Stephen B. Freedman

Relative affinities of drugs acting at cholinoceptors in displacing agonist and antagonist radioligands: the NMS/Oxo-M ratio as an index of efficacy at cortical muscarinic receptors

1 Radioligand binding assays using [3H]‐N‐methylscopolamine (NMS) and [3H]‐oxotremorine M (Oxo‐M) have been devised to predict the efficacy of test compounds at muscarinic receptors in rat cerebral cortex. 2 Muscarinic antagonists, including non‐selective and both M1‐ and M2‐selective compounds, displayed similar affinity for both binding assays. 3 Full agonists such as carbachol and muscarine possessed a ratio of potencies against the antagonist versus the agonist ligand (NMS/Oxo‐M ratio) of > 4000. 4 Compounds which have been shown previously to display partial agonist activity in functional assays e.g. pilocarpine and RS86 had intermediate NMS/Oxo‐M ratios of 100–150. A second group of compounds which included oxotremorine had somewhat higher ratios (500–1400). 5 The ratio of affinity constants for the two assays predicted the ability of agonists to stimulate cortical phosphatidyl‐inositol turnover. 6 These results suggest that the NMS/Oxo‐M ratio may be a useful prediction of efficacy for novel compounds acting at cortical muscarinic receptors.

Muscarinic M1, M2 receptor binding. Relationship with functional efficacy

A comparison has been made between [3H]pirenzepine binding to the M1 receptor population of rat cerebral cortex and [3H]N-methylscopolamine binding to M2 receptors in rat cardiac membranes. Several standard muscarinic antagonists including trihexyphenidyl HCl, benztropine, biperidin and 4-DAMP (4-diphenylacetoxy-N-methyl piperidine methiodide) showed some selectivity for the M1 binding assay. Dicyclomine and hexahydrosiladifenidol were the only antagonists with a selectivity approaching that of pirenzepine. Gallamine and AFDX-116 were the only M2 (cardiac) selective antagonists. Muscarinic agonists displayed profiles which could be classified into two groups, apparently related to their intrinsic activity. One group displayed apparent selectivity for the heart, with low Hill coefficients and contained full agonists such as acetylcholine. The second group displayed less selectivity, intermediate Hill coefficients and contained partial agonists such as pilocarpine. Thus muscarinic agents can distinguish between different tissues not only on the basis of receptor selectivity, but also by recognition of high and low agonist affinity states. Thus the intrinsic activity of a muscarinic agonist may reflect an apparent but not true receptor-mediated selectivity.

Functional expression of human D3 dopamine receptors in differentiated neuroblastoma × glioma NG108–15 cells

This study describes the depression of calcium currents caused by activation of human D3 dopamine receptors which have been stably expressed in the neuroblastoma × glioma NG108–15 cell line. Transfected cells, which had been differentiated with prostaglandin E1 and isobutylmethylxanthine, exclusively expressed D3 receptor mRNA, which was demonstrated by reverse transcription polymerase chain reaction techniques. Transfected cells had high affinity binding sites for iodosulpiride, with a Kd of 0.8 nm and receptor density of 240 fmolmg−1 protein. Calcium currents were recorded using nystatinperforated patch clamp techniques. In contrast to untransfected cells that had been differentiated, high‐threshold calcium currents in differentiated hD3‐NG108–15 cells were depressed by application of dopamine and quinpirole. These responses were abolished by the dopamine receptor antagonist S‐(–)‐sulpiride (1 μm), demonstrating that they were caused by the activation of the transfected dopamine receptors. Coupling of human D3 receptors to calcium currents was sensitive to the action of pertussis toxin, suggesting the involvement of G‐proteins of the Gi and/or Go subtype. These results demonstrate that human D3 receptors represent a functional class of dopamine receptor.

Antinociceptive and toxic effects of (+)-epibatidine oxalate attributable to nicotinic agonist activity

1 Epibatidine is an analgesic substance, isolated from the skin of the poisonous frog Epipedobates tricolor, for which the mechanism of action was previously unknown. 2 The IC50 of synthetic (+)‐epibatidine oxalate (the naturally occurring isomer) for [3H]‐nicotine binding to rat whole‐brain membranes was 0.1 nm. The (−)−isomer also exhibited high affinity (IC50 = 0.2 nm). 3 (+)‐ and (−)−Epibatidine exhibited much lower affinity for displacement of the muscarinic ligand [3H]‐N‐methylscopolamine binding to rat cortical membranes (Kapp − 6.9μm and 16.0 μm respectively). The (+)‐enantiomer of epibatidine had an antagonist/agonist (NMS/oxo‐M) binding ratio of 4.2 This is consistent with a muscarinic antagonist profile. 4 (+)‐Epibatidine oxalate (10 μm) did not cause significant (>30%) displacement of radioligand binding to opioid, excitatory amino acid, benzodiazepine, 5‐HT, dopamine, adrenaline or peptide receptors. 5 (+)‐ and (−)−Epibatidine (5–20 μg kg−1 s.c.) doubled response latency in the mouse hot‐plate test. Antinociception and behavioural depression induced by (+)‐epibatidine (5 μg kg−1) was fully blocked by the nicotinic antagonists mecamylamine (2 mg kg−1 s.c.) or dihydro‐β‐erythroidine (2 mg kg−1 s.c). The muscarinic antagonist scopolamine (0.4 and 10 mg kg−1 s.c.) caused partial reversal of antinociception induced by (+)‐epibatidine in mice, but not in rats. 6 These findings demonstrate that (+)‐epibatidine oxalate salt is a highly selective and potent nicotinic analgesic agent.

Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells

Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, B max = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp‐dependent shifts in [3H]N‐0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1–10 μM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.

Pharmacology of high‐threshold calcium currents in GH4C1 pituitary cells and their regulation by activation of human D2 and D4 dopamine receptors

1 The objective of this study was to characterize the pharmacology of calcium currents in GH4C1 pituitary cells and determine whether activation of heterologously expressed human dopamine receptors can regulate their function. Human D2(short), D3 and D4,2 receptor cDNAs were separately transfected into GH4C1 cells and whole cell calcium currents were recorded by use of nystatin‐perforated patch clamp techniques. 2 High‐threshold calcium currents were antagonized in a biphasic manner by the dihydropyridine, nisoldipine. The half‐maximally effective concentration for each site was 0.2 nm (pIC50 = 9.78 ± 0.21, n = 4) and 339 nm (pIC50 = 6.47 ± 0.12, n = 4). The component of current inhibited by 10 nm nisoldipine was also blocked by ω‐conotoxin GVIA (30 ± 9% at 30 nm, n = 6) or by ω‐agatoxin IVA (34 ± 7% at 100 nm, n = 4). 3 Activation of either D2 or D4 receptors by dopamine (10 μm) or quinpirole (0.1 to 10 μm) reduced the peak calcium current by ca. 20% in the majority of cells studied. No inhibition was observed in control or D3 transfected GH4C1 cell lines. 4 The mobilisation of intracellular calcium by thyrotropin releasing hormone in hD4‐GH4C1 cells was also studied using Fura‐2 AM microspectrofluorimetry. Thyrotropin releasing hormone caused a concentration‐dependent increase in calcium mobilisation with an EC50 of 7 nm. D4 receptor activation had no effect upon either basal or hormone‐induced [Ca2+]i transients. 5 These results demonstrate that GH4C1 pituitary cells have at least two types of dihydropyridine‐sensitive high‐threshold calcium currents and that like D2 receptors, human D4 receptors can also regulate calcium channel function.

Characterisation of a chimeric hD3/D2 dopamine receptor expressed in CHO cells

The D2 dopamine receptor is known to be functionally coupled when expressed in CHO cells, whereas the effector systems for the D3, dopamine receptor remain unclear. A chimeric, human D3/D2 receptor (hD3/D2) was constructed containing the third intracellular loop region of the D2 receptor. CHO cells stably expressing the D2, D3, or hD3/D2 receptors were created and the pharmacology of the receptors was examined. The chimeric hD3/D2 receptor retained D3‐like affinities for dopaminergic ligands. However, in contrast to the D2 receptor neither the D3 receptor nor the hD3/D2 receptor could functionally couple to the adenylate cyclase or arachidonic acid release mechanisms.

Antagonism of the effects of (+)-PD 128907 on midbrain dopamine neurones in rat brain slices by a selective D2 receptor antagonist L-741,626.

1 The ability of PD 128907 to activate dopamine receptors in the ventral tegmental area, substantia nigra pars compacta, and striatum was investigated by use of in vitro electrophysiological recording and fast cyclic voltammetry. The affinity of a novel D2 selective antagonist L‐741,626 for receptors activated by this agonist was measured to determine if its effects were mediated by D2 or D3 receptors. 2 The active (+) enantiomer of PD 128907 bound with high affinity and selectivity to rat D3 dopamine receptors. The Ki values for (+)‐PD 128907 were 620 nM at D2, 1 nM at D3 and 720 nM at D4 receptors. 3 (+)‐PD 128907 inhibited cell firing in both the ventral tegmental area and substantia nigra pars compacta with EC50 values of 33 nM (pEC50 = 7.48 ± 0.10, n= 10) and 38 nM (pEC50 = 7.42 ± 0.15, n = 5), respectively. No effects of (+)‐PD 128907 (100 nM) were observed on glutamate or GABA‐mediated synaptic potentials elicited by focal bipolar stimulation. 4 L‐741,626 antagonized these effects of (+)‐PD 128907 in a concentration‐dependent and surmountable manner with an affinity, determined from Schild analysis, of 20 nM (pKB = 7.71 ± 0.14) in the ventral tegmental area and 11 nM (pKB = 7.95 ± 0.18) in the substantia nigra pars compacta. 5 (+)‐PD 128907 also inhibited dopamine release in the caudate‐putamen with an EC50 of 66 nM (n = 5). The affinity of L‐741,626 for these nerve terminal autoreceptors (pKB = 7.71 ± 0.06; =20 nM) was identical to that observed on midbrain dopamine neurones. 6 These data demonstrate that the D3 receptor ligand (+)‐PD 128907 is a potent agonist on rat midbrain dopamine neurones. However, its lack of regional selectivity, and the high affinity of the selective D2 receptor antagonist L‐741,626 for receptors activated by (+)‐PD 128907, was more consistent with an action on D2 autoreceptors rather than upon a D3 dopamine receptor subtype.

The design of novel muscarinic partial agonists that have functional selectivity in pharmacological preparations in vitro and reduced side-effect profile in vivo

Antagonist/agonist binding ratios (NMS/Oxo-M ratio) were used as an index of the efficacy of novel compounds acting at muscarinic receptors. These binding ratios have been used with a range of functional pharmacological assays to investigate the effects of varying the efficacy of muscarinic agonists. This strategy has been used as a means of obtaining functional receptor selectivity by exploiting differences in effective receptor reserves. The oxadiazole and pyrazine muscarinic agonists L-670,548 (NMS/Oxo-M ratio 1100) and L-680,648 (NMS/Oxo-M ratio 690) are amongst some of the most potent and efficacious agonists known. Decreasing the efficacy of compounds from these series, resulted in compounds with functional selectivity. The chloropyrazine L-689,660 (NMS/Oxo-M ratio 28) was an agonist on the rat superior cervical ganglion (M1), a partial agonist on the guinea-pig ileum (M3), but was an antagonist in the guinea-pig atria (M2). Synthesis of compounds with even lower predicted efficacy, such as the cyclopropyloxadiazole L-687,306 (NMS/Oxo-M ratio 15), maintained agonist activity in the ganglion, but showed antagonist activity in the M3 ileal, as well as the M2 atrial preparations. When tested in vivo these compounds did not produce many of the side effects associated with more efficacious agonists, particularly those associated with the cardiovascular system. However, they were active in reversing scopolamine-induced deficits in a variety of behavioural paradigms. This approach shows how functional selectivity for muscarinic receptor subtypes can be achieved in vitro, that in vivo reduces the dose-limiting side effects normally associated with muscarinic agonists.

Functional Coupling of Human D2, D3, and D4 Dopamine Receptors in HEK293 Cells

The D2 dopamine receptor is known to be functionally coupled to the inhibition of adenylate cyclase when expressed in a number of mammalian cell lines. However, functional coupling of the recently discovered D3 and D4 dopamine receptor subtypes has been more difficult to demonstrate. In this study, human D2, D3 and D4 receptors were stably expressed separately in human embryonic kidney cells (HEK 293). In these cells, activation of D2, D3 or D4 receptors resulted in the inhibition of forskolin-stimulated adenylate cyclase activity in a dose responsive manner. This activation was prevented by pre-incubation of the cells expressing these receptors with the dopaminergic antagonist haloperidol. Radioligand binding studies using [3H]spiperone confirmed that the atypical neuroleptic clozapine has higher affinity for the human D4 receptor than the D3 or D4 receptors, although only 6-fold higher than the D2 receptor in this study. In addition, ribonuclease protection studies demonstrated the presence of D4 dopamine receptor mRNA in human brain regions.