Polymyxin B,a selective inhibitor of protein kinase C,diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-dibutyrate

Summary Slices of the rabbit hippocampus were labelled with ³H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time-and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.Abbreviations PKC protein kinase C - PDB phorbol 12,13-dibutyrate - TPA 12-O-tetradecanoyl 13-acetate     

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     

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.     

Interneurones are probably not involved in the presynaptic dopaminergic control of dopamine release in rabbit caudate nucleus

Summary Slices of rabbit caudate nucleus were preincubated with ³H-dopamine and then superfused. The influence of apomorphine and haloperidol on the overflow of tritium evoked by 20 mmol/l potassium was investigated in the presence and in the absence of tetrodotoxin. The potassium-evoked overflow was largely calcium-dependent and consisted mainly of ³H-dopamine. The dopamine receptor agonist apomorphine 0.01–1.0 mol/l reduced, whereas the antagonist haloperidol 0.1 mol/l enhanced the potassium-evoked overflow of tritium. The effects of apomorphine and haloperidol were as pronounced in the presence as in the absence of tetrodotoxin 0.3 mol/l. It is concluded that the presynaptic dopaminergic modulation of dopamine release is not mediated by a tetrodotoxin-sensitive interneuronal pathway.     

Frequency-dependence of serotonin autoreceptor but not α2-adrenoceptor inhibition of [3H]-serotonin release in rat hypothalamic slices

Summary The overflow of tritium from stimulated rat hypothalamic slices preincubated with [³H]-serotonin (5-HT) was significantly enhanced by reducing the frequency of stimulation from 3 Hz to 1 Hz while keeping the number of impulses constant. The 5-HT receptor agonist 5-methoxytryptamine inhibited in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT with IC₅₀ values of 560 nmol/l and of 34 nmol/l when the stimulations were delivered at 3 Hz and 1 Hz, respectively. The terminal 5-HT autoreceptor antagonist methiothepin enhanced in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT and this effect was greater at a frequency of stimulation of 3 Hz than at 1 Hz. In the same paradigm, the 5-HT reuptake inhibitors citalopram and paroxetine did not alter the overflow of radioactivity elicited by stimulation at 3 Hz but significantly decreased it at 1 Hz. In the presence of 5-HT autoreceptor blockade achieved with methiothepin, citalopram increased the overflow of [³H]-5-HT to the same extent at 1 Hz and at 3 Hz. The IC₅₀ values for inhibition of [³H]-5-HT release by the selective ₂-adrenoceptor agonist UK 14.304 were 35 nmol/l at 3 Hz and 30 nmol/l at 1 Hz. It is concluded that modulation of 5-HT release by 5-HT autoreceptors, but not by ₂-adrenoceptors is dependent on the synaptic concentration of 5-HT as a function of the frequency of depolarization. Send offprint requests to S. Z. Langer at the above address     

Acetylcholine release in rat nucleus accumbens is regulated through dopamine D2-receptors

Summary Experiments in slices of rat nucleus accumbens were carried out in order to investigate whether the release of acetylcholine in this tissue is modulated through dopamine receptors. The slices were preincubated with ³H-choline and then superfused and stimulated electrically twice for 2 min each at a frequency of 3 Hz.The electrically evoked overflow of tritium averaged 2.9–3.9% of the tritium content of the tissue in the various groups. The D₂-selective agonist quinpirole (0.01–1 mol/l) reduced the evoked overflow of tritium by maximally 56%, an effect antagonized by the D₂-selective antagonist (–)-sulpiride (1 mol/l). The D₁-selective agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) caused a slight decrease only at the high concentration of 10 mol/l. (–)-Sulpiride (0.1–10 mol/l) moderately increased the evoked overflow of tritium when given alone. The dopamine uptake inhibitor nomifensine (10 mol/l) caused a decrease, and in its presence the increase produced by (–)-sulpiride became much more marked, amounting to maximally 149%. (+)-Sulpiride (0.1–1 mol/l) failed to change the evoked overflow of tritium in the presence of nomifensine. The dopamine-releasing agent (±)-amphetamine (1 mol/l) also reduced the evoked overflow, an effect abolished by (–)-sulpiride. Finally, bretylium (1 mmol/l), which blocks the release of dopamine, increased the evoked overflow. (–)-Sulpiride (1 mol/l) lost its facilitatory effect in slices treated with bretylium.We conclude that the release of acetylcholine in rat nucleus accumbens, like its release in the nucleus caudatusputamen, is modulated through dopamine D₂-receptors. The receptors are activated by endogenous dopamine under the conditions of these experiments. Send offprint requests to K. Starke at the above address     

Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria

Summary The effect of Bay K 8644 (a dihydropyridine Ca²⁺-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [³H]-noradrenaline. Bay K8644 (3mol/l) significantly increased atrial rate from 206±7 to 259±9 beats·min^–1 (P<0.05) and also tritium outflow (expressed as fractional rate of loss in min^– × 103) from 6.49±0.35 to 8.61±0.74 (P<0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([³H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.Members of Consejo Nacional de Investigaciones Científicas - Técnicas (CONICET), Argentina Send offprint requests to M. C. Camilión de Hurtado at the above address     

Interaction between presynaptic facilitatory angiotensin II receptors and inhibitory muscarinic cholinoceptors on3H-noradrenaline release in the rabbit heart

Summary The existence of a functional interaction between presynaptic receptors modulating the release of noradrenaline was studied in the rabbit heart. Isolated right atria were prelabelled with³H-noradrenaline and the overflow of tritium was induced by field stimulation (2 Hz, 0.1 ms duration, supramaximal voltage for a total of 180 pulses). In atria superfused with Krebs' solution containing 10 mol/l cocaine and 30 mol/l corticosterone, angiotensin II (10 nmol/l) increased the stimulation-evoked overflow of³H-transmitter by 2.8-fold. The addition of atropine (0.3 mol/l) to the perfusion medium, either in the presence or in the absence of uptake inhibitors, further enhanced the facilitatory effect of angiotension II (³H-transmitter release increased by 3.5-fold). Exposure to 1 mol/l carbachol decreased by 65% the stimulation-evoked release of³H-transmitter while the facilitatory effect of angiotensin II determined in the presence of the muscarinic cholinoceptor agonist was enhanced (³H-transmitter release increased by 6.6-fold). Conversely, during sustained activation of presynaptic angiotensin receptors producing a 2.5-fold increase in the release of³H-transmitter, the inhibitory effect of carbachol remained unchanged. These results suggest a functional interaction between presynaptic inhibitory muscarinic cholinoceptors and the presynaptic facilitatory angiotensin receptor which modulate the release of noradrenaline from cardiac noradrenergic nerves.     

Nicotine-induced release of noradrenaline and neuropeptide Y in guinea-pig heart: role of calcium channels and protein kinase C

Summary The role of calcium, calcium influx through calcium channels, and activation of protein kinase C for the nicotine-induced release of noradrenaline and of the sympathetic co-transmitter neuropeptide Y (NPY) was investigated in the guinea-pig isolated perfused heart. In the coronary venous overflow noradrenaline and NPY were determined by high-pressure liquid chromatography and radioimmunoassay, respectively. In the presence of extracellular calcium (1.85 mmol/l) nicotine (1–100 mol/l) evoked a concentration-dependent overflow of both transmitters with a molar ratio of approximately 1500 (noradrenaline):1 (NPY). The nicotine-induced (100 mol/l) overflow of noradrenaline and NPY was in a linear manner related (r = 0.79 and 0.90, respectively; p < 0.05) to the extracellular calcium concentration (0–1.85 mmol/l), and it was prevented by calcium-free perfusion. The L-type calcium channel blocker felodipine (100 nmol/l) did not affect the nicotine-induced (100 mol/l) transmitter overflow. On the other hand, the neuronal (N-type) calcium channel blockers -conotoxin (100 nmol/l) and cadmium chloride (50 mol/l) reduced the nicotine-induced (100 pmol/l) transmitter overflow to 20% of the control value, suggesting a role of N-type calcium channels in mediating the calcium influx for the nicotine-induced transmitter release. The nicotine-induced (30 mol/l) overflow of both transmitters was two- to three-fold increased by activation of protein kinase C (phorbol 12-myristate 13-acetate; 100 nmol/l). The transmitter overflow was unaffected by 4-phorbol 12,13-didecanoate (100 nmol/l), a phorbol ester which does not stimulate protein kinase C. Further supporting a modulatory role of protein kinase C, inhibition of the enzyme by either polymyxin B (100 gmol/I) or by cremophor RH-30 (1mol/l) almost completely suppressed the overflow of noradrenaline and NPY. The results of the present study indicate that nicotine evokes a concentration-dependent exocytotic co-release of noradrenaline and NPY in the guinea-pig isolated perfused heart which is characterized by its dependence on extracellular calcium, calcium influx through N-type calcium channels and activation of protein kinase C.This work was supported by a grant from the Forschungsrat Rauchen und Gesundheit Send of fprint requests to M. Haass at the above address     

Presynaptic β2-adrenoceptors on the sympathetic nerve fibres of the human saphenous vein: no evidence for involvement in adrenaline-mediated positive feedback loop regulating noradrenergic transmission

Summary Spirally cut strips of human saphenous veins preincubated with ³H-noradrenaline were superfused in the presence of corticosterone and, unless stated otherwise, of cocaine or desipramine. Tritium overflow was stimulated electrically (2 Hz). Adrenaline (in the presence of rauwolscine), isoprenaline and the preferential ₂-adrenoceptor agonist procaterol concentration-dependently increased the electrically evoked tritium overflow. Prenalterol, a -adrenoceptor agonist with moderate preference for ₁-adrenoceptors, was ineffective. The concentration-response curve of isoprenaline was shifted to the right by the nonselective -adrenoceptor antagonist propranolol and by the preferential ₂-adrenoceptor antagonist ICI 118,551, but was not affected by the ₁-selective antagonist atenolol. In experiments on strips preexposed to adrenaline 10 nmol/l (i. e. a concentration higher than that which normally occurs in vivo) for 32 min in the absence of cocaine or desipramine, the electrically evoked ³H overflow was not affected 12 and 44 min after withdrawal of adrenaline, irrespective of whether propranolol was absent or present in the superfusion fluid. — In veins incubated with ³H-adrenaline, a considerable amount of the radioactivity was accumulated. During subsequent superfusion with ³H-adrenaline-free solution, electrical stimulation induced tritium overflow in a tetrodotoxin-sensitive manner. Propranolol failed to modify the evoked tritium overflow. — It is concluded that the sympathetic nerve fibres of the human saphenous vein are endowed with facilitatory presynaptic ₂-adrenoceptors. These receptors do not seem to play a substantial role in a local adrenaline (previously taken up)-mediated positive feedback loop regulating noradrenergic transmission, at least under the present in vitro conditions.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert     

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     

3H-noradrenaline release from mouse iris–ciliary body: role of presynaptic muscarinic heteroreceptors

Sympathetic neurotransmitter release and its modulation by presynaptic muscarinic heteroreceptors were studied in mouse iris–ciliary bodies. Tissue preparations were preincubated with ³H-noradrenaline and then superfused and stimulated electrically. Firstly, experimental conditions were defined, allowing study of presynaptic sympathetic inhibition in mouse iris–ciliary body. If tissue was stimulated four times with 36 pulses/3 Hz, tritium overflow peaks were reliably and reproducibly measured. As expected, these stimulation conditions led to marked ₂-autoinhibition as indicated by the release-enhancing effect of the ₂-antagonists phentolamine and rauwolscine. To ensure autoinhibition-free ³H-noradrenaline release, which is optimal for studying presynaptic sympathetic inhibition, ₂-receptors were blocked in all subsequent experiments. Under these conditions, evoked tritium overflow was almost completely abolished in the presence of the sodium channel blocker tetrodotoxin, indicating a neuronal origin of ³H-noradrenaline release. Secondly, muscarinic inhibition of ³H-noradrenaline release was characterized using the conditions described above (36 pulses/3 Hz; phentolamine 1 M and rauwolscine 1 M throughout). The muscarinic receptor agonist oxotremorine M decreased evoked tritium overflow in a concentration-dependent manner with an IC₅₀ of 0.33 M and maximal inhibition of 51%. The concentration–response curve of oxotremorine M was shifted to the right by the muscarinic antagonists ipratropium and methoctramine, whereas pirenzepine was ineffective. The observed rank order of antagonist potencies, ipratropium > methoctramine > pirenzepine, which is typical for the M₂ subtype, indicates that presynaptic muscarinic receptors on sympathetic axons of mouse iris–ciliary bodies are predominantly M₂. Finally, inhibition of ³H-noradrenaline release by endogenously secreted acetylcholine was investigated. Longer pulse trains, 120 pulses/3 Hz and 600 pulses/5 Hz, were used and the cholinesterase inhibitor physostigmine was added to the superfusion medium to increase synaptic levels of endogenous acetylcholine. Under these conditions, ipratropium approximately doubled the evoked overflow of tritium, indicating that endogenously released acetylcholine can activate presynaptic muscarinic heteroreceptors. In conclusion, the present experiments establish measurement of the electrically induced release of ³H-noradrenaline from mouse iris–ciliary bodies. As in other species, noradrenaline release in this preparation was subject to presynaptic muscarinic inhibition. Our results also indicate that the presynaptic muscarinic receptors on sympathetic axons in mouse iris–ciliary body are predominantly M₂. Moreover, these receptors can be activated by both exogenous agonists and endogenously released acetylcholine and, hence, may operate physiologically in the interplay between the parasympathetic and sympathetic nervous system.     

Phorbol ester does not mimic,but antagonizes the alpha-adrenoceptor-mediated positive inotropic effect in the rabbit papillary muscle

Summary The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) was used to examine the hypothesis that phosphoinositide turnover is involved in the regulation of myocardial contractility mediated by stimulation of alpha-adrenoceptors in the mammalian cardiac muscle. Exposure of the isolated rabbit papillary muscle electrically driven at a rate of 1 Hz at a temperature of 37°C to TPA in concentrations of 10–1000 nmol/l for 30 min did not affect the basal force of contraction. The concentration-response curve for the positive inotropic effect of (–)-phenylephrine mediated by stimulation of alpha-adrenoceptors in the presence of (±)-bupranolol (100 nmol/1) was shifted to the right and downward by TPA in concentrations of 30–1000 nmol/l, while the effect of (–)-phenylephrine mediated by stimulation of beta-adrenoceptors in the presence of prazosin (100 nmol/l) was not decreased, but slightly enhanced by exposure of the muscle to relatively low concentrations of TPA (10–100 nmol/l). Incubation of the membrane fraction isolated from the rabbit ventricular muscle with TPA in vitro under the same condition as employed in the physiological experiments decreased the specific binding of [³H]prazosin but not that of [³H]CGP-12177, while the non-tumor promoting phorbol ester, PDD, was ineffective. These results indicate that activation of protein kinase C by TPA does not mimic the positive inotropic effect of catecholamines mediated by activation of myocardial alpha-adrenoceptors. on the other hand, the specific interaction of alpha-adrenoceptor-mediated processes with TPA in the rabbit papillary muscle is in line with the view that the facilitation of phosphoinositide turnover and subsequent activation of protein kinase C may play a certain role in the coupling of alpha-adrenoceptor occupation by agonists to the process leading to the positive inotropic action. Send offprint requests to M. Endoh     

Involvement of adenylate cyclase and protein kinase C in the α2-adrenoceptor-mediated inhibition of noradrenaline and 5-hydroxytryptamine release in rat hypothalamic slices

Summary In superfused rat hypothalamic slices prelabelled with [³H]-noradrenaline, the ₂-adrenoceptor agonist UK 14304 inhibited in a concentration-dependent manner the electrically-evoked release of tritium. This inhibition was antagonized by the ₂-adrenoceptor blocking agent idazoxan, which by itself increased the electrically-evoked tritium overflow. Exposure to forskolin, an adenylate cyclase activator, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of forskolin (1 mol/l), both the inhibitory effect of UK 14304 and the increasing effect of idazoxan on the electrically-evoked release of [³H]-noradrenaline were less pronounced than in the absence of the adenylate cyclase activator. Exposure to forskolin and to the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine shifted to the right the concentration-effect curve for UK 14304 in a similar manner as that observed in the presence of forskolin alone. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l), a drug which activates protein kinase C, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), the concentration effect curve for UK 14304 on tritium overflow was significantly shifted to the right. The increasing effect of idazoxan on tritium overflow was significantly less pronounced in the presence of 1 mol/l phorbol-12,13-dibutyrate.In superfused rat hypothalamic slices prelabelled with [³H]-5-hydroxytryptamine, the ₂-adrenoceptor agonist UK 14304 significantly inhibited the electrically-evoked release of tritium. Exposure to forskolin increased in a concentration-dependent manner [³H]-5-hydroxytryptamine overflow, but did not modify the UK 14304-mediated inhibition. Exposure to 3-isobutyl-1-methylxanthine enhanced the electrically-evoked release of [³H]-5-hydroxytryptamine. In the presence of both forskolin (1 mol/l) and 3-isobutyl-l-methylxanthine (1 mmol/l), the concentration-response curve for UK 14304 was significantly shifted to the right. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l) enhanced in a concentration-dependent manner the electrically-evoked overflow of [³H]-5-hydroxytryptamine. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), UK 14304 was significantly less potent to inhibit tritium release than in the absence of the protein kinase C activator.It is concluded that both cyclic AMP and phosphoinositide turnover are involved in the modulation of noradrenaline and 5-hydroxytryptamine release by presynaptic ₂-adrenoceptors in rat hypothalamic slices. However, these interactions do not represent definitive proof for a cause-effect relationship for the second messengers mediating the ₂-adrenoceptor induced inhibition of transmitter release either as autoreceptor or as heteroreceptor.Send offprint requests to S. Z. Langer at the above address     

Noradrenaline release from rat sympathetic neurons evoked by P2-purinoceptor activation

The effects of ATP and analogues on the release of previously incorporated ³H-noradrenaline were studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Electrical field stimulation (40 mA at 3 Hz) of the neurons for 10 s markedly enhanced the outflow of tritium. ATP applied for 5 s to 2 min at concentrations of 0.01 to 1 mmol/l caused a time- and concentration-dependent overflow with half maximal effects at about 10 s and 100 mol/l, respectively. 2-Methylthio-ATP was equipotent to ATP in inducing ³H-overflow. ADP (100 mol/l), when applied for 2 min, also caused a small ³H-overflow, but , -methylene-ATP (100 mol/l), AMP (100 mol/l), R(–)N⁶-(2-phenylsiopropyl)-adenosine (R(–)-PIA; 10 mol/l) and 5-N-ethylcarboxamidoadenosine (NECA; 1 mol/l) did not. The ³H-overflow induced by 10 s applications of 100 mol/l ATP was abolished by suramin (100 mol/l) and reduced by about 70% by reactive blue 2 (3 mol/l). Electrically evoked overflow, in contrast, was slightly enhanced by suramin, but not modified by reactive blue 2. Xanthine amine congener (10 mol/l) and hexamethonium (10 mol/l) did not alter ATP-evoked release. Removal of extracellular Ca²⁺ from the medium reduced ATP- and electrically induced overflow by about 95%. Tetrodotoxin (1 mol/l) abolished electrically evoked ³H-overflow but inhibited ATP-induced overflow by only 70%. The ₂-adrenoceptor agonist UK 14,304 at a concentration of 1 mol/l diminished both electrically and ATP-evoked tritium overflow by approximately 70%. These results indicate that activation of P₂-purinoceptors stimulates noradrenaline release from rat sympathetic neurons. The release resembles electrically induced transmitter release, but additional mechanisms may contribute. Correspondence to: S. Boehm at the above address     

N-ethylmaleimide (NEM) diminishes α2-adrenoceptor mediated effects on noradrenaline release

Summary The effect of N-ethylmaleimide (NEM), which has been shown to abolish rather selectively inhibition of adenylate cyclase, on the ₂-adrenoceptor modulation of noradrenaline release was studied. Slices of the rabbit hippocampus were loaded with ³H-noradrenaline, superfused continuously and stimulated twice electrically.NEM (30 mol/l) applied for 30 min enhanced both basal and stimulation-evoked tritium overflow significantly. Occupation of the receptor by the ₂-adrenoceptor agonist clonidine prior to and during NEM treatment did not protect the ₂-adrenoceptor-mediated autoinhibitory feedback system from being affected by NEM. Preincubation of the hippocampal slices with NEM was without any influence on ³H-noradrenaline uptake. The inhibitory effect of clonidine on ³H-noradrenaline release was attenuated in a non-competitive manner. In addition, the facilitatory effect of the ₂-adrenoceptor antagonist yohimbine on the stimulusevoked tritium overflow was reduced. The facilitation of the evoked noradrenaline release by yohimbine or yohimbine and NEM converged with increasing concentrations of yohimbine, suggesting that yohimbine and NEM were acting at the same signal-transduction system.These results are compatible with the idea that NEM, by alkylating the N_i-unit of a presynaptically located adenylate cyclase, prevents the ₂-adrenoceptor-mediated modulation of noradrenaline release.Abbreviations NEM N-ethylmaleimide - IAP islet-activating protein     

Modulation of electrically evoked [3H]-noradrenaline release from cultured chick sympathetic neurons

In the present study we attempted a comprehensive characterization of modulation of noradrenaline release from chick sympathetic neurons. To this purpose sympathetic neurons derived from chick lumbosacral paravertebral ganglia and kept in culture for 7 days were loaded with 0.05 mol/l [³H]-noradrenaline and subjected to electrical field stimulation (36 pulses/3 Hz). Since the released transmitter was partially recaptured, superfusion was usually performed in the presence of (+)-oxaprotiline, an inhibitor of noradrenaline re-uptake. [³H]-Noradrenaline was released in a manner which was dependent on extracellular Ca²⁺ and sensitive to tetrodotoxin (TTX). -Conotoxin (-CTX; 100 nmol/l) abolished [³H]-noradrenaline release indicating that influx through -CTX-sensitive Ca²⁺-channels was essential for transmitter release. 1,4-dihydro-2,6-dimethyl-5-nitro4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester ((±)Bay K 8644) and 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylic acid isopropyl ester ((±)-202-791), agonists at L-type voltage sensitive Ca²⁺-channels (VSCCs), increased noradrenaline release and induced, in addition, an overflow of tritium which was Ca²⁺-dependent and prevented by the presence of TTX. The L-type VSCC antagonists (–)-202-791 and (+)-4-(4-benzofurazanyl)-1,4-dihydro2,6-dimethyl-3,5-pyridinedicar boxylic acid methyl, isopropyl ester ((+)-PN 200–110) diminished [³H]-noradrenaline release. These data suggest that L-type VSCCs, probably located on the cell body of the neuron, play an additional role in modulation of release. The full ₂-adrenoceptor agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline ( UK-14,304) and noradrenaline significantly inhibited noradrenaline release, whereas clonidine, a partial a2-agonist, produced only a slight inhibition even at 10 mol/l. The facilitation of noradrenaline release observed in the presence of the ₂-adrenoceptor antagonist rauwolscine was very low in comparison to that obtained with brain slices and isolated smooth muscle tissues. These results corroborate the observation that noradrenaline release from chick sympathetic neurons is regulated by an ₂-adrenoceptor which needs further subtype characterization. The experiments were mostly performed at 25°C, since a rise in temperature to 37°C increased the resting outflow, but not the evoked overflow of tritium, approximately 4-fold. In the presence of pargyline to block monoamine oxidase, however, the temperature-dependent enhancement was diminshed and the release showed properties comparable to those observed at 25°C (with respect to TTX-sensitivity, Ca²⁺ dependence and modulation via ₂-adrenoceptors). In addition to the ₂-adrenoceptors, we detected inhibitory -adrenoceptors, opioid and receptors, and P₂ purinoceptors as well as facilitatory prostaglandin (PG) E receptors. No indication was found for a functional relevance of 5-hydroxytryptamine (5-HT), opioid , PGD, adenosine A₁ or glutamate receptors. In conclusion, electrically evoked noradrenaline release from cultured chick sympathetic neurons shows the properties of action-potential-induced transmitter release and is bidirectionally regulated by various substances. Therefore, sympathetic neurons in culture offer the possibility to investigate directly the mechanisms bringing about receptor-coupled modulation of transmitter release.Abbreviations ATP adenosine 5-triphosphate - Bay K 8644 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester - DAGO (d-Ala²,N-methyl-Phe⁴,Gly-ol⁵)-enkephalin - DPDPE (d-Pen ^2,5)-enkephalin - 5-HT 5-hydroxytryptamine - -CTX -conotoxin - KRBB modified Krebs-Ringer bicarbonate buffer - NMDA N-methyl-d-aspartic acid - PG prostaglandin - PN 200-110 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxy lic acid methyl, isopropyl ester - R-PIA R(–)-N⁶-(2-phenyl-isopropyl)-adenosine - TTX tetrodotoxin - U-50,488H trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzene acetamide - UK-14,304 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline - VSCC voltage sensitive Ca²⁺-channel - 202-791 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylic acid isopropyl ester Correspondence to: C. Allgaier at the above address     

Noradrenaline release from slices of the thalamus of normal and morphine-dependent rats

Summary The effect of morphine on potassium-induced stimulation of (³H)-noradrenaline release from slices of the rat thalamus was investigated. The in vitro addition of morphine (10^–6 M) significantly depressed potassium-induced tritium overflow by 42% and this was prevented by the prior addition of naloxone (3×10^–6 M) to the medium. The stimulation-evoked overflow of tritium from slices of the thalamus of morphine-dependent rats was not significantly different from normal controls. Addition of naloxone (10^–5 M) 10 min before exposure of the tissues to 20 mM K⁺ significantly enhanced noradrenaline release from dependent slices. The results suggest that the basic release mechanism may have adapted to the continuous presynaptic inhibition of release by morphine.     

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     

Adenosine but not an adenine nucleotide mediates tonic purinergic inhibition,as well as inhibition by glutamate,of noradrenaline release in rabbit brain cortex slices

Summary A possible contribution of adenine nucleotides to the endogenous purinergic, A₁-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [³H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A₁-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1–100 nmol/l) as well as the enzyme adenosine deaminase (0.1–10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The ₁-adrenoceptor-selective agonist methoxamine (10 but not 1 mol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mol/l but then not attenuated further by DPCPX100 nmol/l.L-Glutamate (300 mol/l–2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mol/l but greatly, and to the same extent, attenuated by DPCPX 100 mol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg⁺⁺ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mol/l and 1 mol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg⁺⁺). It is concluded that adenosine but not an adenine nucleotide mediates the tonic purinergic presynaptic inhibition of noradrenaline release in rabbit brain cortex. The marked degree of disinhibition by DPCPX and adenosine deaminase underscores the potential physiological role of this inhibition. The purinergic inhibitory tone is reinforced by glutamate, indicating that glutamate releases adenyl compounds in rabbit brain cortex. Again adenosine but not an adenine nucleotide mediates the indirect inhibition by glutamate of the release of noradrenaline. The noradrenaline-releasing effect that glutamate exerts in rat occipito-parietal cortex does not occur in rabbit occipito-parietal cortex. Methoxamine depresses the release of noradrenaline in rabbit brain cortex directly at presynaptic ₂-adrenoceptors rather than by release of purines.Correspondence to I. von Kügelgen at the above address