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.     

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.     

Selective regulation of beta-1 and beta-2 adrenergic receptors by atypical agonists

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)     

Effects of clenbuterol on central beta-1 and beta-2 adrenergic receptors of the rat

Repeated administration of the centrally acting beta adrenoceptor agonist, clenbuterol, to rats reduced the ability of isoproterenol to increase the concentration of cyclic AMP (cAMP) in slices of cerebellum. This reduced responsiveness to isoproterenol was accompanied by a marked reduction in the density of beta adrenoceptors as measured by the binding of the beta adrenoceptor antagonist [125I]iodopindolol. In addition, the agonist-binding properties of remaining cerebellar beta adrenoceptors were altered after clenbuterol treatment. The clenbuterol-induced reduction in the density of beta adrenoceptors in the cerebellum is in marked contrast to its inability to do this in cerebral cortex. Comparison of the ability of clenbuterol to that of isoproterenol to increase levels of cAMP in slices of cerebral cortex or cerebellum showed that clenbuterol is a weakly potent agonist in both brain regions. The increase in cAMP induced by isoproterenol in the cortex was significantly reduced in the presence of the selective beta-1 adrenoceptor antagonist, ICI 89,406. In contrast, the clenbuterol-induced increase in cortical cAMP was unchanged by ICI 89,406 but was reduced significantly by the beta-2 adrenoceptor antagonist, ICI 118,551. In cerebellum, both isoproterenol- and clenbuterol-stimulated accumulation of cAMP were antagonized much more potently by ICI 118,551 than by ICI 89,406. Furthermore, clenbuterol antagonized the cAMP response induced by isoproterenol in the presence of ICI 118,551 in a concentration-dependent manner. In terms of measurement of cAMP in brain slices, clenbuterol is weakly potent as an agonist at beta-2 adrenoceptors and has antagonist properties at beta-1 adrenoceptors.     

Discriminative stimulus properties of clenbuterol: evidence for beta adrenergic involvement

Thirty rats were trained to discriminate the centrally acting beta adrenergic agonist clenbuterol (0.1 mg/kg) from saline using a water-reinforced (fixed-ratio 10 schedule) two-lever operant task. Discrimination acquisition required a mean +/- S.E.M. of 42 +/- 7 training sessions (median of 26 training sessions). The clenbuterol stimulus was dose-dependent (ED50 = 0.03 mg/kg) and stereoselective, and had a rapid onset (5 min) and a duration of approximately 1 hr. The beta adrenergic antagonist propranolol fully antagonized the clenbuterol discriminative stimulus (IC50 = 0.18 mg/kg). Other beta adrenergic agonists such as SOM 1122 (ED50 = 0.01 mg/kg), zinterol (ED50 = 0.03 mg/kg), salbutamol (ED50 = 0.23 mg/kg) and prenalterol (ED50 = 1.91 mg/kg) substituted for clenbuterol. The monoamine uptake inhibitor despiramine (ED50 = 2.25 mg/kg), the psychomotor stimulants amphetamine (ED50 = 0.33 mg/kg) and pentylenetetrazol (ED50 = 0.31 mg/kg), and the dopamine receptor antagonists haloperidol (ED50 = 0.08 mg/kg) and chlorpromazine (ED50 = 2.32 mg/kg) similarly substituted for clenbuterol. However, chlordiazepoxide, pentobarbital, fentanyl, cocaine and fenfluramine produced little or no clenbuterol lever selection up to doses that decreased response rate markedly. The ability of SOM 1122, zinterol, salbutamol, despiramine, amphetamine, pentylenetetrazol and haloperiol to substitute for the clenbuterol stimulus was antagonized by prior treatment with propranolol. Taken together, these results suggest that the discriminative stimulus properties of clenbuterol are mediated, at least in part, through an interaction with beta adrenergic receptors. The same drugs also were assayed for in vitro inhibition of [125I]iodopindolol binding to beta adrenergic receptor preparations of rat cerebral cortex and cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a bromoacetylated derivative of pindolol as a high affinity, irreversible beta adrenergic antagonist in cultured cells

We have utilized cultured cell lines to test the utility of N8-(bromoacetyl)-N1-[3-(4-indolyoxy)-2-hydroxypropyl]-(Z)-1,8-diam ino- p-menthane, BIM, a recently synthesized, irreversible beta adrenergic antagonist. Previously available irreversible antagonists of beta adrenergic receptors have generally exhibited low affinity (typical IC50 values greater than or equal to 1 microM). By contrast, S49 lymphoma cells incubated with 10 nM BIM for 120 min and then washed extensively showed a 70% loss in beta adrenergic receptors, as measured by [125I]iodocyanopindolol binding. This loss, which could be prevented by propranolol, represented a decrease in receptor number without a change in affinity of the remaining receptors for [125I]iodocyanopindolol. The BIM-induced decrease in binding sites was persistent in membranes incubated for several hours after BIM treatment. BIM did not inactivate alpha-1 adrenergic receptors on Madin Darby canine kidney cells, alpha-2 adrenergic receptors on human erythroleukemia cells, nor did BIM treatment alter guanyl-5'-yl-imidodiphosphate-mediated regulation of agonist binding to the beta adrenergic receptors in S49 cell membranes. BIM treatment decreased cyclic AMP (cAMP) accumulation in S49 cells in response to the beta adrenergic agonist isoproterenol, but increased prostaglandin E1-stimulated cAMP accumulation (P = .09) without altering cAMP production in response to forskolin. The inactivation of beta receptors in S49 cells by BIM (IC50 = 0.30 nM) correlated closely with the loss in beta adrenergic receptor-mediated cAMP accumulation in these cells (IC50 = 0.59 nM), implying the absence of substantial receptor reserve for this response. We conclude that BIM is a potent, irreversible, selective beta adrenergic antagonist for the study of beta adrenergic receptors in cultured cells.     

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)     

Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(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.     

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.     

Alteration in sensitivity to isoproterenol and acetylcholine in the rat heart after repeated administration of isoproterenol

Alteration of sensitivity to the inotropic responses to isoproterenol and acetylcholine (ACh) and of saturation constants for the specific binding of [3H]dihydroalprenolol and [3H]quinuclidinyl benzilate to particulate fractions were investigated in rat hearts chronically treated with isoproterenol. The EC50 value for the inotropic response to isoproterenol in the atria of rats injected i.p. with isoproterenol (0.01 or 0.1 mg/kg) twice a day for 10 days, was 9.0-fold higher than in controls. Isoproterenol (0.17 mg/kg i.p.)-induced stimulation of cardiac ornithine decarboxylase activity was significantly (P less than .05) attenuated in atria from animals of the treated groups. Scatchard analysis of specific binding of [3H]dihydroalprenolol revealed that repeated isoproterenol injection produced a significant decrease in the maximum number of binding sites (Bmax), but not in the dissociation constant (KD) for this antagonist. In comparison to the isoproterenol-induced changes in beta adrenergic positive inotropic response, repeated treatment with isoproterenol decreased 2-fold the EC50 value for the negative inotropic response to ACh in isolated atria. Scatchard analysis of specific [3H]quinuclidinyl benzilate binding, however, indicated a significant (P less than .01) decrease in Bmax. Repeated isoproterenol injections also elicited a significant increase in cardiac weight. It is suggested that repeated administration of isoproterenol induces a decrease in density of beta adrenergic receptors which may in part underlie a cardiac hyposensitivity to isoproterenol and an increase in the number of muscarinic ACh receptors and inotropic responsiveness to ACh.     

Coexistence of beta-1 and beta-2 adrenergic receptors in the human heart: effects of treatment with receptor antagonists or calcium entry blockers

The properties of the binding of [125I]iodopindolol ([125I]IPIN) to beta adrenergic receptors on plasma membranes prepared from right atrial tissue removed during cardiac bypass surgery were investigated. Some of the patients from whom the tissue was removed had been treated before surgery with either a beta adrenergic receptor antagonist or a calcium entry blocker or both. The specific binding of [125I]IPIN to beta adrenergic receptors was saturable, stereoselective and rapidly reversible. Studies of the inhibition of the specific binding of [125I]IPIN by drugs selective for beta-1 or beta-2 adrenergic receptors suggested that both beta-1 and beta-2 adrenergic receptors are present in the tissue, with approximately 55% of the receptors having the properties of beta-2 adrenergic receptors. The density of receptors in patients not treated with beta adrenergic receptor antagonists or calcium entry blockers was approximately 80 fmol/mg of protein, whereas the density of beta adrenergic receptors in treated patients was increased by approximately 50%. The relative proportion of beta-1 to beta-2 adrenergic receptors in subjects treated with beta adrenergic receptor antagonists and/or calcium entry blockers was not significantly different from that in untreated subjects. Studies were also carried out with a limited number of samples of human ventricular muscle obtained from untreated subjects at the time of surgery. The density of receptors was lower than that observed in studies with atrial tissue. However, as with atrial tissue, approximately half of the receptors appeared to be beta-2 adrenergic receptors.     

Ability of aged rats to alter beta adrenergic receptors of brain in response to repeated administration of reserpine and desmethylimipramine.

Repeated administration of reserpine to 3-month-old rats produced dose-related increases in [3H]dihydroalprenolol (DHA) binding in pineal gland, cerebral cortex and cerebellum. Reserpine increased DHA binding by increasing the density of beta adrenergic receptors. Brain tissue from 24-month-old rats, however, had an impaired ability to increase receptor density in response to reserpine treatment, even in the pineal gland where the concentration of reserpine was nearly 7 times that found in the glands of young rats given the same dose on the basis of body weight. Repeated administration of desmethylimipramine decreased DHA binding in pineal glands by about 50% and in cerebral cortices by about 25%, but did not alter DHA binding in the cerebellum. The magnitude of these changes was similar in the 3- and 24-month-old rats, although the concentration of desmethylimipramine in the pineal glands and cerebral cortices of the aged rats was significantly higher than that of the young animals. The results indicate that the reserpine-induced decrease in noradrenergic input causes a compensatory increase in beta adrenergic receptor density in rat brain. They suggest further that although aged rats can decrease receptor density in response to increased adrenergic input, they have an impaired ability to increase beta adrenergic receptor density in response to decreased adrenergic input. This finding may explain the decreased density of beta adrenergic receptor found in aged rat brain.     

Thyroid status and adrenergic receptor subtypes in the rat: comparison of receptor density and responsiveness

The density and functional responsiveness of adrenergic receptor subtypes were determined in tissues from control, hyperthyroid and hypothyroid rats. There was a decrease in sensitivity to isoproterenol in spontaneously beating right atria, electrically driven left atria and field-stimulated vas deferens associated with hypothyroidism, with no change in maximum response. Hyperthyroidism increased the potency of isoproterenol in right atria, but not in left atria or vas deferens. The maximal response to isoproterenol was greatly reduced in hyperthyroid left atria. The potency of procaterol, a partial agonist at beta adrenergic receptors in right atria, was unaltered in hyper- or hypothyroidism, although the maximum stimulation by procaterol was increased in hyperthyroidism. Scatchard analysis of specific [125I]pindolol binding showed that beta adrenergic receptor density was greater in hyperthyroidism than in hypothyroidism in left atria, right atria, ventricles, vas deferens and cerebral cortex, although the proportions of beta-1 and beta-2 adrenergic receptor subtypes did not change. There was no change in the responsiveness of alpha-1 adrenergic receptors mediating contraction of caudal artery and vas deferens or mediating [3H]inositol phosphate accumulation in cerebral cortex in hyperthyroid or hypothyroid rats, although the maximal contraction of caudal artery was significantly reduced in hyperthyroidism. Scatchard analysis of specific [125I]BE 2254 binding showed that alpha-1 adrenergic receptor density was significantly decreased in the ventricles from hyperthyroid rats and increased in the ventricles of hypothyroid rats, but was unchanged in vas deferens, caudal artery and cerebral cortex. Alpha-2 adrenergic receptor density in cerebral cortex, determined by Scatchard analysis of specific [3H] rauwolscine binding, was not altered in hyperthyroid or hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Down regulation of beta adrenergic receptors in S49 lymphoma cells induced by atypical agonists

The ability of the atypical agonists celiprolol and pindolol to induce sequestration and down regulation of beta adrenergic receptors was investigated in S49 lymphoma cells. Sequestration was measured as the loss of binding sites for [3H]CGP-12177, a hydrophilic radioligand that binds only to surface beta adrenergic receptors at 6 degrees C. Down regulation was measured as the loss of binding sites for [125I]iodopindolol, a lipophilic radioligand which at 37 degrees C binds to both surface and sequestered receptors. Pindolol and celiprolol do not stimulate adenylate cyclase in membranes from wild-type (WT) S49 cells or do they induce the sequestration of beta adrenergic receptors on intact cells. Incubation of WT S49 lymphoma cells with isoproterenol for 24 hr resulted in the loss of 75% of total cellular beta adrenergic receptors (down regulation). Exposure of WT S49 cells to pindolol or celiprolol for 24 hr resulted in the loss of approximately half of the total cellular beta adrenergic receptors. In mutant S49 cells [cyc- (variant of S49 lymphoma cells which lacks Ns activity) and UNC (variant of S49 lymphoma cells in which Ns is present but cannot interact with beta adrenergic receptors)] in which interaction of beta adrenergic receptors with the stimulatory guanine nucleotide binding regulatory protein (Ns) does not occur, a 24 hr incubation with isoproterenol caused the loss of approximately half of the beta adrenergic receptors, whereas pindolol and celiprolol caused no change in the number of receptors. These results suggest that there are two mechanisms of down regulation of beta adrenergic receptors in S49 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Species differences in the localization and number of CNS beta adrenergic receptors: rat versus guinea pig

The localization and number of beta adrenergic receptors were directly compared in the brains of rats and guinea pigs. The time course of association and saturability of [125I]cyanopindolol (CYP) binding to slide-mounted tissue sections was similar in rats (Kd = 17 pM) and guinea pigs (Kd = 20 pM). The beta-1 and beta-2 receptor subtypes were examined through the use of highly selective unlabeled receptor antagonists, ICI 118,551 (50 nM) and ICI 89,406 (70 nM). Dramatic species differences between rats and guinea pigs were observed in the neuroanatomical regional localization of the beta adrenergic receptor subtypes. For example, in the thalamus prominent beta-1 and beta-2 receptor populations were identified in the rat; however, the entire thalamus of the guinea pig had few, if any, beta adrenergic receptors of either subtype. Hippocampal area CA1 had high levels of beta-2 adrenergic receptors in both rats and guinea pigs but was accompanied by a widespread distribution of beta-2 adrenergic receptors only in rats. Quantitative autoradiographic analyses of 25 selected neuroanatomical regions 1) confirmed the qualitative differences in CNS beta adrenergic receptor localization, 2) determined that guinea pigs had significantly lower levels of beta adrenergic receptors than rats and 3) indicated a differential pattern of receptor subtypes between the two species. Knowledge of species differences in receptor patterns may be useful in designing effective experiments as well as in exploring the relationships between receptor and innervation patterns. Collectively, these data suggest caution be used in extrapolation of the relationships of neurotransmitters and receptors from studies of a single species.     

Labeling in vivo of beta adrenergic receptors in the central nervous system of the rat after administration of [125I] iodopindolol

The amount of radioactivity in vivo in the central nervous system (CNS) of the rat has been studied after tail-vein injections of (-)- [125I] iodopindolol (IPIN). The content of radioactivity in cortex and cerebellum 1 to 4 hr after IPIN administration was significantly reduced in rats pretreated with I-propranolol (1 mg/kg) given i.v. 5 min before IPIN; only a small effect of I-propranolol was seen in brainstem and spinal cord. The maximum reduction in radioactivity caused by I-propranolol was approximately the same in cortex and cerebellum (about 60-65%) and occurred 2 hr after IPIN administration. I-Propranolol was approximately 1500-fold more potent than d-propranolol in reducing radioactivity. Pretreatment of rats with other lipophilic drugs that act at beta receptors was able to reduce the binding of IPIN in vivo; in contrast, pretreatment of rats with drugs which do not have direct agonist or antagonist activity at beta adrenergic receptors (desmethylimipramine, metergoline, diazepam, fluoxetine, phentolamine and haloperidol) had no effect. Experiments using ICI 118, 551, a beta-2 antagonist and betaxolol, a beta-1 antagonist, indicated that the majority of radioactivity in the cortex in vivo was bound specifically to the beta-1 subtype of the receptor whereas in the cerebellum the majority of specific binding was to the beta-2-subtype. When the specific binding of IPIN to beta adrenergic receptors was measured in vitro in seven regions of the CNS, at a ligand concentration of 30 pM, a high correlation was found with the I-propranolol displaceable radioactivity measured in vivo (r = 0.97, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative autoradiography of central beta adrenoceptor subtypes: comparison of the effects of chronic treatment with desipramine or centrally administered l-isoproterenol

This study compares the regulation of the subtypes of central beta adrenoceptors produced by treatment of rats with desipramine (10 mg/kg i.p. twice daily for 10 days) to that caused by central infusion of l-isoproterenol (5 micrograms/hr for 7 days). Beta adrenoceptors were measured by quantitative autoradiography of the binding of [125I]iodopindolol ([125I]IPIN) to coronal sections of rat brain as well as the binding of this radioligand to homogenates of certain areas of brain. Administration of desipramine caused significant reductions in the total specific binding of [125I]IPIN in many areas of the brain, including regions of the amygdala, cerebral cortex, hippocampus, hypothalamus and thalamus. This reduction in the total specific binding of [125I]IPIN was primarily the result of a reduction in its binding to beta-1 adrenoceptors in most brain regions. With the exception of the paraventricular nuclei of the thalamus, no desipramine-induced change in the binding of [125I]IPIN to beta-2 adrenoceptors was observed in any region of the brain. Treatment of rats with desipramine did not alter the binding of [125I]IPIN to the caudate putamen, globus pallidus or the cerebellum. Intracerebroventricular fusion of the nonselective beta adrenoceptor agonist, isoproterenol, reduced the maximum binding site density of the binding of [125I]IPIN to homogenates of cerebellum and hypothalamus, but not to homogenates of cerebral cortex. Autoradiography of the binding of [125I]IPIN to brain sections from rats treated with isoproterenol revealed reductions in many subcortical areas and the cerebellum. In contrast to the effects produced by treatment with desipramine, reductions in the binding of [125I]IPIN after infusions of isoproterenol were a result of a decrease in the binding of [125I]IPIN to beta-2 adrenoceptors in most brain regions. Infusion of isoproterenol reduced the binding of [125I]IPIN to beta-2 adrenoceptors in 10 areas of brain in which the binding of the ligand to beta-1 adrenoceptors was not affected significantly. Inasmuch as there is a selective regulation of beta-1 adrenoceptors by the norepinephrine uptake blocker, desipramine, it may be inferred that these receptors are under the influence of endogenous norepinephrine. By contrast, the density of central beta-2 adrenoceptors are regulated more easily by an exogenous beta adrenoceptor agonist, even one like isoproterenol which is a full agonist at both beta-1 and beta-2 adrenoceptors.