in the myocardium (beta-1) and soleus muscle (beta-2) of the reserpine-pretreated cat were investigated. Each compound fully inhibited the specific binding of [125I] iodohydroxybenzylpindolol in both tissues. Myocardial and soleus muscle adenylate cyclase were equipotently activated by isoproterenol. Prenalterol and procaterol induced no more than marginal elevations in myocardial enzyme activity and only the latter compound produced an increase in soleus muscle adenylate cyclase activity (77% of that produced by isoproterenol). Prenalterol increased myocardial contractility (82% of that of isoproterenol) in a monophasic concentration-dependent manner, as did isoproterenol, whereas procaterol induced a biphasic inotropic response. The high affinity effect (23% of isoproterenol) of procaterol was selectively blocked by IPS 339 (beta-2-selective) and the low affinity component (70% of isoproterenol) was blocked by pamatolol (beta-1-selective). Isoproterenol and procaterol decreased subtetanic soleus muscle contractions equally, whereas prenalterol was devoid of effect in skeletal muscle. From the interrelations between the concentration-effect curves for these drug-induced responses, it was estimated that under the conditions used in these assays a full agonist in the heart has a spare beta-1 adrenoceptor pool of 80 to 90%. A corresponding beta-2 adrenoceptor reserve of 30 to 50% was derived from data obtained in the soleus muscle. On the basis of the present data, prenalterol may be characterized as a nonselective beta adrenoceptor ligand with beta-1 adrenoceptor partial agonistic activity.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Binding, Competitive; Cats; Female; Guanosine Triphosphate; Heart; In Vitro Techniques; Male; Muscle Contraction; Muscles; Myocardial Contraction; Myocardium; Organ Specificity; Pindolol; Receptors, Adrenergic; Receptors, Adrenergic, beta

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Adipose Tissue; Animals; Cell Differentiation; Cell Membrane; Cells, Cultured; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoproterenol; Mice; Pindolol; Receptors, Adrenergic; Receptors, Adrenergic, beta; Receptors, Cell Surface

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

Digitonin-solubilized turkey erythrocyte beta-adrenergic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack beta receptors. Incorporation of turkey beta receptors into acceptor membranes was directly proportional to the quantity of soluble protein added to the reconstitution system. Reconstituted beta receptors demonstrate saturable [125I]iodohydroxybenzylpindolol binding (Bmax = 11.1 +/- 0.8 fmol/mg, K = 77.8 +/- 8.6 pM) and stereospecificity ((-)-propranolol, K = 11.0 nM; (+)-propranolol, K = 2000 nM; (-)-isoproterenol, K = 250 nM; (+)-isoproterenol, K = 82 micro M). Reconstituted beta receptors appear to be incorporated into acceptor membranes as integral proteins. Reconstituted beta receptors cannot be extracted by high salt or pH (3 to 11); detergent is required for resolubilization of reconstituted beta receptors. Adenylate cyclase stimulation was not obtained in reconstituted membranes since acceptor membranes lack a catalytic subunit. However, guanine nucleotide regulation of agonist affinity was observed indicating a functional reconstitution. GTP (100 micro M) produces a 5-fold decrease in the affinity of isoproterenol for reconstituted beta receptors. Experiments with sulfhydryl reagents indicate that the reconstituted beta receptor couples with the guanine nucleotide regulatory protein of the acceptor membranes. These data describe the successful reconstitution of a beta receptor and indicate that the reconstituted beta receptor can interact with the GTP binding protein of human erythrocyte acceptor membranes.

Topics: Animals; Erythrocyte Membrane; Erythrocytes; Guanosine Triphosphate; Humans; Isomerism; Isoproterenol; Kinetics; Pindolol; Propranolol; Receptors, Adrenergic; Receptors, Adrenergic, beta; Species Specificity; Turkeys

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.

Topics: Animals; Cell Membrane; Cells, Cultured; Guanosine Triphosphate; Isoproterenol; Kinetics; Muscles; Pindolol; Propranolol; Receptors, Adrenergic; Receptors, Adrenergic, beta