Andrew T. Kilpatrick

α2‐Adrenoceptor subtypes and imidazoline‐like binding sites in the rat brain

1 The binding of [3H]‐yohimbine and [3H]‐idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]‐yohimbine in the cortex (Bmax 121 ± 10 fmol mg−1, protein; Kd 5.2 ± 0.9 nm) and hippocampus (Bmax 72 ± 6 fmol mg−1 protein; Kd 5.8 ± 0.7 nm). [3H]‐idazoxan labels one site in the cortex (Bmax 87 ± 8 fmol mg−1 protein; Kd 4.1± 0.9 nm) and hippocampus (Bmax 30 ± 6 fmol mg−1 protein; Kd 3.5 + 0.5 nm), when 3 μm phentolamine is used to define non‐specific binding. A second distinct [3H]‐idazoxan binding site (Bmax 110 ± 21fmolmg_1 protein; Kd 3.6 ± 0.07 nm) is identified in rat cortex if 0.3 μm cirazoline is used to define non‐specific binding and 3 μm yohimbine is included to prevent binding to α2‐adrenoceptors. 2 Displacement studies indicate that the α1‐adrenoceptor antagonist prazosin and the 5‐HT1 ligands 8‐OH‐DPAT, RU 24969 and methysergide differentiate [3H]‐yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]‐idazoxan by each ligand was monophasic. 3 The affinities of 8‐OH‐DPAT, RU 24969 and methysergide determined against [3H]‐idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated α2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated α2B. 4 [3H]‐idazoxan, in the presence of 3 μm yohimbine, labels a site that displays high affinity towards cirazoline, naphazoline and guanabenz, but low affinity towards clonidine, p‐aminoclonidine, adrenaline, noradrenaline and the α2‐adrenoceptor antagonists yohimbine, rauwolscine, WY 26703 and BDF 6143. 5 The results of this study indicate that [3H]‐yohimbine labels two sites; the α2A‐ and α2B‐adrenoceptors whereas [3H]‐idazoxan labels an α2‐adrenoceptor with a profile consistent with the α2A‐adrenoceptor subtype. In addition, [3H]‐idazoxan labels an imidazoline binding site in the rat cortex that is pharmacologically distinct from α2‐adrenoceptors. The low affinity of clonidine and p‐aminoclonidine indicates that the imidazoline‐like binding site in rat cortex is different from the site labelled by [3H]‐clonidine and [3H]‐p‐aminoclonidine in human, rat and bovine brain stem, providing evidence of potential heterogeneity within this class of binding sites.

The pharmacology of RS-15385-197, a potent and selective α2-adrenoceptor antagonist

1 RS‐15385‐197 ((8aR, 12aS, 13aS)‐5,8,8a,9,10,11,12,12a,13,13a‐decahydro‐3‐methoxy‐12‐(methylsulphonyl)‐6H‐isoquino [2,1‐g][1,6]‐naphthyridine) was evaluated in a series of in vitro and in vivo tests as an antagonist at α2‐adrenoceptors. 2 RS‐15385‐197 had a pKi of 9.45 for α2‐adrenoceptors in the rat cortex (pA2 in the guinea‐pig ileum of 9.72), whereas the 8aS, 12aR, 13aR enantiomer, RS‐15385‐198, had a pKi of only 6.32 (pA2 6.47) indicating a high degree of stereoselectivity. The racemate RS‐15385‐196 had a pKi of 9.18. 3 RS‐15385‐197 showed unprecedented α2 vs. α1 adrenoceptor selectivity in vitro. In the rat cortex, RS‐15385‐197 had a pKi of 9.45 in displacing [3H]‐yohimbine and 5.29 in displacing [3H]‐prazosin (α2/α1 selectivity ratio in binding experiments > 14000). The compound had a pA2 of 9.72 as a competitive antagonist of the inhibitory effects of UK‐14,304 in transmurally‐stimulated guinea‐pig ileum and 10.0 against BHT‐920‐induced contractions in dog saphenous vein (DSV); this latter value was unaltered by phenoxybenzamine. An apparent pKB of 5.9 was obtained against cirazoline‐induced contractions in DSV, whilst a pA2 of 6.05 was obtained against phenylephrine‐induced contractions in the rabbit aorta (α2/α1 selectivity ratio in functional experiments >4000). 4 RS‐15385‐197 was highly selective for α2‐adrenoceptors over other receptors: the compound showed low affinity for 5‐HT1A (pKi 6.50) and 5‐HT1D (pKi 7.00) receptor subtypes, and even lower affinity (pKi < 5) for other 5‐HT receptor subtypes, dopamine receptors, muscarinic cholinoceptors, β‐adrenoceptors and dihydropyridine binding sites. RS‐15385‐197 was devoid of affinity for the non‐adrenoceptor imidazoline binding site, labelled by [3H]‐idazoxan, which provides further evidence that these sites are not related to α2‐adrenoceptors. In the DSV, contractile responses to 5‐hydroxytryptamine (5‐HT) were unaffected by a concentration of 1 μm RS‐15385‐197. 5 RS‐15385‐197 was non‐selective for the α2A‐ and α2B‐adrenoceptor subtypes in that the pKi for the α2A‐adrenoceptor in human platelets was 9.90 and the pKi for the α2B‐adrenoceptor in rat neonate lung was 9.70. However, RS‐15385‐197 showed lower affinity for the α2‐adrenoceptor subtype in hamster adipocytes (pKi 8.38). 6 In anaesthetized rats, RS‐15385‐197 was a potent antagonist of the mydriasis response induced by UK‐14,304 or clonidine (AD50 5 and 7 μg kg−1, i.v., respectively; 96 μg kg−1, p.o.) and of UK‐14,304‐induced pressor responses in pithed rats (AD50 7 μg kg−1, i.v.); the compound therefore is both centrally and orally active. Even at a high dose (10 mg kg−1, i.v.), RS‐15385‐197 did not antagonize pressor responses to cirazoline in pithed rats, indicating that the selectivity for α2 vs. α1‐adrenoceptors was maintained in vivo. 8 RS‐15385‐197 is therefore a very potent, selective, competitive α2‐adrenoceptor antagonist, both in vitro and in vivo, is orally active and readily penetrates the brain. It will thus be a powerful pharmacological tool for exploring the various physiological roles of α2‐adrenoceptors.

[3H]-RS-15385-197, a selective and high affinity radioligand for α2-adrenoceptors : implications for receptor classification

1 RS‐15385–197 is the most potent and selective α2‐adrenoceptor antagonist available. We have used [3H]‐RS‐15385–197 to define α2‐adrenoceptor subtypes. The binding of [3H]‐RS‐15385–197 to membranes of rat cerebral cortex, rat neonatal lung and human platelets was reversible, saturable and of high affinity. Saturation experiments indicated that [3H]‐RS‐15385–197 bound to a single population of sites in all 3 tissues with high affinity (0.08–0.14 nm). The density of sites labelled by [3H]‐RS‐15385‐197 was greater in the cortex (275 fmol mg−1 protein) than in the neonate lung (174 fmol mg−1 protein) and human platelet (170 fmol mg−1 protein). The density of sites labelled with [3H]‐RS‐15385‐197 in the cortex was significantly greater than that labelled with [3H]‐yohimbine (121 fmol mg−1 protein). 2 The selective α2‐adrenoceptor antagonists, idazoxan, yohimbine, rauwolscine and WY 26703 displaced [3H]‐RS‐15385–197 binding to rat cerebral cortex in a simple manner with Hill slopes close to unity. The affinities derived for these antagonists against [3H]‐RS‐15385–197 were similar to the values obtained for the displacement of [3H]‐yohimbine indicating the α2‐adrenoceptor nature of the binding site. 3 α2A‐Adrenoceptor selective compounds, oxymetazoline and BRL 44409, showed high affinity for [3H]‐RS‐15385–197 binding in the human platelet and lower affinity in the neonate lung, while the α2B‐selective compounds, prazosin and imiloxan, showed high affinity for [3H]‐RS‐15385–197 binding in the neonate lung. This suggests that [3H]‐RS‐15385–197 labels both α2A‐ and α2B‐adrenoceptor subtypes. 4 Prazosin and methysergide inhibited the binding of [3H]‐RS‐15385–197 in the rat cerebral cortex in a simple manner consistent with an interaction at a single site. Although oxymetazoline inhibited [3H]‐RS‐15385–197 with a Hill slope significantly different from unity, the slope was increased to unity in the presence of Gpp(NH)p, suggesting an agonist‐like interaction. 5 The site labelled by [3H]‐RS‐15385–197 in the rat cortex shows high affinity for oxymetazoline and low affinity for prazosin which could be taken as evidence for classifying the site as an α2A‐subtype. However, the affinities of yohimbine, rauwolscine and oxymetazoline at this site do not correspond to the population of sites in the human platelet. Yohimbine and rauwolscine were 20 fold selective for the platelet α2A‐subtype, whereas phentolamine was 2 fold and imiloxan was 10 fold selective for the cortex subtype. Indeed although the site showed some similarities with the α2A‐subtype, the highest degree of homology was observed between this site and the rat submaxillary gland and the RG20 clone, tentatively called the α2D‐adrenoceptor subtype. We propose that the α2‐adrenoceptor in the rat cortex may therefore correspond to the putative α2D‐subtype of the adrenoceptor.

Heterogeneity of α2-adrenoceptors in rat cortex but not human platelets can be defined by 8-OH-DPAT, RU 24969 and methysergide

1 Saturation experiments indicated that [3H]‐yohimbine binding was specific, saturable and labelled a single population of sites in rat cerebral cortex (Kd 5.3 ± 0.9 nm, Bmax 121 ± 10 fmol mg−1 protein) and human platelets (Kd 0.7 ± 0.1 nm, Bmax 152 ± 10 fmol mg−1 protein). 2 The α2‐adrenoceptor antagonists, yohimbine, rauwolscine, WY 26703, idazoxan and BDF 6143 displaced [3H]‐yohimbine binding to each tissue in a simple manner, with high affinity and Hill slopes close to unity. 3 The α1‐adrenoceptor agonist, oxymetazoline and the antagonist prazosin inhibited the binding of [3H]‐yohimbine to rat cortex in a complex manner consistent with an interaction at more than one site. However, indoramin and WB 4101 only appeared to interact with one site. In contrast, in human platelets, all antagonists gave rise to monophasic displacement curves with Hill slopes close to unity suggesting a single site of interaction. 4 The 5‐hydroxytryptamine (5‐HT) receptor ligands, 8‐hydroxy‐2‐(di‐n‐propylamino)‐tetralin (8‐OH‐DPAT), RU 24969, and methysergide inhibited the binding of [3H]‐yohimbine to rat cortex with high and low affinity, consistent with an interaction with two populations of binding sites. However, inhibition of [3H]‐yohimbine binding to human platelets suggested a single site of interaction. The low affinity of 5‐HT, 5‐carboxyamidotryptamine (5‐CT) and dipropyl‐5‐CT indicated that [3H]‐yohimbine was not labelling a 5‐HT1‐like site in rat cortex. 5 The ability of 8‐OH‐DPAT, RU 24969 and methysergide in addition to prazosin and oxymetazoline to differentiate [3H]‐yohimbine binding provides additional pharmacological evidence for heterogeneity within rat cortical α2‐adrenoceptors. However, if the two sites in rat cortex that are differentiated by the 5‐HT ligands represent α2A‐ and α2B‐adrenoceptor subtypes as defined by prazosin and oxymetazoline, then they do not correspond to the population of sites in human platelets. As receptor classification should be linked to affinity of drugs rather than tissue distribution, the current classification of α2‐adrenoceptor subtypes does not appear to be satisfactory.

[3H]‐idazoxan binds with high affinity to two sites on hamster adipocytes: an α2‐adrenoceptor and a non‐adrenoceptor site

1 [3H]‐idazoxan labels a single population of high affinity sites (Kd 2.26 ± 0.02 nM; Bmax 372 ± 25 fmol mg−1 protein) in hamster adipocyte membranes. In the presence of 1 μm yohimbine to preclude binding to α2‐adrenoceptors, the density of [3H]‐idazoxan binding sites was reduced (287 ± 18 fmol mg−1 protein) without an apparent decrease in the affinity (Kd 2.19 ± 0.24 nm) of the radioligand. 2 Displacement studies indicate that α‐adrenoceptor ligands with an imidazoline side chain completely inhibit [3H]‐idazoxan binding to hamster adipocyte membranes; in contrast, the α2‐adrenoceptor antagonists yohimbine, rauwolscine, BDF 6143 and phentolamine inhibited only 20–30% of the specific binding with affinity values consistent with an interaction at α2‐adrenoceptors. 3 The low potency of noradrenaline and adrenaline in displacing [3H]‐idazoxan binding to the second site on hamster adipocyte membranes indicates that it is unlikely that this site is a type of adrenoceptor. 4 These results suggests that [3H]‐idazoxan binds with high affinity to two sites in hamster adipocytes: an α2‐adrenoceptor and a non‐adrenoceptor imidazoline site.

Modulation of central noradrenergic function by RS-15385-197

1 RS‐15385‐197, a highly potent and selective α2‐adrenoceptor antagonist, was examined in a variety of in vitro and in vivo functional tests to assess the selectivity of its interaction with central noradrenergic neurones in the rat. 2 In hypothalamic slices, RS‐15385‐197 was potent in augmenting K+‐evoked release of [3H]‐noradrenaline, with an EC50 of 9 nm. Idazoxan and yohimbine showed 100 fold less activity. This was due to its antagonist action at presynaptic α2‐adrenoceptors, as RS‐15385‐197 (10 μm), did not directly release [3H]‐noradrenaline from cortical slices unlike reserpine (10 μm), and did not inhibit noradrenaline re‐uptake into cortical synaptosomes. 3 In vivo, RS‐15385‐197 (0.5 mg kg−1, p.o.) increased levels of 3‐methoxy‐4‐hydroxy‐phenylglycol (MHPG) in the cerebral cortex without modifying levels of 5‐hydroxyindoleacetic acid (5‐HIAA). This dose, but not a lower dose (0.1 mg kg−1, p.o.) caused β‐adrenoceptor down‐regulation in the cortex when administered once daily for 14 days whereas 5‐HT2 receptor number was unaltered, indicating a selective effect on noradrenergic transmission. 4 Selective depletion of cortical 5‐HT by administration of p‐chlorophenylalanine (PCPA; 100 mg kg−1, i.p. for 14 days) or 5,7‐dihydroxytryptamine (5,7‐DHT; 150 μg i.c.v.) prevented the β‐adrenoceptor down‐regulation caused by RS‐15385‐197, indicating that a tonic 5‐hydroxytryptaminergic input was required for it to elicit β‐adrenoceptor down‐regulation. It was not possible to prevent the loss of activity of RS‐15385‐197 in these 5‐HT‐depleted animals by co‐administration with the 5‐HT1A partial agonist, 8‐hydroxy‐n‐dipropyl aminotetralin (8‐OH‐DPAT, 0.3 mg kg−1, i.p. twice daily for final 3 days). 5 At a dose (1 mg kg−1, p.o.) which completely prevented the hypoactivity produced by clonidine (0.1 mg kg−1, p.o.), RS‐15385‐197 did not affect behavioural stereotypy induced by 8‐OH‐DPAT (0.3 mg kg−1, s.c.). Similarly, following chronic dosing with the racemate, RS‐15385‐196 (3 mg kg−1, p.o., once daily for 14 days), there was no effect on the behavioural and hypothermic response to 8‐OH‐DPAT (0.5 mg kg−1, s.c.). Therefore, RS‐15385‐197 was selective for central α2‐adrenoceptors over 5‐HT1A receptors in in vivo functional tests. 6 Thus, RS‐15385‐197 was highly selective in interacting with central noradrenergic neurones in the rat in vitro and in vivo. It is therefore currently the agent of choice for investigations of the role of α2‐adrenoceptors in the CNS.

Selective antagonism of calcium channel activators by fluspirilene

1 Fluspirilene has been claimed to bind to a high affinity site in the calcium channel in skeletal muscle. We have investigated its calcium‐antagonistic effects in smooth muscle and affinity for the channel in radioligand binding assays. 2 Fluspirilene was weakly active as an antagonist of Ca2+‐induced contractions in K+‐depolarized taenia preparations from the guinea‐pig caecum, with threshold antagonism starting from concentrations of 30 nm. Nitrendipine, nicardipine and nimodipine were very potent antagonists in this model (threshold antagonism, > 1 nm). 3 In contrast, fluspirilene (10–1000 nm) was a potent non‐competitive antagonist of the effects of Bay K 8644 (1–3000 nm) on Ca2+‐induced contractions and, at 10 nm, selectively antagonised the effects of Bay K 8644, abolished the Ca2+‐channel activator effects of CGP 28392, without changing the calcium antagonist effects of nitrendipine, or modifying the sensitivity of the tissues to Ca2+. In contrast, the dihydropyridines were more effective as antagonists of Ca2+ than of Bay K 8644. Fluspirilene therefore selectively antagonised the effects of dihydropyridine Ca2+ channel activators without affecting the antagonist potency. 4 In radioligand binding experiments, fluspirilene was a potent displacer of [3H]‐PN‐200‐110 binding to rat cerebral cortical membranes (EC50 30 nm), albeit with a low Hill slope (0.66), and was more potent than other lipophilic diphenylalkylamines such as flunarizine and lidoflazine. Fluspirilene interacted non‐competitively with [3H]‐PN‐200‐110 and increased dissociation of the radioligand.

Cerebral ischaemia reduces the density of 5-HT2 binding sites in the frontal cortex of the gerbil

The 5-HT2 antagonist [3H]ketanserin labels a single population of high affinity sites (Kd 0.48 +/- 0.03 nM; Bmax 206 +/- 20 fmol/mg protein) in the frontal cortex of the gerbil. Specific binding of [3H]ketanserin was displaced by a number of 5-HT2A antagonists ritanserin, cyproheptadine and methysergide) but not by the 5-HT1A agonist, 8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT) or the 5-HT1A/1B agonists 5-carboxyamidotryptamine (5-CT) or RU 24969, indicating that the labelled site probably represents the 5-HT2 receptor. Cerebral ischaemia induced in either a 3 hr unilateral non-recovery model or a 5 min bilateral, 3-day recovery model, resulted in a significant decrease in the density of 5-HT2 binding sites in the ischaemic frontal cortex without an apparent change in their affinity for the ligand. The decrease in density was not simply related to levels of 5-HT because occlusion of the right carotid artery for 3 hr resulted in bilateral depletion of 5-HT but only in an ipsilateral reduction in the density of binding sites. In addition, a significant decrease in the density of 5-HT2 binding sites occurred in the recovery model at a time when the levels of 5-HT in the cortex were unaltered.

Characterisation of sodium-dependent uptake of 5-hydroxytryptamine into guinea-pig brain.

The uptake of 5-hydroxytryptamine (5-HT) was studied using isolated nerve endings (synaptosomes) prepared from the corpus striatum of the guinea pig. Analysis of temperature-dependent uptake revealed that two processes were involved, a sodium-dependent and sodium-independent mechanism. Kinetic studies indicated that the affinity of 5-HT (Ku) and the transport capacity (Vu) of the uptake process were dependent upon the external concentration of sodium. The transport capacity increased linearly with increasing concentrations of sodium up to 140 mmol/l, whereas the affinity for 5-HT was enhanced at concentrations of sodium up to 100 mmol/l, but reduced at 130 mmol/l sodium. The findings of this study suggest that uptake of 5-HT occurs as a result of a mobile carrier transport system which, depending upon the external concentration of sodium proceeds by a single or double displacement mechanism.

The α2-adrenoceptor antagonist SK & F 104078 has high affinity for 5-HT1A and 5-HT2 receptors

Abstract The affinity of SK & F 104078, a putative selective postjunctional α 2 -adrenoceptor antagonists, was determined at 5-HT 1A and 5-HT 2 receptors in rat brain. SK & F 104078 had moderate affinity towards α 2 -adrenoceptors (pK i 6.7) but displayed higher affinity at 5-HT 1A (pK i 8.1) and 5-HT 2 (pK i 7.6) receptors. If SK & F 104078 is used in functional studies to define heterogeneity within of α 2 -adrenoceptors, care must be taken to define the role of 5-HT 1A and 5-HT 2 receptors in the response being measured.