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.     

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.     

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)     

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.     

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.     

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)     

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)     

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.     

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.     

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)     

Effects of clenbuterol and antidepressant drugs on beta adrenergic receptor/N-protein coupling in the cerebral cortex of the rat

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)     

Quantitative analysis of the selectivity of radioligands for subtypes of beta adrenergic receptors

In tissues with two classes of binding sites for a drug, it is common to estimate the proportion of each class of binding site by inhibiting the binding of a radioligand with a selective unlabeled ligand. Accurate estimates of the density of each class of binding site, however, will be obtained only if the radioligand is nonselective or used at a concentration that saturates both classes of binding sites. A method of simultaneous regression analysis of multiple inhibition curves, using the program MLAB on the PROPHET system, was used to quantify the selectivity of radioligands for beta-1 or beta-2 adrenergic receptors. The selectivity of [125I]iodopindolol, [125I]iodocyanopindolol, [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol for beta-1 and beta-2 adrenergic receptors was assessed by inhibiting the binding of each radioligand with the beta-1-selective unlabeled ligand ICI 89,406 at increasing concentrations of the radioligand, using membranes prepared from C6 glioma cells, which have both beta-1 and beta-2 adrenergic receptors. Scatchard plots for all four radioligands were linear, with correlation coefficients greater than 0.95. [125I]Iodopindolol and [125I]iodocyanopindolol were 3.2- and 2-fold selective, respectively, and [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol were 5.8- and 2.3-fold selective, respectively, for beta-2 adrenergic receptors. Values obtained for the densities of beta-1 and beta-2 adrenergic receptors and the affinities of the receptors for ICI 89,406 were independent of the radioligand used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of beta adrenergic antagonists with isolated rat alveolar type II pneumocytes. II. Receptor-independent accumulation of beta adrenergic antagonists and other cationic amphiphilic drugs in lamellar bodies

Accumulation of basic drugs by pulmonary tissue is well known. The cationic amphiphilic nature of many of these compounds suggests that they may be sequestered within an acidic and/or phospholipid-rich compartment. We described previously receptor-independent, concentrative, temperature- and pH-dependent sequestration of the beta adrenergic antagonists [125I]iodocyanopindolol and [125I]iodopindolol by intact rat type II pneumocytes in primary culture. The present study reveals that type II pneumocytes sequester [125I]iodocyanopindolol to an extent greater than other cell types (type II cells greater than polymorphonuclear leukocytes greater than S49) within 80-min incubations. Localization of fluorescence into large granular structures was observed after incubation of type II cells with 9-aminoacridine-propranolol (9-AAP). The distribution of fluorescence coincides with surfactant-containing lamellar bodies (LB) visualized with tannic acid/osmium staining. The large granule localization of 9-AAP fluorescence decreases with time in primary culture in parallel with changes in cell morphology and decreased numbers of LB. Comparison of patterns of fluorescence after incubation with 9-AAP, acridine orange and 9-aminoacridine (all 1 microM) indicates that localization is not a property of the acridine moiety, but requires the propranolol side-chain. Association of 9-AAP fluorescence with LB is inhibited completely by chlorpromazine (10 microM); the same concentration of propranolol or chlorquine produces less extensive, but detectable, inhibition.(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)     

Pharmacological characterization of chick and frog beta adrenergic receptors in primary cultures of myocardial cells.

In membrane preparations derived from primary cultures of chick myocardial cells, beta adrenergic receptors modeled for a single low-affinity site for both betaxolol (beta-1-selective) and ICI 118551 (beta-2-selective) displacement of [125I]iodocyanopindolol (ICYP), indicating that the chick beta receptor is pharmacologically distinct from both mammalian beta-1 and beta-2 adrenergic receptors with respect to these antagonists. However, the highly beta-1-selective compound CGP 20712A was able to distinguish two binding sites on ICYP competition curves, a high-affinity "beta-1 site" (75%) and a low-affinity "beta-2 site" (25%). Also, in chick heart cell membranes the relative ability of agonists to displace ICYP produced a profile typical of beta-1 adrenergic receptors with a rank order of potency or efficacy of: isoproterenol greater than epinephrine = norephinephrine. When agonist-mediated adenylyl cyclase stimulation was assessed the order of potency was slightly different, isoproterenol greater than epinephrine greater than or equal to norepinephrine. Additionally, antagonism of isoproterenol stimulation of adenylyl cyclase by CGP 20712A yielded a Kb value (1.16 +/- 0.35 x 10(-7) M) intermediate between the high and low-affinity binding sites of CGP 20712A, suggesting that the low-affinity site is coupled to adenylyl cyclase. In membrane preparations of frog myocardial cells, ICYP/antagonist competition curves modeled for a mixed population of receptors, with subtype percentages varying from 50:50 beta-1:beta-2 to 100% beta-2 depending on the specific antagonist used and the individual cell preparation. For ICYP/agonist competition binding experiments the relative ability to displace ICYP was isoproterenol greater than epinephrine = norepinephrine, a profile typical of beta-1 adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. I. High- and low-affinity states of agonist binding to membrane-bound beta adrenergic receptors

The properties associated with ligand interactions with membrane-bound beta adrenergic receptors prepared from L6 myoblasts were examined at five temperatures between 10 degrees and 30 degrees C. The interactions of antagonists with membrane-bound receptors were insensitive to temperature, whereas the interactions of agonists were temperature-dependent. The affinity constants for the low-affinity binding states of agonists (KL) decreased slightly with decreasing assay temperature. The small temperature-dependent changes in KL values were similar to the changes in Kd values observed in studies of the binding of agonists in the presence of GTP. The high-affinity dissociation constants (KH) for binding of full agonists to membrane-bound receptors in the absence of GTP were approximately 50-fold lower at 10 degrees than at 30 degrees C. The KH values for partial agonists also decreased with decreasing temperature, but the changes were smaller in magnitude. Thermodynamically, the binding of antagonists was primarily entropy-driven, whereas the binding of agonists was enthalpy-driven. The energetics of the low-affinity component of agonist binding to membrane-bound receptors were similar to the energetics for binding of agonists to membrane-bound receptors in the presence of GTP. Under these conditions, standard enthalpy change (delta H degree) and standard entropy change (delta S degree) values for binding of agonists were more negative than the corresponding values for binding of antagonists, possibly reflecting a conformational change in the receptor or an increased ordering of the lipids surrounding the receptor. The interaction of the receptor with the guanine nucleotide-binding protein to form the high-affinity component of agonist binding was thermodynamically described by larger negative changes in enthalpy and entropy than the values for formation of the low-affinity component of agonist binding. There was a correlation between the efficacies of ligands in activating adenylate cyclase and the delta H degree and delta S degree values for high-affinity binding of agonists. Thus, the extent or nature of the interaction between the guanine nucleotide-binding protein and the receptor may determine the efficacies of ligands.     

Alpha adrenergic receptors in the rabbit bladder base smooth muscle: alpha-1 adrenergic receptors mediate contractile responses

Alpha adrenergic receptors in rabbit bladder base smooth muscle were investigated by in vitro responses of smooth muscle strips to exogenous alpha agonist stimulation and in radioligand binding assays. Norepinephrine and phenylephrine caused significantly greater maximal contractile responses than did clonidine. Also, the contractile response was only inhibited weakly by the alpha-2 selective antagonist yohimbine but was potently inhibited by alpha-1 selective antagonists prazosin and BE2254, suggesting that the response is mediated predominantly by alpha-1 adrenergic receptors. The alpha-1 selective antagonist [125I]BE2254 was used to specifically label a single class of binding sites with a dissociation constant of 131.0 +/- 5.9 pM and a maximal binding capacity of 17.6 +/- 1.9 fmol/mg of protein. Catecholamines compete for [125I]BE2254 binding stereospecifically and with the characteristic alpha adrenergic potency series of (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The alpha-1 selective antagonist prazosin (Kd = 2.4 nM) is much more potent in competing for [125I]BE2254 binding than is the alpha-2 selective antagonist yohimbine (Kd = 2900 nM). Also, this dissociation constant of prazosin of [125I]BE2254 binding for bladder base smooth muscle membranes was similar to prazosin's pA2 value of 8.23 to 8.58 in the contraction experiments. The results suggest that alpha-1 rather than alpha-2 receptors predominantly mediate catecholamine-induced contraction in the rabbit bladder base. Also, these receptor sites can be measured directly with the specific antagonist radioligand, [125I]BE2254.     

Presynaptic modulation of beta adrenergic receptors in rat cerebral cortex after treatment with antidepressants.

Treatment with desmethylimipramine (DMI), a tricyclic antidepressant, for 7 to 21 days resulted in a 35 to 45% decrease in the accumulation of adenosine cyclic 3':5'-monophosphate (cAMP) in response to a maximally effective concentration of (-)-isoproterenol (ISO) in rat cerebral cortical slices. The EC50 for ISO-stimulated cAMP accumulation was not affected by DMI administration. The diminution in responsiveness to catecholamines was accompanied by a 35 to 40% decrease in the density of beta adrenergic receptors as measured by the binding of [125I]iodohydroxybenzylpindolol. Decreases in ISO-sensitive cAMP accumulation and in beta adrenergic receptor density were temporally correlated, maximal decreases being observed within 5 to 7 days. Within 7 days after cessation of chronic DMI treatment ISO-stimulated cAMP accumulation and beta adrenergic receptor density returned to normal. The role of presynaptic nerve terminals in mediating these phenomena was also investigated. Treatment of newborn rats with 6--hydroxydopamine inhibited the development of noradrenergic nerve terminals in the cerebral cortex and blocked the effects of DMI on cortical cAMP accumulation and on beta adrenergic receptor density. The administration of the beta adrenergic receptor antagonist propranolol led to increases in maximal ISO-stimulated cAMP accumulations and beta adrenergic receptor density in the rat cerebral cortex. This increase was not affected by the simultaneous administration of propranolol and DMI. Thus, the effect of DMI appears to be mediated through an action of norepinephrine at beta adrenergic receptors. Chronic treatment with two other clinically effective antidepressants, pargyline and iprindole, led to effects similar to those observed with DMI administration. Pretreatment of neonates with 6-hydroxydopamine blocked the effect of iprindole on beta adrenergic receptors. Preincubation of cortical membranes with guanosinetriphosphate before determination of the density of beta adrenergic receptors had no effect on the decreased number of receptors had no effect on the decreased number of receptors seen in DMI-treated animals. These experiments suggest that antidepressants, acting presynaptically, increase the concentration of transmitter at noradrenergic synapses and induce a compensatory decrease in the density of beta adrenergic receptors.     

Noradrenergic denervation alters serotonin2-mediated behavior but not serotonin2 receptor number in rats: modulatory role of beta adrenergic receptors

Recent behavioral evidence suggests that enhancement of noradrenergic neurotransmission may alter the functional sensitivity of serotonin2 (5-HT2) receptors in the central nervous system. The present studies have examined the effects of two types of noradrenergic denervation [neurotoxic: via N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) treatment; and pharmacologic: via chronic beta adrenergic receptor blockade] on the 5-HT2-mediated head shake response and cortical beta adrenergic and 5-HT2 receptor number in the rat. No changes in quipazine-induced head shakes were observed 3 days after DSP4 lesion. However, the frequency of head shakes was significantly enhanced 10 days after DSP4 treatment in the presence of a 39% up-regulation of beta adrenergic receptors. Pretreatment with propranolol 10 days after DSP4 lesion selectively antagonized the enhancement of the behavioral response to quipazine without altering base-line response rate, whereas pretreatment with the 5-HT2 antagonist ketanserin totally blocked head shakes in both control and DSP4-treated rats. Pharmacologic denervation achieved by continuous (14 day) administration of the beta adrenergic antagonist propranolol also resulted in a potentiation of the head shake response (274% of control) and an upregulation of beta adrenergic receptors (44%). Conversely, continuous treatment with the beta adrenergic agonist clenbuterol resulted in a marked reduction in head shakes (36% of control) with a concomitant 29% down-regulation of beta adrenergic receptors. 5-HT2 receptor binding was not modified by either DSP4 lesion or continuous administration of beta adrenergic agonists or antagonists. These studies demonstrate that changes in cortical beta adrenergic receptor density may modify 5-HT2-mediated behavior in a manner that is independent of changes in 5-HT2 receptor number.