Effects of (+)-tubocurarine on [3H]acetylcholine release from the rat phrenic nerve at different stimulation frequencies and train lengths

Summary The effect of (+)-tubocurarine (TC) on the release of [³H]acetylcholine from the rat phrenic nerve-hemidiaphragm preincubated with [3H]choline was investigated at different stimulation frequencies and train lengths.At 0.5 Hz (100 pulses) TC failed to modulate the evoked acetylcholine release. A slight (30%) inhibition was observed at 1 Hz (100 pulses). Release of acetylcholine evoked at 5, 25 and 50 Hz (100 pulses) or 100 Hz (200 pulses) was markedly reduced by TC. The degree of inhibition (60%) was similar between 5 Hz and 100 Hz. A concentration of 1 mol/l TC was a maximal effective concentration at 5 Hz whilst at all higher stimulation frequencies a 10-fold higher concentration was necessary for the maximal effect. When 300 pulses were continuously applied at 5 Hz or 50 Hz TC caused only a slight inhibition (20%). Additionally, the phrenic nerve was stimulated intermittently. Trains of 15 pulses were repeated 10 times with an interval of 3 s between each train. Under this latter stimulation condition TC failed to reduce acetylcholine release.It is concluded that nicotinic autofacilitation of acetylcholine release from the motor nerve operates at frequencies and stimulation conditions similar to the pattern of nerve activity under in vivo conditions. At least more than 15 pulses are required before the nicotinic autofacilitation becomes apparent. It appears unlikely that the TC induced fading of end-organ responses can only be attributed to a blockade of the presynaptic nicotine receptors. Send offprint requests to I. Wessler at the above address     

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.     

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     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid 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     

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     

Nitroimipramines — Synthesis and pharmacological effects of potent long-acting inhibitors of [3H] serotonin uptake and [3H] imipramine binding

Summary A series of nitro derivatives of imipramine have been prepared by nitration of imipramine. Several of the products, especially 2-nitroimipramine (2) and 2,8-dinitroimipramine (4) were found to be very potent inhibitors of [³H] serotonin uptake and high affinity [³H] imipramine binding in human platelets. In contrast to the parent antidepressant, imipramine, the inhibition of platelet [³H] serotonin uptake and [³H] imipramine binding by the nitro derivatives of imipramine was long-acting and essentially irreversible at low temperatures. These compounds should prove to be valuable tools for subsequent studies on the purification and characterization of the transport protein(s) involved in serotonin uptake and may have novel behavioral and clinical effects.     

Glycine-evoked release of [3H]acetylcholine from rat striatal slices is independent of the NMDA receptor

Summary KCl-, NMDA-, and glycine-evoked release of [³H]acetylcholine was studied in superfused rat striatal slices. KCl-evoked release of [³H]acetylcholine was inhibited by 1.2 mM MgC12 and 100 M lidocaine. Similarly, NMDA-evoked release was inhibited by MgCl₂ and lidocaine as well as 10 M CGS 19755, a competitive antagonist at NMDA receptors, and 10 nM MK-801, a noncompetitive antagonist of NMDA-induced responses. Glycine-evoked release was calcium-dependent and was inhibited by 0.1 M strychnine whereas KCl- and NMDA-evoked release were resistant to strychnine. In addition, lidocaine inhibited the glycine-induced response. Cross-tachyphylaxis was not observed between NMDA- and glycine-evoked release. These results indicate that the strychnine-sensitive, glycine-evoked release of [³H]acetylcholine is independent of the NMDA receptor.     

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     

Purinoceptor-mediated modulation by endogenous and exogenous agonists of stimulation-evoked [3H]noradrenaline release on rat iris

Summary To investigate whether endogenous purinoceptor agonists affect the sympathetic neurotransmission in the rat isolated iris, and to classify the purinoceptors modulating exocytotic [³H]-noradrenaline release, we have determined the effect of adenosine receptor antagonists on, and the relative potency of selected agonists in modulating, the field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow. In addition, the apparent affinity constants of 8-phenyltheophylline (8-PT) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in antagonizing the prejunctional effects of purinoceptor agonists were estimated.The relatively A₁-selective DPCPX 10 and 100 nmol/l increased the evoked [³H]-noradrenaline overflow by about 25%–35%a indicating a minor inhibition of evoked release by endogenous purinoceptor agonists probably via an A₁ adenosine receptor. Whereas the A₁/A₂-antagonist 8-PT failed to increase the evoked [³H]-noradrenaline overflow in the absence of exogenous agonists (without or with dipyridamole 1 pmol/l present), the relatively A₂-selective antagonist CP-66,713 (4-amino-8-chloro -1-phenyl(1,2,4)triazolo(4,3-a)quinoxaline) 100 nmol/l decreased it by 20%–30% in the absence and continuous presence of DPCPX. This may be compatible with a minor A₂-mediated facilitation by an endogenous purinoceptor agonist.All exogenous agonists tested (except UTP 100 mol/l) inhibited the evoked [³H]-noradrenaline overflow. The relative order of agonist potency (IC4o, concentration in mol/l for inhibition of evoked release by 40%) was CPA (N⁶-(cyclopentyl)adenosine, 0.004) > R-PIA (R(–)N6-(2phenylisopropyl)adenosine, 0.066) = CHA (N⁶-(cyclohexyl)adenosine, 0.082) > NECA (N⁵-(ethyl-carboxamido)adenosine 0.44) > ADO (adenosine, 4.1). ATP was n early equipotent with ADO. Maximum inhibition was 70%–80% and similar for all agonists. Adenosine deaminase 1 u/ml failed to affect the ATP-induced, but abolished the adenosine-induced prejunctional inhibition. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine (NBTG) failed to enhance the potency of ADO and ATP. The A₁-selective antagonist DPCPX 10 nmol/l did not reduce the ATP potency indicating an effect of ATP per se not mediated via an A₁ purinoceptor.Prejunctional affinity constants of 8-PT were 6.07 when tested against adenosine (in the presence of dipyridamole), and 6.60 against CHA. The apparent -log K_B of DPCPX tested against CPA was 9.71. The high DPCPX affinity is compatible with an A₁ adenosine receptor mediating inhibition of sympathetic neurotransmission in rat iris. This receptor may not be the only prejunctional purinoceptor on rat iris sympathetic nerves. The receptor by which ATP acts prejunctionally in this tissue remains to be determined.This study was supported by the Deutsche Forschungsgemeinschaft (Fu 163/2 and 163/3) Send offprint requests to H. Fuder at the above address     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

[3H]resiniferatoxin binding by the vanilloid receptor: species-related differences,effects of temperature and sulfhydryl reagents

Summary Specific binding of [³H]resiniferatoxin (RTX) is thought to represent the vanilloid (capsaicin) receptor. In the present study, we have used this binding assay to elucidate the contribution of differential receptor expression to the capsaicin-resistance of hamsters and rabbits; binding parameters were compared to those of species (rats, mice) regarded as capsaicin-sensitive. Whereas the 5-fold lower affinity for [³H]RTX binding in the hamster (100 pM) as compared to the rat (20 pM) is unlikely to account for the 100-fold difference in the in vivo responses of RTX-induced inflammation and hypothermia, the lack of detectable specific [³H]RTX binding sites in the rabbit might represent the predominant mechanism of capsaicin-resistance in this species. Regulation of the vanilloid receptor was further characterized in the rat. In accord with the temperature dependence of both in vivo and in vitro capsaicin actions, we found a marked temperature dependence for association rates. Dissociation turned out to have complex kinetics dependent on time and receptor occupancy. Low pH (5.5–7.0) did not affect receptor binding. Preincubation with heavy metal cations and other sulfhydryl-reactive agents inhibited specific [³H]RTX binding indicating that the vanilloid receptor is a thiol-protein, and that free sulfhydryl groups play an essential role in agonist binding activity. Preliminary characterization suggested noncompetitive inhibition.Correspondence to P. M. Blumberg at the above address     

[3H]-Spiroperidol labels dopamine receptors in membranes from rabbit mesenteric artery

Summary The potent dopamine receptor antagonist [³H]-spiroperidol was used to label binding sites in a membrane fraction derived from rabbit mesenteric artery which had characteristics expected for dopamine receptors. The binding was of high affinity with an equilibrium dissociation constant (K_D) of 13.1 nM; it was saturable with 110 fmol of [³H]-spiroperidol bound/mg protein at maximal occupancy of the sites. Binding at 37° C was rapid and readily reversible with rate constants of 0.0154 nM^–1 min^–1 and 0.114 min^–1 for forward and reverse reaction, respectively. Dopamine receptor antagonists were about 100–200 times more potent than -adrenolytic drugs in competing for the [³H]-spiroperidol binding sites and dopamine was much more potent than (–)-noradrenaline, adrenaline, (–)-isoprenaline, clonidine or serotonin. It is concluded that in a membrane fraction of the rabbit mesenteric artery there exist binding sites for [³H]-spiroperidol indistinguishable from dopamine receptors. Thus the present results support the view that in vascular smooth muscle there exist specific dopamine receptors.This work was supported by the Deutsche Forschungsgemeinschaft     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Light-dark differences in [3H]-paroxetine binding to rabbit platelet membranes

Summary [³H]-Paroxetine binding to rabbit blood platelet membranes from samples obtained under light and dark conditions was examined. Animals were kept on a 14 h light (L) — 10 h dark (D) schedule and blood samples were collected at L + 7 and D + 5 h. Significant differences were found for B _max values of [³H]-paroxetine binding, with low B _max values during the light period and high B _max values during the dark period. The K _d values were not significantly different. These results confirm previous observations on light-dark differences of [³H]-imipramine binding in rabbit blood platelets suggesting the existence of a circadian rhythm for the 5-HT transporter complex.Send offprint requests to S. Z. Langer at the above address     

Effects of 5-HT4 receptor stimulation on basal and electrically evoked release of acetylcholine from guinea-pig myenteric plexus

Summary The effects of 5-methoxytryptamine and 5-hydroxytryptamine (5-HT) on both basal and electrically evoked outflow of tritium were studied in guinea-pig myenteric plexus preparations preincubated with [³H]-choline. Basal outflow. 5-Methoxytryptamine caused a transient and calcium-dependent increase in basal outflow of [³H]acetylcholine that was abolished by tetrodotoxin. Ondansetron (1 mol/1) did not affect the stimulatory response of 5-methoxytryptamine but ICS 205-930 (1 and 3 mol/1) produced parallel rightward displacements of the concentration-response curve to 5-methoxytryptamine. The PK_B value for ICS 205-930 was 6.6 suggesting an involvement of 5-HT₄ receptors. 5-HT caused an increase in basal outflow of [³H]acetylcholine and a biphasic concentration-response curve was obtained. The maximal response of the first phase to 5-HT (release of 0.98% of tissue tritium) and the maximal response to 5-methoxytryptamine (0.94% of tissue tritium) were similar but 5-methoxytryptamine (-log EC₅₀: 6.9) was less potent than 5-HT (-log EC₅₀ of the high affinity component: 7.9). ICS 205-930 (0.01–1.0 mol/1) acted as a competitive antagonist against the low affinity component of the 5-HT concentration-response curve with a pA₂ value of 8.0. It is concluded that stimulation of both 5-HT₄ receptors (by 5-methoxytryptamine and submicromolar concentrations of 5-HT) and 5-HT₃ receptors (by micromolar concentrations of 5-HT) causes a release of acetylcholine which in turn leads to smooth muscle contraction. Electrically evoked outflow. This outflow of [³H]acetylcholine was concentration-dependently inhibited by both 5-methoxytryptamine and 5-HT. ICS 205-930 (1 mol/1) reinforced the inhibitory effect of 5-methoxytryptamine but not that of 5-HT. In the presence of methiothepine (0.1 mol/1) 5-methoxytryptamine enhanced the evoked outflow of [³H]acetylcholine, an effect which was attenuated by 3 mol/1 ICS 205-930. These results suggest that 5-methoxytryptamine may both inhibit (via 5-HT₁ receptors) and facilitate (via 5-HT₄ receptors) the evoked release of acetylcholine from guinea-pig myenteric neurones. The facilitatory action is unmasked when the 5-HT₁ receptor is blocked by methiothepine. Send offprint requests to H. Kilbinger at the above address     

Circadian rhythm of the B max of [3H]-imipramine binding in rabbit platelets

Summary [³H]-imipramine binding was measured in rabbit blood platelet membranes on a 24 h cycle. Animals were kept on a 14 h light (L) 10 h dark (D) schedule, and blood samples were collected at L + 2, L + 8, D + 2, D + 8 and L – 2 h on a following cycle. Significant differences were found for B_max values of [³H]-imipramine binding, with highest values during the dark phase and lowest during the light phase. No significant differences were found in K _d values. These results suggest the existence of a circadian rhythm for the B_max of [³H]-imipramine binding in blood platelets. Send offprint requests to S. Z. Langer     

Characterization of nicotinic acetylcholine receptors on cultured bovine adrenal chromaffin cells using modified l-[3H]nicotine binding assay

Summary To characterize the properties of nicotinic acetylcholine receptors (nAChRs) in autonomic ganglia, we examined l-[³H]nicotine binding to membrane fraction prepared from cultured bovine adrenal chromaffin cells, using a modified filtration method. Binding of l-[³H]nicotine to non-treated glass fiber filters interfered with the detection of specific binding to the membrane fraction. Presoaking glass fiber filters in 3% or higher concentrations of polyethyleneimine (PEI) solution (sixty times higher than earlier used concentration) for at least 5 h could reduce the binding of l-[³H]nicotine to the filters to the background level. Specific l-[³H]nicotine binding to the membrane fraction was detected only when the membrane fraction was prepared in Ca²⁺- and Mg²⁺ (EDTA, EGTA and protease inhibitors were added)-free buffer. Specific binding of l-[³H]nicotine was saturable and reversible. Both computer program and Scatchard analysis revealed a single class of high affinity binding sites with an average K_d of 8.9 nM and a B_max of 42.5 fmol/mg protein. The Hill coefficient was 0.98. In inhibition studies, both cholinergic agonists (carbachol and l-nicotine) and ganglionic agonists (lobeline and 1,1-dimethyl-4-phenylpiperazinium iodide) were much effective in inhibiting l-[³H]nicotine binding, whereas both neuromuscular blocking (-bungarotoxin and d-tubocurarine) and ganglionic blocking agents were less effective. These results suggest that high affinity nicotinic binding sites on adrenal chromaffin cells are nAChRs of the ganglion-type, which have properties different from nAChRs on the neuromuscular junction but similar to nAChRs in the brain. Send offprint requests to K. Lee at his present address     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.