Stereospecificity of beta adrenergic antagonists: R-enantiomers show increased selectivity for beta-2 receptors in ciliary process

The (+)-stereoisomers of arylethanolamine beta adrenergic agonists and antagonists are usually much less active in biological systems than their corresponding (-)-forms. In the eye, however, prior physiological studies have shown that these (+)-stereoisomers are unexpectedly potent in altering intraocular pressure, results which could be due to a difference in distribution and metabolism or to a difference in receptor interaction. The present experiments evaluated six stereoisomeric pairs of beta adrenergic antagonists for their ability to block rabbit ciliary process and cardiac beta adrenergic receptors activating adenylate cyclase, in vitro, under conditions in which the effects of drug metabolism, distribution and membrane lipid solubility were minimized. In the heart, all six pairs of antagonists demonstrated the expected increased potency of (-)-forms, with isomeric activity ratios of: 33 for metoprolol, 44 for timolol; 48 for bunitrolol; 76 for t-butyl-betaxolol; 100 for t-butyl-didesmethyl-ICI-118,551; and 530 for betaxolol. Under identical assay conditions in the ciliary process, (+)-enantiomers were much more potent relative to (-)-forms, with isomeric activity ratios of: 0.82 for timolol; 3.3 for bunitrolol; 7.4 for t-butyl-didesmethyl-ICI-118,551; 10 for metoprolol; 16 for t-butyl-betaxolol; and 190 for betaxolol. With the exception of metoprolol, all (+)-enantiomers demonstrated a substantially higher absolute affinity for ciliary process receptors (known to be almost exclusively of the beta-2 subtype) than for cardiac receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of the interactions of agonists and antagonists with beta adrenergic receptors

The affinity of the beta adrenergic receptor for antagonists is frequently higher than that for agonists. It has been assumed that the binding of agonists and antagonists is diffusion limited and that the high affinity of the receptor for typical antagonists is due to slow rates of dissociation. To test this hypothesis, the kinetics of binding of unlabeled agonists and antagonists were determined using the method described by Motulsky and Mahan (Mol. Pharmacol. 25: 1-9, 1984). The time course of the binding of a radioligand in the presence of a competing unlabeled ligand was analyzed in terms of rate constants of association (kon) and dissociation (koff) for binding of the radioligand and the competitor. This approach was validated by showing that the rate constants for binding of [3H]dihydroalprenolol and [3H]CGP-12177 [[3H]-4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on ] determined directly were similar to values determined when the binding of [125I]iodopindolol was measured in the presence of [3H]dihydroalprenolol or [3H]CGP-12177. Computer simulations suggested that this method was experimentally limited to competing ligands with rate constants of dissociation below approximately 0.50 min-1. The apparent rate constants for binding of four unlabeled agonists and eight antagonists were determined experimentally at 10 degrees C. Although the values of koff for agonists and antagonists were similar, the values for kon for binding of agonists were consistently lower than the values for binding of antagonists. The relatively slow rate constant for association of agonists may be explained by a two-step mechanism or may involve agonist-induced isomerization of the receptor.     

Interaction of beta adrenergic agonists and antagonists with brain beta adrenergic receptors in vivo

There is interest in knowing whether beta adrenergic antagonists or agonists, when administered systemically, can enter the brain to interact with central beta adrenergic receptors. To study this, the reduction in the radioactive content in the brain of rats after administration of (-)-[125I]iodopindolol (IPIN) by systemically administered beta agonists or antagonists was measured. Previous studies show that after the i.v. administration of IPIN the binding in vivo to various areas of the central nervous system has the characteristics expected of binding to beta adrenergic receptors. Of the antagonists tested, pindolol and butylpindolol showed potent interactions with beta receptors in both cortex and cerebellum whereas atenolol and practolol did not interact at doses up to 30 mg/kg. CGP-12177 showed moderate potency in inhibiting IPIN binding in vivo. We have shown previously that propranolol and alprenolol inhibit IPIN binding with high potency in cortex and cerebellum. At high doses, butoxamine, a beta-2 antagonist, reduced the binding of IPIN in the cerebellum but not in the cortex. Of the agonists tested, clenbuterol and prenalterol caused a significant dose-dependent reduction of the binding of IPIN, with clenbuterol being more potent. Isoproterenol, salbutamol, salmefamol and dobutamine had no effect. With the exception of CGP-12177, the affinity of the drugs for central beta adrenergic receptors measured in vitro was correlated significantly with their ability to inhibit IPIN binding in vivo whereas their degree of lipophilicity was not correlated significantly with potency in vivo. The inhibition of IPIN binding in vivo from brain areas can be used to evaluate whether drugs penetrate into brain and interact with central beta adrenergic receptors.     

Evidence for two subtypes of alpha adrenergic receptors in canine airway smooth muscle

The contractile response of canine tracheal muscle to i.a. phenylephrine, clonidine and norepinephrine was studied isometrically in situ in 32 dogs after beta adrenergic and ganglionic blockade. Intra-arterial phenylephrine caused dose-related tracheal contraction which was not altered by yohimbine (5 microgram/kg i.a.). Prazosin (4 mg/kg i.v.) caused a 77 +/- 3% decrease in tracheal response to i.a. phenylephrine. Clonidine also caused dose-related tracheal contraction, which was not altered by prazosin (4 mg/kg i.v.) but was 95 +/- 2% blocked by 5 microgram/kg i.a. of yohimbine. Norepinephrine caused tracheal muscle contraction which was greater than both phenylephrine (P less than .05) and yohimbine (P less than .001). Prazosin (4 mg/kg i.v.) caused 53 +/- 6% blockade and yohimbine (5 microgram/kg i.a.) caused 76 +/- 2% blockade of the response to i.a. norepinephrine; prazosin plus yohimbine caused greater than 98% blockade of the response to i.a. norepinephrine. The dose-response curve to i.a. acetylcholine was not altered by treatment with prazosin (4 mg/kg i.v.) plus yohimbine (5 microgram/kg i.a.). These results demonstrate that tracheal contraction induced by sympathomimetic amines is mediated by two subtypes of alpha adrenergic receptors on tracheal muscle, alpha-1 for phenylephrine, alpha-2 for clonidine and both alpha-1 and alpha-2 for norepinephrine.     

Desensitization of beta adrenergic receptors linked to adrenocorticotropin secretion

Stimulation of beta adrenergic receptors on AtT-20 cells increases intracellular cyclic AMP levels and adrenocorticotropin hormone (ACTH) release. Pretreatment of these cells with catecholamines reduces the ability of (-)-isoproterenol to stimulate both cyclic AMP formation and ACTH secretion. This beta receptor desensitization is time- and dose-dependent and is reversible. Various beta adrenergic agonists can induce this desensitization with a rank order of potency of salmefamol greater than or equal to (-)-isoproterenol greater than or equal to epinephrine greater than or equal to norepinephrine greater than or equal to (+)-isoproterenol. (+/-)-Propranolol but not practolol can block the (-)-isoproterenol-induced beta receptor desensitization. Long-term treatment of AtT-20 cells with (-)-isoproterenol reduces the density of beta receptors but does not affect the affinity of these sites for [3H]dihydroalprenolol. In addition to desensitizing beta receptors, (-)-isoproterenol pretreatment enhances basal ACTH secretion. This effect was dose-dependent and blocked by (+/-)-propranolol. Forskolin-stimulated cyclic AMP formation and ACTH secretion was not altered by (-)-isoproterenol treatment indicating that the desensitization of beta receptors on AtT-20 cells is the result of receptor-adenylate cyclase uncoupling. No cross-desensitization of corticotropin releasing factor or vasoactive intestinal peptide receptors occurred as (-)-isoproterenol treatment did not alter the effect of these peptides on cyclic AMP synthesis or ACTH secretion.     

Genetic influence on the regulation of beta adrenergic receptors in mice

The regulation of beta adrenergic receptors was investigated in inbred mouse strains in which previous studies revealed differences in the regulation of dopamine receptors. The density of beta adrenergic receptors in the cerebral cortex of BALB/J mice was about one-third of that in CBA/J and C57BL/6J mice. Strain differences in the binding of [125I]iodohydroxypindolol to beta adrenergic receptors were due to changes in the density of beta-1 adrenergic receptors. Chronic administration of propranolol did not result in an increase in the density of beta adrenergic receptors receptors in cortices of C57BL/6J and BALB/cJ mice were observed. In contrast, pretreatment with 6-hydroxydopamine resulted in increases in the density of beta adrenergic receptors in the cerebral cortex of all three strains. Analysis of the effects of these treatments on the subtypes of beta adrenergic receptors revealed that the changes were restricted to changes in the density of beta-1 receptors. The failure to observe a response to propranolol in CBA/J mice expands the extent of deficits reported previously in this strain for striatal dopamine receptor supersensitivity after chronic treatment with haloperidol (Severson et al., Brain Res. 210: 201-215, 1981). CBA/J mice may be a useful model for genetic analysis of mechanisms for the control of receptor sensitivity and to investigate the impairments of the regulation of catecholaminergic receptors observed in aged rodents.     

Agonist interactions with beta adrenergic receptors in rat brain

Agonist interactions with beta adrenergic receptors on membranes prepared from rat brain were examined by measuring agonist inhibition of [125I]iodopindolol binding in the absence or presence of GTP. When rat cerebral cortical membranes were prepared with 1 mM EDTA in the homogenization medium and 2.5 mM MgCl2 was included in the binding reaction, then 250 microM GTP increased the Hill coefficient for isoproterenol from 0.77 to 0.99 and increased the IC50 from 88 to 213 nM. By contrast, I-propranolol competition curves were steep (Hill coefficient = 0.98) and were not affected by GTP. It was inferred from the results of computer-modeling that, in the absence of GTP, isoproterenol bound to two states of the receptor; GTP converted isoproterenol binding to a single low-affinity state. I-Propranolol bound to a single state in the absence or presence of GTP. The effect of GTP on I-epinephrine inhibition of [125I]iodopindolol binding was essentially identical to its effect on isoproterenol inhibition. GTP and GDP were the most potent of all the nucleotides tested. Guanylylimidodiphosphate (1 mM) produced only partial shifts in the isoproterenol competition curves and GMP and ATP were inactive. In membranes prepared from rat hippocampus and hypothalamus, isoproterenol competition curves and GTP effects were qualitatively similar to those observed in cerebral cortex. However, GTP produced only partial shifts of I-isoproterenol competition curves in cerebellum and neostratium. It appears that agonists, but not antagonists, can stabilize a high-affinity ternary complex with the beta adrenergic receptor and the guanine nucleotide binding regulatory protein in membranes prepared from various regions of the rat brain.     

Binding and functional characteristics of beta adrenergic receptors in the intact neutrophil

Beta adrenergic receptors have been previously characterized in human neutrophil sonicates. In the present study the intact neutrophil has been assessed for the number and affinity of beta adrenergic binding sites by using the antagonist DNA. Agonist and antagonist potencies, characterized by their effect on DHA binding and cyclic AMP accumulation, are compared with agonist inhibition of lysosomal enzyme (beta glucuronidase) release. Criteria for beta adrenergic receptor identification were successfully demonstrated. At 30 degrees C, beta adrenergic binding was rapid (t 1/2 2 min) and reversible (t 1/2 9 min). Receptor binding was saturable, revealing approximately 900 high-affinity receptors per neutrophil with DHA concentrations of 0.1 to 10 nM. By utilizing both equilibrium and kinetic techniques, the KD was determined to be approximately 0.6 nM. Agonists and antagonists competed for DHA binding in a manner consistent with their effect on cyclic AMP generation. Rank order potency was suggestive of a beta-2 receptor: isoproterenol greater than epinephrine greater than norepinephrine. Stereoselectivity was shown by the greater potency of L-propranolol compared to the D isomer. A high degree of receptor-adenylate cyclase coupling efficiency was suggested by the observation that with only 1% receptor occupancy isoproterenol stimulated 50% maximal cyclic AMP generation. Finally, there was an excellent correlation between the isoproterenol concentration which resulted in 50% of maximal inhibition of beta glucuronidase release (Ki) and that causing 50% maximal cyclic AMP stimulation (Kact), suggestive of a close relationship between beta adrenergic-induced adenylate cyclase activation and beta adrenergic regulation of neutrophil lysosomal enzyme release. The data presented suggest that the use of the intact neutrophil for study of the beta adrenergic receptor is feasible and may provide information which is considerably more closely related to modulation of physiological function by neurohormones than is possible with disrupted cell preparations.     

Psychopharmacological consequences of activation of beta adrenergic receptors by SOM-1122

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.     

Relative importance of alpha and beta adrenergic receptors during resuscitation.

Successful resuscitation from cardiac arrest in the asphyxiated dog model has been ascribed to the use of artificial ventilation, closed chest cardiac massage, and administration of a vasopressor. Controversy remains over whether the most commonly employed vasopressor, epinephrine, exerts its effects primarily by elevating diastolic pressure and reestablishing coronary flow, or by exciting cardiac pacemaker cells and enhancing myocardial contractility. To observe pure alpha and beta adrenergic receptor influences during resuscitation, three groups (alpha-blocked, beta-blocked, unblocked) of dogs were studied. beta-blocked dogs resuscitated with phenylephrine and unblocked dogs resuscitated with epinephrine experienced 100% successful resumption of spontaneous circulation after 5 min of asphyxia-induced arrest. Only 27% of alpha-blocked animals resuscitated with isoproterenol were successfully revived. The appearance of the ECG during cardiac arrest and resuscitation could in no way be used to predict the outcome of resuscitation attempts. Results suggest that, initially, alpha receptor stimulation with concomitant diastolic pressure elevation is more important to the success of resuscitation than beta receptor stimulation.     

The beta adrenergic receptors of chromatophores of the frog, Rana pipiens.

The isolated skin of Rana pipiens was found to be a suitable model for the quantitative study of chromatophore beta adrenergic receptors uninfluenced by prejunctional phenomena. Cumulative concentration-response curves for adrenergic agonists were obtained in preparations in which effective alpha adrenergic blockade had been produced with phenoxybenzamine. The beta adrenergic agonists darkened the preparation, as did melanocyte-stimulating hormone, but the maximum effects differed. The maximum of the l-isoproterenol cumulative concentration-response curve was approximately 50% less than that of melanocyte-stimulating hormone, while the maxima for l-epinephrine and l-norepinephrine were significantly less than that for isoproterenol. Microscopic examination revealed a qualitative difference: while maximal darkening produced by melanocyte-stimulating hormone was associated with maximal changes in both interspot melanophores and iridophores, maximal adrenergic-induced darkening was associated with maximal iridophore granule concentration only. No qualitative differences could be observed in the darkening caused by the three adrenergic agonists. The beta adrenergic potencies of l-norepinephrine and l-isoproterenol relative to l-epinephrine were determined by four-point bioassay. Isoproterenol was found to be 138 times as potent as epinephrine, while norepinephrine was 4 times as potent. Similarly, antagonism of isoproterenol-induced darkening of phenoxybenzamine-pretreated skin samples by the beta adrenergic blocking agents dl-propranolol, dl-sotalol, dl-practolol, l-butoxamine and d-butoxamine was studied, and their KB and pA2 values, respectively, were found to be: dl-propranolol (1.44 X 10(-8)M, 7.81); dl-sotalol (7.25 X 10(-8)M, 7.23); l-butoxamine (6.92 X 10(-6)M, 5.10); dl-practolol (1.91 X 10(-5)M, 4.96); d-butoxamine (no activity). Comparison of the potency ratios and pA2 values cited above with similar parameters obtained by other investigators in several mammalian tissues suggests that there is wide variation among beta adrenergic receptors.     

Beta adrenergic receptor subtypes in the atria: evidence for close coupling of beta-1 and beta-2 adrenergic receptors to adenylate cyclase

The relationship between occupancy of beta adrenergic receptors and stimulation of adenylate cyclase in dog atrial tissue was examined by studying the binding of [125I]iodopindolol and the activation of adenylate cyclase. Computer-assisted nonlinear regression analysis was used to analyze the inhibition of isoproterenol-stimulated adenylate cyclase activity by beta-1- or beta-2-selective antagonists. The Ki values for each subtype of receptor for the selective antagonists resulting from studies of the inhibition of adenylate cyclase activity were similar to those determined in studies of the inhibition of the binding of [125I]iodopindolol. To compare further the occupancy of beta-1 or beta-2 adrenergic receptors with the activation of adenylate cyclase mediated by each class of receptor, computer modeling of the stimulation of adenylate cyclase by the beta-1-selective agonist norepinephrine was carried out. The EC50 values of norepinephrine for each receptor subtype, as measured in studies of norepinephrine-stimulated adenylate cyclase activity, were similar to the Ki values for the inhibition by norepinephrine of the binding of [125I]iodopindolol to each receptor subtype. The data led to the conclusion that beta-1 adrenergic receptors make up about 70% of the total number of beta adrenergic receptors and mediate 70% of the increase in adenylate cyclase activity produced by isoproterenol. These results suggest that the relationship between occupancy of each class of receptor and activation of adenylate cyclase is linear and that, when agonist-stimulated adenylate cyclase activity is used as a functional response, neither spare beta-1 nor spare beta-2 adrenergic receptors exist in the atrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. II. Agonist binding to soluble beta adrenergic receptors

The thermodynamic parameters associated with the interactions of agonists and antagonists with digitonin-solubilized beta adrenergic receptors were determined. A rapid method for measuring the binding of [125I]iodopindolol to soluble receptors using glass-fiber filters was developed. The binding of [125I]iodopindolol, an antagonist with intrinsic sympathomimetic activity, to soluble receptors was temperature-sensitive as is the binding of the ligand to membrane-bound receptors. The interactions of propranolol and timolol with soluble receptors were independent of temperature. In contrast, the binding of agonists to soluble receptors was sensitive to temperature, although insensitive to GTP. Thermodynamically, the interactions of the antagonists timolol and propranolol with soluble beta adrenergic receptors were entropy-driven, with little contribution from changes in enthalpy. This is consistent with a hydrophobic interaction between the receptor and the antagonist. The binding of [125I]iodopindolol was enthalpy-driven. The binding of full agonists with soluble receptors was described thermodynamically by changes in enthalpy and entropy that were negative relative to the values for propranolol and timolol, suggesting that the guanine nucleotide-binding protein required for stimulation of adenylate cyclase activity and an intact lipid environment are not involved in the thermodynamics of formation of the low-affinity component of agonist binding. These results are consistent with an agonist-induced change in the conformation of the receptor.     

Effects of pindolol and propranolol on beta adrenergic receptors on human lymphocytes

The cardiovascular supersensitivity observed after discontinuation of administration of beta adrenergic receptor antagonists may be explained by an increase in the density of beta adrenergic receptors. Thus, a change in the density of receptors has been observed in human lymphocytes after administration of propranolol (Aarons et al., 1980). The effects of pindolol, a beta adrenergic receptor antagonist with intrinsic sympathomimetic activity, were compared with those of propranolol or placebo. Pindolol (10 mg q.i.d.), propranolol (40 mg q.i.d.) or placebo were administered to 12 subjects for 8 days. The density of beta adrenergic receptors was determined by Scatchard analysis of the specific binding of [125I]iodopindolol on membranes prepared from human lymphocytes. Administration of pindolol resulted in a 30 to 50% decrease in the density of beta adrenergic receptors. This decrease was apparent within 1 day of beginning pindolol administration and it persisted for at least 8 days after discontinuation of drug administration. The reversibility of the decrease in receptors observed after pindolol administration was studied in 27 subjects given propranolol, pindolol or placebo for 4 days in a double-blind cross-over trial. Propranolol consistently induced a small increase in the density of beta adrenergic receptors. The density of receptors returned to predrug values within 2 days after discontinuation of propranolol administration. Pindolol induced a 30 to 50% decrease in the density of receptors which, as observed previously, persisted for at least 10 days after discontinuation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)