Dihydroalprenolol Research Articles

Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β2-adrenergic receptor

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix–helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2–H4 in the β2-adrenergic receptor (β2-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly3157.42 and Ser3197.46, on H7 for structure-function analysis. Replacing Ser3197.46 with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15–20% agonist-independent activity. Replacement of Ser3197.46 with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly3157.42 and Ser3197.46 are stabilizing β2-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly3157.42 with Trp2866.48 and hydrogen bonding interactions of Ser3197.46 with amino acids on H1–H2–H7 and with structural water.

No Evidence of the Existence of Beta Adrenoceptors in Human Sperm Using Radioligand Binding Technique/Kein Anhalt für die Existenz von β-Adrenorezeptoren an menschlichen Spermatozoen bei Verwendung der Radioligand-Bindungs-Technik

The investigations were carried out on 20 freshly ejaculated and pooled samples of human semen with normozoospermia and motility of 70-80%. In the present study no difference was found between total and non-specific binding to semen membranes using [3H]-dihydroalprenolol (DHA) as a radioligand. Our results suggest that beta adrenoceptors could not be found in human sperm.

Putrescine modulation of acute activation of the β-adrenergic system in the left atrium of rat

Endogenous polyamines mediate acute metabolic effects and cardiac hypertrophy associated to β-adrenoceptor stimulation. The aim of this study is to characterize the role of polyamines on β-adrenoceptor system mediated responses. To this end, the functional interaction of polyamine modifying drugs on isoproterenol-elicited cardiotonic effect, in isolated left atria of male Wistar rats, and their effects on [ 3H]dihydroalprenolol (DHA) binding on β-adrenoceptors and on adenylyl cyclase activity of membrane heart were studied. Polyamines interact with β-adrenoceptors in rat heart, as shown by the displacement of [ 3H]DHA binding. Furthermore, putrescine (but not spermidine or spermine) increased adenylyl cyclase activity, elicited a positive inotropism and increased intracellular cAMP. The putrescine effect on adenylyl cyclase was not antagonized by the β-adrenoceptors blockers, alprenolol and ICI-118,551, and facilitated the isoproterenol effect. Neither alprenolol, atenolol nor ICI-118,551 antagonized putrescine-elicited positive inotropism. However, the effect was abolished in preparations with desensitized β-adrenoceptors. α-Difluoromethylornithine, an inhibitor of ornithine decarboxylase, antagonized the effect of isoproterenol on inotropism and cAMP increase. In addition, putrescine might elicit effects by mechanisms independent of β-adrenoceptor system, since in left atria with functional desensitized receptors an interaction with ouabain-elicited cardiotonic effect was observed. These results suggest that putrescine may act as a low affinity agonist on β-adrenoceptors and modulate acute responses mediated by β-adrenoceptors. These findings may be of importance in the physiology and in diseases involving cardiac β-adrenoceptors.

Identification of beta-adrenoceptor subtypes in lower urinary tract of the female pig.

We investigated the presence and functional role of beta-adrenoceptor subtypes in the bladder base and proximal urethra of the female pig. Saturation experiments were done with 7 concentrations (0.25 to 16 nM.) of [(3)H]-dihydroalprenolol (NEN Life Science Products, Boston, Massachusetts). Competition experiments with [(3)H]-dihydroalprenolol were performed with unlabeled antagonists (beta1 selective CGP20712A, beta2 selective ICI118551 and beta3 selective SR59230A). In functional studies concentration-relaxation curves to the beta3-agonist BRL37344 were obtained and antagonist affinities for SR59230A were determined. CGP20712A displaced [(3)H]-dihydroalprenolol with low affinity, suggesting that beta1-adrenoceptors were not present. Displacement with ICI118551 in the bladder base and urethra best fitted a 2-site model with 20% and 28% high affinity sites (beta2), respectively. Displacement experiments with SR59230A in the bladder base demonstrated that 59% of binding sites had high affinity (beta3). In the urethra displacement with SR59230A best fitted a 1-site model but with a pK(i) of 7.2 that was intermediate between that expected for beta2 and beta3-adrenoceptors. In functional studies BRL37344 induced relaxation with pEC50 values of 5.5 and 8, and a maximum relaxation response relative to 30 microM. isoprenaline of 79% and 90% in the bladder base and urethra, respectively. The affinity value of SR59230A for the response to BRL37344 was 7.87 and 7.71 in the bladder base and urethra, respectively, which were intermediate between those of beta2 and beta3-adrenoceptors. Apparently beta3-adrenoceptors are the predominant beta-adrenoceptor subtype present in the lower urinary tract of the pig.

Antipsychotic drugs influence transport of the β-adrenergic antagonist [3H]-dihydroalprenolol into neuronal and blood cells

SummaryThe amine hypothesis suggests that the cause of schizophrenic or depressive psychosis is dysfunction of noradrenergic or serotonergic neurotransmission. We investigated pharmacological properties of [3H]-dihydroalprenolol (DHA) transport into C6, IMR32, native lymphocytes, B-lymphoblastoids and MOLT-3 cells. DHA transport was inhibited by a heterogeneous group of structurally related compounds exhibiting an amine group and various aromatic ring structures. It was verified on cells of neuronal/glial and blood cell origin but in detail on B-lymphoblastoids. The latter once showed strongest inhibition of DHA transport using tricyclic antidepressants (amitriptyline: IC50=2.86 μM, imipramine: IC50=3.33 μM) and haloperidol (IC50=3.98 μM) as a neuroleptic. Antipsychotics like clozapine (IC50=11 μM), olanzapine (IC50=15 μM), spiperone (IC50=66 μM) and EMD 49980 (IC50 ≫ 100 μM) were less effective. In contrast to cells of blood origin, a stimulation of DHA transport by antipsychotics was not detectable using neuronal cells.As antipsychotics showed a distinct inhibition and, concerning cells of blood origin, a stimulation of transport after pre-incubation, further investigations seem to be of interest in respect to its involvement in the cellular uptake of drugs and therefore its impact on the quality of therapy of psychiatric patients.

Evaluation of drug release from gels on pig nasal mucosa in a horizontal Ussing chamber

In this study, controlled release gel formulations containing dihydroalprenolol (DHA), hydrocortisone (HC) or testosterone (TS) in Carbopol 934P (C934) were evaluated using pig nasal mucosa in a horizontal Ussing chamber. The controlled release gel formulations were designed by including DHA in vesicle bilayers formed with sodium dodecyl sulphate (SDS) (1.4 and 36 mM) and by partitioning TS to the core of Brij 58 (B58, 1%) micelles. For comparison, unmodified gels and solutions of the drugs and additives were examined in parallel experiments. The viability and toxicity were evaluated with electrophysiological measurements and light microscopy. The results showed that C934 did not affect the viability of the mucosa and that the rate and profile of the appearance on the receiver side was independent of whether the substances were released from an unmodified gel or an unmodified solution. Continuous electrophysiological measurements made during exposure showed that B58 (1%) and SDS (1.4 mM) inactivated the mucosa, whereas SDS (36 mM) activated it. Investigations made after a 90-min exposure to the formulations showed that all the modified gels had inactivated the mucosa and had negative effects on the morphology. For the TS-B58 (1%) and the DHA-SDS (36 mM) gels, the rate-limiting step in transport was the release from the formulation. The results confirmed that gels from C934 are suitable for nasal administration and also clearly indicated the different degrees of toxicity of the controlled release formulations evaluated in this study. The horizontal Ussing chamber method was a suitable tool for the evaluation of gels for nasal administration.

The role of beta(3)-adrenoceptors in mediating relaxation of porcine detrusor muscle.

1. beta-adrenoceptors mediate relaxation of bladder detrusor smooth muscle. This study investigates the contribution of beta(3)-adrenoceptors to relaxation of the pig urinary bladder. 2. Cell membranes were prepared from detrusor muscle of the pig bladder dome and competition experiments with [(3)H]-dihydroalprenolol (DHA), a non-selective beta-adrenoceptor antagonist was used as a specific radioligand to determine the presence of beta-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the affinity of antagonists. 3. In competition binding experiments, CGP20712A (beta(1)-adrenoceptor selective) displaced [(3)H]-DHA from a single binding site with a low affinity. In contrast, displacement data for ICI 118551 (beta(2)-adrenoceptor antagonist) and SR59230A (beta(3)-adrenoceptor antagonist) best fitted a two-site model suggesting a predominant (70%) population of beta(3)-adrenoceptors. 4. In functional studies, isoprenaline and salbutamol (beta(2)-adrenoceptor agonist) relaxed KCl precontracted muscle strips with high potency (pEC(50) 7.7 and 7.2, respectively), whilst CGP12177 and BRL37344 (beta(3)-adrenoceptor agonists) had low potency and were partial agonists. CGP20712A and atenolol (beta(1)-adrenoceptor antagonists) antagonised responses with a low affinity. ICI118551 antagonized responses to isoprenaline and salbutamol with a high affinity (pK(B)=7.8 and 8.7, respectively), but the Schild slopes were low suggesting that responses were mediated by more than one beta-adrenoceptor. The Schild plot for SR59230A was biphasic, apparent pK(B) values for 3 - 10 nM SR59230A being 8.6 and those for 30 nM - 1 microM being 7.7. 5. These data suggest that beta(3)-adrenoceptors are the predominant beta-adrenoceptor subtype present in the pig bladder and that beta-adrenoceptor mediated responses of this tissue are mediated via both the beta(2)- and beta(3)-adrenoceptor subtypes.

Pharmacological actions of the antidepressant venlafaxine beyond aminergic receptors.

The present study examines the effects of the antidepressant venlafaxine, a dual amine reuptake inhibitor, on (a) in vivo regulation of the densities of high- and low-affinity dihydroalprenolol (DHA) binding sites in the cortex of normal and reserpinized Sprague-Dawley rats and (b) targets beyond the beta adrenoceptor. While venlafaxine (30 mg/kg i.p. b.i.d.) administered for 4 d did not alter the DHA-binding parameters in the cortex of normal rats, it significantly reduced, in reserpinized animals, the number of up-regulated low-affinity sites (R(L)) which have been tentatively identified as serotonin(1B) sites. The drug did not influence the up-regulated high-affinity (R(H)) DHA-binding sites (beta-adrenoceptor sites). Venlafaxine failed to alter the up-regulated R(L) sites in brains of rats depleted of serotonin (5-HT) by p-chlorophenylalanine (PCPA) indicating that the normalization by venlafaxine of the up-regulated R(L) receptor population is mediated by increased synaptic 5-HT. Venlafaxine, given for a short period of time, thus mimicked the action of fluoxetine. While venlafaxine (20 mg/kg i.p. b.i.d.) given for 10 d did not change protein kinase A activity as assessed by the phosphorylation of kemptide in the 900 g supernatant or particulate fractions, the drug significantly reduced phosphorylated cAMP response-element binding protein (CREB-P) in nuclear lysates of cortex after chronic but not acute administration. Depletion of 5-HT by PCPA did not alter the venlafaxine-induced change in nuclear CREB-P. Lastly, analysis of reverse transcribed cortical CREB mRNA by competitive PCR indicated that the mean steady-state levels of CREB mRNA in venlafaxine vs. saline-treated animals were not significantly different. Therefore, since the phosphorylation status of CREB determines its transcriptional activity the reduction of nuclear CREB-P may be venlafaxine's most relevant action beyond the adrenoceptor.

Tannin inhibits the cAMP-beta-adrenergic receptor pathway in bovine tracheal epithelium.

Tannin, isolated from cotton bracts, inhibits chloride secretion in airway epithelium. In bovine tracheal epithelial cells, tannin (25 micrograms/ml) blunted isoproterenol (Iso)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Inhibition was time and dose dependent, with 52 +/- 5% (mean +/- SE, n = 6) inhibition at 60 min and 82 +/- 9% (n = 3) inhibition at 8 h. Inhibition was reversible starting at 4 h. Low-molecular-mass tannin (1,000-5,000 Da) had no effect on Iso-stimulated cAMP accumulation, whereas N-acetylcysteine, which interacts with cysteine residues, blocked the effects of tannin on Iso-stimulated cAMP accumulation. Tannin exposure (25 micrograms/ml for 30 min) had no effect on the dissociation constant (Kd) for [3H]dihydroalprenolol (DHA) (0.41 +/- 0.03 nM, n = 3) but decreased maximal binding from 252 +/- 32 to 162 +/- 36 fmol/mg protein. Using single-point analysis and [3H]CGP-12177, we determined that tannin (25 micrograms/ml for 4 h) decreased surface beta-adrenergic receptor density from 26.4 +/- 4.3 (n = 12) to 11.9 +/- 3.0 fmol/mg protein and that the decrease was dose dependent. Agonist binding affinity by Iso displacement of DHA demonstrated a two-site model (Kd values = 27 +/- 9 and 2,700 +/- 600 nM) and a ratio of high- to low-affinity receptors of 1:1. Tannin (25 micrograms/ml) steepened the curve and shifted it to the right, as did Gpp(NH)p. Gpp(NH)p had no further effect on the shape or position of the displacement curve in the presence of tannin. In contrast, when polymer length was decreased by oxidation, tannin had no effect on the DHA displacement curve. These data demonstrate that tannin reversibly desensitizes bovine tracheal epithelial cells to Iso, decreases beta-adrenergic receptor density, and uncouples the receptor from its stimulatory G protein. These data also suggest that the polymer length of tannin and its interaction with cysteine residues are important for these effects. These studies provide additional evidence for the role of tannin in the occupational lung disease byssinosis.

SEX DIFFERENCES IN FUNCTION AND DISTRIBUTION OF beta-ADRENOCEPTORS IN RABBIT URINARY BLADDER

Purpose To examine whether there are sex-related differences in urinary bladder function, we evaluated the function and distribution of beta-adrenoceptor in the male and female rabbit urinary bladder. Materials and Methods Ten male and ten female rabbits were studied. The function and distribution of beta-adrenoceptors were studied by measuring changes in contractile forces from isoproterenol, a beta-adrenoceptor agonist, and forskolin, an adenylate cyclase activator, and by measuring beta-adrenoceptor densities using radioligand receptor binding techniques. Results Isoproterenol caused significantly greater relaxation in female trigonal muscles than in male trigonal muscles. It caused similar relaxation responses in the male and female detrusors. The B max values for [sup 3 H]dihydroalprenolol (DHA) binding in trigonal muscles were significantly higher in the female than in the male, whereas B max values for [sup 3 H]DHA binding in the male and the female detrusor were similar. In contrast, forskolin caused similarly marked relaxations in the male and the female detrusor and trigonal muscles. Conclusion These findings demonstrate that there are sex-related differences in the function and the distribution of beta-adrenoceptors in rabbit trigonal smooth muscles.

Beta-Adrenergic receptors in rat fat cells and their relationship with lipolysis.

Norepinephrine stimulated lipolysis in rat fat cells while (-)-alprenolol completely inhibited this lipolysis. (-)-Alprenolol competed for (-)-[3H] dihydroalprenolol (DHA) binding sites on rat fat cells. The specific (-)-[3H]DHA binding sites identified by competition with (-)-alprenolol were found to be transferred to the solubilized supernatant during preparation of endogenous lipid droplets from the fat cells. Although the lipid droplets did not exhibit specific (-)-[3H]DHA binding, norepinephrine induced lipolysis in a cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). Norepinephrine-induced lipolysis in the cell-free system was inhibited by propranolol and (-)-alprenolol, but not by phenoxybenzamine. The lipolytic action of norepinephrine and the anti-lipolytic actions of propranolol and (-)-alprenolol disappeared after sonication of the lipid droplets in the cell-free system. These results suggest that the adrenergic receptor concerned with lipolysis in fat cells may not be a specific (-)-[3H]DHA binding site, but may be closely related to the lipid droplets.

Regulation of Lens β-Adrenergic Receptors by Receptor Occupancy and Dexamethasone

Beta-adrenergic binding sites in primary cultures of chick lens annular pad (CLAP) cells were characterized with dihydroalprenolol (DHAP). Binding site affinities and densities were similar to beta-adrenergic receptors (BARs) previously characterized on crude membranes from freshly isolated cells. In competitive displacement studies, the beta-blocker propranolol was shown to increase the number of available binding sites in a concentration dependent manner. Acute exposure of CLAP cells to propranolol prior to DHAP binding also resulted in an increase in the number of available binding sites. Finally, lens beta-adrenergic binding site levels could be modulated by dexamethasone treatment. These results indicate that lens BARs are subject to common regulatory mechanisms and further implicate ophthalmic pharmaceuticals as possible cataractogenic agents.

Roles of Adrenoceptors in Isolated Canine Parietal Cells

Functional roles of adrenoceptors in parietal cells were pharmacologically investigated using isolated canine parietal cells. In the crude membranes obtained from preparations highly purified in parietal cells (> 95% of purity), the specific binding of [3H]dihydroalprenolol (DHA) was observed with a Kd value of 2.9 nM and Bmax of 234 fmol/mg protein, while the specific binding of [3H]prazosin and [3H]rauwolscine were not attained. Propranolol concentration-dependently reduced the specific binding of [3H]dihydroalprenolol with a Ki value of 2.6 nM. Isoproterenol concentration-dependently stimulated [14C]aminopyrine accumulation in preparations enriched in parietal cells (about 70% purity) with the maximum at 10 nM. Isoproterenol increased the content of cyclic AMP in preparations enriched in parietal cells (70%) with the maximum at 100 nM. The isoproterenol-induced stimulatory effect of [14C]aminopyrine accumulation in preparations enriched in parietal cells (70%) was completely abolished by 1 microM propranolol but not by 1 microM phentolamine. In the presence of 1 microM propranolol, 100 microM noradrenaline did not affect carbachol- and histamine-induced [14C]aminopyrine accumulation in preparations enriched in parietal cells (70%). The present study suggests that stimulation of beta-adrenoceptors located on canine parietal cells evokes acid production in a cyclic-AMP-dependent manner. Furthermore, a possibility arises that canine parietal cells are not the site of action of alpha-adrenoceptors in mediating inhibition of gastric acid secretion.

The Reversal Effect of Insulin on Diabetes-Induced Alterations In Beta Adrenergic and Muscarinic Receptors in Rat Prostate

Previous studies from our laboratory demonstrated that 8 weeks after the induction of diabetes by the administration of streptozotocin (STZ) there was a downregulation of beta adrenergic and muscarinic cholinergic receptors in rat prostate, and that early insulin treatment (started 3 days after the onset of diabetes) prevented these alterations from occurring. In the present study, the effects of later insulin treatment (started 8 weeks after the onset of diabetes) on the reversibility of diabetes-induced alterations in beta adrenergic and muscarinic receptors in rat prostate were investigated. Three groups of rats were maintained for 16 weeks: 1) diabetics, 2) insulin-treated diabetics (subcutaneously injected with 5 to 8 U per day starting 8 weeks after the onset of diabetes) and 3) age matched controls. Binding studies with [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB) showed a significantly lower density of beta adrenergic and muscarinic cholinergic receptors in the diabetic rat prostate than in prostate from either controls or insulin-treated diabetic animals. Inhibition of [3H]DHA binding by isoproterenol, a beta adrenergic agonist, and binding of [3H]QNB by carbachol, a muscarinic agonist, indicated the presence of low and high affinity agonist binding sites for each receptor. The relative proportion of high affinity to total binding sites as well as the low and high affinity constants were similar in all groups. These data indicate that insulin treatment, begun 8 weeks after the onset of diabetes, can reverse the diabetes-induced downregulation of both beta adrenergic and muscarinic cholinergic receptors in STZ-diabetic rat prostates.

Characterization of β-Adrenoceptors in Pig Basilar Artery from Functional and Radioligand Binding Studies

β-Adrenoceptors in pig basilar arteries were investigated by measuring the relaxation responses to norepinephrine and by a radioligand binding assay with [3H]-dihydroalprenolol (DHA). Norepinephrine induced concentration-dependent relaxations. The relaxation responses were independent of the presence of endothelial cells, and they were competitively antagonized by (±)-propranolol, atenolol, butoxamine and ICI 118,551. Specific [3H]-DHA binding to β-adrenoceptors was saturable, reversible and high affinity (Kd= 1.4 nM), with a Bmax of 48.7 fmol/mg protein. Computer analysis of inhibition of [3H]-DHA binding by atenolol, butoxamine and ICI 118,551 gave a β1:β2-adrenoceptor ratio of approximately 65:35. The pA2 values of these antagonists were significantly correlated with the Ki values for β1-adrenoceptor determined by the radioligand binding assay. The present findings indicate that the relaxation responses to norepinephrine are predominantly mediated through the stimulation of β1-adrenoceptors on vascular smooth muscle cells in a pig basilar artery.

Diurnal variation of amylase secretion is coupled with alterations of β-adrenoceptors in the rat parotid gland

Diurnal changes in the neurotransmitter receptors are important for studying the receptor function in neurophysiology. The purpose of this study is to gain an insight into the regulatory mechanisms of the diurnal variation of amylase secretion. Rat salivary amylase levels showed a diurnal variation with two peaks, a marked peak at 13 h and a lesser peak at 21 h. This increase in salivary amylase levels was completely inhibited by pretreatment of rats with the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. Amylase level in parotid tissue homogenate also showed a diurnal change, but there was only one peak, at 13 h. The number of maximal binding sites (Bmax) for [3H]dihydroalprenolol (DHA) in parotid membranes showed a diurnal variation with two marked peaks at 13 and 21 h, but the affinity of parotid beta-adrenoceptors for agonists or antagonists did not show any diurnal changes. Phosphorylation of nuclear non-histone proteins in the rat parotid gland showed diurnal variation with two marked peaks at 13 and 21 h. These results indicate that a diurnal variation in the number of rat parotid beta-adrenoceptors, which is presumably regulated by gene expression, is coupled with a change in salivary amylase secretion.

A possible role of decreased relaxation mediated by beta-adrenoceptors in bladder outlet obstruction by benign prostatic hyperplasia.

1. To explore mechanisms of urinary obstruction in benign prostatic hyperplasia (BPH), the features of contraction and relaxation in human hyperplastic and non-hyperplastic (control) prostatic tissues were investigated for beta- and alpha 1-adrenoceptors by radioligand binding and in vitro isometric tension experiments. 2. Hyperplastic and control prostatic tissues had a similar number (per mg protein) of prazosin binding sites with similar affinities. Noradrenaline (NA) induced dose-dependent contraction in both tissues. Contraction induced by either exogenous NA or transmural stimulation was inhibited by prazosin in both tissues, indicating that the same contractile mechanisms mediated by alpha 1-adrenoceptors exist in hyperplastic and control tissues. 3. The number of dihydroalprenolol (DHA) binding sites (per mg protein) was less in hyperplastic tissues than in controls, whereas the affinity to the ligand was identical in both tissues. Isoprenaline caused a marked relaxation of the tonic contraction induced by KCl in control tissues, but not in hyperplastic tissues. Propranolol enhanced exogenous NA- or transmural stimulation-induced contraction more in control tissues than in hyperplastic tissues. Both tissues, however, similarly responded to forskolin by relaxation. 4. These results indicate that decreased beta-adrenoceptor-mediated relaxation in hyperplastic prostatic tissues, which is attributable at least in part to the decreased number of beta-adrenoceptors, may play a role in the urinary obstruction of BPH in addition to mechanical compression of the urethra by the enlarged prostate.

Down-regulation of cortical beta-adrenoceptors by chronic treatment with functional NMDA antagonists.

Down-regulation of cortical beta-adrenoceptors is observed in laboratory animals following chronic treatment with many clinically effective antidepressant therapies. [3H]Dihydroalprenolol (DHA) binding to cortical beta-adrenoceptors was examined in mice treated with the functional NMDA antagonists 1-aminocyclopropane-carboxylic acid (ACPC) and MK-801. ACPC and MK-801 reduced [3H]DHA binding by 19 (P less than 0.05) and 21% (P less than 0.05), respectively, while imipramine produced a 23% (P less than 0.05) reduction. No corresponding changes in the KD of [3H]DHA were observed. These findings are consistent with the observation that functional NMDA antagonists are active in animal models commonly used to evaluate antidepressants and may represent a novel approach to the treatment of depression.

Effect of forced-running stress on beta-adrenergic receptors in rat brain regions and liver.

Rats were exposed to forced-running stress for 1 day, 3 days or a long term (approximately 2 weeks), and beta-adrenergic receptor binding was then assayed using [3H]dihydroalprenolol (DHA) in six brain regions and the liver. In the pons + med.obl., hypothalamus and midbrain, a reduction in beta-adrenergic receptor density was first evident on day 1. In contrast, a decrease in beta-adrenergic receptor density in the cerebral cortex and hippocampus was first evident on day 3. Decreased [3H]DHA binding in the pons + med.obl., cerebral cortex and hippocampus subsequently plateaued for the duration of the forced-running stress. In the midbrain and hypothalamus, however, decreased [3H]DHA binding subsequently returned to control levels despite the exposure to the forced-running stress. In the cerebellum and the liver, [3H]DHA binding did not change significantly throughout the stress. These results indicate that the forced-running stress induces both the time- and region-specific changes in beta-adrenergic receptors. Moreover, the rats showed either a behavioral depression or a spontaneous recovery of running activity during the 2 weeks following the end of the long-term stress. Thus, we also examined the relationship of beta-adrenergic receptors to these behavioral differences. [3H]DHA binding for the behavioral depression group was lower in the hippocampus and higher in the liver than for the spontaneous recovery group.

Ligand binding to the porcine adipose tissue beta-adrenergic receptor.

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