Age-dependent modulation of neuropeptide Y on adrenergic transmission of guinea pig vas deferens.

1. The effect of neuropeptide (NPY) on [3H]noradrenaline ([3H]NA) release- and on contractions evoked by field electrical stimulation (FES) was studied in vitro in vas deferens from mature and immature guinea pigs. 2. The evoked tritium overflow (which reflected [3H]NA release) was determined by liquid scintillation spectrometry. 3. Field electrical stimulation of 5 Hz (trains of 50 pulses in 20 sec intervals) evoked guanethidine-sensitive contractions. 4. NPY (0.01-1 microM) dose-dependently inhibited the evoked contractions in both groups of animals. NPY, 1 microM, almost completely inhibited the evoked contractions in mature animals, while those in immature guinea pigs were inhibited but only by 80.4 +/- 3.6%. 5. The amount of tritium overflow evoked by 5 Hz stimulation (300 pulses: 15 trains of 20 pulses in 20 sec intervals) was higher in immature guinea pigs (0.46 +/- 0.03%) compared with the amount of the evoked tritium overflow in mature guinea pigs (0.39 +/- 0.02%). 6. NPY, 1 microM, inhibited the evoked tritium overflow. The NPY inhibition was more pronounced in vas deferens of mature (45.3 +/- 2.0%) than in immature (25.1 +/- 3.5%) guinea pigs. 7. The results suggest that NPY modulation of adrenergic transmission at the prejunctional level increases with the maturity.     

Effects of clonidine on the stimulation-evoked release of3H-noradrenaline from superfused rat brain slices as a function of the biophase concentration

The influence of clonidine on the stimulation-evoked overflow of tritium was studied in brain slices preincubated with 3H-noradrenaline. The slices were prepared from parietal cortex (Cx), nucleus anterior hypothalami (nah) and nucleus tractus solitarii (nts). After preincubation, the tissues were superfused at 23 degrees C or 37 degrees C with a medium containing the noradrenaline uptake inhibitor desipramine. Electrical field stimulation was applied using stimulation frequencies of 0.3-10 Hz. At 23 degrees C/0.3 Hz, clonidine concentration-dependently inhibited the evoked overflow of tritium in all three brain regions. In contrast, at 23 degrees C/3 Hz the inhibitory effect of the drug in the Cx was abolished and a facilitation was observed in the nah and nts. When tested at increasing frequencies of stimulation in the nts at 23 degrees C, clonidine exerted a dual action, characterized by a reduction of electrically evoked responses at frequencies below 1 Hz and a facilitation at frequencies above 1 Hz. At 37 degrees C, clonidine concentration-dependently decreased the evoked overflow in all brain regions studied, this effect being more pronounced at 0.3 Hz than at 3 Hz. The apparent lack of an effect of clonidine on the stimulation-evoked overflow of tritium in the Cx at 23 degrees C/3 Hz was turned to a facilitation when noradrenaline (0.01 mumol/l) was included in the superfusion medium. Conversely, an inhibitory effect of clonidine was seen when the uptake blocker desipramine (as well as noradrenaline) was omitted from the superfusion medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leucine-enkephalin- and neuropeptide Y-modulation of [3H]noradrenaline release in the oviduct of mature and juvenile rabbits

1. The effects of leucine-enkephalin and neuropeptide Y (NPY) on [3H]noradrenaline release induced by electrical field stimulation were studied in the isthmic part of the oviduct of juvenile and mature rabbits. 2. [3H]noradrenaline and total tritium overflow in the presence of cocaine, corticosterone and hyoscine were determined by liquid scintillation spectrometry. 3. Tritium overflow evoked by electrical stimulation (1 or 4 Hz, 1 msec) was calcium dependent. [3H]noradrenaline content (measured by ion exchange chromatography) accounted for 85% of the total tritium overflow. 4. Leucine-enkephalin (1 microM) in the presence of the peptidase inhibitor bacitracin reduced the stimulation-evoked tritium overflow in mature rabbits by 26.1 +/- 1.6% and in juvenile rabbits by 11.9 +/- 1.9%. Naloxone (1 microM) antagonized the effect of leucine-enkephalin. 5. NPY (0.2 microM) reduced the evoked tritium overflow in mature rabbits by 23.4 +/- 2.4% and in juvenile rabbits by 17.2 +/- 4.3%. 6. It is concluded that leucine-enkephalin and NPY inhibited [3H]noradrenaline release in rabbit oviduct and the modulatory effect of leucine-enkephalin depends on maturity while NPY modulation is a more independent system.     

Chloroethylclonidine: an irreversible agonist at prejunctional alpha 2-adrenoceptors in rat vas deferens.

1. The possibility that chloroethylclonidine (CEC) activates prejunctional alpha 2-adrenoceptors was studied in the isolated vas deferens of the rat. Tissues were stimulated electrically and both the stimulation-evoked overflow of tritium (after preincubation with [3H]-noradrenaline) and the purinergic contraction component (isolated by prazosin 0.3 microM) were measured. 2. CEC (0.1-3 microM) concentration-dependently reduced the overflow of tritium evoked by trains of 6 pulses/100 Hz. The inhibition by CEC was not altered by prazosin (0.3 microM) but was prevented by pre-exposure to rauwolscine (0.3 microM). The inhibition, once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 3. CEC (0.1-3 microM) concentration-dependently reduced the purinergic component of contractions elicited by single pulses. The inhibition, again, was prevented by pre-exposure to rauwolscine (0.3 microM) and once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 4. CEC (3 microM) reduced the overflow of tritium evoked by 20 pulses/10 Hz, did not alter the overflow evoked by 100 pulses/10 Hz and increased the overflow evoked by 500 pulses/10 Hz. 5. CEC (3 microM) reduced the early peak, but increased the late plateau phase, of purinergic contractions elicited by 100 pulses/10 Hz. 6. It is concluded that CEC reduces the release of noradrenaline and a purinergic co-transmitter by irreversible activation of prejunctional alpha 2-adrenoceptors. CEC seems to be a partial alpha 2-agonist with an efficacy lower than that of noradrenaline. The prejunctional inhibitory effect limits the suitability of CEC for the characterization of postjunctional alpha 1-adrenoceptors mediating responses to sympathetic nerve stimulation.     

Differences between the neuronal handling of 3H-7- and 3H-7,8-(−)noradrenaline: implications for the release of the labelled neurotransmitter by nerve stimulation

Neighbouring rabbit aortic strips were exposed to a tracer concentration of 3H-7- or 3H-7,8-(-)noradrenaline (bearing 30-35% of its label in position 8) and to 0.5 mumol/l unlabelled (-)noradrenaline for 60 min and then washed in amine-free Krebs solution. Catechol-O-methyl transferase and extraneuronal amine uptake were inhibited throughout. After 114 min of wash-out, the tissue contained less tritium when loaded with 3H-7-(-)noradrenaline than when loaded with 3H-7,8-(-)noradrenaline, and the fractional rate of loss of tritium was greater for the former than for the latter tissues. In the presence of cocaine (to prevent neuronal re-uptake), the same percentage of tissue tritium was released by nerve stimulation (six consecutive periods of stimulation at 1 Hz for 5 min each) in spite of the above difference between tissue tritium levels of the two differently labelled amines. In the absence of cocaine, a higher percentage of tissue tritium was released by nerve stimulation (1 or 3 Hz, 5 min each) for 3H-7- than 3H-7,8-(-)noradrenaline. Unchanged 3H-(-)noradrenaline amounted to 35% of tritium in the stimulation-evoked overflow for 3H-7- and to 50% for 3H-7,8-(-)noradrenaline (frequency of stimulation, 1 Hz). When monoamine oxidase (MAO) was inhibited, no differences were observed between the neuronal handling of 3H-7- and 3H-7,8-(-)noradrenaline, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further study of prerequisites for the enhancement by α-adrenoceptor antagonists of the release of noradrenaline

Summary Segments of the rabbit ear artery were preincubated with (–)-³H-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of ³H-noradrenaline and total tritium was determined.In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 M present). Thirteen pulses (0.25 Hz) elicited an overflow of ³H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 M did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 M decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 M decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 M was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses.In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of ³H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 M did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses. TEA 100 M increased the evoked overflow at all pulse numbers. Similar results were obtained in the presence of cocaine 5 M.The results demonstrate that the enhancement by -adrenoceptor antagonists of the release of noradrenaline depends on the biophase concentration of noradrenaline. Under the present conditions, graded increases in biophase noradrenaline concentration led to graded increases in the effect of the antagonists. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train. Under the present conditions, graded increases in train length up to about 20s led to graded increases in the effect of the antagonists, even though the average biophase concentration of noradrenaline did not change with the pulse train length. This pattern of effects of the -antagonists is not shared by at least one other release-enhancing drug, namely TEA.     

Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery

1. Interactions between A(2A)-adenosine receptors and alpha(2)-, A(1)- and P2- release-inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [(3)H]-noradrenaline, superfused with desipramine-containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz). 2. Blockade of alpha(2)-autoreceptors with yohimbine (1 microM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz. 3. The selective A(2A)-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 1-100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA; 100 nM) or by the P2-receptor agonist 2-methylthioadenosine triphosphate (2-MeSATP; 80 microM). 4. CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304; 30 nM), the A(1)-receptor agonist CPA (100 nM) or the P2-receptor agonist 2-MeSATP (80 microM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow. 5. Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow. 6. It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A(2A)-adenosine receptors is favoured by activation of release inhibitory receptors.     

Evidence for negative feedback control of the release of [3H]serotonin from superfused mouse cerebellum slices induced by electrical field stimulation

Slices of mouse cerebellum preloaded with [3H]serotonin were superfused with a solution of Krebs-Ringer phosphate. The effects of exogenous serotonin, serotonin antagonists, fluoxetine, Ca2+ absence, Ca2+ chelation and frequency of stimulation on basal and electrically induced tritium overflow were investigated. Exogenous unlabeled serotonin decreased the stimulus-evoked tritium overflow in a concentration-related manner. This effect was blocked by simultaneous administration of methiothepin, but not by methysergide. When given alone, methiothepin did not alter the electrically induced tritium overflow at 50 Hz, but did potentiate the increased tritium overflow produced at 100 Hz. The basal tritium efflux was increased by exogenous serotonin, but this effect was reversed by the simultaneous administration of fluoxetine. Under this condition exogenous serotonin reduced the basal tritium efflux in a concentration-related manner. Superfusion of the slices with a Ca2+-free solution alone or in the presence of EGTA, reduced the basal tritium efflux and the stimulated tritium overflow. These results support the existence of serotoninergic presynaptic inhibitory autoreceptors in the cerebellum of the mouse.     

Presynaptic beta-adrenoceptors in guinea pig papillary muscle: evidence for adrenaline-mediated positive feedback on noradrenergic transmission.

Guinea pig papillary muscles were preincubated in the presence of 5 x 10(-9) mol/L unlabeled noradrenaline or adrenaline then incubated with (3H)-noradrenaline and superfused. Electrical field stimulation with 180 pulses delivered at 1 or 3 Hz was used to induce overflow of radioactivity. Comparison of the effects of preexposure of the tissue to adrenaline or noradrenaline revealed that adrenaline incubation caused an enhancement of stimulation-evoked overflow of (3H)noradrenaline and a reduction of the effect of exogenously added isoprenaline. Furthermore, the selective beta 2-adrenoceptor antagonist ICI 118,551 (10(-7) mol/L), but not the selective beta 1-adrenoceptor antagonist ICI 89,406 (10(-7) mol/L), reduced electrically evoked overflow of (3H)noradrenaline in tissue preincubated with adrenaline but not in tissue preincubated with noradrenaline. The overflow-reducing effect of ICI 118.551 occurred at stimulation with 3 Hz but not at stimulation with 1 Hz. The present results support the hypothesis that noradrenergic transmission in guinea pig papillary muscle is facilitated via beta 2-adrenoceptors, and that adrenaline may serve as transmitter in this positive feedback mechanism after its incorporation into sympathetic nerves.     

Effect of α-adrenoceptor agonists and antagonists on cholinergic transmission in guinea-pig isolated atria

Evidence was sought for the existence on cholinergic nerve terminals in guinea-pig atria of alpha-adrenoceptors subserving inhibition of acetylcholine release. The experiments were performed with atria which had been incubated with 3H-choline and transmitter release was deduced from the efflux of radioactivity elicited by field stimulation. In preparations which had been incubated with 3H-choline, field stimulation (60 pulses, 2 Hz) evoked release and radioactivity which was inhibited by 1.0 mumol/l noradrenaline, in the presence of propranolol (1.0 mumol/l), but was unaltered by clonidine (1.0 and 10.0 mumol/l). The inhibitory effect noradrenaline on the stimulation-induced efflux of radioactivity was blocked by idazoxan (0.3 mumol/l), and phentolamine (1.0 mumol/l) but not by prazosin (0.3 mumol/l). In the presence of propranolol (1.0 mumol/l), neither phentolamine (1.0 mumol/l), idazoxan (0.3 mumol/l) nor prazosin (0.3 mumol/l) had any effect on stimulation-induced efflux of radioactivity. Stimulation of the extrinsic vagus nerve of atrial preparations with trains of pulses at frequencies of 2, 4, 8, and 16 Hz produced graded decreases in the rate of atrial beating. The negative chronotropic responses to vagus stimulation were unaffected by noradrenaline (1.0 mumol/l) in the presence of propranolol (1.0 mumol/l). These findings indicate that the release of acetylcholine from the cholinergic terminals in guinea-pig atria can be inhibited by a mechanism apparently involving prejunctional alpha 2-adrenoceptors. However, under the experimental conditions used here the chronotropic responses of atria to stimulation of the extrinsic vagus nerve was not affected by activation of the prejunctional alpha 2-adrenoceptors associated with the cholinergic terminals.     

Evidence for autoinhibition of stimulation-induced noradrenaline release from vasa deferentia of the guinea-pig and rat.

Both phenoxybenzamine and idazoxan increased the efflux of radioactivity elicited by a train of stimulation (4 pulses at 5 Hz) in vasa deferentia preincubated with [3H]-noradrenaline. Phenoxybenzamine increased the release of radioactivity from vasa stimulated with a single pulse, whereas idazoxan did not. The contractile response in both guinea-pig and rat vasa was biphasic: phenoxybenzamine enhanced the initial twitch component and reduced the second component in guinea-pig vasa stimulated with a single pulse or a train of pulses. Idazoxan enhanced both phases of the response of guinea-pig vasa stimulated with a train of pulses but did not affect the response to stimulation with a single pulse. The effect of phenoxybenzamine in increasing the efflux of radioactivity produced by a single pulse of stimulation was abolished by cocaine, indicating that the increase in efflux was due to blockade of noradrenaline uptake. Contractile responses of guinea-pig vasa stimulated with a single pulse in the presence of cocaine were unaltered by phenoxybenzamine, whereas with a train of stimulation the twitch component was enhanced and the second phase was reduced. The effects of phenoxybenzamine or idazoxan on the efflux of radioactivity from rat vasa portions were qualitatively the same as were observed in whole vasa. The contractile response of the prostatic portion consisted of a rapid twitch with a single pulse of stimulation, but was biphasic with a train of stimulation; the response of the epididymal portion was biphasic with either a single pulse or a train of pulses. These results suggest that there is no inhibitory feedback modulation of noradrenaline release with a single pulse of stimulation in guinea-pig and rat vasa deferentia whereas, with a train of stimulation, there is autoinhibition of noradrenaline release.     

Effects of prostaglandin E1 and indomethacin on the stimulation- evoked overflow of 3H-noradrenaline from guinea-pig taenia caecum

Prostaglandin E₁ (5.8 × 10^?8 M) markedly and reversibly reduced the stimulation-evoked overflow of total tritium, while it had no effects on basal outflow. Indomethacin (8.4 × 10^?6 M) increased the stimulation-evoked overflow of total tritium at low frequencies (2–5 Hz), while it had no effect at high frequencies of stimulation (more than 10 Hz). It was concluded that endogenous prostaglandin E₁ also plays a regulatory role in adrenergic inhibitory neurotransmission by inhibiting the noradrenaline release from adrenergic nerve terminals of the guinea-pig taenia caecum.     

Release of noradrenaline and ATP by electrical stimulation and nicotine in guinea-pig vas deferens

Summary Effects of electrical stimulation and nicotine on ATP and tritium outflow and smooth muscle tension were studied in the guinea-pig isolated vas deferens preincubated with [³H]-noradrenaline. ATP was measured using the luciferase technique.Electrical stimulation caused biphasic contractions and an acceleration of ATP and tritium outflow. The contraction amplitude and the overflow of ATP increased markedly, whereas the overflow of tritium increased only slightly with the frequency of stimulation (1–10 Hz; constant number of 60 pulses). The contraction amplitude did not increase with an increase in pulse number (20–540 pulses; constant frequency of 5 Hz), whereas the overflow of ATP increased slightly, and that of tritium markedly. Nicotine caused monophasic, transient contractions and, again, an acceleration of ATP and tritium outflow. Contractions, ATP and tritium overflow increased with the concentration of nicotine (56–320 mol/l) in an approximately parallel manner. The influence of some drugs on responses to electrical stimulation (60 pulses, 5 Hz) and nicotine (180 mol/l) was investigated. Tetrodotoxin blocked all effects of electrical stimulation but did not change those of nicotine. The reverse was true for hexamethonium. Neither electrical stimulation nor nicotine caused contraction or an increase in ATP outflow after pretreatment with 6-hydroxydopamine. The main effects of prazosin 0.3 mol/l were to reduce electrically evoked contractions (above all second phase) as well as nicotine-evoked contractions and the nicotine-evoked overflow of ATP (the latter by about 81 %). Prazosin also tended to diminish the electrically evoked overflow of ATP. ,ß-Methylene-ATP 10 mol/l elicited a transient contraction and ATP overflow on its own. The main change in the subsequent state of desensitization was a decrease of the first phase of electrically evoked contractions. The main effects of prazosin combined with desensitization by ,ß-methylene-ATP were marked decreases of electrically evoked contractions (by 94%), the electrically evoked overflow ATP (by 66%), nicotine-evoked contractions (by 97%) and the nicotinee-voked overflow of ATP (by 70%).It is concluded that both electrical stimulation and nicotine release noradrenaline and ATP in guinea-pig vas deferens. Only part of the evoked overflow of ATP (about 32%) is neural in origin. Another part probably originates from smooth muscle cells where it is released by neurogenic noradrenaline acting at ₁-adrenoceptors. Corelease leads to cotransmission: electrically as well as nicotine-evoked contractions consist of adrenergic and purinergic components. Varying types of stimulation release cotransmitter mixtures of varying composition. Electrical stimulation at high frequency (for example 10 Hz) and with low pulse numbers (for example 20 pulses) seems to release the cotransmitters at a relatively high ATP/noradrenaline ratio. Activation of prejunctional nicotine receptors seems to release the cotransmitters at a relatively low ATP/noradrenaline ratio. Send offprint requests to Ivar von Kügelgen at the above address     

The effects of amiodarone, an alpha and beta receptor antiagonist, on adrenergic transmission in the cat spleen.

The anti-anginal drug amiodarone produced a dose dependent reduction in the overflow of transmitter from the isolated blood perfused cat spleen following nerve stimulation at 30 Hz.In the presence of phenoxybenzamine (30 μg/ml) the normal increase in overflow of transmitter, following 200 stimuli at 10 Hz was prevented. This effect occurred whatever the order of addition of phenoxybenzamine and amiodarone, indicating that amiodarone did not reduce the overflow by stimulation of inhibitory presynaptic α receptors.In experiments in which the transmitter stores were labelled with [³H](?)-noradrenaline, amiodarone inhibited the release of label following nerve stimulation but had no effect on release induced by tyramine. Responses of the spleen to both nerve stimulation and tyramine were reduced by amiodarone but uptake of [³H](?)-noradrenaline given as injections (pulses) or as infusions, was not significantly affected. The effects of amiodarone on nerve evoked overflow of transmitter are not therefore related to changes in uptake of noradrenaline or to selective stimulation of presynaptic α receptors but probably reflect a neurone blocking action of the drug.     

Efflux of 3H-5-hydroxytryptamine from rat hypothalamic slices by continuous electrical stimulation: Frequency-dependent responses to serotonergic antagonists and 5-hydroxytryptamine

Rat hypothalamic slices were incubated with 3H-5-hydroxytryptamine and superfused in the presence of paroxetine to inhibit 5-hydroxytryptamine (5-HT) reuptake. The slices were continuously stimulated electrically with rectangular pulses at varying frequencies. Continuous stimulation for up to 42 min at 1 Hz or at 3 Hz evoked a steady efflux of tritium that slowly decayed with time. The efflux produced by continuous stimulation at 5 Hz declined more rapidly with time. Continuous stimulation at 1 Hz in the presence of increasing concentrations of unlabelled 5-HT produced a concentration-dependent decrease in tritium efflux. The presence of methiothepin (0.5 mumol/l), quipazine (10 mumol/l) and (-)- but not (+)-propranolol (1 mumol/l) attenuated this response to 5-HT. From these data, the apparent pA2 values were calculated and found to be in agreement with published values. Frequency-dependent responses were determined using a "cumulative stimulation" protocol whereby the slices were subjected to three consecutive 14 min periods of stimulation at increasing frequencies (1, 3 and then 5 Hz). Unlabelled 5-HT (1 mumol/l) inhibited electrically-evoked tritium efflux more at 1 than at 5 Hz. Methiothepin (0.5 mumol/l) and quipazine (10 mumol/l) enhanced the stimulated efflux in a manner inversely related to the frequency of stimulation. Neither (+)- nor (-)-propranolol enhanced stimulated tritium efflux at any of the three frequencies tested. It is concluded that continuous electrical stimulation of rat hypothalamic slices at a low frequency provides a rapid means of obtaining apparent affinities and intrinsic activities of drugs that modify the serotonergic autoreceptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of the biophase concentration of noradrenaline at presynaptic α2-adrenoceptors in brain slices

Summary The aim of the present study was to determine the local concentrations of noradrenaline existing at presynaptic ₂-adrenoceptors during electrical pulse train stimulation of brain slices at different frequencies. The experiments are based on the assumption that the concentration of released noradrenaline at the ₂-adrenoceptors exerting a certain autoinhibition should be equal to the concentration of exogenous noradrenaline causing the same inhibition under conditions in which any influence of the released transmitter is excluded. In order to avoid autoinhibition, hippocampus and cortex slices of the rabbit and the rat, prelabelled with [³H]noradrenaline and superfused in presence of an uptake inhibitor, were electrically stimulated using 4 pulses delivered at 100 Hz (POP stimulation). Exogenous noradrenaline diminished the overflow of tritium elicited by POP stimulation in a concentration-dependent manner. In rabbit brain tissues the EC₅₀ value and maximum inhibition of noradrenaline release were found to be approximately 6 nmol/l and more than 95%, respectively, whereas in rat tissues the corresponding values were between 20 and 30 nmol/l and approximately 90%. When electrical stimulation was performed with trains of 36 pulses delivered at 0.1, 0.3 or 3 Hz in absence or presence of an uptake inhibitor, the ₂-adrenoceptor antagonist yohimbine (1 or 10 mol/l) enhanced the evoked tritium overflow in a manner which was dependent on the frequency of stimulation and on blockade of the re-uptake mechanism. The facilitatory effects of yohimbine reflected an extent of autoinhibition which was between 53% (36 pulses/0.1 Hz, no uptake inhibitor) and 85% (36 pulses/3 Hz, uptake inhibitor present) in rabbit and between 16% (36 pulses/0.3 Hz, no uptake inhibitor) and 71% (36 pulses/3 Hz, uptake inhibitor present) in rat brain slices. Accordingly, the corresponding estimated biophase concentrations of noradrenaline were generally higher in rat than in rabbit tissues (they were between 32.5 and 74.5 or 5.1 and 51.6 nmol/l in the presence or absence of an uptake inhibitor, respectively, in the rat, and between 15 and 23.1 or 6.1 and 18.6 nmol/l in the rabbit). The observed frequency dependence of the effect of re-uptake blockade on the calculated biophase concentrations of noradrenaline would be compatible with the idea of a dependence of the effectiveness of the re-uptake mechanism on the firing rate of the neurone in being more effective at lower frequencies. Moreover, the stikingly low biophase concentrations of noradrenaline suggest that also in brain tissue noradrenaline causes lateral inhibition of release as has recently been shown for guinea-pig vas deferens. Send offprint requests to C. Allgaier at the above address     

Transmitter release patterns of noradrenergic,dopaminergic and cholinergic axons in rabbit brain slices during short pulse trains,and the operation of presynaptic autoreceptors

Summary Slices of rabbit brain were field-stimulated either by single electrical pulses or by trains of 4 or 8 pulses at 1 or 100 Hz in order to study transmitter release patterns and the autoinhibition of transmitter release. The slices were preincubated with ³H-noradrenaline (cortex), ³H-dopamine (caudate nucleus) or ³H-choline (caudate nucleus).Slices preincubated with ³ H-noradrenaline were superfused with medium containing desipramine 1 gmol/l. The overflow of tritium elicited by single pulses amounted to 0 .19% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was similar, whereas that elicited by 4 pulses/100 Hz was 5.1-fold higher. Yohimbine 101000 nmol/l increased up to 2.5-fold the overflow evoked by 4 pulses/1 Hz but did not change the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-dopamine were superfused with medium containing nomifensine 1 mol/l. The overflow of tritium elicited by single pulses was 0.39% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was 1.3-fold and the overflow elicited by 4 pulses/100 Hz 1.4-fold higher. Domperidone 1–100 nmol/l and sulpiride 10–1000 nmol/1 increased up to 2.4-fold the overflow evoked by 4 pulses/ 1 Hz but increased only slightly the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 mol/l. In physostigmine-free medium, atropine did not increase the evoked overflow of tritium at any stimulation condition. In physostigmine-containing medium, the overflow elicited by single pulses was 0.18% of the tritium content of the tissue. The overflow elicited by 8 pulses/1 Hz was 2.0-fold and the overflow elicited by 8 pulses/100 Hz 2.2-fold higher. Atropine 2–200 nmol/1 increased up to 2.4-fold the overflow evoked by 8 pulses/1 Hz but increased only slightly the overflow evoked bysingle pulses or 8 pulses/100 Hz. In physostigmine-free medium, sulpiride 10–1000 nmol/1 did not change the single-pulse-evoked overflow of tritium in the absence but increased it in the presence of nomifensine 1 mol/l.Single pulses elicit a large release of ³H-noradrenaline, ³H-dopamine and ³H-acetylcholine under the conditions of these experiments. Release elicited by single pulses is not subject to autoinhibition except for a small inhibition by spontaneously released transmitter in the case of dopaminergic and cholinergic axons. When 3 or 7 further pulses follow the first one at intervals of 1 s, they elicit much smaller release. At least a great part of the fall is due to autoreceptor mediated inhibition (for 3H-acetylcholine release in the presence of physostigmine only). When 3 or 7 further pulses follow at intervals of 10 ms, they elicit release that is either similar to that evoked by the first pulse (³H-noradrenaline) or much smaller (³H-dopamine, ³H-acetylcholine). However, the fall is not due to stimulation-dependent, auto-receptor-mediated inhibition; autoinhibition does not develop in these short high-frequency trains. Overall, the results are in accord with the autoreceptor theory. They demonstrate the role of autoinhibition in determining the transmitter release patterns of central noradrenergic, dopaminergic and cholinergic neurones. Send offprint requests to N. Limberger at the above address     

Stimulation-evoked release of [3H]-noradrenaline by 1, 10 or 100 pulses and its modification through presynaptic alpha 2-adrenoceptors

1 Mice isolated vasa deferentia were loaded with 1-[7,8-3H]-noradrenaline and subsequently field stimulated with 1, 10 or 100 pulses (2 ms pulse width, 1 Hz). The tritium overflow was separated into [3H]-noradrenaline and its 3H-metabolites. 2 The resting release of tritium contained about 7% [3H]-noradrenaline, 33% [3H]-3, 4-dihydroxyphenylglycol ([3H]-DOPEG) and 60% 3H-non-catechols with usually less than 1% [3H]-dihydroxymandelic acid ([3H]-DOMA). The proportion of the tritium as [3H]-noradrenaline increased with stimulation train length to 35% with 100 pulses; this increase in [3H]-noradrenaline was associated with falls in [3H]-DOPEG and 3H-non-catechols. Generally the proportional increase in [3H]-noradrenaline on stimulation was about 10 x total tritium when compared with the resting release. 3 The fractional release of [3H]-noradrenaline per pulse was independent of train length, averaging about 6 x 10(-6). This was reduced by the alpha 2-adrenoceptor agonist clonidine (0.3 - 30 nM) with an IC50 of 4.8 nM (10 pulses at 1 Hz). 4 The alpha 2-adrenoceptor antagonist, yohimbine (10 - 100 nM), did not alter the fractional release of [3H]-noradrenaline elicited by 1 pulse. The antagonist did not change the amount or composition of the resting or evoked tritium overflow. However, yohimbine (1 - 100 nM) increased the fractional release of [3H]-noradrenaline per pulse for trains of 10 or 100 pulses (1 Hz) in a concentration-dependent fashion. An increase above controls was significant only with 100 pulses and yohimbine, 30 nM. 5 The results show that the release of noradrenaline during trains of pulses in the mouse vas deferens can be regulated through presynaptic alpha 2-adrenoceptors. There was no evidence of inhibition by noradrenaline of its own release following a single pulse.     

Corelease of noradrenaline and ATP by brief pulse trains in guinea-pig vas deferens

Contractions and overflow of tritium and ATP elicited by single electrical pulses or short pulse trains were studied in the guinea-pig isolated vas deferens preincubated with [³H]-noradrenaline. ATP was measured using the luciferase technique.A single pulse caused only a small contraction and minimal tritium and ATP overflow. In contrast, trains of 6 pulses elicited marked contractions as well as tritium and ATP overflow. In experiments with 6 pulses/100 Hz, prazosin 0.3 M reduced the contraction by 73 %, did not change the evoked overflow of tritium, and reduced the evoked overflow of ATP by 85%. Suramin 300 M reduced the contraction by 69% but changed neither the evoked overflow of tritium nor that of ATP. The combination of prazosin 0.3 gM and suramin 300 M abolished the contraction, did not change the evoked overflow of tritium, and reduced the evoked overflow of ATP by 70%. When 6 pulses were applied at frequencies of 1, 2, 10 or 100 Hz, all responses increased with frequency up to a maximum at 10 Hz, but contractions and the evoked overflow of ATP increased with frequency to a greater extent than the evoked overflow of tritium. A similar frequency overflow relationship was observed when the medium contained prazosin 0.3 M and suramin 300 M (and evoked ATP overflow was greatly reduced). Yohimbine 1 M did not affect the overflow of tritium evoked by 6 pulses/100 Hz but increased that evoked by 6 pulses/10 Hz.The results demonstrate an overflow of both noradrenaline and ATP in response to short pulse trains. As observed previously for prolonged pulse trains, the major part of the evoked overflow of ATP was derived from non-neural cells. The ATP overflow remaining during ₁-adrenoceptor blockade by prazosin and P₂-purinoceptor blockade by suramin is likely to reflect neural release of ATP. The results support the view that release of ATP increases with frequency to a greater extent than release of noradrenaline. The latency for the onset of prejunctional ₂-autoinhibition in guinea-pig vas deferens is between 50 and 500 ms. Correspondence to: I. von Kügelgen at the above address     

The effect of amitriptyline on presynaptic mechanisms in noradrenergic nerves.

1 Electrically evoked and resting overflow of tritium was measured from mouse vas deferens previously incubated with [3H]-(--)-noradrenaline. 2 At low concentrations (1.6 X 10(-7) to 4 X 10(-6)M) amitriptyline increased only the evoked tritium overfow while higher concentrations increased both evoked and resting overflow. 3 In the presence of atropine (1 X 10(-6 M) amitriptyline still produced an increase in evoked tritium overflow. 4 In the presence of a concentration of cocaine (1.1 X 10(-5) M) which produced a maximal blockade of the uptake of exogenous noradrenaline the application of amitriptyline still produced an increase in evoked tritium overflow. 5 In the presence of a concentration of phentolamine (1 X 10(-5) M) that produced complete blockade of the presynaptic alpha-adrenoceptors, amitriptyline still produced an increase in evoked tritium overflow. 6 In the presence of cocaine and phentolamine together the effect of amitriptyline on evoked overflow was abolished. 7 It is concluded that amitriptyline may raise synaptic levels of noradrenaline by blocking presynaptic alpha-adrenoceptors controlling noradrenaline release and by blocking its uptake into sympathetic neurones.