Nigel R. Newberry

Inhibition of human α7 nicotinic acetylcholine receptors by open channel blockers of N‐methyl‐D‐aspartate receptors

Human α7 nicotinic acetylcholine (ACh) receptors were expressed in Xenopus oocytes and the effects of the N‐methyl‐D‐aspartate (NMDA) receptor open channel blockers memantine and cerestat on this receptor were examined using two‐electrode voltage‐clamp recordings and 125I‐α‐bungarotoxin (125I‐α‐bgtx) binding. Memantine and cerestat produced complete inhibition of ACh‐induced inward currents with affinities similar to that reported for native NMDA receptors. Cerestat, IC50 1.7 (−1; +2) μM, was more potent than memantine, IC50 5 (−3;+8) μM, and the effects of both drugs were fully and rapidly reversible. Inhibition of α7 receptor function was voltage‐independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of 125I‐α‐bgtx to α7 receptors, suggesting that the effects of memantine or cerestat are noncompetitive. These results provide evidence that human α7 receptors are inhibited by memantine and cerestat and suggest that caution should be applied when using these compounds to study systems in which NMDA and nACh receptors co‐exist.

Evidence that the 5-HT3 receptors of the rat, mouse and guinea-pig superior cervical ganglion may be different.

1 Using grease‐gap recordings from the isolated superior cervical ganglion of mouse, rat and guinea‐pig, we have compared the depolarization evoked by 5‐hydroxytryptamine (5‐HT) with that evoked by the selective 5‐HT3 receptor agonist 2‐methyl‐5‐HT (2‐Me‐5‐HT). 2 The maximum depolarization induced by 2‐Me‐5‐HT was smaller than that induced by 5‐HT in all three species, and particularly in the guinea‐pig. 3 The 5‐HT2 receptor antagonist ketanserin (1 μm) caused a clear rightward shift of the dose‐response curve to 5‐HT on the guinea‐pig ganglion, but not on the mouse or rat ganglion. Spiperone (0.03 μm) had a quantitatively similar action to ketanserin (0.1 μm) on the 5‐HT dose‐response curve of the guinea‐pig ganglion. Ketanserin had no significant effect on the dose‐response curve to 2‐Me‐5‐HT on any of these ganglia. 4 Using 2‐Me‐5‐HT as the agonist, we determined the pA2 values for two 5‐HT3 receptor antagonists. The potency of ICS 205–930 varied by approximately 100 fold between the species and that of (+)‐tubocurarine varied by over 1000 fold. The differences in the pA2 values of these compounds varied independently among the species. 5 We conclude that 5‐HT3 receptors are present on the superior cervical ganglion from the rat, mouse and guinea‐pig, but these receptors may be pharmacologically distinct from each other. In addition, the depolarization of the guinea‐pig superior cervical ganglion by low concentrations of 5‐HT is largely mediated by ketanserin‐sensitive receptors.

Pharmacological differences between two muscarinic responses of the rat superior cervical ganglion in vitro

1 Pharmacological differences have been observed between the muscarinic agonist‐induced depolarizing and hyperpolarizing responses of the rat isolated superior cervical ganglion. 2 Pirenzepine (0.3 μm) selectively reduced the depolarizing response and unmasked the hyperpolarizing response. No such selectivity was observed with a concentration of N‐methylatropine which was equipotent with pirenzepine in antagonizing the depolarizing response. 3 The neuromuscular blocking agents gallamine (10 μm) and pancuronium (3 μm) exhibited the oppositive selectivity to pirenzepine, both dramatically reduced the hyperpolarization but only slightly antagonized the depolarization. 4 The potencies of a range of agonists in evoking the depolarizing and hyperpolarizing responses, the latter in the presence of 0.3 μm pirenzepine, have been determined. Methylfurmethide failed to hyperpolarize the ganglion at concentrations which evoked maximal depolarizations. 5 The muscarinic hyperpolarization did not appear to be mediated by the secondary release of catecholamines. 6 It was concluded that the two muscarinic responses on the rat superior cervical ganglion, the slow depolarization and faster hyperpolarization, are mediated by different muscarinic receptor subtypes.

Interaction of the atypical neuroleptic clozapine with 5-HT3 receptors in the cerebral cortex and superior cervical ganglion of the rat.

Clozapine, an atypical neuroleptic drug devoid of extrapyramidal side effects, was a moderately potent, competitive inhibitor of the binding of [3H]quaternised ICS 205-930 to 5-HT3 receptor sites in rat cortical membranes, possessing a pKi value of 7.0. In contrast, several other antipsychotic agents, including fluphenazine, alpha-flupenthixol, haloperidol, spiperone and (-)-sulpiride were essentially inactive. Clozapine also antagonised the 2-methyl 5-HT-induced depolarisation of the rat isolated superior cervical ganglion, a response known to be mediated via 5-HT3 receptors. Clozapine (0.1-1 microM) induced parallel displacements to the right of the dose-response curve to 2-methyl 5-HT in this tissue, possessing a pKb value of 7.3. These data suggest that the atypical antipsychotic profile of clozapine may be related, at least, in part to its ability to interact with central 5-HT3 receptor sites.

5-HT1A receptors activate a potassium conductance in rat ventromedial hypothalamic neurones

The extracellularly recorded firing rate of rat ventromedial hypothalamic neurones in vitro was inhibited by 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and buspirone, 2-methyl-5-HT was relatively ineffective. At 1 microM, spiperone and cyanopindolol antagonised the 5-CT induced inhibition, MDL 72222 (10 microM) and ketanserin (1 microM) did not. Intracellular recordings with voltage-clamp revealed that 5-HT and 5-CT evoked a tetrodotoxin-resistant outward current with a reversal potential of ca. -100 mV. 5-HT1A receptors likely activate a potassium conductance on these neurones.

A novel series of non‐quaternary oxadiazoles acting as full agonists at muscarinic receptors

1 A novel series of non‐quaternary oxadiazole‐based muscarinic agonists demonstrated high affinity for muscarinic receptors. 2 These agonists possessed high efficacy in the nanomolar range at muscarinic receptors in the superior cervical ganglion, atrium and ileum but did not show selectivity across the tissue preparations. 3 Two amino oxadiazoles, one from a quinuclidine series (L‐660,863) and one from a 1‐azanorbornane series (L‐670,207) possessed a high ratio of potency for displacing the binding of [3H]‐N‐methylscopolamine ([3H]‐NMS) to potency for displacing the agonist [3H]‐oxotremorine‐M ([3H]‐oxo‐M) (NMS/oxo‐M ratio) predictive of high efficacy in the cortex. 4 The two azanorbornane derivatives L‐670,548 and L‐670,207 stimulated the turnover of phosphatidylinositol in the cortex with a potency higher than that obtained with any other known muscarinic agonist (ED50 0.26 and 0.18 μm respectively). 5 The maximum response obtained with L‐670,207 was greater than that observed for carbachol but was comparable to that of the natural ligand acetylcholine. 6 These oxadiazole muscarinic agonists are among the most potent and efficacious non‐quaternary muscarinic agonists ever described.

L‐689,660, a novel cholinomimetic with functional selectivity for M1 and M3 muscarinic receptors

1 L‐689,660, 1‐azabicyclo[2.2.2]octane, 3‐(6‐chloropyrazinyl)maleate, a novel cholinomimetic, demonstrated high affinity binding (pKD (apparent) 7.42) at rat cerebral cortex muscarinic receptors. L‐689,660 had a low ratio (34) of pKD (apparent) values for the displacement of binding of the antagonist [3H]‐N‐methylscopolamine ([3H]‐NMS) compared with the displacement of the agonist [3H]‐oxotremorine‐M ([3H]‐Oxo‐m), in rat cerebral cortex. Low NMS/Oxo‐M ratios have been shown previously to be a characteristic of compounds that are low efficacy partial agonists with respect to stimulation of phosphatidyl inositol turnover in the cerebral cortex. 2 L‐689,660 showed no muscarinic receptor subtype selectivity in radioligand binding assays but showed functional selectivity in pharmacological assays. At M1 muscarinic receptors in the rat superior cervical ganglion, L‐689,660 was a potent (pEC50 7.3 ± 0.2) full agonist in comparison with (±)‐muscarine. At M3 receptors in the guinea‐pig ileum myenteric plexus‐longitudinal muscle or in trachea, L‐689,660 was again a potent agonist (pEC50 7.5 ± 0.2 and 7.7 ± 0.3 respectively) but had a lower maximum response than carbachol. In contrast L‐689,660 was an antagonist at M2 receptors in guinea‐pig atria (pA2 7.2 (95% confidence limits 7, 7.4)) and at muscarinic autoreceptors in rat hippocampal slices. 3 The putative M1‐selective muscarinic agonist, AF102B (cis‐2‐methylspiro‐(1,3‐oxathiolane 5,3′)‐quinuclidine hydrochloride) was found to have a profile similar to L‐689,660 but had up to 100 times less affinity in binding and functional assays. RS‐86 (2‐ethyl‐8‐methyl‐2,8‐diazospiro[4,5]decan 1,3‐dione hydrochloride) also had lower affinity than L‐689,660, and had no binding selectivity for muscarinic receptor subtypes. RS‐86 had a higher NMS/Oxo‐M ratio than L‐689,660 and was a full agonist at M1, M2 and M3 receptors in the functional pharmacological assays. 4 The functional selectivity of L‐689,660 in muscarinic pharmacological assays is consistent with the effects of a low efficacy partial agonist in tissues with different effective receptor reserves.

5-Hydroxytryptamine evokes three distinct responses on the rat superior cervical ganglion in vitro

In addition to the 5-HT3-mediated fast depolarisation, 5-hydroxytryptamine (5-HT) evoked two additional responses on the rat superior cervical ganglion: a hyperpolarisation and a slow depolarisation. These responses appeared to be direct actions on 5-HT receptors since they were present in a low calcium medium containing tetrodotoxin and were not abolished by a variety of non-serotonin antagonists. The hyperpolarisation was not antagonised by 5-HT3 or 5-HT2 antagonists. The 5-HT1 ligands 5-carboxamidotryptamine (5-CT) and 8-OH-DPAT also evoked a hyperpolarisation. The hyperpolarisation was antagonised by six 5-HT1A antagonists including WB-4101 and spiroxatrine. It was therefore concluded to be mediated by a 5-HT1A receptor. The slow depolarisation was only evoked by 5-HT. The receptor involved in this response, however, could not be determined. We conclude that in addition to 5-HT3 receptors the rat superior cervical ganglion possesses 5-HT1A receptors and another uncharacterised 5-HT receptor.

L-687,306: a functionally selective and potent muscarinic M1 receptor agonist

The oxadiazole L-687,306 is a high affinity muscarinic agonist with a N-methylscopolamine/oxotremorine-M binding profile predictive of a partial agonist. L-687,306 showed marked selectivity in functional pharmacological assays. L-687,306 was a partial agonist at muscarinic M1 receptors in the rat ganglion but a high affinity competitive antagonist at guinea-pig cardiac M2 and ileal M3 muscarinic receptors. This compound gives an opportunity to study receptor reserve involved in muscarinic receptors in vitro and in vivo.

A 5‐HT1‐like receptor mediates a pertussis toxin‐sensitive inhibition of rat ventromedial hypothalamic neurones in vitro

5‐ Hydroxytryptamine (5‐HT), 5‐carboxamidotryptamine and 8‐hydroxy‐2‐(di‐n‐propylamino)‐tetralin inhibited rat ventromedial hypothalamic neurones in vitro in a concentration‐dependent manner. The agonist‐induced inhibition was reduced by spiperone (1 μM) and by pertussis toxin, but not by MDL 72222 (10 μM) or ketanserin (1 μM). The inhibition appeared to be mediated via 5‐HT1A‐receptors and a pertussis toxin‐sensitive pathway.