Specificity evaluation of antibodies against human β3-adrenoceptors

β(3)-Adrenoceptors are a promising drug target for the treatment of urinary bladder dysfunction, but knowledge about their expression at the protein level and their functional role is limited, partly due to a lack of well validated tools. As many antibodies against G-protein-coupled receptors, including those against β(3)- and other β-adrenoceptor subtypes, lack selectivity for their target, we have evaluated the specificity of five antibodies raised against the full-length protein of the human β(3)-adrenoceptor (H155-B01), its N-terminus (LSA4198 and TA303277) and its C-terminus (AB5122, Sc1472) in immunoblotting and immunocytochemistry. Our primary test system were Chinese hamster ovary cells stably transfected to express each of the three human β-adrenoceptor subtypes at near physiological levels (100-200?fmol/mg protein). None of the five antibodies exhibited convincing target specificity in immunoblotting with Sc1472 apparently being least unsuitable. In immunocytochemistry, LSA4198 and Sc1472 appeared most promising, exhibiting at least some degree of specificity. As these two antibodies have been raised against different epitopes (N- and C-terminus of the receptor, respectively), we propose that concordant staining by both antibodies provides the most convincing evidence for β(3)-adrenoceptor labelling in cyto- or histochemistry studies.     

Ligand-directed signalling at β-adrenoceptors

β-Adrenoceptors (ARs) classically mediate responses to the endogenous ligands adrenaline and noradrenaline by coupling to Gsα and stimulating cAMP production; however, drugs designed as β-AR agonists or antagonists can activate alternative cell signalling pathways, with the potential to influence clinical efficacy. Furthermore, drugs acting at β-ARs have differential capacity for pathway activation, described as stimulus trafficking, biased agonism, functional selectivity or ligand-directed signalling. These terms refer to responses where drug A has higher efficacy than drug B for one signalling pathway, but a lower efficacy than drug B for a second pathway. The accepted explanation for such responses is that drugs A and B have the capacity to induce or stabilize distinct active conformations of the receptor that in turn display altered coupling efficiency to different effectors. This is consistent with biophysical studies showing that drugs can indeed promote distinct conformational states. Agonists acting at β-ARs display ligand-directed signalling, but many drugs acting as cAMP antagonists are also able to activate signalling pathways central to cell survival and proliferation or cell death. The observed complexity of drug activity at β-ARs, prototypical G protein-coupled receptors, necessitates rethinking of the approaches used for screening and characterization of novel therapeutic agents. Most studies of ligand-directed signalling employ recombinant cell systems with high receptor abundance. While such systems are valid for examining upstream signalling events, such as receptor conformational changes and G protein activation, they are less robust when comparing downstream signalling outputs as these are likely to be affected by complex pathway interactions.This article is part of a themed section on Molecular Pharmacology of GPCR. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00695.x     

Are there functional β3-adrenoceptors in the human heart?

β₃-Adrenoceptor mRNA is expressed in the human heart, but corresponding receptor protein has not yet consistently been demonstrated. Furthermore, their physiological role remains highly controversial. For example, in human atria these receptors apparently do not promote cAMP formation. Evidence presented in this issue of the BJP suggests that a previously reported β₃-adrenoceptor-mediated stimulation of Ca²⁺ channels at room temperature is absent at physiological temperatures, and that β₃-adrenoceptors have no effect on atrial contraction. Drugs classified as β₃-adrenoceptor agonists cause contraction in human atria but in most cases this involves β₁- and/or β₂-adrenoceptors. In contrast, in human ventricles β₃-adrenoceptor agonists can exhibit negative inotropic effects, potentially involving Pertussis toxin-sensitive G-proteins and activation of a NO synthase. However, firmer pharmacological evidence is required that these effects indeed occur via β₃-adrenoceptors. Whether the expected future use of β₃-adrenoceptor agonists in the treatment of urinary bladder dysfunction is associated with adverse events related to cardiac function remains to be determined from clinical studies.

LINKED ARTICLE

This article is a commentary on Christ et al., pp. 823–839 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2010.00996.x     

Binding of agonists and antagonists to β-adrenoceptors in rat vas deferens: relationship to functional response

Summary The properties of -adrenoceptors in rat vas deferens were examined using radioligand binding assays of ¹²⁵I-pindolol (¹²⁵IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. ¹²⁵IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of ₂-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC₅₀ of 18.0±2.1 nM. K _B values for antagonists in competitively antagonizing this response correlated well (r ²=0.99) with the K _D values for inhibition of ¹²⁵IPIN binding. Inhibition of ¹²⁵IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl₂, agonists inhibited specific ¹²⁵IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 M guanosine-5-triphosphate, agonists inhibited specific ¹²⁵IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 M BAAM produced a 45% decrease in the B _max for ¹²⁵IPIN with no change in the apparent K _D. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol folowed by a decreased maximum response. K _A values for isoprenaline and salbutamol in activating the functional -adrenoceptors were compared with K _I values for agonist inhibition of specific ¹²⁵IPIN binding. The K _A values for both agonists were not significantly different from the low affinity K _I values, but were significantly different from the high affinity K _I values. These data suggest that 1) a homogeneous population of ₂-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with ¹²⁵IPIN, 2) there is a substantial -adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by -adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.Supported by HL29871 and an Advanced Predoctoral Fellowship from the Pharmaceutical Manufacturers Association Foundation to J.M.M.Portions of this work were presented at the FASEB meeting April 4, 1984, St. Louis, MO, USA, at the ASPET meeting August 18–22, 1985, Boston, MA, USA, and appeared in Fed Proc 43:744     

Different steric characteristics of β1- and β2-adrenoceptors

Summary -Adrenoceptors of lung (75% ₂) and heart (95% ₁) of calf were labelled with ³H-(–)-propranolol. The stereoisomers of 10 ligands were used to inhibit the binding of ³H-(–)-propranolol to membrane particles. The affinity ratio of sereoisomers is consistently greater for ₁-adrenoceptors than for ₂-adrenoceptors, regardless of whether the ligands are agonists, partial agonists or antagonists. The ₁-adrenoceptor appears to possess stricter steric requirements than the ₂-adrenoceptor. This property may prove helpful in differentiating the -adrenoceptor subtypes during receptor solubilization and purification.     

Agonist-mediated conformational changes of β-adrenoceptors could occur independent of functional coupling to Ns

Summary Agonist and antagonist binding characteristics of -adrenoceptors in turkey erythrocyte ghosts were determined at different temperatures ranging between 7°C and 42°C. [³H]-DHA saturation binding experiments revealed that the antagonist-receptor interaction is entropy-driven with a small enthalpic contribution. Isoproterenol/[³H]-DHA competition binding followed the law of mass action at all the investigated temperatures. The agonist-receptor interaction is enthalpy driven with a small unfavorable decrease in entropy. This is consistent with the agonist's ability to favor an endoenergetic transconformation of the receptors.Only part of the agonist-bound receptors can undergo functional coupling to the stimulatory component of the adenylate cyclase system (Ns). This coupling process is associated with locking-in of the agonist and becomes persistent in the presence of the alkylating reagent N-ethylmaleimide. The number of agonist/N-ethylmaleimide-sensitive sites (i.e. coupling-prone receptors) increases with the temperature until it reaches a plateau value of 50% between 27–32°C. Qualitatively similar data were obtained for rat lung and turkey erythrocyte membranes. These observations suggest that the whole receptor population can undergo agonist-mediated conformational changes but that only part of them can couple to Ns.     

Sodium regulation of agonist and antagonist binding to β-adrenoceptors in intact and Ns-deficient membranes

Summary Agonist binding to various hormone receptors mediating adenylate cyclase inhibition is decreased by sodium ions. We studied the influence of Na⁺ on agonist and antagonist binding to -adrenoceptors in membrane preparations of guinea pig lung, S49 lymphoma wild-type cells (WT) and their N_s-deficient cyc^– variants by measuring binding of the antagonist, [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP). At 37° C, sodium decreased the receptor affinity for the agonist, isoproterenol, in all three membrane preparations. In lung and WT membranes, Na⁺ steepened the shallow agonist competition curves in a manner similar to and synergistic with guanine nucleotides. When binding was performend at 4° C, sodium regulation but not guanine nucleotide regulation of agonist binding was preserved. At the low temperature, [¹²⁵I]CYP affinity was reduced, and sodium increased [¹²⁵I]CYP binding in both N_s-containing and N_s-deficient membranes by increasing the antagonist affinity without significant change in total receptor number. Compared to Na⁺, Li⁺ and K⁺ were much less potent and efficient in decreasing agonist and increasing antagonist binding. Na⁺ and Mg²⁺ had opposite effects on agonist binding in the N_s-containing lung and WT membranes but not in the N_s-deficient cyc^– membranes. The data indicate that sodium not only regulates binding of inhibitory hormone receptors but also agonist and antagonist binding to the adenylate cyclase stimulatory -adrenoceptor. The finding that sodium regulation of -adrenoceptor binding is also observed in the N_{s 2} cyc^– membranes, furthermore, indicates that the target of sodium is not the -subunit of N_s but possibly a component common to both types of receptor systems regulating adenylate cyclase activity.     

Previously unsuspected widespread cellular and tissue distribution of β-adrenoceptors and its relevance to drug action

The discovery of β-adrenoceptors in previously unsuspected cell types is contributing to the rethinking of new drug targets. Recent developments in β-adrenoceptor pharmacology might have excited and surprised James Black, given his interest in developing drugs based on the selective manipulation of receptors to alter physiological responses. β-adrenoceptors continue to generate surprises at molecular and pharmacological levels that often require knowledge of receptor location to interpret. In this review, we emphasize the use of fluorescent ligands as the most selective means of demonstrating receptor localization. Fluorescent ligand binding in live tissues can provide quantitative pharmacological data, under carefully controlled conditions, relevant to other signalling parameters. Consideration of the role of β-adrenoceptors in many cell types (previously ignored) is needed to understand the actions of drugs at β-adrenoceptors throughout the body, particularly in the lung epithelium, vascular endothelium, immune cells and other 'structural' and 'restorative' cell types.     

Interactions of the enantiomers of 3-O-methyldobutamine with α- and β-adrenoceptors in vitro

Summary The enantiomers of 3-O-methyldobutamine, a metabolite of dobutamine, were evaluated for their - and -adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed ₁-adrenoceptor agonist activity in isolated guinea pig aorta. However, both enantiomers of 3-O-methyldobutamine were competitive ₁-adrenoceptor antagonists, with the (+)-enantiomer being approximately 10-fold more potent than the (-)-enantiomer as assessed either in guinea pig aorta or by displacement of ³H-prazosin binding from ₁-adrenoceptors in rat cerebral cortex. The ₁-adrenoceptor blocking activity of (+)-3-O-methyldobutamine was relatively potent and corresponded to a pA₂ of 7.33 in guinea pig aorta and a-log K _i of 7.72 in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed ₂-adrenoceptor agonist activity in field-stimulated guinea pig ileum. Although (+)-3-O-methyldobutamine weakly inhibited the twitch response in field-stimulated guinea pig ileum, the response was not blocked by the selective ₂-adrenoceptor antagonist, yohimbine, and was found to result from weak anticholinergic activity (pA₂=5.06). Neither enantiomer of 3-O-methyldobutamine possessed ₁-adrenoceptor agonist activity in guinea pig atria, however the (+)-enantiomer was a weak noncompetitive antagonist at ₁-adrenoceptors. In contrast, both enantiomers of 3-O-methyldobutamine were weak ₂-adrenoceptor agonists in rat uterus, however these weak effects were not highly stereoselective, which was also confirmed in radioligand binding studies. The results of the present study indicate that 3-O-methyldobutamine is a potent and highly selective ₁-adrenoceptor antagonist, with minimal activity at ₂-, ₁- and ₂-adrenoceptors. It is hypothesized that the potent ₁-adrenoceptor antagonist activity of 3-O-methyldobutamine, which resides predominantly in the (+)-enantiomer, may play a role in the hemodynamic effects of dobutamine, by contributing, in part, to the decrease in total peripheral vascular resistance observed following administration of dobutamine.     

Pharmacological evidence for the presence of functional β3-adrenoceptors in rat retinal blood vessels

The aim of this study was to examine whether stimulation of β₃-adrenoceptors dilates rat retinal blood vessels and how diabetes affects the vasodilator responses. Images of ocular fundus were captured with an original high-resolution digital fundus camera in vivo. The retinal vascular responses were evaluated by measuring diameter of retinal blood vessels contained in the digital images. Both systemic blood pressure and heart rate (HR) were continuously recorded. The β₃-adrenoceptor agonist CL316243 (0.3–10 μg/kg/min, i.v.) increased diameter of retinal arterioles (at 10 μg/kg/min, a 31% increase) and decreased mean blood pressure (at 10 μg/kg/min, a 21% decrease) in a dose-dependent manner. CL316243 produced a small but significant increase in HR (at 10 μg/kg/min, a 9% increase). Both SR59230A (1 mg/kg, i.v.) and L-748337 (50 μg/kg, i.v.), β₃-adrenoceptor antagonists, significantly prevented CL316243-induced retinal vasodilator responses. Similar observations were made with another β₃-adrenoceptor agonist, BRL37344. The β₂-adrenoceptor agonist salbutamol also increased diameter of retinal arterioles (at 10 μg/kg/min, a 43% increase), whereas the drug produced greater decrease in blood pressure (at 10 μg/kg/min, a 46% decrease) and increase in HR (at 10 μg/kg/min, a 16% increase), compared with β₃-adrenoceptor agonists. The retinal vasodilator responses to CL316243 and BRL37344 observed under blockade of β₁/β₂-adrenoceptors with propranolol (2 mg/kg, i.v. bolus followed by 100 μg/kg/min infusion) were unaffected 2 weeks after induction of diabetes by the combination of streptozotocin treatment and d-glucose feeding. On the other hand, the vasodilator responses to salbutamol of retinal arterioles were significantly reduced in diabetic rats. These results suggest that stimulation of β₃-adrenoceptors causes the vasodilation of retinal arterioles in vivo and the vasodilator responses are unaffected at the early stage of diabetes.     

MH-3: evidence for non-competitive antagonism towards the low-affinity site of β1-adrenoceptors

β-Adrenoceptor antagonists are important drugs for the treatment of cardiovascular diseases and some of those drugs also block the so-called low-affinity site of β₁-adrenoceptors although at much higher concentrations. This low-affinity site, also identified in vivo and in human tissue, may come into play under certain pathophysiological situations including arrhythmias. The aim of our study was to determine the potency of 14 compounds chemically related to bupranolol or bevantolol and two xanthone derivatives at the low-affinity site of the β₁-adrenoceptor. The potency of the compounds at the low- and high-affinity site of β₁-adrenoceptors (β_1L and β_1H; both increasing heart rate) was compared in the pithed rat. One compound was also studied in the isolated rat heart and its α₁-adrenolytic effect determined in the isolated rat mesenteric artery. In the pithed rat, four compounds blocked the β_1L-adrenoceptor at a ≥10-fold lower potency than the β_1H-adrenoceptor whereas the xanthone derivative (?)-MH-3 was equipotent. In the spontaneously beating right atrium (?)-MH-3 was a non-competitive antagonist of comparable potency at either receptor; its apparent pD′₂ value for the β_1L-adrenoceptor ranged from 5.6 to 6.4 under various conditions, including the Langendorff preparation. Its apparent pA₂ at the α₁-adrenoceptor in the mesenteric artery was 8.4. (?)-MH-3 is the first compound with virtually the same potency at the low- and high-affinity site of β₁-adrenoceptors in vivo; it appears to be a non-competitive antagonist at either site in vitro.     

Functional investigation of β-adrenoceptors in human isolated detrusor focusing on the novel selective β3-adrenoceptor agonist KUC-7322

This study aimed to characterize the β-adrenoceptor (β-AR) subtype mediating relaxation of isolated human bladder strips and to explore relaxation by the novel β3-AR-selective agonist KUC-7322 for its relaxant effect on the human isolated detrusor and for its effect on the carbachol (CCh)-induced contractile response. In two parallel studies, relaxation of isolated human bladder strips was tested for the β-AR agonists isoproterenol, clenbuterol, BRL 37344, and KUC-7322. For the isoproterenol and KUC-7322 responses, antagonism by CGP 20712A, ICI 118551, and SR59230A was determined. The potency and efficacy of the reference agonists for detrusor relaxation was in line with their known β3-AR activity. KUC-7322 relative to isoproterenol was a full agonist with a pEC(50) of 5.95 ± 0.09 and 5.92 ± 0.11 in the two studies. SR59230A exhibited antagonism of the expected potency against isoproterenol (apparent pK (B) 7.2) but not against KUC-7322. Neither isoproterenol nor KUC-7322 nor forskolin significantly attenuated CCh-induced contraction. These results suggest that KUC-7322 displays full agonistic activity in relaxing the human detrusor without inhibiting the contraction induced by cholinergic stimulation. These characteristics, if proven in vivo, may be beneficial for the treatment of overactive bladder, as increased bladder capacity with a negligible effect on voiding contractions may be anticipated.     

Presence of functionally active β-adrenoceptors in rat mast cells

Summary The correlation between the binding of a -adrenoceptor antagonist, (–)[³H]-dihydroalprenolol (DHAP), and the adrenergic inhibition of histamine release by acetylcholine and by compound 48/80 was studied with isolated purified rat mast cells and in rat mast cell crude membrane fractions.Acetylcholine-evoked histamine release was inhibited by catecholamines, in the order isoprenaline > adrenaline > noradrenaline. Pretreatment of cells with (–)alprenolol antagonized the inhibitory effect of isoprenaline on acetylcholine-induced histamine release.40/80-evoked histamine release was bocked by isoprenaline at significantly higher concentrations than those required to inhibit cholinergic histamine release. The inhibitory effect of isoprenaline was equally antagonized by preincubating mast cells with (–)alprenolol.Specific binding sites for DHAP have been demonstrated in rat mast cell membranes. The specific binding of DHAP was inhibited by adrenoceptor agonists and antagonists according to the stereospecificity of these compounds.A close correlation between the binding-inhibitory potency of various adrenergic compounds and the data obtained in the pharmacological experiments was found, thus indicating the presence of -adrenoceptors in rat mast cells.     

The β-adrenoceptors and catecholamine levels in lead poisoned rats

To investigate β-adrenoceptor dysfunction upon exposure to lead, we measured (a) β-adrenoceptor density in brain, heart, blood vessels and lymphocytes and (b) plasma catecholamine levels in rats with lead poisoning. Wistar rats were given drinking water containing lead acetate (2% w/v) for a period of 60 days. The radioligand [(125)I]iodocyanopindolol was used for determining the density of β-adrenoceptors in membrane fragments in vitro and a high performance liquid chromatography (HPLC) for measuring plasma catecholamine levels. Plasma norepinephrine levels were found to be significantly higher in lead-exposed rats than in control animals (4.69 ± 0.58 μg/l vs. 3.67 ± 0.53 μg/l, p < 0.01). In lead-exposed animals the density of β-adrenoceptors in brain (36%), heart (68%), blood vessels (57%) and lymphocytes (48%) was significantly less than in controls (p < 0.001), whereas the K(d) did not vary between the two groups. We have found that β-adrenoceptor dysfunction in lead-poisoned rats was brought about by a decline in β-adrenoceptor density.     

Celiprolol exerts microvascular dilatation by activation of β2-adrenoceptors

Summary In order to clarify the question whether the ₁-selective adrenoceptor antagonist celiprolol possesses vasodilating properties, isolated vascular networks were perfused with increasing concentrations of celiprolol (in a cumulative manner) ranging from 10^–8 to 10^–4 mol/l. The study was carried out using the isolated mesenteric vascular bed of the guinea pig mesenterium coli. Vascular diameters of four different vascular regions [vessels classified as G1 (585 ± 30 m), G2 (403 ± 25 m), G3 (282 ± 27 m) and G4 (197 ± 13 m)] were assessed by means of microscopic videoangiometry. Perfusion with celiprolol resulted in concentration dependent vasodilation which was more pronounced in G3 and G4 vessels. In addition, cumulative concentration-response curves were determined from responses obtained in the presence of 10^–8, 10^–7, 10^–6 and 10^–4 mol/l ICI 118,551 (a highly selective adrenoceptor antagonist). In the presence of ICI 118,551 at concentrations 10^–6 mol/l, no celiprolol response could be observed. Lower concentrations of ICI 118,551 shifted the celiprolol concentration-response curve to the right in a concentration-dependent manner. Therefore, it is concluded (a) that celiprolol has a vasodilating effect, (b) that this vasodilation is produced by stimulation of ₂-adrenoceptors and (c) that the vasodilating effect is more pronounced in smaller than in larger vessels (G3, G4 vs G1, G2). Send offprint requests to S. Dhein at the above address     

Evidence that (+)-bupranolol interacts directly with myocardial β-adrenoceptors

Summary Melting points measured with the capillary method were 150.5°C, 150.5°C and 224.0°C for hydrochlorides of (+)-bupranolol, (–)-bupranolol and (±)-bupranolol, respectively. The large difference in melting points of 73.5°C prompted us to determine possible contaminations of (+)-bupranolol with traces of (–)-bupranolol using differential scanning calorimetry. We detected as little as 0.001% (–)-bupranolol in a standard mixture of (+)-bupranolol and (–)-bupranolol. A batch of (+)-bupranolol not measurably contaminated with (–)-bupranolol (optically purity>99.999%) was used in pharmacological and biochemical assays.The affinities of (–)-bupranolol and (+)-bupranolol were determined functionally by the blockade of isoprenaline stimulation of spontaneously beating rat right atria and electrically driven kitten papillary muscles; and directly by the inhibition of binding of ³H-(–)-propranolol to kitten ventricle membrane particles. In all 3 systems the enantiomeric (–)/(+) affinity ratio was 50–120 for bupranolol. These experiments prove that (+)-bupranolol itself binds to the -adrenoceptors of mammalian myocardium.     

A dose-ranging study to evaluate the β1-adrenoceptor selectivity of bisoprolol

Summary A dose-ranging study was performed to compare the ₁-adrenoceptor selectivity of bisoprolol with that of atenolol and nadolol. Seven normal subjects (mean age 26 y) were given single oral doses of bisoprolol 5 mg (B5), 10 mg (B10), 20 mg (B20); atenolol 50 mg (A50), 100 mg (A100); nadolol 40 mg (N40); and placebo (PL), in a single blind randomised cross-over design. ₂-adrenoceptor responses were assessed by attenuation of finger tremor and cardiovascular responses to graded isoprenaline infusions. Dose-response curves were constructed, and doses of isoprenaline required to increase finger tremor by 100% (IT₁₀₀), heart rate by 25 beats/min (IH₂₅), SBP by 25 mm Hg (IS₂₅), cardiac output by 35% (IC₃₅), and decrease DBP by 10 mm Hg (ID₁₀), after each treatment were calculated. These indices were compared with placebo response and expressed as dose-ratios. Exercise heart rate (EHR) was used to assess ₁-adrenoceptor blockade.There were dose-related increases in plasma concentrations of bisoprolol and atenolol. Reduction of EHR was significantly less with B5 (16.8%) in comparison with all other treatments: B10 21.9%, B20 23.1%; A50 22.5%, A100 22.6%; N40 22.9%. There were small but significant reductions in isoprenaline-induced tachycardia with bisoprolol and atenolol, although mean dose-ratios were considerably less in comparison with N40 (IH₂₅ dose-ratios): B5 2.55, B10 3.18, B20 3.93, A50 2.91, A100 4.89, N40 17.23. There were similar patterns for the other isoprenaline responses.These results show that conventional doses of bisoprolol (10 mg) and atenolol (50 mg) produced equal antagonism of ₁ and ₂-adrenoceptors, and therefore possess equal degrees of ₁-adrenoceptor selectivity. Increasing doses of bisoprolol and atenolol were associated with partial loss of selective ₁-adrenoceptor blockade, although antagonism of ₂-adrenoceptors was significantly less compared with the effects of nadolol.     

Do gene polymorphisms alone or in combination affect the function of human β3-adrenoceptors?

Background and purpose

β₃-Adrenoceptors mediate many important physiological functions, for example, in the urinary bladder. The corresponding gene is polymorphic, and the W64R (Trp64Arg) single nucleotide polymorphism has been associated with disease states such as obesity, type 2 diabetes and bladder dysfunction. While these clinical data suggest that the 64R variant is hypofunctional, previous in vitro studies in which this variant was generated by site-directed mutagenesis and subsequent transfection have not consistently confirmed this.

Experimental approach

We transfected the wild-type human β₃-adrenoceptor and the 64R variant and also the more recently discovered 265M and 306F variants as well as 64R/265M and 64R/306F double mutants into human embryonic kidney cells and selected clones expressing the receptors at a density of about 100 fmol mg protein⁻¹. Receptor activation was measured by cAMP accumulation and ligand affinity by radioligand binding. Desensitisation was assessed as alterations of cAMP responses after prolonged agonist treatment.

Key results

Neither mutated receptor exhibited alterations in efficacy or potency for cAMP accumulation for any of five agonists (isoprenaline, noradrenaline, YM 178, FK 4664, CGP 12 177). In competition binding studies, the mutations did not affect the ability of any agonist to bind to the receptor. Wild-type receptors and the 64R variant exhibited similar isoprenaline-induced functional desensitization during a 24 h treatment.

Conclusions and implications

None of the polymorphisms tested here significantly altered the interaction of isoprenaline, noradrenaline, YM 178, FK 4664 or CGP 12 177 with the human β₃-adrenoceptor when expressed at near physiological levels in a human cell line.