Isabelle Van Liefde

Regional Distribution of α2A-and α2B-Adrenoceptor Subtypes in Postmortem Human Brain

Abstract: The newly available and highly selective radiolabeled antagonist [3H]RX 821002 was used to examine the distribution of α2 adrenoceptors in human brain. High densities of α2 adrenoceptors were found in the hippocampus, frontal cortex, thalamus, amygdala, pons, and medulla oblongata. Intermediate densities were observed in the striatum (nucleus accumbens, nucleus caudatus, and putamen), globus pallidus, and substantia nigra. The KD values for [3H]RX 821002 were similar in all regions (ranging from 2.8 to 7.5 nM). On the basis of their different affinities for prazosin and oxymetazoline, the α2 adrenoceptors have been divided into α2A and α2B subtypes. To examine the α2A/α2B‐adrenoreceptor ratio in the different brain regions, we performed oxymetazoline and prazosin/[3H]RX 821002 competition binding experiments. In frontal cortex membranes, the competition curves with prazosin were steep, indicating a single class of binding sites, whereas the competition curves with oxymetazoline were shallow and fitted by computer best to a two‐site model. However, in the presence of GTP, the high‐affinity sites for oxymetazoline were partially converted into low‐affinity sites, indicating that this agonist interacts with high‐ and low‐affinity states of the α2 adrenoceptors. This implies that oxymetazoline is not very suitable for discriminating the α2A‐and α2B‐receptor subtypes in radioligand binding studies. Therefore, prazosin/[3H]RX 821002 competition binding experiments were used to investigate the distribution of the α2‐adrenoceptor subtypes in human brain. The α2A‐receptor subtype was detected in all brain regions examined. In contrast, α2B receptors were only observed in striatum and globus pallidus.

Long-lasting angiotensin type 1 receptor binding and protection by candesartan: comparison with other biphenyl-tetrazole sartans.

Background The ability of biphenyl-tetrazole angiotensin type 1 (AT1) receptor antagonists (BTsartans) to block angiotensin II (Ang II)-mediated responses has been extensively investigated in vascular tissues and, more recently, in cell lines expressing the human AT1-receptor. When pre-incubated, BTsartans acted surmountably (shifting the Ang II concentration–response curve to the right) or insurmountably (also decreasing the maximal response). It was shown that their insurmountable behaviour is due to the formation of tight, long-lasting complexes with the receptor. Partial insurmountable antagonism is due to the co-existence of tight and loose complexes. The proportion of insurmountable antagonism, the potency and the dissociation rate of the BTsartans decreases in the order: candesartan > EXP3174 (losartans active metabolite) > valsartan > irbesartan ≫ losartan. Objective It is of interest to explore how tight AT1-receptor binding of BTsartans such as candesartan might contribute to their long-lasting clinical effect. Methods Computer-assisted simulations (COPASI program) were performed to follow the receptor-occupation and protection by different antagonists as a function of time. Free antagonist concentrations were allowed to decrease exponentially with time. Results The simulations suggest that slow dissociation does not tangibly prolong receptor occupancy if the free antagonist is eliminated at a slower pace (as is the case for BTsartans). Yet when surmountable and insurmountable antagonists occupy the same amount of receptors, insurmountable antagonists offer appreciably better protection against fluctuations in natural messenger concentration. Conclusion Slow receptor dissociation and slow antagonist elimination are likely to act in synergy to produce long-lasting receptor protection.

No functional atypical β-adrenergic receptors in human omental adipocytes

Abstract Isoproterenol stimulates lipolysis in human omental adipocytes with an EC50 (concentration at which an agonist produces half-maximal stimulation) of 120 nM. CGP12177 (dl-4-3 [(1,1-dimethylethyl) amino]- 2-hydroxylpropoxy]1,3- dihydro-2H-benzimidazol-2-one hydrochloride), a potent β 1 -β 2 -adrenergic receptor (AR) antagonist but being an agonist for atypical β-AR, fails to stimulate lipolysis in these cells, even at a concentration as high as 0.1 mM. Since CGP12177 is a partial agonist, its failure to stimulate lipolysis may result from a poor stimulus-response coupling, so that it can not be excluded that atypical β-AR are actually present and even functional in these cells. To evaluate this hypothesis, we estimated the potency of CGP12177 to inhibit the isoproterenol-stimulated lipolysis. This inhibition curve reflects a single class of sites and the IC50- value (concentration at which an antagonist produces half-maximal inhibition) of CGP12177 (3.8 nM) is in good agreement with what should be expected for β1-AR/β2-AR. Moreover, metoprolol and atenolol, two β 1 - AR- selective antagonists, shift the isoproterenol dose-response curve to the right with high potency as well. These potencies are similar to the ones found for β1-AR in the human heart but appreciably higher than those which should be expected for atypical β-AR. The present study suggest that atypical β-AR are not functional in human omental adipocytes.

Radioligand dissociation measurements: potential interference of rebinding and allosteric mechanisms and physiological relevance of the biological model systems.

Introduction: In many situations, optimal drug therapy requires continuing high levels of target occupancy and this notion has led pharmacologists to focus their attention on the rate by which drug candidates dissociate from their target. To this end, radioligand dissociation experiments are often carried out on in vitro models, such as intact cells and the membranes thereof, but the interpretation of the collected data is sometimes ambiguous. Areas covered: Pharmacodynamics is concerned about what the drug does to the target and, in this respect, allosteric modulation constitutes a quite novel, very promising research topic. The ability of unlabeled drugs to accelerate radioligand dissociation is often advocated to be a hallmark of such mechanism. Yet, the present computerized simulations reveal that competitive drugs produce the same effect by preventing hindered diffusion- and “forced proximity”-related rebinding of the radioligand. Herein, the authors provide hints to discern among those mechanisms. Expert opinion: A critical, but constructive appraisal of radioligand dissociation binding data leads to the viewpoint that, from a physiological perspective, dissociation from confluent target-expressing plated cells, when in a naïve medium, is likely to provide the most pertinent insight in that ligands in vivo residence time.

Isoproterenol and selective agonists stimulate similar atypical β-adrenoceptors in rat adipocytes

We have demonstrated previously that (-)isoproterenol triggers lipolysis in rat epididymal fat cells by stimulating both classical (beta 1, beta 2) and atypical beta-adrenoceptors. The contribution of the classical beta-adrenoceptors can be blocked by addition of 3 nM CGP12177(di-4-3[(1,1-dimethylethyl)amino]-(2-hydroxylpropoxy )1,3-dihydro-2H-benzimidazol-2-one hydrochloride). At higher concentrations, CGP12177 triggers lipolysis also, but by stimulating atypical beta-adrenoceptors only. To find out whether (-)isoproterenol and CGP12177 stimulate similar atypical beta-adrenoceptors, we compared their interaction with recognised beta 3-adrenoceptor antagonists: CGP20712 (1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl- 4-trifluoromethyl-2-imidazolyl)phenoxy]-propan-2-ol) (beta 1-selective), ICI118551 [erythro-1-(7-methylindan-4-yloxy)-3- (isopropylamine)-butan-2-ol] (beta 2-selective) and the stereoisomers as well as the racemic mixture of propranolol (non-beta 1/beta 2-subtype selective) and of metoprolol (beta 1-selective). There was a highly significant relationship (r = 0.93) between the potencies of these antagonists for inhibiting the lipolytic response to (-)isoproterenol (in the absence of classical beta-adrenoceptor stimulation) and CGP12177. In both cases, propranolol and metoprolol showed also the same degree of stereoselectivity. These findings suggest that (-)isoproterenol and CGP12177 stimulate the same type and/or form of atypical beta-adrenoceptors in rat epididymal adipocytes.

α2A Adrenoceptors and non-adrenergic idazoxan binding sites in calf brain and retina are distinct from those in human brain

alpha 2 Adrenoceptors in membrane preparations of human and calf frontal cortex and of calf retina can be labelled by the antagonists [3H]idazoxan, [3H]rauwolscine and [3H]RX 821002. Present and previous data indicate that [3H]idazoxan possesses the highest affinity for the alpha 2 adrenoceptors in the calf tissues, whereas [3H]rauwolscine displays the highest affinity for those in the human frontal cortex. Competition binding experiments with adrenergic and serotonergic drugs further support the notion that the alpha 2 adrenoceptors in calf frontal cortex and retina are similar, but distinct from the receptors in human frontal cortex. The alpha 2 adrenoceptors in the three investigated tissues display low affinity for the antagonist prazosin, which suggests that they all belong to the alpha 2A subclass. The competition binding curves of the alpha 2A adrenoceptor subtype-selective agonist oxymetazoline are shallow, but undergo a rightward shift and steepening in the presence of GTP. The shallow curves can therefore be attributed to the coupling of the alpha 2 adrenoceptors to G proteins. The different binding characteristics of the alpha 2A adrenoceptors from the investigated human and bovine tissues are likely to reflect species-related differences in protein structure. [3H]Idazoxan binds also to non-adrenergic sites in membrane preparations from the three tissues. However, the affinity of [3H]idazoxan for these sites in calf cortex and retina is appreciably lower than for those in human cortex. The species-related differences of the non-adrenergic idazoxan binding sites may be due to differences in protein structure or even to differences in gene-product.

Radioligand binding to intact cells as a tool for extended drug screening in a representative physiological context.

Radioligand binding assays on intact cells offer distinct advantages to those on membrane suspensions. Major pharmacological properties like drug affinity and binding kinetics are more physiologically relevant. Complex mechanisms can be studied with a wider choice of experimental approaches and so provide insights into induced-fit type binding, receptor internalisation and even into pharmacomicrokinetic phenomena like drug rebinding and partitioning into the membrane. Hence, intact cell binding constitutes a valuable addition to the pharmacologists toolbox.