A Systematic Review of Inverse Agonism at Adrenoceptor Subtypes.

As many, if not most, ligands at G protein-coupled receptor antagonists are inverse agonists, we systematically reviewed inverse agonism at the nine adrenoceptor subtypes. Except for ß3-adrenoceptors, inverse agonism has been reported for each of the adrenoceptor subtypes, most often for ß2-adrenoceptors, including endogenously expressed receptors in human tissues. As with other receptors, the detection and degree of inverse agonism depend on the cells and tissues under investigation, i.e., they are greatest when the model has a high intrinsic tone/constitutive activity for the response being studied. Accordingly, they may differ between parts of a tissue, for instance, atria vs. ventricles of the heart, and within a cell type, between cellular responses. The basal tone of endogenously expressed receptors is often low, leading to less consistent detection and a lesser extent of observed inverse agonism. Extent inverse agonism depends on specific molecular properties of a compound, but inverse agonism appears to be more common in certain chemical classes. While inverse agonism is a fascinating facet in attempts to mechanistically understand observed drug effects, we are skeptical whether an a priori definition of the extent of inverse agonism in the target product profile of a developmental candidate is a meaningful option in drug discovery and development.

Desensitization of cAMP Accumulation via Human ß3-Adrenoceptors Expressed in Human Embryonic Kidney Cells by Full, Partial, and Biased Agonists.

ß3-Adrenoceptors couple not only to cAMP formation but, at least in some cell types, also to alternative signaling pathways such as phosphorylation of extracellular signal-regulated kinase (ERK). ß3-Adrenoceptor agonists are used in long-term symptomatic treatment of the overactive bladder syndrome; it is only poorly understood which signaling pathway mediates the clinical response and whether it undergoes agonist-induced desensitization. Therefore, we used human embryonic kidney cells stably transfected with human ß3-adrenoceptors to compare coupling of ligands with various degrees of efficacy, including biased agonists, to cAMP formation and ERK phosphorylation, particularly regarding desensitization. Ligands stimulated cAMP formation with a numerical rank order of isoprenaline ≥ L 755,507 ≥ CL 316,243 > solabegron > SR 59,230 > L 748,337. Except for the weakest agonist, L 748,337, pretreatment with any ligand reduced cAMP responses to freshly added isoprenaline or forskolin to a similar extent. On the other hand, we were unable to detect ERK phosphorylation despite testing a wide variation of conditions. We conclude that a minor degree of efficacy for cAMP formation may be sufficient to induced full desensitization of that response. Transfected human embryonic kidney cells are not suitable to study desensitization of ERK phosphorylation by ß3-adrenoceptor stimulation.

Muscarinic type-1 receptors contribute to IK,ACh in human atrial cardiomyocytes and are upregulated in patients with chronic atrial fibrillation.

BACKGROUND: Basal and acetylcholine-gated inward-rectifier K+-currents (IK1 and IK,ACh, respectively) are altered in atrial fibrillation (AF). Gi-protein-coupled muscarinic (M) receptors type-2 are considered the predominant receptors activating IK,ACh. Although a role for Gq-coupled non-M2-receptor subtypes has been suggested, the precise regulation of IK,ACh by multiple M-receptor subtypes in the human atrium is unknown. Here, we investigated M1-receptor-mediated IK,ACh regulation and its remodeling in chronic AF (cAF). METHODS AND RESULTS: M1-receptor mRNA and protein abundance were increased in atrial cardiomyocyte fractions and atrial homogenates from cAF patients, whereas M2-receptor levels were unchanged. The regulation of IK,ACh by M1-receptors was investigated in right-atrial cardiomyocytes using two applications of the M-receptor agonist carbachol (CCh, 2µM), with pharmacological interventions during the second application. CCh application produced a rapid current increase (Peak-IK,ACh), which declined to a quasi-steady-state level (Qss-IK,ACh). In sinus rhythm (Ctl) the selective M1-receptor antagonists pirenzepine (10nM) and muscarinic toxin-7 (MT-7, 10nM) significantly inhibited CCh-activated Peak-IK,ACh, whereas in cAF they significantly reduced both Peak- and Qss-IK,ACh, with no effects on basal inward-rectifier currents in either group. Conversely, the selective M1-receptor agonist McN-A-343 (100µM) induced a current similar to the CCh-activated current in Ctl atrial cardiomyocytes pretreated with pertussis toxin to inhibit M2-receptor-mediated Gi-protein signaling, which was abolished by MT-7. Computational modeling indicated that M1- and M2-receptors redundantly activate IK,ACh to abbreviate APD, albeit with predominant effects of M2-receptors. CONCLUSION: Our data suggest that Gq-coupled M1-receptors also regulate human atrial IK,ACh and that their relative contribution to IK,ACh activation is increased in cAF patients. We provide novel insights about the role of non-M2-receptors in human atrial cardiomyocytes, which may have important implications for understanding AF pathophysiology.

The new radioligand [(3)H]-L 748,337 differentially labels human and rat ß3-adrenoceptors.

As no suitable radioligand exists for the detection of ß3-adrenoceptors, we have explored the radioligand binding properties of a tritiated version of the selective ß3-adrenoceptor antagonist L 748,337. Kinetic and equilibrium saturation and competition binding experiments were performed with [(3)H]-L 748,337 on membrane fractions of HEK and CHO cells stably transfected with human and rat ß-adrenoceptor subtypes. Based on both association/dissociation kinetic and equilibrium saturation binding studies in transfected HEK cells, [(3)H]-L 748,337 exhibited an affinity of approximately 2 nM for human ß3-adrenoceptors. Competition studies with agonists and subtype-selective antagonists validated its binding to ß3-adrenoceptors. In CHO cells transfected with human ß3-adrenoceptors similar saturable high-affinity of [(3)H]-L 748,337 was observed. While some isoprenaline-sensitive [(3)H]-L 748,337 binding was also observed in CHO cells transfected with human ß1- or ß2-adrenoceptors, this was not saturable in a similar concentration range and/or not sensitive to the antagonists propranolol and SR 59,230, indicating that it did not primarily involve ß-adrenoceptors. In CHO cells transfected with rat ß3-adrenoceptors [(3)H]-L 748,337 exhibited a considerably lower affinity than with the human subtype (12-95 nM). Low affinity for the rat ß3-adrenoceptor was also found with unlabelled L 748,337 in rat bladder strip relaxation experiments. We conclude that L 748,337 apparently has lower affinity for the rat than the human ß3-adrenoceptors and that [(3)H]-L 748,337 can bind to a low-affinity site distinct from the orthosteric pocket of ß-adrenoceptors. Nevertheless, [(3)H]-L 748,337 appears to be the most promising radioligand for the selective labelling of human ß3-adrenoceptors reported to date.

Agonist-induced desensitization of human ß3-adrenoceptors expressed in human embryonic kidney cells.

ß3-Adrenoceptors are resistant to agonist-induced desensitization in some cell types but susceptible in others including transfected human embryonic kidney (HEK) cells. Therefore, we have studied cellular and molecular changes involved in agonist-induced ß3-adrenoceptor desensitization in HEK cells. Cells were treated with isoprenaline or forskolin, and following wash-out, cyclic adenosine monophosphate (cAMP) accumulation in response to freshly added agonist was quantified. Receptor and G protein expression were quantified by radioligand binding and immunoblot experiments, respectively. Treatment with isoprenaline induced a concentration- and time-dependent desensitization of cAMP accumulation in response to freshly added isoprenaline. This functional desensitization primarily consisted of reduced maximum responses with little change of agonist potency. Maximum desensitization was achieved by pre-treatment with 10 µM isoprenaline for 24 h. It was not accompanied by changes in ß3-adrenoceptor density as assessed in saturation radioligand-binding studies. The desensitization was associated with a small reduction in immunoreactivity for α-subunits for Gs and Gi1, whereas that for Gi2, Gi3, and Gq/11 was not significantly altered. In cells treated with pertussis toxin, isoprenaline-induced cAMP accumulation as well as desensitization by isoprenaline pre-treatment remained unchanged. Isoprenaline pre-treatment also reduced forskolin-induced cAMP accumulation; conversely, pre-treatment with forskolin caused a similar desensitization of isoprenaline-induced cAMP accumulation. We conclude that agonist-induced ß3-adrenoceptor desensitization in HEK cells does not involve reduced receptor numbers and small, if any, reduction of Gs expression; changes at the level of adenylyl cyclase function can fully explain this desensitization.

The muscarinic receptor antagonist propiverine exhibits α(1)-adrenoceptor antagonism in human prostate and porcine trigonum.

PURPOSE: Combination therapy of male lower urinary tract symptoms with α(1)-adrenoceptor and muscarinic receptor antagonists attracts increasing interest. Propiverine is a muscarinic receptor antagonist possessing additional properties, i.e., block of L-type Ca(2+) channels. Here, we have investigated whether propiverine and its metabolites can additionally antagonize α(1)-adrenoceptors. METHODS: Human prostate and porcine trigone muscle strips were used to explore inhibition of α(1)-adrenoceptor-mediated contractile responses. Chinese hamster ovary (CHO) cells expressing cloned human α(1)-adrenoceptors were used to determine direct interactions with the receptor in radioligand binding and intracellular Ca(2+) elevation assays. RESULTS: Propiverine concentration-dependently reversed contraction of human prostate pre-contracted with 10 µM phenylephrine (-log IC(50) [M] 4.43 ± 0.08). Similar inhibition was observed in porcine trigone (-log IC(50) 5.01 ± 0.05), and in additional experiments consisted mainly of reduced maximum phenylephrine responses. At concentrations ≥1 µM, the propiverine metabolite M-14 also relaxed phenylephrine pre-contracted trigone strips, whereas metabolites M-5 and M-6 were ineffective. In radioligand binding experiments, propiverine and M-14 exhibited similar affinity for the three α(1)-adrenoceptor subtypes with -log K (i) [M] values ranging from 4.72 to 4.94, whereas the M-5 and M-6 did not affect [(3)H]-prazosin binding. In CHO cells, propiverine inhibited α(1)-adrenoceptor-mediated Ca(2+) elevations with similar potency as radioligand binding, again mainly by reducing maximum responses. CONCLUSIONS: In contrast to other muscarinic receptor antagonists, propiverine exerts additional L-type Ca(2+)-channel blocking and α(1)-adrenoceptor antagonist effects. It remains to be determined clinically, how these additional properties contribute to the clinical effects of propiverine, particularly in male voiding dysfunction.

Lack of evidence that nebivolol is a ß3-adrenoceptor agonist.

Nebivolol is a selective ß1-adrenoceptor antagonist which, in addition, displays endothelium-dependent vasodilating properties in humans and other species. ß3-adrenoceptors have been proposed to be a molecular target of nebivolol-induced vasodilatation. Therefore, we have investigated possible ß3-adrenoceptor agonism by nebivolol for relaxation of the human and rat urinary bladder (prototypical ß3-adrenoceptor-mediated responses) as well as for cAMP accumulation in Chinese hamster ovary cells stably transfected with the human ß-adrenoceptor subtypes. Nebivolol concentration-dependently relaxed both human and rat isolated urinary bladder strips but with low potency, similar to that reported for vasodilatation. However, nebivolol-induced bladder relaxation in either species was not inhibited by the ß3-adrenoceptor antagonist SR 59,230A (10µM), although this compound inhibited the isoprenaline-induced relaxation with the expected potency. In radioligand binding studies nebivolol had lower affinity for human ß3-adrenoceptors than the other two ß-adrenoceptor subtypes, but this low affinity was in line with its potency to relax the bladder or isolated blood vessels. In functional studies nebivolol even in high concentrations did not stimulate cAMP formation via any of the three cloned human ß-adrenoceptors or in rat bladder smooth muscle cells. Taken together these data demonstrate that nebivolol can relax not only vascular but also urinary bladder smooth muscle. However, they do not support the hypothesis that nebivolol is an agonist at cloned human ß3-adrenoceptors or in rat or human urinary bladder.

WITHDRAWN: Is nebivolol a beta(3)-adrenoceptor agonist?

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Comparison of three radioligands for the labelling of human beta-adrenoceptor subtypes.

We have compared the ability of three radioligands, [(125)I]-cyanopindolol, [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, to label the three human beta-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [(3)H]-CGP 12,177 had very similar affinity for beta(1)- and beta(2)-adrenoceptors (about 40 pM), [(125)I]-cyanopindolol and [(3)H]-dihydroalprenolol had 4- to 6-fold higher affinity for beta(2)- as compared to beta(1)-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [(125)I]-cyanopindolol at beta(3)-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The beta(3)-adrenoceptor affinity of [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label beta(3)-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [(125)I]-cyanopindolol appears the least unsuitable to label beta(3)-adrenoceptors. There is a need for high-affinity radioligands which are either selective for beta(3)-adrenoceptors or reasonably non-selective among all three beta-adrenoceptor subtypes.

Effects of ageing on muscarinic receptor subtypes and function in rat urinary bladder.

We compared the density and function of M2 and M3 muscarinic acetylcholine receptor subtypes in the urinary bladder of young adult (3 months) and old (23 months) male Wistar rats. Old rats had a reduced density of muscarinic receptors (96+/-10 vs. 156+/-21 fmol/mg protein), but competition experiments with the M3-selective darifenacin did not indicate alterations in the relative roles of M2 and M3 receptors, with the former being more abundant. The amount of immunodetectable alpha-subunits of various G-proteins potentially linked to muscarinic receptor function was unchanged. The potency of carbachol to contract bladder strips was also unaltered; its maximum effects as well as those of a single KCl concentration were unchanged if raw data or those corrected for strip length were analysed, but somewhat reduced when those corrected for strip weight were analysed. Antagonistic effects of atropine, the M2-selective Ro 320-6206 and the M3-selective darifenacin were unchanged. Agonistic effects of the M3-sparing agonist 4-(2-oxo-2,3-dihydro-benzoimidazol-1-yl)-[1,4']bipiperidinyl-1'-carboxylic acid ethyl ester were similarly poor in young and old rats. Additional experiments were concomitantly performed in submandibular glands from the same animals. While total muscarinic receptor density in submandibular glands was not significantly affected by age (56+/-5 vs. 61+/-4 fmol/mg protein), the relative contribution of M3 receptors significantly declined from 68+/-3% to 57+/-2% based upon darifenacin competition curves. We conclude that aged Wistar rats express fewer muscarinic receptors in their urinary bladder, but there is no change in the relative abundance of M2 and M3 receptors; this is accompanied by only minor if any alterations in receptor responsiveness. In contrast, submandibular gland expresses similar receptor numbers in young and old rats, but slightly fewer M3 receptors in old animals.