Increased uptake of [3H]choline by rat superior cervical ganglion: An effect of dexamethasone

The high affinity uptake of [³H]choline by the superior cervical ganglion, isolated from the rat, was found to be increased by dexamethasone. Maximal increase (60–65% above control values) occurred at the steroid concentration of 5 × 10^?5 M. Other glucocorticoids (triamcinolone, corticosterone and hydrocortisone) were without an effect on the [³H]choline uptake. Following administration of dexamethasone (25 mg/kg, i.p.), there was a marked increase in the level of choline in the ganglion. The increase was 3-fold at 1 hr and 10-fold at 6 hr, and by 24 hr the choline levels still remained higher in the steroid-treated animals than in the controls. Levels of acetylcholine in the ganglion were also increased, beginning at thr after the injection of steroid. The increase was 85% by 3 hr and 60% by 6hr. Triamcinolone, a glucocorticoid that was without an effect on [³H]choline uptake in vitro, was also ineffective in altering the levels of choline and acetylcholine in vivo. It seems probable that the increase of choline uptake in the ganglion induced by dexamethasone may, at least in part, occur in the preganglionic cholinergic terminals, leading to increased synthesis of acetylcholine. Such an effect of dexamethasone provides another case of a selective steroid acting directly on nerve terminals by altering a transport mechanism.     

Kinetics of [3H]choline uptake in abdominal ganglia of Limulus polyphemus.

The uptake of [³H]choline by isolated ganglia of the ventral nerve cord of Limulus potyphemus was studied. The uptake process was linear for 90 min and was concentration dependent. Kinetic analysis suggested the existence of a high affinity uptake process (K_t 14μM and V_max 0.19 pmole/mg/min) and a low affinity uptake process (K_t 14μM and V_max 0.89 pmole/mg/min). The high affinity uptake system showed a greater dependence on sodium ions and was more sensitive to inhibition by hemicholinium-3. Neither uptake system was greatly influenced by the absence of calcium or potassium. It is suggested that this system may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.     

Kinetics of [3H]choline and [3H]acetylcholine metabolism in several regions of rat brain following intracerebroventricular injection of [3H]choline: Effects of haloperidol

Intracerebroventricular (icv.) injection of [³H]choline in conscious rats produced a rapid, efficient labeling of brain choline and acetylcholine (ACh) stores without altering steady-state levels of endogenous ACh. The kinetics of [³H]choline and [³H]ACh metabolism were measured in seven brain regions for up to 10 min following icv. administration of [³H]choline. The initial rate of formation of [³H]ACh varied in different brain areas, being greatest in the striatum and least in the hypothalamus. In contrast, the rate of [³H]choline metabolism was similar in all regions of the brain. Pretreatment of rats with haloperidol resulted in an increase in the apparent synthesis rate of ACh only in the striatum and rostral hypothalamus, pointing to possible dopaminergic-cholinergic interaction in these regions.     

Effect of soman on N-methyl-d-aspartate-stimulated [3H]norepinephrine release from rat cortical slices

Effects of soman on N-methyl-d-aspartate (NMDA) evoked [³H]norepinephrine (NE) release were examined in rat brain cortical slices. NMDA increased [³H]NE release in a concentration-dependent manner. Soman could inhibit the increase evoked by NMDA, but carbachol, an agonist of cholinergic receptor, could potentiate the increase evoked by NMDA. Atropine (a selective muscarinic antagonist) attenuated the release of [³H]NE induced by NMDA in the presence of carbachol or acetylcholine (ACh), but had no effect on the release of [³H]NE induced by NMDA alone. Both d-tubocurarine (an antagonist of nicotinic receptor) and atropine had no effect on the release of [³H]NE induced by NMDA in the presence of soman. These results suggested that soman has a direct action at non-cholinergic sites, probably at NMDA receptors.     

Epithelium-derived inhibition of [3H]acetylcholine release from the isolated guinea-pig trachea

Summary To investigate presynaptic, regulatory mechanisms on parasympathetic nerve fibres innervating the airways, the release of newly-synthesized [³H]acetylcholine from the isolated trachea was studied. Reverse phase HPLC followed by liquid scintillation spectrometry was used to separate and quantify the radioactive compounds choline, phosphorylcholine and acetylcholine in the incubation medium and the tissue.During the incubation of the tracheae with [³H]choline a significant synthesis of [³H]acetylcholine (35,000 dpm/preparation) and [³H]phosphorylcholine (500,000 dpm/preparation) occurred. In epithelium-deficient tracheae the formation of [³H]phosphorylcholine was enhanced, whereas the content of [³H]acetylcholine remained unchanged. The spontaneous outflow of tritium consisted mainly of [³H]phosphorylcholine (900 dpm/3 min) and [³H]choline (800 dpm/3 min); [³H]acetylcholine was only a minor fraction (50 dpm/3 min). Electrical stimulation of tracheae with intact epithelium caused only a small release of [³H]acetylcholine (460 dpm in the sample obtained during stimulation), but a considerable outflow of [³H]phosphorylcholine (1,900 dpm) without affecting the outflow of [³H]choline. Electrical stimulation of epithelium-deficient tracheae, however, induced a substantial release of [³H]acetylcholine (2,400 dpm), but only a small outflow of [³H]phosphorylcholine. Chemical stimulation (30 mol/1 veratridine) also caused a large release of [³H]acetylcholine (1,700 dpm) without affecting the outflow of [³H]phosphorylcholine or [³H]choline. Indomethacin (3 mol/1) enhanced the electrically-evoked release of [³H]acetylcholine from tracheae with intact epithelium by 89%.The present experiments demonstrate a strong inhibition by the epithelium of the electrically-evoked release of [³H]acetylcholine from the isolated guinea-pig trachea. Cyclooxygenase products of arachidonic acid do not appear as the main mediators of the epithelium-derived inhibition of acetylcholine release. Send offprint requests to I. Wessler at the above address     

Involvement of phospholipase A2 in the regulation of [3H]hemicholinium-3 binding

We have examined the effects of exogenous phospholipase A2 (PLA2) on the sodium-dependent high-affinity choline uptake mechanism as assessed by the specific binding of [3H]hemicholinium-3 ([3H]HCh-3). Incubation of striatal synaptic membranes with bee venom PLA2 resulted in a concentration-dependent increase in the specific binding of [3H]HCh-3. The effect of PLA2 on [3H]HCh-3 binding was inhibited by quinacrine, a PLA2 inhibitor, and by removal of calcium. Scatchard analysis revealed that the observed changes in binding reflected a 2-fold increase in both the capacity and affinity of [3H]HCh-3 for its binding site. Choline and N-butylcholine inhibited the specific binding of [3H]HCh-3 in both control and PLA2-treated membranes with similar potency. When a low concentration of PLA2 was incubated with the striatal synaptosomes, a small but significant increase in high-affinity [3H]choline uptake was observed. However, higher concentrations of PLA2, which further increased the specific binding of [3H]HCh-3, caused a reduction of [3H]choline uptake, apparently due to disruption of synaptosomal integrity by PLA2. Finally, potassium depolarization- and PLA2-induced increases in specific [3H]HCh-3 binding were not additive. These results suggest a possible role for endogenous PLA2 in the calcium-dependent regulation of sodium-dependent high-affinity choline uptake.     

Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation

Summary Two different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors.When the whole nerve-muscle preparation (110–180 mg) was incubated with [³H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow.Incubation of the end-plate preparation (16–29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [³H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [³H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [³H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [³H]acetylcholine tissue content per pulse.It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [³H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used. Removal of most of the muscle fibres reduces the possibility for [³H]choline to be captured by them and then more radioactive choline can enter the end-plate region. From this end-plate preparation a calcium-dependent release of radioactive transmitter can be measured in the absence of cholinesterase inhibitors.     

Effects of phencyclidine on [3H]catecholamine and [3H]serotonin uptake in synaptosomal preparations from rat brain

Phencyclidine inhibited uptake in vitro of [³H]norepinephrine (ic₅₀ 0.52 μM), [³H]dopamine (ic₅₀ 0.73 μM) and [³H]serotonin (ic₅₀ 0.80 μM) in crude synaptosomal preparations from rat brain through a competitive mechanism. Phencyclidine was fairly similar in potency to d-amphetamine and methylphenidate in inhibiting catecholamine uptake but was 8 times more potent than d-amphetamine and 34 times more potent than methylphenidate in inhibiting [³H]serotonin uptake.     

Evaluation by reverse phase HPLC of [3H]acetylcholine release evoked from the myenteric plexus of the rat

Summary Myenteric plexus-longitudinal muscle strips isolated from the small intestine of rats were incubated with [³H]choline to measure the synthesis and the release of [³H]acetylcholine. To separate different radioactive compounds (acetylcholine, choline, phosphorylcholine) from both the tissue and the overflow a new method, the reverse phase HPLC, was used.The radiochromatogram following the injection of a [³H]choline-standard and a [¹⁴C]acetylcholine-standard onto the HPLC showed a clear separation of both isotopes with a recovery rate of roughly 100%. Incubation of the muscle strips with [³H]choline caused the synthesis of [³H]acetylcholine (30,000 dpm/preparation) that increased 2-fold, when the electrical field stimulation during labelling was increased from 0.2 Hz to 1 Hz. Electrical field stimulation (3 Hz, 2 min) caused an increase in tritium efflux that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. Analysis by reverse phase HPLC of the overflow showed that the stimulated increase in tritium overflow was balanced by the enhanced release of [³H]acetylcholine, whereas the overflow of [³H]choline was not affected by the electrical field stimulation. Oxotremorine (1 mol/l) suppressed the release of [³H]acetylcholine by 60%. Scopolamine (0.1 mol/l) prevented this inhibition and, given alone, enhanced the release of [³H]acetylcholine by 43%. The release of [³H]acetylcholine evoked at 0.2, 2 or 20 Hz did not consistently decline at increasing frequencies.The present experiments show the synthesis and the calcium-dependent release of [³H]acetylcholine from the myenteric plexus-longitudinal muscle preparation of rats correspondingly to the same in-vitro preparation isolated from guinea-pigs. Muscarinic autoinhibition operates also in the small intestine of rats. However, some differences (frequency-dependency of [³H]acetylcholine release, spontaneous neuronal activity) are evident between both species. Reverse phase HPLC is a useful method to separate radioactive choline and acetylcholine with a high recovery rate.Send offprint requests to I. Wessler at the above address     

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.     

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.     

Release of [3H]acetylcholine from the isolated rat or guinea-pig trachea evoked by preganglionic nerve stimulation; a comparison with transmural stimulation

Summary Basal and stimulated outflow of radioactive acetylcholine, phosphorylcholine and choline from rat and guinea-pig isolated tracheae were measured by reverse phase HPLC followed by liquid-scintillation-spectrometry. Tracheae were stimulated either by an electrical field (transmural stimulation) or by a local stimulation of the innervating parasympathetic nerves (preganglionic stimulation). Epithelium was removed in most experiments, as the epithelium inhibits acetylcholine release.The basal tritium efflux (1,600 dpm/3min) from rat isolated tracheae incubated with [³H]choline consisted of 56% [³H]phosphorylcholine and 38% [³H]choline. Preganglionic stimulation (15 Hz, 1,200 pulses) caused a 2-fold increase in tritium outflow that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. The stimulated outflow of tritium induced by preganglionic nerve stimulation was caused by an exclusive release of [³H]acetylcholine, whereas the efflux of [³H]phosphorylcholine and [³H]choline remained unaffected by this stimulation mode. Transmural stimulation of the rat or guinea-pig trachea, however, caused, in addition to the release of [³H]acetylcholine, the outflow of [³H]phosphorylcholine. Hexamethonium (300 mol/l) or tubocurarine (100 mol/l) inhibited (80%) the increase in tritium outflow evoked by preganglionic stimulation, but did not affect tritium outflow evoked by transmural stimulation. Oxotremorine reduced [³H]acetylcholine release evoked by both stimulation modes, but oxotremorine was less potent with transmural stimulation. Scopolamine (0.3 mol/l) enhanced (120%) the release of [³H]acetylcholine evoked by preganglionic nerve stimulation indicating the blockade of an endogenous negative muscarinic feedback mechanism. Epithelium-dependent inhibition of [³H]acetylcholine release was evident with both preganglionic and transmural stimulation.The present experiments demonstrate the release of [³H]acetylcholine evoked from the isolated trachea by stimulation of the preganglionic trunk of the parasympathetic cholinergic nerves. Qualitative and quantitative differences were observed in comparison to transmural stimulation. Preganglionic nerve stimulation allows a selective excitation of pulmonary, parasympathetic nerve fibres, mimics the physiological excitation of intramural neurones and is not followed by the liberation of phosphorylcholine from non-neuronal cells. Send offprint requests to I. Wessler at the above address     

Potassium activation of [3H]-choline accumulation by isolated sympathetic ganglia of the rat.

1 The effect of K-depolarization on the uptake of low and high concentrations of [3H]-choline by isolated superior sympathetic ganglia of the rat has been studied. 2 In unstimulated ganglia, the uptake of [3H]-choline (0.1 microM) ('high affinity uptake') was unaffected by denervation or by hemicholinium-3 (HC-3), suggesting uptake by structures other than cholinergic nerve terminals. 3 K-depolarization of the ganglia increased [3H]-choline accumulation by the high affinity uptake process but in contrast the 'low affinity' accumulation of [3H]-choline (100 microM) was decreased. 4 The K-activated, 'high affinity' component of choline uptake was highly sodium-dependent, inhibited by HC-3, and was abolished by denervation. 5 In incubation conditions designed to prevent transmitter release (Ca-free medium and high-Mg medium), the K-activated uptake of [3H]-choline was abolished. 6 It is concluded that in unstimulated ganglia, there is little choline uptake by nerve terminals. However, when the terminals are depolarized, choline uptake is increased by the activation of a sodium-dependent, HC-3-sensitive transport process. The activation of this uptake process is apparently associated with the release of acetylcholine from the terminals, or by changes in ionic fluxes, and not by the depolarization per se.     

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     

Rapid in vitro modulation of [3H]hemicholinium-3 binding sites in rat striatal slices

The effects of depolarizing concentrations of potassium chloride on the modulation of [3H]hemicholinium-3 binding sites and high affinity choline uptake were examined in vitro. When rat striatal slices were incubated in Krebs buffer for 20 min, [3H]hemicholinium-3 binding sites diminished to 60% of binding measured in fresh un-incubated tissue, and remained stable for 60 min. Upon addition of Krebs buffer containing 40 mM KCl, the number of binding sites increased during a 20 min period, and remained stable for 40 min. Changes in [3H]hemicholinium-3 binding sites closely paralleled changes in high affinity choline uptake. Scatchard analysis revealed that changes in binding result from alterations in the number of binding sites (Bmax), and not in the affinity (KD). These results suggest that neuronal depolarization rapidly alters the velocity of choline transport into cholinergic neurons by increasing the number of available carriers.     

Actylcholine output from the cerebral cortex,choline uptake and muscarinic receptors in morphine-dependent,freely-moving rats

Spontaneous acetylcholine (ACh) output from the cerebral cortex, choline high affinity uptake and [³H]-QNB binding to muscarinic receptors in the cerebral cortex and caudate nucleus in freely moving rats made morphine-dependent by morphine pellet subcutaneous implantation were investigated before and during naloxone-induced withdrawal syndrome. The frequency and intensity of the withdrawal signs were also assessed.No significant change in ACh output was found in tolerant rats when compared with that of placebopellet implanted rats. During naloxone-induced withdrawal syndrome a 60% increase in ACh output occurred.In rats made dependent after a large septal lesion or treated for ten days with calcium gluconate (10 mg/kg i.m.) no increase in ACh output was found during the withdrawal syndrome. The intensity of some of its signs was also reduced.During the withdrawal syndrome a marked increase in choline high affinity uptake in the cerebral cortex and caudate nucleus was detected.The affinity of muscarinic receptors (K_D) for [³H]-QNB was significantly increased in the cerebral cortex and caudate nucleus of morphine-dependent rats before naloxone administration. It returned to normal during the withdrawal syndrome. In the caudate nucleus the number of binding sites (B_max) was decreased before and after the withdrawal syndrome.These findings emphasize the role of cholinergic mechanisms in opiate addiction.     

Presynaptic GABAB autoreceptor regulation of nicotinic acetylcholine receptor mediated [H]-GABA release from mouse synaptosomes

Activation of nicotinic acetylcholine receptors (nAChRs) can elicit neurotransmitter release from presynaptic nerve terminals. Mechanisms contributing to cell-and-terminal specific regulation of nAChR-mediated neurotransmitter exocytosis are not fully understood. The experiments discussed here examine how activation of GABA_B auto- and hetero-receptors suppress nAChR-mediated release of [³H]-GABA and [³H]-dopamine (³H-DA) from mouse striatal synaptosomes. Activation of presynaptic GABA_B receptors with (R)-baclofen decreased both [³H]-GABA and [³H]-DA release evoked by potassium depolarization. However, when nAChRs were activated with ACh to evoke neurotransmitter release, (R)-baclofen had no effect on [³H]-DA release, but potently inhibited ACh-evoked [³H]-GABA release. Inhibition of nAChR-evoked [³H]-GABA release by (R)-baclofen was time sensitive and the effect was lost after prolonged exposure to the GABA_B agonist. The early inhibitory effect of GABA_B activation on ACh-evoked [³H]-GABA release was partially attenuated by antagonists of the phosphatase, calcineurin. Furthermore, antagonists of protein kinase C (PKC) prevented the time-dependent loss of the inhibitory (R)-baclofen effect on [³H]-GABA release. These results suggest that α4β2*-nAChRs present on GABAergic nerve terminals in the striatum are subject to functional regulation by GABA_B autoreceptors that is apparently cell-type specific, since it is absent from DAergic striatal nerve terminals. In addition, the functional modulation of α4β2*-type nAChRs on striatal GABAergic nerve terminals by GABA_B autoreceptor activation is time-sensitive and appears to involve opposing actions of calcineurin and PKC.     

Increased release of [3H]acetylcholine in vitro from the mouse hippocampus by a convulsant barbiturate

The effects of the convulsant barbiturate, 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB), on the spontaneous release of [³H]acetylcholine (ACh) from mouse hippocampal slices in an in vitro superfusion system have been evaluated. The pattern of the release of [³H]ACh by a single treatment with CHEB or an elevated potassium concentration was similar, with the peak release occurring in the same fraction. A maximally effective concentration of CHEB (500 μM) caused a 177% stimulation of spontaneous release of [³H]ACh, while 50 mM KCl increased the release by 2100% above the baseline. The stimulation of the spontaneous release of [³H]ACh by CHEB was concentration-dependent with an EC₅₀ of 180 μM. The pattern of release of [³H]ACh induced by multiple treatment with CHEB and elevated potassium appeared to differ, suggesting that different pools of [³H]ACh in the cholinergic neurons might be affected by these treatments. The action of CHEB on the sponaneous release of [³H]ACh was unique among some other convulsant drugs that were studied. Another convulsant barbiturate, S(+)-1-methyl-5-phenyl-5-propyl barbituric acid [S(+)-MPPB], pentylenetetrazol and a convulsant benzodiazepine 1,3-dihydro-5-methyl-2H-1,4-benzodiazepine-2-one (Ro-5-3663) did not affect the spontaneous release of [³H]ACh. The relationship between the stimulation of release of ACh and the convulsant action of the barbiturates is discussed.     

3H]Dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. A reassessment using the selective radioligands [3H]prazosin, [3H]yohimbine, and [3H]rauwolscine

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These binding studies employed the ligand [³H]dihydroergocryptine ([³H]DHE), an alpha-adrenergic antagonist that does not select between alpha₁- and alpha₂-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [³H]prazosin (alpha₁-selective), [³H]yohimbine (alpha₂-selective), and [³H]rauwolscine (alpha₂-selective), and we have compared the binding of these selective radioligands with that of [³H]DHE. [³H]Yohimbine and [³H]rauwolscine both bound with high affinity (K_D = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient ～1.0) of sites (～300 per platelet), but [³H]yohimbine yielded lower nonspecific binding than did [³H]rauwolscine. In paired experiments, [³H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [³H]yohimibine or [³H]rauwolscine. Unlabeled vohimbine and epinephrine competed for fewer [³H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha₂-adrenergic receptors identified by [³H]yohimbine and [³H]rauwolscine, [³H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [³H]prazosin did not identify alpha₁-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [³H]yohimbine and [³H]DHE binding at higher concentrations than usually observed for alpha₁-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha₂ subtype, (2) [³H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [³H]yohimbine and [³H]rauwolscine, and (3) [³H]yohimbine is the preferred antagonist radioligand for studying the alpha₂-adrenergic receptors on human platelets.     

Effect of a sulfonium analog of dopamine on the depolarization-induced release of [3H]acetylcholine from mouse striatal slices

P3 have synthesized a chemical analog or dopamine in which the amino group has been replaced by a charged dimethylsulfonium group. The dopaminergic activity of this drug was evaluated by determining its ability to inhibit the depolarization-evoked release of [3H]acetylcholine from mouse striatal slices. The slices were preincubated with [3H]choline (0.1 microM) and then superfused in physiological medium. [3H]Acetylcholine release was induced by exposure of the slices to a high potassium medium (12.5 mM) for 5 min. The sulfonium analog of dopamine, dopamine, and apomorphine inhibited the evoked [3H]acetylcholine release with IC50 values of approximately 10, 2.0, and 0.3 microM respectively. The inhibition by the sulfonium analog was reversed by fluphenazine (1 microM), suggesting that the inhibition of [3H]acetylcholine release was due to the activation of dopaminergic receptors. The sulfonium analog also inhibited the uptake of [3H]dopamine into striatal slices and caused the release of exogenously taken up [3H]dopamine from these slices. The release of [3H]dopamine by the sulfonium analog was inhibited by cocaine (3 microM), suggesting that the drug-induced release of [3H]dopamine was dependent on the carrier-mediated uptake of the sulfonium analog into dopaminergic neurons. The inhibition of the evoked [3H]acetylcholine release by high concentrations (30 and 60 microM) of the sulfonium analog did not appear to be mediated by endogenous dopamine release, since the analog still inhibited [3H]acetylcholine release from slices after reserpine-alpha-methyl-p-tyrosine treatment. However, the inhibitory effect of the sulfonium analog at 10 microM was reduced by reserpine-alpha-methyl-p-tyrosine treatment, suggesting that the inhibition at lower concentrations was mediated through endogenous DA release. These results suggest that a charged compound can act as a substrate for the dopamine carrier and can activate the dopamine receptor regulating acetylcholine release. They also indicate that the nitrogen on the dopamine molecule is not essential for dopamine agonist activity.