Staffan Uhlén

Delineation of rat kidney α2A- and α2B-adrenoeeptors with [3H]RX821002 radiollgand binding: computer modelling reveals that guanfacine is an α2A-selective compound

We developed a method for the simultaneous determination of drug affinity constants for rat alpha 2A- and alpha 2B-adrenoceptor subtypes by using [3H]RX821002 radioligand binding in the kidney. Three competition curves were obtained for each drug: one for the test compound in the absence of ARC 239 (a drug found to have 108-fold higher affinity for alpha 2B- than for alpha 2A-adrenoceptors), one in the presence of ARC 239, and one for ARC 239. It is possible to determine the Kds of a tested drug for both alpha 2A- and alpha 2B-adrenoceptors by simultaneous computer modelling because of the increased constraint in the calculations given by the inclusion of ARC 239 into the assay. Using this approach, we found guanfacine and oxymetazoline to be highly alpha 2A-selective. The most alpha 2B-selective were ARC 239, prazosin and corynanthine. A number of other drugs, for example UK-14,304, rilmenidine and clonidine, were non-selective or showed minor selectivity for alpha 2A- or alpha 2B-adrenoceptors. Moreover, using Monte Carlo simulations, we showed that the three-curve method gives more accurate estimates of drug binding constants for assays when two receptor sites are present than methods analysing only one competition curve.

Further Characterization of the Guinea Pig Cerebral Cortex Idazoxan Receptor: Solubilization, Distinction from the Imidazole Site, and Demonstration of Cirazoline as an Idazoxan Receptor-Selective Drug

Abstract: We have demonstrated previously that [3H]idazoxan, besides being able to bind to α2‐adrenergic receptors, may also bind to a nonadrenergic idazoxan‐receptor site with high affinity. The idazoxan receptor is tightly bound to cellular membranes, and we have now developed a method to solubilize it from the guinea pig cerebral cortex by using the detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS). The CHAPS‐solubilized receptor retains its binding properties for drugs: the membrane‐bound, as well as the solubilized, idazcxan receptor shows high affinities for a number of imidazolines (cirazoline, idazoxan, tolazoline, naphazoline, tramazoline, clonidine, and oxymetazoline), some imidazoles (medetomidine, detomidine), and guanfacine. By contrast, catecholamines (adrenaline, noradrenaline, isoprenaline, and dopamine) and a number of other neurotransmitters and neuromodulators (serotonin, histamine, glutamic acid, γ‐aminobutyric acid, glycine, and adenosine) show negligible affinities for the idazoxan receptor. Moreover, the idazoxan receptor shows grossly different binding properties for histamine, cimetidine, and imidazole‐4‐acetic acid compared to what has been described for the nonadrenergic imidazole site labeled by p‐[3H]aminoclonidine, indicating that the two receptor sites are distinct. Radioligand binding data further indicate that cirazoline is an idazoxan receptor‐selective drug (KD= 1 nM) showing a 50–210‐fold selectivity for binding to the idazoxan receptor when compared to α2‐adrenergic receptors and an about 500‐fold selectivity when compared to α1‐adrenergic receptors. We have also reviewed the literature for possible nonadrenergic actions of idazoxan and cirazoline, and we suggest that idazoxan receptors might be involved in the control of prolactin release from the pituitary.

[3H]‐MK 912 binding delineates two α2‐adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d α2‐adrenoceptor

1 Simultaneous computer modelling of control and guanfacine‐masked [3H]‐MK 912 saturation curves as well as guanfacine competition curves revealed that the drugs bound to two α2‐adrenoceptor subtypes in the rat cerebral cortex with very different selectivities. These α2‐adrenoceptor subtypes were designated α2A and α2C. The Kd value of [3H]‐MK 912 for the α2A‐subtype was 1.77 nm and for the α2C‐subtype 0.075 nm; the receptor sites showing capacities 296 and 33 fmol mg−1 protein, respectively. The Kds of guanfacine were 19.9 and 344 nm, respectively. 2 Binding constants of 26 compounds for the two rat cerebral cortex α2‐adrenoceptor subtypes were determined by simultaneous computer modelling of control and guanfacine‐masked drug competition curves as well as plain guanfacine competition curves using [3H]‐MK912 as labelled ligand (i.e. a ‘3‐curve assay’). Of the tested drugs WB4101, corynanthine, rauwolscine, yohimbine, ARC 239 and prazosin were found to be clearly α2C‐selective with selectivities ranging from 16 to 30 fold whereas guanfacine, oxymetazoline, BRL 44408 and BRL 41992 were found to be α2A‐selective with selectivities ranging from 9 to 22 fold. 3 The Kds of compounds obtained for the cerebral cortex α2C‐adrenoceptors showed an almost 1:1 correlation with the corresponding Kds for α2‐adrenoceptors expressed by the pA2d‐gene (the rat ‘α2‐C4’ adrenoceptor) in CHO‐cells. The cerebral cortex α2A‐adrenoceptors did not correlate well with the pA2d α2‐adrenoceptor Kds. 4 In the rat spinal cord [3H]‐MK 912 bound to α2A‐ and α2C‐adrenoceptor sites with similar affinities as in the cerebral cortex and with densities 172 and 7.4 fmol mg−1 protein, respectively. Drug affinities for some compounds showing major selectivity for α2A‐ and α2C‐adrenoceptors were fully compatible with the notion that the spinal cord sites were α2A‐ and α2C‐adrenoceptors.

Medetomidine stereoisomers delineate two closely related subtypes of idazoxan (imidazoline) I-receptors in the guinea-pig

A number of imidazoline, imidazole and guanidinium compounds and other drugs were compared for their ability to bind to non-adrenergic idazoxan (imidazoline) I-receptors in particulate guinea pig cerebral cortex and ileum smooth muscle fractions. Radioligand binding with [3H]idazoxan was used for the experiments. Computer modelling of the binding data gave dissociation constants for drug binding to both I-receptors and alpha 2-adrenoceptors. Most drugs showed similar affinities for I-receptors in cortex and ileum. However, medetomidine stereoisomers as well as a few other drugs clearly delineated the I-receptors in cortex and ileum as different.

Improved approach for proteochemometrics modeling: application to organic compound---amine G protein-coupled receptor interactions

MOTIVATION Proteochemometrics is a novel technology for the analysis of interactions of series of proteins with series of ligands. We have here customized it for analysis of large datasets and evaluated it for the modeling of the interaction of psychoactive organic amines with all the five known families of amine G protein-coupled receptors (GPCRs). RESULTS The model exploited data for the binding of 22 compounds to 31 amine GPCRs, correlating chemical descriptions and cross-descriptions of compounds and receptors to binding affinity using a novel strategy. A highly valid model (q2 = 0.76) was obtained which was further validated by external predictions using data for 10 other entirely independent compounds, yielding the high q2ext = 0.67. Interpretation of the model reveals molecular interactions that govern psychoactive organic amines overall affinity for amine GPCRs, as well as their selectivity for particular amine GPCRs. The new modeling procedure allows us to obtain fully interpretable proteochemometrics models using essentially unlimited number of ligand and protein descriptors.

Delineation of three pharmacological subtypes of α2‐adrenoceptor in the rat kidney

1 Simultaneous computer modelling of plain and ARC 239‐ and guanoxabenz‐masked [3H]‐RX821002 saturation curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239‐masked guanoxabenz competition curves revealed that the drugs bound to three α2‐adrenoceptor subtypes in the rat kidney with grossly differing selectivities. These α2‐adrenoceptor subtypes were termed α2A, α2B1 and α2B2. The order of affinities for [3H]‐RX821002 for the adrenoceptor sites was α2A > α2B1 > α2B2, the Kd s being 0.62 ± 0.05, 2.52 ± 0.11 and 6.74 ± 1.21 nm, respectively. The order of affinities for ARC 239 was α2B1 > α2B2 ≫ α2A with Kd s 4.78 ± 1.04, 28.8 ± 4.1 and 1460 ± 270 nm, respectively. For guanoxabenz the order of affinities was α2A > α2B1 ≫ α2B2 with Kd s 99.7 ± 15.1, 508 ± 135 and 25,400 ± 2400 nm, respectively. 2 Binding constants for 14 compounds for the three rat kidney α2‐adrenoceptor subtypes were determined by the simultaneous computer modelling of plain and ARC 239‐ and guanoxabenz‐masked drug competition curves, plain ARC 239 and guanoxabenz competition curves as well as ARC 239‐masked guanoxabenz competition curves. Of the 14 compounds tested, oxymetazoline and guanfacine were found to bind with low affinities to both of the α2B1‐ and α2B2‐adrenoceptors but with high affinity to the α2A‐adrenoceptor. Prazosin instead bound with high affinities to the α2B1‐ and α2B2‐adrenoceptors but with low affinity to the α2A‐adrenoceptor. By contrast, guanoxabenz and ARC 239 delineated clearly between all the three α2‐adrenoceptor subtypes. Notably the affinities of guanoxabenz for α2B1‐ and α2B2‐adrenoceptors differed 72 fold and for α2A‐ and α2B2‐adrenoceptors 380 fold. The selectivities of a number of other drugs were less marked but their Kd s were consistent with all three sites being α2‐adrenoceptors. 3 (−)‐Adrenaline and (−)‐noradrenaline showed dissimilar order of affinities for the three α2‐adrenoceptors. For (−)‐adrenaline the order of affinities was α2B1 ≥ α2A > α2B2 and for (−)‐noradrenaline α2B2 ≥ α2B1 > α2A. All three α2‐adrenoceptors showed the expected stereoselective binding for adrenaline enantiomers, the (+)‐form being 7–10 fold less potent than the (−)‐form. 4 [3H]‐yohimbine was also used as radioligand. The data with this ligand were fully compatible with the [3H]‐RX821002 data. However, [3H]‐yohimbine appeared to label only α2B1‐ and α2B2‐adrenoceptors presumably because it had too low an affinity for α2A‐adrenoceptors. 5 We conclude that three pharmacological subtypes of α2‐adrenoceptors are labelled by [3H]‐RX821002 in the rat kidney. Guanoxabenz and ARC 239 may be used in competition studies to delineate between these three α2‐adrenoceptor subtypes. Moreoever, we here present a method allowing the determination of binding constants for an arbitrary drug to the three α2‐adrenoceptor subtypes.

Autoradiographic studies of central α2A- and α2C-adrenoceptors in the rat using [3H]MK912 and subtype-selective drugs

Abstract In the present study we examined the distribution of α2A- and α2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between α2A- and α2C-adrenoceptors, the slices were incubated with [3H]MK912 in the presence of graded concentrations of the α2A-selective drug, BRL44408, or the α2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [3H]MK912 plus 185 nM BRL44408 selectively labeled α2C-adrenoceptors, while 0.4 nM [3H]MK912 plus 220 nM spiroxatrine selectively labeled α2A-adrenoceptors. Using this approach, α2C-adrenoceptors were detected in the striatum, while α2A-adrenoceptors predominated in the cortical layers 1–4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.

Antinociceptive Actions of α2‐Adrenoceptor Agonists in the Rat Spinal Cord: Evidence for Antinociceptive α2‐Adrenoceptor Subtypes and Dissociation of Antinociceptive α2‐Adrenoceptors from Cyclic AMP

Abstract: The antinociceptive actions of intrathecal injections of two α2‐adrenergic agonists, UK‐14,304 and guanfacine, were investigated in rats after pretreatment of the animals with the noradrenaline neurotoxin N‐2‐chloroethyl‐N‐ethyl‐2‐bromobenzylamine (DSP4) 14 days in advance. The chronic noradrenaline depletion induced by DSP4 caused a marked increase in sensitivity of the antinociceptive action of UK‐14,304 in the tail‐flick test. By contrast, the antinociceptive effect of guanfacine was not appreciably affected by the DSP4 treatment. The antinociceptive effects of both UK‐14,304 and guanfacine were blocked by intraperitoneal injections of yohimbine, a result indicating that both drugs induced their actions by activating α2‐adrenoceptors. Both UK‐14,304 and guanfacine were found to reduce the production of cyclic AMP (cAMP) in the spinal cord, as determined using an in vitro radioisotopic method. The cAMP inhibitory effects of both agonists were effectively blocked by yohimbine, but not by prazosin, a finding indicating the α2‐adrenergic nature of the response. However, the cAMP inhibitory effect of UK‐14,304 was not potentiated by pretreatment with DSP4, a finding in marked contrast with the strong potentiation of the antinociceptive action of UK‐14,304 induced by the chronic depletion of endogenous noradrenaline. Moreover, intrathecal injections of forskolin, which increased the endogenous levels of spinal cord cAMP fivefold, did not modify the antinociceptive effects of UK‐14,304 or guanfacine in neither normal nor DSP4‐treated animals. It is suggested that there exist pharmacologically differing α2‐adrenergic receptor pathways capable of mediating antinociceptive effects at the level of the spinal cord. The cAMP inhibitory actions of spinal cord α2‐adrenoceptors appear not to be directly linked with the antinociceptive actions of these receptors.

Reduced natriuretic response to acute sodium loading in COMT Gene deleted mice

BackgroundThe intrarenal natriuretic hormone dopamine (DA) is metabolised by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). Inhibition of COMT, as opposed to MAO, results in a potent natriuretic response in the rat. The present study in anaesthetized homozygous and heterozygous COMT gene deleted mice attempted to further elucidate the importance of COMT in renal DA and sodium handling. After acute intravenous isotonic sodium loading, renal function was followed.ResultsCOMT activity in heterozygous mice was about half of that in wild type mice and was zero in the homozygous mice. MAO activity did not differ between the genotypes. Urinary sodium excretion increased 10-fold after sodium loading in wild type mice. In heterozygous and homozygous mice, the natriuretic effects of sodium loading were only 29 % and 39 %, respectively, of that in wild type mice. Arterial pressure and glomerular filtration rate did not differ between genotypes. Baseline norepinephrine and DA excretions in urine were elevated in the homozygous, but not in heterozygous, COMT gene deleted mice. Urinary DA excretion increased after isotonic sodium loading in the wild type mice but not in the COMT gene deleted mice.ConclusionsMice with reduced or absent COMT activity have altered metabolism of catecholamines and are unable to increase renal DA activity and produce normal natriuresis in response to acute sodium loading. The results support the hypothesis that COMT has an important role in the DA-mediated regulation of renal sodium excretion.

Identification of drugs subtype-selective for α2A-, α2B-, and α2C-adrenoceptors in the pig cerebellum and kidney cortex

Abstract The radioligands [ 3 H]MK912 and [ 3 H]RX821002 were used to label α 2A -, α 2B -, and α 2C -adrenoceptors of the pig cerebellum and kidney cortex. By inclusion of the α 2A -adrenoceptor-selective drug, BRL44408, and using a ‘multi-curve’ experimental design all the three porcine α 2 -adrenoceptor subtypes could be characterized pharmacologically. The data indicate that the pig α 2 -adrenoceptor subtypes are pharmacologically more related to human α 2 -adrenoceptor subtypes than to the rodent α 2 -adrenoceptors. We suggest a set of drugs that are useful for the delineation of the pig α 2 -adrenoceptor subtypes.