J.M. Young

Histamine H3 receptor activation selectively inhibits dopamine D1 receptor-dependent [3H]GABA release from depolarization-stimulated slices of rat substantia nigra pars reticulata

The release of [3H]GABA from slices of rat substantia nigra pars reticulata induced by increasing extracellular K+ from 6 to 15 mM in the presence of 10 microM sulpiride was inhibited by 73 +/- 3% by 1 microM SCH 23390, consistent with a large component of release dependent upon D1 receptor activation. The histamine H3 receptor-selective agonist immepip (1 microM) and the non-selective agonist histamine (100 microM) inhibited [3H]GABA release by 78 +/- 2 and 80 +/- 2%, respectively. The inhibition by both agonists was reversed by the H3 receptor antagonist thioperamide (1 microM). However, in the presence of 1 microM SCH 23390 depolarization-induced release of [3H]GABA was not significantly decreased by 1 microM immepip. In rats depleted of dopamine by pretreatment with reserpine, immepip no longer inhibited control release of [3H]GABA, but in the presence of 1 microM SKF 38393, which produced a 7 +/- 1-fold stimulation of release, immepip reduced the release to a level not statistically different from that in the presence of immepip alone. Immepip (1 microM) also inhibited the depolarization-induced release of [3H]dopamine from substantia nigra pars reticulata slices, by 38 +/- 3%. The evidence is consistent with the proposition that activation of histamine H3 receptors leads to the selective inhibition of the component of depolarization-induced [3H]GABA release in substantia nigra pars reticulata slices which is dependent upon D1 receptor activation. This appears to be largely an action at the terminals of the striatonigral GABA projection neurons, which may be enhanced by a partial inhibition of dendritic [3H]dopamine release.

THE BINDING OF [3H]‐PROPYLBENZILYLCHOLINE MUSTARD BY LONGITUDINAL MUSCLE STRIPS FROM GUINEA‐PIG SMALL INTESTINE

1 The synthesis of tritium labelled propylbenzilylcholine mustard ([3H]‐PrBCM; N‐2′‐chloroethyl‐N‐[2″, 3″‐3H2] propyl‐2‐aminoethyl benzilate) is described. 2 The uptake by muscle strips was measured and shown to be considerably increased by previous immersion of the muscle in distilled water. 3 A considerable part of the uptake is inhibited selectively by atropine, but not by nicotinic antagonists. A number of muscarinic agonists also inhibit uptake and their apparent affinity constants have been determined. 4 The uptake by atropine‐sensitive sites is temperature‐insensitive, whereas the other sites are temperature‐sensitive. Recovery is highly temperature‐sensitive and there is good agreement between recovery of sensitivity to agonists and loss of radioactivity from the muscle.

THE PROPERTIES OF MUSCARINIC RECEPTORS IN MAMMALIAN CEREBRAL CORTEX

1 The reaction of tritiated propylbenzilylcholine mustard ([3H]‐PrBCM; N‐2′‐chloroethyl‐N‐[2″,3″‐3H2]‐propyl‐2‐aminoethylbenzilate) with homogenates of mammalian brain has been studied. 2 The uptake can be divided into an atropine‐sensitive component of fixed capacity (380 pmol/g protein in the rat) and an atropine‐insensitive part. 3 The atropine‐sensitive portion is identified as muscarinic receptor by its insensitivity to nicotinic antagonists and anticholinesterases and its sensitivity to a range of muscarinic antagonists. 4 The uptake of [3H]‐PrBCM is also inhibited by muscarinic agonists and there is reasonable quantitative agreement between the affinities of agonists estimated in this way and in intact tissues by physiological responses. 5 The fraction of [3H]‐PrBCM uptake inhibited by muscarinic antagonists and agonists is the same. 6 The amount of receptor found in six mammalian species was inversely related to the size of the brain, but the rates of alkylation and the sensitivity to atropine were not dissimilar.

HISTAMINE H1‐RECEPTORS IN THE BRAIN OF THE GUINEA‐PIG AND THE RAT: DIFFERENCES IN LIGAND BINDING PROPERTIES AND REGIONAL DISTRIBUTION

1 The equilibrium dissociation constant, Kd, for mepyramine binding to a particulate fraction from rat brain, 9.1 nm, determined from inhibition of the binding of 1 nm [3H]‐mepyramine, was distinctly higher than that, 0.83 nm, measured on an equivalent preparation from guinea‐pig brain. 2 In rat brain the dissociation constant for mepyramine, determined from the binding of [3H]‐mepyramine sensitive to inhibition by 2 × 10−6 m promethazine, was higher than the constant obtained from the inhibition of the binding of 1 nm [3H]‐mepyramine by non‐radioactive mepyramine. This suggests that the promethazine‐sensitive binding of [3H]‐mepyramine includes a lower affinity non‐receptor component, which becomes apparent at higher concentrations of [3H]‐mepyramine. 3 In the guinea‐pig the dissociation constant for mepyramine determined from inhibition of [3H]‐mepyramine binding was in good agreement with the value obtained from inhibition of the contractile response of intestinal smooth muscle to histamine. No similar comparison was possible in the rat. Rat ileum was much less sensitive to histamine and the contraction produced was not inhibited by 10−6 m mepyramine, indicating that it is not mediated by H1‐receptors. 4 Low levels of promethazine‐sensitive [3H]‐mepyramine binding were present in membrane fractions prepared from the longitudinal muscle from rat small intestine, but the characteristics of this binding suggest that it may be largely to lower affinity, non‐receptor sites. 5 Promethazine was practically equipotent as an inhibitor of [3H]‐mepyramine binding in rat and guinea‐pig brain. Chlorpheniramine snowed stereospecificity in the rat as in the guinea‐pig, although the potency of the (+)‐isomer in the rat was only a tenth of that in the guinea‐pig. Histamine had nearly the same IC50 in both species. 6 The evidence suggests that the high‐affinity [3H]‐mepyramine binding sites in rat brain can be described as H1‐receptors, but that these differ structurally from H1‐receptors in the guinea‐pig. 7 The regional distribution of [3H]‐mepyramine binding in rat brain was not the same as that in guinea‐pig brain, the most notable difference being the very much lower level in rat cerebellum compared to guinea‐pig cerebellum.

Histamine H3 receptor-mediated inhibition of depolarization-induced, dopamine D1 receptor-dependent release of [3H]-γ-aminobutyric acid from rat striatal slices

A study was made of the regulation of [3H]‐γ‐aminobutyric acid ([3H]‐GABA) release from slices of rat striatum by endogenous dopamine and exogenous histamine and a histamine H3‐agonist. Depolarization‐induced release of [3H]‐GABA was Ca2+‐dependent and was increased in the presence of the dopamine D2 receptor family antagonist, sulpiride (10 μM). The sulpiride‐potentiated release of [3H]‐GABA was strongly inhibited by the dopamine D1 receptor family antagonist, SCH 23390 (1 μM). Neither antagonist altered basal release. The 15 mM K+‐induced release of [3H]‐GABA in the presence of sulpiride was inhibited by 100 μM histamine (mean inhibition 78±3%) and by the histamine H3 receptor‐selective agonist, immepip, 1 μM (mean inhibition 81±5%). The IC50 values for histamine and immepip were 1.3±0.2 μM and 16±2 nM, respectively. The inhibitory effects of histamine and immepip were reversed by the H3 receptor antagonist, thioperamide, 1 μM. The inhibition of 15 mM K+‐induced [3H]‐GABA release by immepip was reversed by the H3 receptor antagonist, clobenpropit, Kd 0.11±0.04 nM. Clobenpropit alone had no effect on basal or stimulated release of [3H]‐GABA. Elevated K+ caused little release of [3H]‐GABA from striatal slices from reserpinized rats, unless the D1 partial agonist, R(+)‐SKF 38393, 1 μM, was also present. The stimulated release in the presence of SKF 38393 was reduced by 1 μM immepip to the level obtained in the absence of SKF 38393. These observations demonstrate that histamine H3 receptor activation strongly inhibits the dopamine D1 receptor‐dependent release of [3H]‐GABA from rat striatum; primarily through an interaction at the terminals of GABA neurones.

HISTAMINE H1‐AGONIST POTENTIATION OF ADENOSINE‐STIMULATED CYCLIC AMP ACCUMULATION IN SLICES OF GUINEA‐PIG CEREBRAL CORTEX: COMPARISON OF RESPONSE AND BINDING PARAMETERS

1 A range of histamine analogues have been examined as potentiators of the adenosine‐stimulated accumulation of cyclic adenosine 3′,5′‐monophosphate (cyclic AMP) in slices of guinea‐pig cerebral cortex. Dose‐response curves were constructed for the 6 most active compounds and characterized in terms of the IC50, the slope and the maximum response attainable relative to that of histamine. 2 Histamine, 2‐thiazolylethylamine and Nα‐methylhistamine produced a maximal or near maximal response. Nα,Nα‐dlmethylhistamine and 2‐methylhistamine appear to be partial agonists. 3 The response to all the agonists was practically abolished by mepyramine 1 μm, indicating that the response is mediated largely or wholly via histamine H1‐receptors. 4 The relative potencies of the agonists on cyclic AMP accumulation were in general similar to relative potencies in causing contraction of intestinal smooth muscle. The biggest difference was observed with Nα‐methylhistamine. 5 The histamine analogues were also examined as inhibitors of [3H]‐mepyramine binding in homogenates of guinea‐pig cerebral cortex. The inhibition curves were characterized in terms of IC50, the slope and the maximum percentage inhibition. This last value was compared with the inhibition produced by promethazine 2 μm. 6 For the 6 most potent agonists, the EC50 for cyclic AMP accumulation was compared with the IC50 against [3H]‐mepyramine binding, corrected for inhibition of non‐receptor binding and for competition with [3H]‐mepyramine. With the possible exception of 2‐pyridyletfiylamine, the values did not differ by more than a factor of 3.

The binding of doxepin to histamine H1‐preceptors in guinea‐pig and rat brain

1 The affinity constant for doxepin obtained from inhibition of histamine‐induced contraction of guinea‐pig intestinal smooth muscle at 30°C was 2.6 ± 0.18 ± 1010m−1. The slope of a Schild plot was not significantly different from unity. 2 The affinity constant of doxepin did not vary markedly with temperature. At 37°C it was 3.75 ± 0.02 ± 10m−1 and at 25°C 2.1 × 10m−1. 3 Doxepin was a competitive inhibitor of [3H]‐mepyramine binding to guinea‐pig cerebellar homogenates. The affinity constant derived for doxepin at 30°C was 1.12 ± 0.45 ± 10m−1. 4 Hill coefficients for curves of doxepin or mepyramine inhibition of [3H]‐mepyramine binding in guinea‐pig cerebellum, cerebral cortex and hippocampus did not differ significantly from unity. 5 The mean affinity of mepyramine for histamine H1‐receptors in rat brain homogenates at 30°C was 3.5 × 108m−1. Hill coefficients for curves of doxepin or mepyramine inhibition of [3H]‐mepyramine binding to homogenates of rat cerebral cortex or rat whole brain were near unity. 6 These studies provide no evidence that doxepin binds preferentially to a sub‐class of histamine H1‐receptors in rat brain.

Ligand binding to muscarinic receptors in intact longitudinal muscle strips from guinea-pig intestine.

1 The binding of ligands to muscarinic receptors in intact longitudinal muscle strips from guinea‐pig small intestine has been determined by measuring the inhibition of the irreversible binding of [3H]‐propylbenzilylcholine mustard ([3H]‐PrBCM). 2 The IC50 values for inhibition of [3H]‐PrBCM binding by a given ligand were generally higher in intact strips than those reported for broken‐cell preparations. This effect is probably due, at least in part, to the presence of an access‐limitation factor in the kinetics of the irreversible binding of [3H]‐PrBCM to the intact tissue. 3 The mean Hill coefficients for antagonist binding approached unity, but those for strong agonists were significantly less than unity. There was, with the possible exceptions of hexyltrimethylammonium and oxotremorine, reasonably good agreement with the Hill coefficients reported for brain homogenates.

Calcium-dependence of histamine- and carbachol-induced inositol phosphate formation in human U373 MG astrocytoma cells: comparison with HeLa cells and brain slices

1 Histamine (1 mm) induced an accumulation of inositol monophosphate ([3H]‐IP1) in the U373 MG human astrocytoma cell line which increased with time in the presence of 30 mm Li+. After a 30 min incubation period with 1 mm histamine [3H]‐IP1 was the major product detected (84 ± 1 % of total [3H]‐IPx) and was present at a level 11 (±1) fold of basal accumulation. 2 Concentration‐response curves for histamine‐induced [3H]‐IP1 accumulation in U373 MG cells (EC50 5.4 ± 0.5 μm) were shifted to the right in a parallel fashion by mepyramine (slope of a Schild plot 0.99 ± 0.08), yielding a Kd for mepyramine of 3.5 ± 0.3 nm, consistent with the involvement of histamine H1‐receptors. 3 The temelastine‐sensitive binding of [3H]‐mepyramine to a membrane fraction from U373 MG cells was hyperbolic and had a mean Kd of 2.5 ± 1.0 nm. The maximum amount of temelastine‐sensitive binding was 86 ± 19 pmol g−1 membrane protein. 4 Carbachol also induced [3H]‐IP1 accumulation in U373 MG cells, 2.8 (± 0.1) fold of basal with 1 mm carbachol, with an EC50 of 48 ± 8 μm. Pirenzepine shifted carbachol concentration‐response curves to the right (slope of Schild plot 0.89 ± 0.07) giving a Kd for pirenzepine of 0.10 ± 0.01 μm, suggesting that phosphoinositide hydrolysis in U373 MG cells is mediated by the M3‐, rather than the M1‐, muscarinic receptor subtype. 5 [3H]‐IP1 accumulation induced by both 1 mm histamine and by 1 mm carbachol increased when the Ca2+ concentration of the medium was increased from ‘zero’ (no added Ca2+) to 0.3 mm. Histamine‐stimulated [3H]‐IP1 accumulation was further increased, although not so markedly, as the Ca2+ was raised to 4 mm. The same pattern was apparent with histamine‐induced accumulations of [3H]‐IP2 and [3H]‐IP3. In contrast, [3H]‐IPx accumulation in response to carbachol increased between 0.3 and 1.3 mm, but thereafter remained unchanged ([3H]‐IP1) or declined ([3H]‐IP2 and [3H]‐IP3). 6 In HeLa cells, [3H]‐IP1 accumulations induced by 1 mm histamine and 1 mm carbachol showed the same pattern of Ca2+ dependence and were independent of extracellular Ca2+ above 0.3 mm (histamine) or 1.3 mm (carbachol). The response to carbachol appeared to be mediated by an M3‐muscarinic receptor (apparent Kd for pirenzepine 0.09 μm). 7 In cross‐chopped slices of guinea‐pig cerebral cortex and guinea‐pig cerebellum, [3H]‐IP1 accumulation induced by 1 mm histamine in the presence of 10 mm Li+ increased as the extracellular Ca2+ was increased from 0.3 to 2.5 mm, but a further increase to 4 mm had no further effect. In contrast the response to histamine in rat cerebral cortex increased markedly between 1.3 and 4 mm Ca2+. Accumulations of [3H]‐IP1 induced by carbachol in guinea‐pig or rat cerebral cortical slices were not increased as extracellular Ca2+ was raised from 0.3 to 4 mm. 8 Nimodipine (100 nm) and ω‐conotoxin (3 μm) had no significant effect on histamine‐induced [3H]‐IP1 accumulation in rat cerebral cortical slices or in U373 MG cells. 9 We conclude that histamine‐induced [3H]‐IP1 accumulation in U373 MG cells does appear to have a component dependent on the extracellular Ca2+ concentration. The degree of Ca2+‐dependence approaches that observed in guinea‐pig cerebral cortex but is much less than in rat cerebral cortex. Whether U373 MG cells will be of use as a model system for the apparent Ca2+‐entry component observed in guinea‐pig or rat brain slices remains to be established.

The interaction of amine local anaesthetics with muscarinic receptors.

1 Amine local anaesthetics inhibited the binding of (‐)‐[3H]‐quinuclidinyl benzilate ((‐)‐[3H]‐QNB) to muscarinic receptors in crude synaptosomal preparations from guinea‐pig brain. The order of potency was SKF 525A > tetracaine > procaine ⋍ quinidine > procainamide > bupivacaine > lignocaine > prilocaine. 2 The concentration of tetracaine or prilocaine causing 50% inhibition of the receptor‐specific binding of [3H]‐QNB varied linearly with the concentration of [3H]‐QNB present for the range of concentrations of prilocaine used and at lower concentrations of tetracaine, thus providing evidence for a competitive interaction. The affinity constant for tetracaine was 2.6 ± 0.2 × 105 m−1 and that for prilocaine 2.6 ± 0.8 × 103 m−1. At higher concentrations of tetracaine the interaction appears to diverge from simple competitive kinetics. 3 The log dose‐response curve for the contractile response of longitudinal muscle strips from guinea‐pig intestine to carbachol was shifted in a parallel fashion by low concentrations of tetracaine, but flattened by higher doses. A similar effect was observed for both lignocaine and prilocaine. The affinity constants for tetracaine and prilocaine calculated from the parallel shifts, 1 × 105 m−1 and 4 × 103m‐1, respectively, were in reasonable accord with the binding data. 4 The curve for the inhibition of [3H]‐QNB binding by carbachol was not significantly altered, either in position or shape, in the presence of 1 mm prilocaine. Thus there is no evidence that prilocaine, which increases the affinity of nicotinic acetylcholine receptors for agonists, has any similar effect on agonist binding to muscarinic receptors.