Effect of potassium depolarization and preganglionic nerve stimulation on the metabolism of [3H]-choline in rat isolated sympathetic ganglia

1 The effects of potassium depolarization and preganglionic nerve stimulation on the metabolism of [³H]-choline in the isolated superior sympathetic ganglion of the rat have been studied.

2 When unstimulated (resting) ganglia were incubated for 10 min with a low concentration (0.1 μM) of [³H]-choline (high affinity uptake), approximately 75% of the accumulated radioactivity was present as [³H]-phosphorylcholine, 11% was [³H]-acetylcholine ([³H]-ACh) and the remainder was unchanged [³H]-choline.

3 Depolarization of the ganglia with K (46 mM) before their incubation with [³H]-choline, increased [³H]-choline uptake by 70% and increased [³H]-ACh synthesis by more than 700%, so that [³H]-ACh represented almost 50% of the total radioactivity recovered. In contrast, the proportion of [³H]-phosphorylcholine fell to 36% of the total radioactivity recovered.

4 The striking effect of K-depolarization on [³H]-ACh synthesis in ganglia occurred at a concentration of 30 mM or above, and the maximum effect was seen at 45-50 mM.

5 Chronic denervation of the ganglia abolished all the effects of high-K on [³H]-choline metabolism. In resting ganglia, [³H]-ACh formation was reduced by over 80% but [³H]-phosphorylcholine synthesis and the level of unchanged [³H]-Ch were not affected by denervation.

6 Exposure of the ganglia to low-Na or hemicholinium-3 (HC-3) greatly reduced [³H]-ACh synthesis in control resting ganglia and almost abolished the effects of high-K on [³H]-ACh synthesis.

7 Prevention of transmitter release with high-Mg or low-Ca medium also prevented K-depolarization from stimulating [³H]-ACh synthesis.

8 Preganglionic nerve stimulation had an effect on [³H]-choline metabolism similar to that of K-depolarization. Thus, at all the frequencies studied (1-30 Hz), [³H]-ACh synthesis was greatly increased and [³H]-phosphorylcholine was reduced, the maximum effects occurring at 3 Hz.

9 When ganglia were incubated with a high concentration (100 μM) of [³H]-choline (low affinity uptake), a different pattern of metabolism was observed. Most of the radioactivity in resting ganglia was present as unchanged [³H]-choline (70%) with [³H]-phosphorylcholine and [³H]-ACh representing 23% and 6% of the total radioactivity respectively. K-depolarization decreased [³H]-choline uptake but increased the proportions of [³H]-phosphorylcholine and [³H]-ACh to 32% and 24% of the total radioactivity respectively.

10 It is concluded that in unstimulated (resting) rat sympathetic ganglia most of the [³H]-choline transport and metabolism occurs in postsynaptic structures. However, depolarization of the presynaptic nerve terminals appears to trigger a sodium-dependent, HC-3 sensitive, high-affinity uptake process, and causes a dramatic increase in presynaptic [³H]-ACh synthesis together with a fall in postsynaptic [³H]-phosphorylcholine synthesis. These changes in choline metabolism cannot be due to the depolarization of the nerve terminals per se, because they were abolished by high-Mg or low-Ca, i.e. when transmitter release was prevented. Thus, the increase in ACh synthesis may be triggered by a fall in the intraterminal concentration of ACh or by the changes in Ca flux induced by depolarization. Our experiments do not provide evidence on these possible mechanisms.

     

On the mechanism by which veratridine causes a calcium-independent release of γ-aminobutyric acid from brain slices

1 The mechanisms by which veratridine increases the release of γ-aminobutyric acid (GABA) from brain slices have been studied.

2 Exposure of superfused cerebro-cortical, nigral or cerebellar slices to veratridine (5 μM) or KCl (50 mM) caused large increases in the efflux of [³H]-GABA.

3 Reduction of the external Ca concentration [Ca]_o to zero had strikingly different effects on the veratridine and K-evoked release of [³H]-GABA. The K-evoked release from all three areas was greatly reduced in Ca-free medium, but the veratridine-evoked release from cerebeller slices was not affected, and the release of [³H]-GABA from cortical and nigral slices was increased three fold. The potentiation of the veratridine evoked release of GABA which occurred in Ca-free medium was not due to the reduction in divalent ions, because it still occurred in medium in which the Ca was replaced by an equivalent amount of Mg.

4 The veratridine-evoked release of [¹⁴C]-glycine from slices of spinal cord was also significantly increased in Ca-free medium. In contrast, the release of cortical [³H]-noradrenaline and [¹⁴C]-acetylcholine caused by the alkaloid was greatly diminished in Ca-free medium.

5 The veratridine but not the K-evoked release of [³H]-GABA was abolished when the external Na concentration [Na]_o was reduced to zero and by tetrodotoxin (TTX) (0.2 μM). Cl-free medium did not affect the veratridine-evoked release of [³H]-GABA or its potentiation by Ca-free medium.

6 Exposure of the tissue to depolarizing concentrations of external K ([K]_o = 120 mM) did not abolish the veratridine evoked release of [³H]-GABA or its potentiation by Ca-free medium.

7 Pre-incubation of cortical slices with L-2,4, diaminobutyric acid (DABA), or substitution of Na in the superfusion medium with Li, did not affect the veratridine-evoked release of [³H]-GABA, indicating that the alkaloid does not stimulate GABA efflux by a carrier-mediated transport process.

8 Exposure of the tissue to ruthenium red (10 μM) increased the veratridine evoked release of [³H]-GABA in both normal and in Ca-free medium but almost abolished the K-evoked release.

9 It is suggested that veratridine causes GABA release by increasing the permeability of the nerve terminals to Na. In normal medium, the resulting influx of Ca²⁺ ions through voltage-dependent Ca²⁺ channels may be involved in triggering the release of GABA. However, a major part of the GABA efflux appears to be triggered by the release of Ca²⁺ ions from intraterminal mitochondria, which results from the increase in[Na]_i. Since Ca²⁺ ions antagonize the action of veratridine, the potentiation of the drug-evoked release of GABA that occurs in Ca-free medium, might be due to the absence of the antagonistic Ca²⁺ ions. The resulting greater increase in Na entry and [Ca]_i caused by Ca release from intracellular stores, must presumably more than balance the contribution normally made by any influx of extracellular Ca²⁺.

     

Effects of the histamine H2-receptor blocking drugs burimamide and cimetidine on noradrenergic transmission in the isolated aorta of the rabbit and atria of the guinea-pig

1 In rabbit aortic strips, concentration-response curves to noradrenaline (NA) were shifted to the right in a parallel and concentration-dependent manner by the α-adrenoceptor blocking drug, phentolamine and also by the histamine H₂-receptor blocking drugs, burimamide and cimetidine. Responses to 5-hydroxytryptamine were not affected by these drugs.

2 Burimamide had the properties of a competitive antagonist of noradrenaline, possessing about one-hundredth the potency of phentolamine. Cimetidine was weaker than burimamide and did not fulfil the requirements for competitive antagonism of noradrenaline.

3 In guinea-pig isolated atria, in which noradrenergic transmitter stores were labelled with [³H]-noradrenaline, phentolamine (3 μM), burimamide (30 μM) and cimetidine (30 μM), in decreasing order of effectiveness, each enhanced stimulation-induced efflux of [³H]-noradrenaline, indicating that their blocking effects on prejunctional α-adrenoceptors in this tissue are in the same order of relative potency as on postjunctional α-adrenoceptors in rabbit aortic strips.

4 In the concentrations used (30 μM), neither burimamide nor cimetidine interfered with the neuronal uptake of noradrenaline. Burimamide, and to a much lesser extent, cimetidine, increased the resting efflux of [³H]-noradrenaline from guinea-pig atria.

5 The effect of clonidine, a partial agonist on prejunctional α-adrenoceptors in guinea-pig atria, in increasing stimulation-induced efflux of [³H]-noradrenaline when stimulated with 150 pulses at 5 Hz was blocked by cimetidine (30 μM) and reversed by phentolamine (3 μM) and burimamide (30 μM).

     

Catecholamine transport in isolated lung parenchyma of pig

1 Lung parenchyma strips of the pig incubated at 37°C with [³H]-(-)-noradrenaline ([³H]-NA) or [³H]-(±)-isoprenaline ([³H]-Iso), accumulated radioactivity via saturable, high affinity uptake processes. Apparent saturation constants (K_m) for [³H]-NA and [³H]-Iso were 1.34 × 10^-6 M and 1.63 × 10^-6 M respectively, while apparent transport maxima (V_max) were 4.86 and 1.63 × 10^-9 mol min^-1 g^-1 respectively.

2 Cellular accumulation of radioactivity from radiolabelled catecholamines was greatly reduced by lowering the temperature to 7°C, pretreatment with ouabain (100 μM), phentolamine (15 μM) or phenoxybenzamine (80 μM). However, accumulation of radioactivity derived from (³H]-NA was inhibited selectively by cocaine (10 μM) and desipramine (1 μM), while normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM) caused much greater reductions in cellular radioactivity from [³H]-Iso than from (³H]-NA. Taken together with information from kinetic studies, the results indicate that these amines are transported by separate uptake processes.

3 Cocaine (50 μM) which selectively reduced [³H]-NA transport, had no significant effect on the sensitivity (EC₅₀) of isolated parenchyma lung strips of the pig to the contractile effects of cumulative concentrations of NA. The catechol-O-methyl transferase (COMT) inhibitor, U-0521 (60 μM), also failed to alter the potency of NA, while normetanephrine (80 μM) caused a 2 fold decrease in potency.

4 Phentolamine (15 μM), which reduced the cellular accumulation of radioactivity derived from [³H]-Iso by 64%, caused a small potentiation of Iso-induced relaxations of porcine lung strips. Normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM), which also depressed the accumulation of cellular radioactivity from [³H]-Iso by > 50%, caused rightward shifts in Iso concentration-effect curves as a result of β-adrenoceptor blockade. In sharp contrast, cortisol (80 μM) and U-0521 (60 μM), which caused smaller reductions in the cellular accumulation of radioactivity derived from [³H]-Iso, both caused an approximately 9 fold potentiation of responses to Iso in isolated lung strips.

5 The results indicate that the major sites of uptake and metabolism of NA in porcine parenchyma strip are remote from α-adrenoceptors mediating NA-induced contraction. Similarly, some major sites of uptake of Iso are remote from β-adrenoceptors mediating Iso-induced relaxation. However, β-adrenoceptors are apparently in close proximity to a compartment containing COMT activity.

     

Pharmacological characterization of the inhibitory effects of neurotensin on the rabbit ileum myenteric plexus preparation.

1 [³H]-amezinium is taken up selectively into noradrenergic axons and their transmitter-storing vesicles and is released from these axons by action potentials. We used it as a non-α-adrenergic marker in order to study the α-adrenergic autoinhibition of noradrenaline release.

2 Rat occipitocortical slices were preincubated with [³H]-amezinium 0.03 μM and then superfused and stimulated electrically (3 Hz for 3 min). The stimulation-evoked overflow of tritium was measured in six groups of slices: from saline-pretreated rats; from saline-pretreated rats, the slices being exposed to exogenous noradrenaline before preincubation with [³H]-amezinium; from saline-treated rats, slices from which were exposed simultaneously to noradrenaline and cocaine before preincubation with [³H]-amezinium; from rats in which noradrenaline stores had been depleted by pretreatment with α-methyltyrosine (α-MT); from α-MT-treated rats, the slices being exposed to noradrenaline before preincubation with [³H]-amezinium; and from α-MT-treated rats, slices from which were exposed to noradrenaline plus cocaine before preincubation with [³H]-amezinium.

3 The stimulation-evoked overflow of tritium, expressed as a percentage of the tritium content of the tissue, was 1.15% in slices from saline-pretreated rats, and was similar in slices from saline-pretreated rats after exposure to noradrenaline or noradrenaline plus cocaine. It was 2.56% in slices from α-MT-treated rats, 1.20% from α-MT-treated rats after exposure to noradrenaline, and 2.88% from α-MT-treated rats after exposure to noradrenaline plus cocaine.

4 Yohimbine 0.1 and 1 μM increased the stimulation-evoked overflow of tritium in slices from all groups of saline-pretreated rats and in those slices from α-MT rats that had been in contact with exogenous noradrenaline. Yohimbine did not change the evoked overflow in slices from α-MT rats that had not been exposed to noradrenaline, or had been exposed to noradrenaline plus cocaine.

5 Clonidine 0.01-1 μM decreased the stimulation-evoked overflow of tritium moderately in slices from saline-pretreated rats, markedly in slices from α-MT-treated rats, and moderately again when the latter slices had been exposed to noradrenaline.

6 It is concluded that the action potential-evoked release of [³H]-amezinium as well as the modulation of this release by yohimbine and clonidine depend on the presence or absence of α-adrenergic autoinhibition caused by the co-secretion of noradrenaline. When there is co-secretion of noradrenaline, the evoked release of [³H]-amezinium is relatively small, yohimbine increases the release, and clonidine can cause only moderate inhibition. When there is no or very little co-secretion of noradrenaline, the evoked release of [³H]-amezinium is at least doubled, yohimbine causes no further increase and clonidine produces strong inhibition.

     

Accumulation and overflow of 3H following incubation of the guinea-pig gall bladder with [3H]-noradrenaline

1 Strips of guinea-pig gall bladder readily accumulate ³H following incubation in the presence of 5 × 10^-8 M (-)-[³H]-noradrenaline. This accumulation was reduced by lowering the incubation temperature (from 37° to 23°C), by cocaine (10^-6 M), by nortriptyline (10^-8, 10^-6 and 10^-4 M) and following incubation of the tissues with 6-hydroxydopamine (10^-3 M for 3 h). At 10^-6 M, (-)-noradrenaline and (-)-adrenaline, but not (-)-isoprenaline, inhibited the accumulation of ³H.

2 Following preloading of strips of guinea-pig gall bladder with 3.6 × 10^-7 M (-)-[³H]-noradrenaline for 1 h, the spontaneous overflow of ³H was observed. Cocaine (10^-4 M), nortriptyline (10^-6 M), (-)-isoprenaline (10^-5 M), acetylcholine (10^-5 M) and adenosine 5′-triphosphate (ATP, 10^-4 M) had no effect on the spontaneous overflow of ³H. KCl (10^-1 M), (-)-noradrenaline (10^-5 M), (-)-adrenaline (10^-5 M), and tyramine (10^-5 M) increased the overflow of ³H. These results illustrate similar characteristics of the guinea-pig gall bladder to other noradrenergically-innervated tissues in accumulating and releasing ³H following incubation in the presence of [³H]-noradrenaline.

3 Following incubation in the presence of 3.6 × 10^-7 M (-)-[³H]-noradrenaline, field stimulation, at 5 Hz, of strips of gall bladder, in the absence or presence of 10^-6 M atropine, increased the overflow of ³H and, simultaneously, induced contractions. The contractile responses to 5 Hz were smaller in the presence than in the absence of 10^-4 M lignocaine. Lignocaine (10^-4 M) reduced the overflow of ³H evoked by field stimulation at 5 Hz. It is suggested that the contractile responses to 5 Hz are due to nerve stimulation and that the increased overflow of ³H is due to the stimulation of noradrenergic nerves.

4 The overflow of ³H evoked by field stimulation at 5 Hz was unaltered and increased by propranolol (10^-6 M) and phentolamine (10^-6 M), respectively. Clonidine (5 × 10^-5 M) had no effect in the absence but reduced the amount of ³H which overflowed in response to field stimulation at 5 Hz in the presence of 10^-6 M atropine. The contractile responses to field stimulation at 5 Hz were reduced by phentolamine (10^-6 M) or clonidine (5 × 10^-6, 10^-5 and 5 × 10^-5 M) whether or not atropine (10^-6 M) was present. These results illustrate the presence of postsynaptic α-adrenoceptors and suggest the presence of presynaptic α-adrenoceptors in the gall bladder of the guinea-pig.

     

Inhibition of K+-stimulated [3H]dopamine and [14C]acetylcholine release by the putative dopamine autoreceptor agonist,B-HT 920

Summary The inhibition of K¹-stimulated [³H]dopamine and [¹⁴C]acetylcholine release from preloaded rat striatal slices was used to examine the presynaptic selectivity of the putative dopamine autoreceptor agonist, B-HT 920. In the micromolar range, B-HT 920 caused a concentration-dependent inhibition of the release of both labeled neurotransmitters as evoked by 20 mM K⁺. The effect of B-HT 920 on both [³H]dopamine and [¹⁴C]acetylcholine release was completely blocked by (+) butaclamol but not by (–) butaclamol. Sulpiride, a selective D₂ antagonist, similarly blocked the inhibitory effect of B-HT 920 on the release of both labeled neurotransmitters indicating both responses were mediated by D₂ receptors. (+) Butaclamol alone elevated stimulated [³H]dopamine release suggesting a significant amount of autoreceptor occupancy by endogenously released dopamine. Experiments with tolazoline and the alpha₂ agonist, B-HT 933, did not suggest any involvement of alpha-adrenoceptor activity in the inhibitory effects of B-HT 920 on the release of either transmitter. Inhibition of release was a selective effect of B-HT 920 as the drug was without effect on the K⁺-stimulated release of [³H]serotonin. The results indicate that in vitro B-HT 920 is active of both pre-and postsynaptic dopamine receptors in contrast to the pattern of effects observed after its in vivo administration.     

Mechanism of nicotine-induced release of noradrenaline from adrenergic nerve endings

1 A study of the mechanism of release of [³H]-noradrenaline ([³H]-NA) by nicotine from isolated vas deferens of the rat was made using incubation media of different ionic composition.

2 Nicotine (20 μg/ml)-induced release of [³H]-NA was significantly potentiated in K⁺-free Krebs solution as compared to that in normal Krebs-Ringer solution.

3 Nicotine-induced release of [³H]-NA was significantly reduced in Na⁺-deficient Krebs solution (containing only 11 mM Na⁺) and was abolished in Na⁺-free Krebs solution.

4 In totally depolarized tissues, nicotine failed to cause an outflow of [³H]-NA but Ca²⁺ (5 mM) did so.

5 Nicotine required the presence of Ca²⁺ in the incubation medium to cause release of [³H]-NA from adrenergic nerve terminals, the magnitude of release being dependent upon the concentration of Ca²⁺.

6 Nicotine-induced release of [³H]-NA was demonstrated in high Ca²⁺, Na⁺-free Krebs solution in which all Na⁺ had been replaced with Ca²⁺.

7 It is concluded that nicotine increases the membrane permeability to both Na⁺ and Ca²⁺. It is also suggested that the increase in permeability to Ca²⁺ alone is not sufficient but a local depolarizing action of nicotine is necessary to cause release of noradrenaline from adrenergic nerve endings.

     

Inhibition by apomorphine of the potassium-evoked release of [3H]-γ-aminobutyric acid from the rat substantia nigra in vitro

1 The spontaneous and potassium-evoked release of tritium from the rat substantia nigra prelabelled with [³H]-γ-aminobutyric acid [³H]-GABA were assessed in vitro under conditions of superfusion.     

Spectrum of the μ-, δ- and κ-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ-, δ- and κ-binding sites of the opiate receptor determined by saturation analysis.

2 In competition experiments, binding of the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin at the μ-site was displaced by [D-Ala²,D-Leu⁵]enkephalin with rather low affinity (K_I = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K_I = 3.1 nM) and (-)-bremazocine (K_I = 0.32 nM), which also displaced the binding of [³H]-[D-Ala²,D-Leu⁵]enkephalin from the δ-site. In contrast, the binding to the κ-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the μ-ligand [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (K_I = 2000-3000 nM) or the δ-ligand [D-Ala²,D-Leu⁵]enkephalin (K_I > 20,000 nM).

3 The dissociation equilibrium constant (K_D) and the binding capacity (pmol/g) of the μ-binding site were determined with the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin. For the δ-site, [³H]-[D-Ala²,D-Leu⁵]enkephalin was used in the presence of unlabelled [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin in order to suppress cross-reactivity to the μ-binding site. For the estimation of κ-binding, [³H]-(±)-ethylketazocine or [³H]-(-)-bremazocine were used in the presence of unlabelled μ- and δ-ligands for the suppression of cross-reactivities to the μ- and δ-binding sites.

4 In rat brain the capacity of the μ-binding site was 7.3 pmol/g brain, that of the δ-binding site 6.7 pmol/g brain and that of the κ-binding site 2.0 pmol/g brain. Thus, the κ-binding site had the lowest value whereas in the guinea-pig brain the capacity of the μ-binding site was lower than that of the δ- or κ-binding site.

     

The binding of [3h]-oestradiol-17β in the immature rat uterus during the sequential administration of nonsteroidal anti-oestrogens

1 The binding of [³H]-oestradiol-17β (0.08 μg) in the uterus, vagina, liver and heart of immature female rats has been studied in vivo and the effect of daily administrations of the non-steroidal anti-oestrogens, tamoxifen and monohydroxytamoxifen, on the 2 h accumulation of [³H]-oestradiol-17β in the uterus has been determined.     

Inhibitory actions of catecholamines on electrically induced contractions of the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus

1 The submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus was used to study the actions of catecholamines on the twitch responses to electrical stimulation.

2 When the preparation was stimulated coaxially (0.1 Hz, 0.5 ms, supramaximal voltage), stable twitch-like contractions were obtained. These were abolished by tetrodotoxin (0.1 μM) and atropine (0.1 μM), potentiated by physostigmine (0.1 μM), and were mediated presumably by stimulation of intramural cholinergic nerves.

3 The twitch contractions of the muscularis mucosae were inhibited by catecholamines, in a concentration-dependent manner. The order of potency was isoprenaline > adrenaline > noradrenaline > dopamine.

4 The inhibitory actions of noradrenaline (1 μM) and adrenaline (1 μM) were partly reversed by phentolamine (1 μM) or by propranolol (1 μM), and completely abolished by both antagonists together. The inhibitory effect of dopamine (300 μM) was largely reversed by phentolamine (1 μM), but not by propranolol (1 μM), while the inhibitory action of isoprenaline was competitively antagonized only by propranolol (pA₂ of 7.6).

5 The contraction of the muscularis mucosae to exogenously applied acetylcholine (ACh, 20 nM) which was comparable in magnitude with that to electrical stimulation was also inhibited by isoprenaline (0.1 μM), adrenaline (1 μM) and noradrenaline (1 μM), but not by dopamine (300 μM). In the presence of propranolol (1 μM), noradrenaline, adrenaline and dopamine potentiated the ACh-induced contraction, while the effect of isoprenaline was mainly antagonized. The potentiating effects were antagonized by further treatment with phentolamine (1 μM).

6 Adrenaline, noradrenaline and dopamine but not isoprenaline, produced a weak contraction of the longitudinal muscularis mucosae in the presence of propranolol (3 μM). The contractile responses were completely inhibited by phentolamine (3 μM). Tone in the muscularis mucosae induced by carbachol (3 μM) in the presence of phentolamine (10 μM) was inhibited by catecholamines, in a concentration-dependent manner, an effect that was competitively antagonized by propranolol.

7 In the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus there are three types of adrenoceptor, inhibitory prejunctional α-adrenoceptors, excitatory postjunctional α-adrenoceptors and inhibitory postjunctional β-adrenoceptors, and cholinergic neurotransmission is inhibited by catecholamines acting at both prejunctional α- and postjunctional β-adrenoceptors.

     

Cholinergic modulation of [3H]dopamine release from dendrosomes of rat substantia nigra

Summary Dendrosomes prepared from substantia nigra are able to take up and release [³H]dopamine in a Ca²⁺-dependent manner. The V_max values of [³H]dopamine uptake in substantia nigra dendrosomes was about 5 times lower than that in caudate putamen synaptosomes. The pattern of the K⁺-dependency of the [³H]dopamine release in substantia nigra dendrosomes was significantly different from that found in caudate putamen synaptosomes. The release of [³H]dopamine evoked by 15 mmol/l KCl from superfused dendrosomes was increased in a concentration-dependent manner by acetylcholine. The maximal potentiation produced by acetylcholine was about 40%. The potentiation of [³H]dopamine release by 10 µmol/l acetylcholine was insensitive to mecamylamine but antagonized by atropine and by pirenzepine. The effects of acetylcholine on the release of [³H]acetylcholine from substantia nigra nerve endings was also studied. Exogenous acetylcholine added to the superfusion medium decreased in a concentration-dependent manner the release of acetylcholine. This effect was not antagonized by mecamylamine or pirenzepine but fully antagonized by atropine. The data suggest the existence, in the substantia nigra of the rat, of two distinct muscarinic receptor subtypes regulating respectively dopamine release from dopamine dendrites and acetylcholine release from cholinergic nerve terminals.Part of this work was presented at a satellite meeting of the 11th International Congress of Pharmacology: Dopamine '90 held in Como, Italy (July 1990) Send offprint requests to M. Raiteri at the above address     

The novel nicotinic receptor antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), inhibits nicotine-evoked [H]norepinephrine overflow from rat hippocampal slices

Smoking is a significant health concern and strongly correlated with clinical depression. Depression is associated with decreased extracellular NE concentrations in brain. Smokers may be self-medicating and alleviating their depression through nicotine stimulated norepinephrine (NE) release. Several antidepressants inhibit NE transporter (NET) function, thereby augmenting extracellular NE concentrations. Antidepressants, such as bupropion, also inhibit nicotinic receptor (nAChR) function. The current study determined if a recently discovered novel nAChR antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), inhibits nicotine-evoked NE release from superfused rat hippocampal slices. Previous studies determined that bPiDDB potently (IC₅₀ = 2 nM) inhibits nicotine-evoked striatal [³H]dopamine (DA) release in vitro, nicotine-evoked DA release in nucleus accumbens in vivo, and nicotine self-administration in rats. In the current study, nicotine stimulated [³H]NE release from rat hippocampal slices (EC₅₀ = 50 μM). bPiDDB inhibited (IC₅₀ = 430 nM; I_max = 90%) [³H]NE release evoked by 30 μM nicotine. For comparison, the nonselective nAChR antagonist, mecamylamine, and the α7 antagonist, methyllycaconitine, also inhibited nicotine-evoked [³H]NE release (IC₅₀ = 31 and 275 nM, respectively; I_max = 91% and 72%, respectively). Inhibition by bPiDDB and mecamylamine was not overcome by increasing nicotine concentrations; Schild regression slope was different from unity, consistent with allosteric inhibition. Thus, bPiDDB was 200-fold more potent inhibiting nAChRs mediating nicotine-evoked [³H]DA release from striatum than those mediating nicotine-evoked [³H]NE release from hippocampus.     

Correlation between catecholamine secretion from bovine isolated chromaffin cells and [3H]-ouabain binding to plasma membranes

1 Secretion of catecholamines (CA) evoked by ouabain, chlormadinone acetate (CMA), phenoxybenzamine (Pbz) and vanadate, four agents known to inhibit Na⁺, K⁺-dependent Mg²⁺-activated adenosine triphosphatase (ATPase) activity has been studied in suspensions of bovine isolated adrenal medullary cells.

2 Acetylcholine (ACh) evoked a 5 fold increase of the basal CA secretion from isolated cells suspended in oxygenated Krebs-bicarbonate solution kept at 27°C. Secretion was antagonized by Ca²⁺-deprivation or hexamethonium, indicating good functional viability of the cells.

3 Ouabain (10^-7 to 10^-4 M) evoked a progressive, dose-dependent release of CA from cell suspensions. Study of the time course of the secretory response for 2 h allowed the separation of two components in the secretory response at all doses studied: a slow initial component (0.011 pg/min CA) and a second faster component (0.032 pg/min CA).

4 CMA evoked a clear-cut CA secretory response. The ED₅₀ for CMA was 10^-4 M, as compared to 3 × 10^-6 M for ouabain. Pbz and vanadate did not induce CA release.

5 [³H]-ouabain was taken up and bound to intact isolated cells by a non-saturable binding process. However, in semi-purified plasma membranes from bovine adrenal medulla a saturable specific [³H]-ouabain binding process was observed with a K_D of 8.1 nM. Binding to the membranes was ATP-dependent and antagonized by K⁺.

6 [³H]-ouabain specific binding to membranes was antagonized by ouabain and CMA, but not by Pbz or vanadate; the ID₅₀ for ouabain and CMA were 10^-6 and 10^-5 M respectively.

7 Ouabain partially inhibited, in a dose-dependent manner, Na⁺, K⁺-Mg²⁺ ATPase activity of the semi-purified plasma membranes.

8 The results demonstrate a good correlation between the ability of different drugs, known to inhibit ATPase activity, to displace [³H]-ouabain binding to adreno—medullary plasma membranes and their capacity to evoke a CA secretory response from isolated chromaffin cells. The data also suggest that the CA secretory effects of ouabain may not be due simply to inhibition of the Na⁺ pump and the subsequent ionic redistribution across the plasma membrane; a second mechanism may also be involved.

     

Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151-2

Summary In order to label dopamine D₂ receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [³H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K _D of 57 pmol/l and B _max of 36 pmol/g tissue as determined by Scatchard analysis. The K _D, but not the B _max, of [³H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [³H]-YM-09151-2 labeled 40% more sites than [³H]-spiperone in the same tissue homogenate. [³H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D₂ receptor profile. Thus, dopamine agonists inhibited [³H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [³H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [³H]-YM-09151-2, D ₂ ^high and D ₂ ^low . The D ₂ ^high state of the receptor could be converted to D ₂ ^low by guanine nucleotides and sodium ions as is the case for [³H]-spiperone binding to D₂ receptors. [³H]-YM-09151-2 appears to be a more selective ligand for dopamine D₂ receptors than [³H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S₂) receptors. [³H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D₂ receptors.Abbreviations used (±)ADTN (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene - NPA N-n-propylnorapomorphine - Gpp(NH)p 5-guanylylimidodiphosphate     

The effect of antiparkinsonian drugs on oxidative stress induced pathological [3H]dopamine efflux after in vitro rotenone exposure in rat striatal slices

An in vitro model of mitochondrial dysfunction with subsequent oxidative stress was elaborated and utilized to study the effect of drugs, currently used for the treatment of Parkinson's disease, on pathological H₂O₂-evoked [³H]dopamine efflux and the formation of toxic dopamine metabolites in rat striatal slices. 60 min rotenone (0.1–10 μM) pretreatment decreased dopamine content and [³H]dopamine uptake, as well as ATP level and energy charge of the slices. In addition, a robust potentiation of H₂O₂-evoked [³H]dopamine efflux and the formation of dopamine quinone in the effluent was detected. l-DOPA (200 μM) markedly elevated resting but not 100 μM H₂O₂-evoked and electrically-induced [³H]dopamine efflux. Furthermore, l-DOPA promoted the formation of dopamine quinone. Ropinirole (100 nM) did not affect resting and H₂O₂-evoked [³H]dopamine efflux and inhibited the electrically evoked release only in untreated slices. l-deprenyl, at concentration of 0.01 μM potentiated, whilst between 1 and 50 μM diminished H₂O₂-evoked [³H]dopamine efflux. Rasagiline (0.01–50 μM) slightly inhibited H₂O₂-evoked [³H]dopamine efflux, and it was able to prevent the generation of dopamine quinone. Neither of the drugs was able to suppress both the pathological H₂O₂-evoked [³H]dopamine efflux and the formation of dopamine quinone with simultaneous augmentation of electrically evoked [³H]dopamine release what should be a future concept of antiparkinsonian drug-design.     

The effect of amperozide on uptake and release of [3H]-dopamine in vitro from perfused rat striatal and limbic brain areas

Amperozide, a putatively antipsychotic drug, was studied for its effects on uptake and release of [³H]-dopamine in rat brain in vitro. Amperozide inhibited uptake of [³H]-dopamine in striatal chopped tissue in vitro with an IC₅₀ of 18 μM. It also increased basal release of [³H]-dopamine from perfused rat striatal and limbic tissue in vitro at concentrations above 5 μM. Release of [³H]-dopamine from perfused rat striatal and limbic tissue stimulated with 5 μM amphetamine, was inhibited by 1 μM amperozide to 46%. No significant difference was found for the effect of amperozide on in vitro release of [³H]-dopamine from corpus striatum compared to tissue from limbic brain regions; neither on basal release nor on amphetamine-stimulated release of dopamine.     

Differential labelling of dopamine receptors in rat brain in vivo: Comparison of [3H]piribedil, [3H]S 3608 and [3H]N,n-propylnorapomorphine

The in vivo distribution of radioactivity in brain and labelling of cerebral dopamine receptors in rats derived from the administration of the atypical dopamine agonists [³H]piribedil and [³H]S 3608 has been compared to that of the classical dopamine agonist [³H]N,n-propylnorapomorphine (NPA). Radioactivity derived from [³H]piribedil accumulated in the substantia nigra, nucleus accumbens and cervical spinal cord, and this accumulation was prevented by administration of (+)-butaclamol and apomorphine only in substantia nigra and the nucleus accumbens. Radioactivity derived from [³H]S 3608 accumulated in the same areas and, additionally, in the frontal cortex and tuberculum olfactorium; this accumulation was prevented by administration of (+)-butaclamol and apomorphine only in substantia nigra, nucleus accumbens and tuberculum olfactorium. Neither ligand caused accumulation of radioactivity in the striatum. In contrast, radioactivity derived from [³H]NPA accumulated in the substantia nigra, nucleus accumbens, striatum, and tuberculum olfactorium. Radioactivity derived from [³H]NPA was prevented from accumulating in all these areas by (+)-butaclamol and by apomorphine, but piribedil only prevented accumulation in the substantia nigra and nucleus accumbens and S 3608 only prevented accumulation in substantia nigra, tuberculum olfactorium and nucleus accumbens. Neither piribedil nor S 3608 prevented accumulation of radioactivity derived from [³H]NPA in the striatum. Piribedil and S 3608 showed equal capacity in vitro to displace [³H]spiperone and [³H]NPA from striatal or nucleus accumbens tissue preparations. These results suggest that, in vivo piribedil and S 3608 selectively interact with dopamine receptors in the substantia nigra and nucleus accumbens, but not with those in striatum, perhaps due to differential distribution within brain.     

Effects of sulphasalazine and its metabolites on prostaglandin synthesis, inactivation and actions on smooth muscle

1 We have investigated the effects of sulphasalazine and of its principal colonic metabolites (5-aminosalicylic acid and sulphapyridine) on prostaglandin inactivation, synthesis and actions on gastrointestinal smooth muscle.

2 Sulphasalazine inhibits prostaglandin F_2α breakdown in 100,000 g supernatants in all organs so far tested from 7 species with an ID₅₀ of approx. 50 μM; it has a selective action on prostaglandin 15-hydroxydehydrogenase and does not inhibit prostaglandin Δ-13 reductase, prostaglandin 9-hydroxydehydrogenase or `enzyme X' at millimolar concentrations. Enzyme activities were measured radiochemically or by bioassay.

3 Sulphapyridine and 5-aminosalicylic acid do not inhibit prostaglandin inactivation in vitro (4 species tested). A methyl analogue of sulphasalazine is a more potent inhibitor than the parent compound. Rabbit colon prostaglandin F_2α metabolism in vitro was inhibited by the following drugs with ID₅₀ values (μM) of: diphloretin phosphate 20, sulphasalazine 50, indomethacin 220, frusemide 1000 and aspirin 10,000. A similar rank order of potencies was obtained with rabbit kidney.

4 Sulphasalazine at 50 to 100 μM inhibited inactivation of prostaglandin E₂ in the perfused rat and guinea-pig lung by 3 to 40% (rat) and 32 to 100% (guinea-pig) when measured by superfusion cascade bioassay and of prostaglandin F_2α by 43.6 ± 6.5% in rat lung perfused with 50 μM sulphasalazine and assayed radiochemically.

5 Prostaglandins E₁ and E₂ were 97.0 ± 8.2% and 92.3 ± 6.8% inactivated in the lungs after intravenous injection in the anaesthetized rat as measured by reference to their vasodepressor potencies when injected intra-arterially. Prostaglandin A₂ was not similarly inactivated. Pulmonary inactivation was prevented in the presence of an intravenous infusion of 16.3 μg kg^-1 min^-1 sulphasalazine and partially inhibited at a lower infusion rate.

6 Prostaglandin biosynthesis from arachidonic acid was measured in microsomal preparations from four sources by bioassay and radiochemical methods. Indomethacin was a potent inhibitor (ID₅₀ 0.8 to 4.1 μM) but sulphasalazine and its methyl analogue were very weak inhibitors (ID₅₀ 1500 to > 5000 μM), 5-aminosalicylic acid was weaker still and sulphapyridine inactive.

7 Sulphasalazine at 50 μM did not affect the actions of prostaglandins on five smooth muscle preparations; at 500 μM there was a rapidly reversible and probably non-specific antagonism of responses to low doses of prostaglandins.

8 The specificity and selectivity of the interaction of sulphasalazine and its metabolites with the formation, breakdown and actions of prostaglandins are discussed.