Neural ATP release and its α2-adrenoceptor-mediated modulation in guinea-pig vas deferens

Summary Contractions, release of previously stored [³H]-noradrenaline (measured as overflow of total tritiated compounds) and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) were studied in the superfused vas deferens of the guinea pig. Prazosin and suramin were used to suppress non-neural ATP release, and effects of bromoxidine and rauwolscine on the neural release thus isolated were examined.Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. Both prazosin (0.03–3 M) and suramin (30–300 M) reduced contractions as well as the evoked overflow of ATP. No visible contraction remained in 21 of 28 tissues exposed to prazosin 0.3 M combined with suramin 300 M. The evoked overflow of ATP under these conditions was about 17% of that observed in the absence of drugs. In the presence of prazosin 0.3 M and suramin 300 M, bromoxidine (0.01–1 M) decreased and rauwolscine (0.1–10 M) increased the evoked overflow of both tritium and ATP. Rauwolscine increased the evoked overflow of tritium to a significantly greater extent than the overflow of ATP.It is concluded that the overflow of ATP elicited by electrical (neural) stimulation in the presence of prazosin 0.3 M and suramin 300 M reflects purely neural release of ATP. This release of ATP, like the release of noradrenaline, is modulated through prejunctional ₂-adrenoceptors. The ₂-adrenoceptor modulation of the release of noradrenaline seems to be more marked than the modulation of the release of ATP. Correspondence to B. Driessen at the above address     

Phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex

Summary Possible antagonist effects of phentolamine at presynaptic serotonin autoreceptors were studied in slices of the occipito-parietal cortices of the rabbit and the rat. The slices were preincubated with ³H-serotonin and then superfused and stimulated electrically with single pulses or pulse trains. Nitroquipazine 1 mol/l, a compound that inhibits the high affinity neuronal uptake of serotonin, was present in the superfusion medium in all one pulse-experiments as well as in experiments in which the effect of unlabelled serotonin was examined.In rabbit cortical slices, unlabelled serotonin reduced the single pulse-evoked overflow of tritium. Its concentrationresponse curve was not changed by the selective ₂-adrenoceptor antagonist idazoxan 1 mol/l but was shifted to the right by phentolamine 1 and 10 mol/l. Phentolamine 10 mol/l also shifted to the right the concentration-inhibition curve of the selective 5-HT₁-receptor agonist 5-carboxamidotryptamine. When the slices were stimulated by trains of 30 pulses at 3 Hz, phentolamine 1 and 10 mol/l but not 0.1 mol/l increased the evoked overflow of tritium, the maximal increase amounting to 178%; its effect was enhanced in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l (a drug combination that, when given alone, slightly increased the evoked overflow of tritium). The serotonin receptor antagonist metitepin at concentrations of 0.01–1 mol/l also increased the overflow of tritium elicited by 30 pulses/3 Hz, the maximal increase amounting to 280%; its effect was potentiated in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l but was abolished or almost abolished in the presence of nitroquipazine 1 mol/l plus phentolamine 10 mol/l (a drug combination that, given alone, greatly increased the evoked overflow of tritium). When slices were stimulated by trains of 360 pulses at 3 Hz, there was no apparent antagonism of phentolamine 10 mol/l against the inhibitory effect of unlabelled serotonin. In rat brain cortex slices, unlabelled serotonin reduced the overflow of tritium elicited by 4 pulses delivered at 100 Hz. Again, phentolamine 10 mol/l shifted the concentration-response curve to the right.It is concluded that phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex with pA₂ values of 6.44 and 5.95, respectively. Previous failures to detect the antagonistic effect against exogenous agonists were probably due to stimulation conditions that led to marked endogenous autoinhibition of serotonin release. At least the major part of the increase by phentolamine of the release of serotonin is due to autoreceptor blockade rather than blockade of the presynaptic a2-adrenoceptors at the cortical serotoninergic axons.Send offprint requests to N. Limberger at the above address     

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [³H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 M and even more so by prazosin 0.3 M combined with suramin 300 M. They were also reduced by nifedipine 10 M and even more so by nifedipine 10 M combined with ryanodine 20 M (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [³H]-noradrenaline, exogenous noradrenaline 10 M and ,-methylene ATP 10 M elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 M but not suramin 300 M and were greatly reduced by nifedipine 10 M and in Ca²⁺-free medium. Responses to ,-methylene ATP were blocked by suramin 300 M but not prazosin 0.3 M were reduced by nifedipine 10 M (effect on ATP overflow not significant) and were reduced even more in Ca²⁺-free medium. Neuropeptide Y 0.3 M caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both ₁-adrenoceptor and P₂-purinoceptor activation. Ca²⁺ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A purely neural overflow of ATP was not isolated under the conditions of the experiments. Correspondence to: R. Bültmann at the above address     

Functional consequences of inhibition of nucleotide breakdown in rat vas deferens: a study with Evans blue

The effect of Evans blue on nucleotide breakdown, nucleotide-evoked contractions and electrically evoked contractions, overflow of ATP and overflow of tritium (after labelling with [³H]-noradrenaline) was studied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methylthio ATP (all 100 M) over 30 min. Evans blue (100 M) reduced this degradation to 22 to 26%. Nucleotides elicited contraction with potency declining in the order , \-methylene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 M) shifted the concentration-response curve of , \-methylene ATP to the right and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and responses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > , \-methylene ATP > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited contraction and an overflow of tritium and ATP. Evans blue (100 M) did not alter the contraction and the evoked overflow of tritium but increased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an — albeit impure — ecto-nucleotidase inhibitor in functional experiments. Such experiments demonstrate that the low potency of ATP (and also ADP and 2-methylthio ATP) in eliciting contraction, and the small size of the overflow of ATP upon sympathetic nerve stimulation, are due to rapid breakdown.     

Opposite modulation of cotransmitter release in guinea-pig vas deferens: increase of noradrenaline and decrease of ATP release by activation of prejunctional β-adrenoceptors

Effects of isoprenaline on contraction, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) as well as on contractions elicited by exogenous noradrenaline and ATP were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique.In [³H]-noradrenaline-pretreated tissues, electrical stimulation elicited an overflow of tritium and ATP and a biphasic contraction. Isoprenaline (1–100 nM) reduced the contraction, mainly phase I, and enhanced the evoked overflow of tritium; evoked overflow of ATP was not changed significantly. No, or almost no, contraction remained in [³H]-noradrenaline-pre-treated tissues exposed to both prazosin (0.3 M) and suramin (300 M), and the evoked overflow of ATP was reduced by about 82%. Under these conditions, isoprenaline (1–100 nM) again enhanced the evoked overflow of tritium, but it now decreased the evoked overflow of ATP. Propranolol (1 M), when added on top of prazosin and suramin, prevented the effects of isoprenaline (1–100 nM). In some tissues not pretreated with [³H]-noradrenaline, purinergic and adrenergic components of the neurogenic contraction (again to 210 pulses, 7 Hz) were isolated by exposure to prazosin (0.3 M) and suramin (300 M), respectively. Isoprenaline (1–100 nM) decreased the isolated purinergic component but did not change significantly the isolated adrenergic component. Contractions elicited by ATP (1000 M) were not changed and contractions elicited by noradrenaline (100 M) were slightly increased by isoprenaline (1–100 nM). Isoprenaline (100 nM) did not change the degradation of ATP (100 M) by pieces of the vas deferens.It is concluded that, in the guinea-pig vas deferens, activation of prejunctional -adrenoceptors modulates the neural release of noradrenaline and ATP in opposite directions: release of noradrenaline is enhanced, whereas release of ATP is decreased.     

α-Adrenoceptor-mediated inhibition of noradrenaline release in rabbit brain cortex slices

Summary Brain cortex slices from rabbits were preincubated with [³H]noradrenaline and then superfused and stimulated electrically at 3Hz. In the presence of cocaine 30 M, unlabelled noradrenaline, -methylnoradrenaline, clonidine, oxymetazoline, xylazine and guanabenz decreased, whereas yohimbine, corynanthine, phentolamine, tolazoline and azapetine increased the stimulation-evoked overflow of tritium. Phenylephrine and prazosin had no effect on the evoked overflow except at concentrations that greatly accelerated the basal outflow of tritium. The results indicate that the noradrenergic axons of rabbit brain cortex are endowed with presynaptic -adrenoceptors which are exclusively of the ₂-type. Addition of various concentrations of cocaine, addition of pargyline, or stimulation at different current strengths was used to obtain either a high or a low stimulation-evoked overflow of tritium. Independently of the method used, a low evoked overflow coincided with a large percentage inhibition produced by 0.1M clonidine, whereas a high evoked overflow coincided with a smaller percentage inhibition produced by clonidine. The results indicate that drugs which block the re-uptake of noradrenaline diminish the presynaptic inhibitory effect of -adrenergic agonists by increasing the biophase concentration of released noradrenaline.     

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     

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.     

Mutual interaction of histamine H3-receptors and α2-adrenoceptors on noradrenergic terminals in mouse and rat rain cortex

Summary Brain cortex slices were preincubated with ³H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the inhibition of the electrically evoked tritium overflow caused by histamine in the presence of -adrenoceptor ligands (mouse and rat brain cortex), and the inhibition caused by talipexole (the former B-HT 920) in the presence of H₃-receptor ligands (mouse brain cortex).In mouse brain cortex slices, the inhibitory effect of histamine on the tritium overflow evoked by 36 pulses, 0.3 Hz was not changed by the ₁-adrenoceptor antagonist prazosin, but increased by the ₂-adrenoceptor antagonist rauwolscine. When the current strength or the duration of electrical pulses was reduced to compensate for the increase in evoked tritium overflow produced by rauwolscine, the latter still. enhanced the effect of histamine. The histamine-induced inhibition of tritium overflow evoked by 360 pulses, 3 Hz was not affected by the ₁-adrenoceptor agonist phenylephrine but attenuated by the ₂-adrenoceptor- agonist talipexole. Finally, the inhibition by histamine of the tritium overflow evoked by 3 pulses, 100 Hz was attenuated by talipexole but not affected by rauwolscine. Conversely,. the inhibitory effect of talipexole on tritium overflow elicited by 360 pulses, 3 Hz was slightly attenuated by the H₃-receptor agonist R-(–)--methylhistamine but not, affected by the H₃- receptor antagonist thioperamide. In rat brain cortex slices, histamine only tended to inhibit tritium overflow evoked by 360 pulses, 3 Hz, both in the absence of -adrenoceptor antagonists and in the presence of prazosin. However, histamine markedly inhibited the evoked overflow in the presence of rauwolscine. Again, enhancement of the histamine-induced inhibition also occurred when the current strength or the duration of pulses was reduced in order to compensate for the increase in evoked tritium overflow produced by rauwolscine.The results suggest that the ₂-autoreceptors and the H₃-heteroreceptors at the noradrenergic nerve endings in the brain of mouse and rat interact with each other. Activation of the ₂-autoreceptors decreases, whereas blockade of the activated (but not of the non-activated) ₂-autoreceptors increases, the inhibitory effect of histamine. Activation of the H3-heteroreceptors slightly decreases, whereas blockade of the H₃-receptors fails to affect, the inhibitory effect of talipexole.Send offprint requests to E. Schlicker at the above address     

Blockade of α2-adrenoceptors permits the operation of otherwise silent opioid κ-receptors at the sympathetic axons of rabbit jejunal arteries

Summary We sought for presynaptic, release-inhibiting opioid receptors at the postganglionic sympathetic axons innervating the jejunal arteries of rabbits. Evoked excitatory junction potentials (e.j.p.s; trains of 15 pulses at 1 Hz) as well as the evoked overflow of tritium after preincubation with [³H]-noradrenaline (trains of 120 pulses at 1 Hz) were used to estimate transmitter release. In otherwise untreated tissues ethylketocylazocine reduced neither the e.j.p. amplitudes nor the evoked overflow of tritium; [Met⁵]-enkephalin depressed the evoked overflow of tritium. Ethylketocyclazocine reduced e.j.p. amplitudes, however, in tissues exposed to either yohimbine, tolazoline or phentolamine, but not in tissues exposed to prazosin. Ethylketocyclazocine also depressed the evoked overflow of tritium when yohimbine was present. The inhibition produced by ethylketocyclazocine in the presence of yohimbine was antagonized by (-)-3-furylmethyl)--noretazocine (MR 2266) but not by N,N-diallyl-Tyr--aminoisobutyric acid--aminoisobutyric acid-Phe-Leu-OH (ICI 174864). It is concluded that the sympathetic neurones of rabbit jejunal arteries possess presynaptic -receptors in addition to the previously identified -receptors. The -receptors become operative only when presynaptic ₂-adrenoceptors have been blocked.     

Presynaptic imidazoline receptors and α2-adrenoceptors in the human heart: discrimination by clonidine and moxonidine

The involvement of presynaptic ₂-autoreceptors and imidazoline receptors in the modulation of noradrenaline release was investigated in strips from human atrial appendages preincubated with [³H]noradrenaline and superfused with medium containing desipramine and corticosterone. Electrical impulses were applied transmurally at 2 Hz. The imidazoline derivatives moxonidine and clonidine reduced the evoked tritium overflow in a concentration-dependent manner. Moxonidine was 18-fold more potent than clonidine. The concentration-response curve for moxonidine, but not for clonidine was shifted to the right by the ₂-adrenoceptor antagonist rauwolscine. The apparent pA₂ value of rauwolscine against moxonidine was 8.41. An inhibitory effect was also observed for the imidazoline derivative BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), a mixed ₂-adrenoceptor antagonist/imidazoline receptor agonist; BDF 6143 was about 2-fold more potent than clonidine. Rauwolscine (1 M) did not substantially shift the concentration-response curve of BDF 6143.It is concluded that noradrenaline release in the human atrium is inhibited not only via presynaptic ₂-autoreceptors but also via presynaptic non-I₁, non-I₂ imidazoline receptors. The failure of rauwolscine to antagonize the inhibitory effect of clonidine suggests that clonidine preferentially stimulates the presynaptic imidazoline receptors; the ₂-adrenoceptor component of its action is probably suppressed by an inhibitory interaction between the two receptors. In contrast, moxonidine acts via presynaptic ₂-autoreceptors, leaving the presynaptic imidazoline receptor unaffected.     

Only activated but not non-activated presynaptic α2-autoreceptors interfere with neighbouring presynaptic receptor mechanisms

Summary Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic ₂-autoreceptors of effects mediated by presynaptic opioid - and adenosine A₁-receptors requires activation of the ₂-receptors. The slices were preincubated with ³H-noradrenaline and then superfused with medium containing desipramine 1 mol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz.The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca²⁺-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 mol/l or yohimbine 0.3 mol/l. Ethylketocyclazocine (EK; 0.1–10 nmol/l) and R-(–)-N⁶-phenylisopropyladenosine (PIA; 1–1,000 nmol/1) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. — The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 mol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca²⁺ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine.The results demonstrate that there is no ₂-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic ₂-adrenoceptor does not interfere with presynaptic opioid - and adenosine A₁-receptor mechanisms. It is only when the autoreceptor is activated by released noradrenaline that it attenuates neighbouring presynaptic receptor mechanisms, and this attenuation is removed by ₂-adrenoceptor antagonists.Send offprint requests to N. Limberger at the above address     

Comparison of corelease of noradrenaline and ATP evoked by hypogastric nerve stimulation and field stimulation in guinea-pig vas deferens

Contractions and overflow of tritium and ATP elicited by hypogastric nerve stimulation (HNS) and field stimulation (FS) were studied in the guinea-pig isolated vas deferens preincubated with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique.HNS and FS elicited contraction, tritium overflow and ATP overflow. HNS at supramaximal current strength produced smaller responses than did FS at supramaximal current strength (210 pulses/7 Hz). Supramaximal HNS and submaximal FS were used in the remainder of the study. Prazosin (0.3 mol/l) reduced contractions and the overflow of ATP elicited by both HNS and FS; the evoked overflow of tritium was not changed (210 pulses/7 Hz). Combined administration of prazosin (0.3 mol/l) and suramin (300 mol/l) abolished contractions and reduced the overflow of ATP elicited by both HNS and FS slightly more than did prazosin alone; tritium overflow again was not changed (210 pulses/7 Hz). Contractions, tritium overflow and ATP overflow increased with the frequency of both HNS and FS (from 7 to 25 Hz; 210 pulses); the increase in ATP overflow with frequency was more marked than the increase in tritium overflow. The preferential increase of ATP overflow with the frequency of HNS and FS persisted in the combined presence of prazosin (0.3 mol/l) and suramin (300 mol/l).The study confirms for HNS, a more physiologic way of sympathetic nerve stimulation, several observations previously obtained with FS. First, HNS-evoked ATP release is detectable as an overflow of ATP into the superfusion fluid. Second, a large part of the HNS-evoked release of ATP is postjunctional in origin, due to activation of post-junctional ₁-adrenoceptors and presumably P₂-purinoceptors. Third, the average neural release of ATP per pulse facilitates with the frequency of stimulation to a greater extent than the average release of noradrenaline per pulse.     

P1-purinoceptor-mediated modulation of neural noradrenaline and ATP release in guinea-pig vas deferens

The effect of P₁-purinoceptor activation on contractions, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) was studied in the superfused vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferinluciferase technique.Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. In the absence of other drugs, adenosine (10–100 M) did not change evoked contractions but reduced the evoked overflow of tritium and ATP. In subsequent experiments ₁-adrenoceptors were blocked by prazosin, P₂-purinoceptors by suramin and ₂-adrenoceptors by rauwolscine. No or almost no contraction remained under these conditions. The evoked overflow of tritium was 505% and the evoked overflow of ATP 34% of that observed in the absence of prazosin, suramin and rauwolscine. Adenosine (1–100 M) again reduced the evoked overflow of tritium and ATP, and so did the A₁-selective agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA; 0.032–0.32 M). Adenosine and CCPA decreased the evoked overflow of ATP to a greater extent than the evoked overflow of tritrium.It is concluded that neural release of both postganglionic sympathetic cotransmitters, noradrenaline and ATP, is decreased upon activation of prejunctional P₁- (A₁-) purinoceptors in guinea-pig vas deferens. The A₁-receptor-mediated inhibition of the release of ATP is more marked than the inhibition of the release of noradrenaline, a pattern opposite to the inhibition produced by activation of prejunctional ₂-autoreceptors. Correspondence to: B. Driessen at the above address     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

Electrically evoked noradrenaline release from cultured chick sympathetic neurons: modulation via presynaptic α2-adrenoceptors and lack of autoinhibition

Summary Sympathetic neurons from twelve day old chick embryos were plated on polystyrol discs and kept in culture for five days. After incubation with ³H-noradrenaline the discs were transferred to small chambers and superfused. Electrical field stimulation (36 pulses at 3 Hz) increased the outflow of tritium. The evoked overflow of tritium was abolished in the absence of extracellular calcium and was diminished by about 90% in the presence of tetrodotoxin (1 mol/l). The ₂-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14,304) caused a concentration-dependent decrease in overflow, whereas the ₁-adrenoceptor agonist methoxamine was ineffective at up to 1 mol/l. The concentration-response curve of UK-14,304 was shifted to the right by the ₂-adrenoceptor antagonist yohimbine (0.03 mol/l). Yohimbine on its own caused no significant change. The noradrenaline reuptake inhibitor cocaine (10 mol/l) caused a small (20%) increase in evoked overflow. The results indicate that cultured chick sympathetic neurons possess release-modulating ₂-adrenoceptors and that the electrically induced overflow of transmitter occurs under conditions virtually free of autoinhibition. Send offprint requests to E.A. Singer at the above address     

Failure of tyramine to release neuronal ATP as a cotransmitter of noradrenaline in the guinea-pig vas deferens

Contractions, release of noradrenaline and r elease of ATP elicited by the indirectly acting sympathomimetic amine tyramine and responses elicited by exogenous noradrenaline were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique.In tissues pretreated with pargyline 1 mM, tyramine 300 M, when added to the superfusion medium for 2 min, elicited contraction and an overflow of tritium (mainly [³H]-noradrenaline) and ATP. Contraction and ATP overflow responses were prevented and tritium overflow was greatly reduced by desipramine 10 M Prazosin 0.3 M abolished contractions and evoked ATP overflow without changing tritium overflow. Blockade of postjunctional P₂-purinoceptors by suramin 300 M caused a marked decrease of tyramine-evoked contractions and a slight reduction of tritium overflow whereas evoked ATP overflow was markedly increased. The effect on contraction was not shared by two other P₂-purinoceptor antagonists, namely pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS) 32 M and diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) 32 M: PPADS increased contractions about fourfold, whilst DIDS had no effect at all. When the vas deferens was superfused for 24 min with medium containing tyramine 300 M, evoked contractions and tritium overflow continued throughout whereas ATP overflow faded rapidly to basal values. In the presence of prazosin 0.3 M, tyramine 300 M again failed to elicit contractions as well as an overflow of ATP. Application of noradrenaline 10 M instead of tyramine also resulted in prolonged contraction and an overflow of ATP that declined rapidly.It is concluded that all ATP released by tyramine is non-neuronal in origin, secondary to the activation of postjunctional ₁-adrenoceptors by released noradrenaline. The non-neural ATP does not seem to play a functional role in smooth muscle contraction and derives from a postjunctional source which is subject to a rapid depletion upon sustained ₁-adrenoceptor activation.     

Inhibition by nucleotides acting at presynaptic P2-receptors of sympathetic neuro-effector transmission in the mouse isolated vas deferens

Summary Effects of nucleotides and nucleosides on smooth muscle tension and the release of previously stored [³H]-noradrenaline were studied in the mouse isolated vas deferens. The tissue was stimulated twice by 20 electrical field pulses delivered at 2 Hz (S₁, S₂)., \-Methylene-ATP, ATPS, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine (up to 100 mol/1) and uridine (up to 1 mmol/1). The electrically evoked overflow of tritium was reduced by the drugs in the following order of potency: ATPS > ATP = adenosine > UTP; ,\-meth-ylene-ATP (up to 10 µmol/l) and uridine (up to 1 mmol/1) did not significantly change the evoked overflow. 8-(p-Sulphophenyl)theophylline did not alter the contractile responses to the nucleotides; it prevented the overflow-inhibiting effect of adenosine and reduced that of UTP; the overflow-inhibiting effects of ATP and ATPS were not significantly attenuated. After prolonged exposure to ,-methylene-ATP, all contractile nucleotide effects were abolished; in contrast, the depression by adenosine and the nucleotides of the evoked overflow of tritium persisted. None of the effects was changed by indometacin, yohimbine or reactive blue 2.It is concluded that ATP, ATPS, ,\-methylene-ATP and UTP produce contraction of the vas deferens by activation of P_2x-receptors. Moreover, the nucleotides inhibit per se the release of [3H]-noradrenaline (and presumably the co-transmitter mixture of noradrenaline and ATP); the effect of ATP is not, or only to a small extent, due to breakdown to adenosine. The presynaptic site of action of the purine nucleotides is a P₂-receptor which differs from the P_2X-receptor and may be a reactive blue 2-resistant P_2y-like receptor.     

Modulation of N-methyl-d-aspartate (NMDA)-stimulated noradrenaline release in rat brain cortex by presynaptic α2-adrenoceptors

Summary Rat brain cortex slices and synaptosomes preincubated with [³H]noradrenaline were used to investigate whether the NMDA-evoked noradrenaline release is modulated by agonists or antagonists at presynaptic ₂-adrenoceptors.In experiments on slices, noradrenaline and the preferential ₂-adrenoceptor agonists talipexole (former B-HT 920) and clonidine inhibited the NMDA evoked tritium overflow whereas the selective ₁-adrenoceptor agonists cirazoline and methoxamine were ineffective. The ₂-adrenoceptor antagonists rauwolscine and idazoxan facilitated the NMDA-evoked tritium overflow whereas the preferential ₁-adrenoceptor antagonist prazosin was ineffective. The concentration-response curve of talipexole for its inhibitory effect on NMDA-evoked overflow was shifted to the right by idazoxan (apparent pA₂ = 7.5). The EC₅₀ of NMDA (97 mol/l) for its stimulating effect on tritium overflow was not substantially changed by blockade of ₂-autoreceptors with 1 mol/l rauwolscine (EC₅₀ of NMDA in the presence of the ₂-adrenoceptor antagonist, 155 mol/l), but the maximal overflow of tritium was increased 2.5 fold by this rauwolscine concentration. In experiments on synaptosomes, talipexole and noradrenaline inhibited the NMDA-evoked tritium overflow. The inhibitory effect of talipexole was abolished by idazoxan which, given alone, was ineffective, as was prazosin. Talipexole did also not produce an inhibition when tritium overflow was evoked by NMDA in the presence of -conotoxin GVIA 0.1 mol/l; the latter, by itself, decreased the response to NMDA by about 55%. It is concluded that the NMDA-evoked noradrenaline release in the cerebral cortex is modulated via presynaptic ₂-adrenoceptors on the noradrenergic neurones. Stimulation of these autoreceptors in slices by endogenous noradrenaline does not result in a decreased potency of NMDA, but in a decreased maximum effect, in stimulating noradrenaline release. The inhibitory effect of ₂-adrenoceptor agonists on the NMDA-evoked release is at least partially due to a functional interaction between the NMDA receptors and ₂-autoreceptors at the level of the same varicosities. The results obtained with -conotoxin GVIA suggest that Ca²⁺ influx via the N-type voltage-sensitive calcium channel (VSCC) occurs in response to NMDA receptor stimulation and contributes substantially to the induction of NMDA-evoked noradrenaline release. The inhibitory effect of ₂-adrenoceptor stimulation on this release appears to be ultimately due to an inhibition of the influx of Ca²⁺ via the N-type VSCC. Correspondence to: M. Göthert at the above address     

Electrically-evoked release of taurine in the rat vas deferens: evidence for a purinoceptor-mediated effect

Release of taurine evoked by electrical stimulation (2700 pulses; 5 Hz; 10 mA unless stated otherwise) and its dependence on noradrenaline and ATP was studied in isolated, perifused rat vas deferens. Outflow of noradrenaline was also measured in some experiments.The basal outflow of taurine averaged 3.90±0.32 nmol/g tissue per min. Electrical stimulation increased the outflow to about 4 times basal values. The electrically-evoked overflow averaged 128.0±11.7 nmol/ g. An increase in current strength to 40 mA increased the evoked overflow by about 50%. At either current strength, the evoked overflow of taurine (and noradrenaline) was abolished by tetrodotoxin. Ca²⁺-deprivation blocked the overflow of taurine elicited by 10 mA and increased the overflow elicited by 40 mA pulses (but abolished noradrenaline overflow under either condition). Neither prazosin nor pretreatment of the rats with reserpine reduced electrically-evoked overflow of taurine (although reserpine pretreatment abolished evoked noradrenaline overflow). Tyramine (100 mol/1; 9 min) caused an overflow of taurine 36% of that caused by electrical stimulation (but an overflow of noradrenaline 3 times higher than that evoked by electrical stimulation). Exogenous noradrenaline (9 min) caused a concentration-dependent overflow of taurine with a maximal effect at 162 mol/1, amounting to 33% of the electrically-evoked overflow. ,\-Methylene ATP (19 mol/1) elicited an overflow of taurine that faded despite continued exposure to the drug and amounted to 62% of the response to electrical stimulation. Thirty minutes after the start of application of , \.-methylene ATP, electrically-evoked overflow of taurine was greatly reduced. Suramin (100 mol/1) also reduced taurine overflow in response to electrical stimulation.It is concluded that electrical (neural) stimulation releases taurine in rat vas deferens. The release is mainly postjunctional in origin, secondary to ATP release from sympathetic axon terminals, and a consequence of postjunctional P_2x-purinoceptor activation.