Characterization and presynaptic modulation of stimulation-evoked exocytotic co-release of noradrenaline and neuropeptide Y in guinea pig heart

Summary The relationship between noradrenaline and neuropeptide Y (NPY) release was investigated in the in situ perfused guinea pig heart with intact sympathetic innervation. For determination of NPY concentrations in the perfusate, a specific radioimmunoassay was employed and further characterized. Electrical stimulation of the left stellate ganglion (4, 8, 12, and 50 Hz; for 10 min) evoked a calcium-dependent and frequency-related overflow of noradrenaline and NPY, which was positively correlated (r = 0.83; p < 0.001; n = 25). When two subsequent stimulations (12 Hz; each for 1 min) were performed in the same heart, addition of noradrenaline (10 M) 5 min prior to the second stimulation reduced NPY overflow by 43 ± 10%. The stimulated release of noradrenaline and NPY was increased by the alpha₂-adrenoceptor antagonist yohimbine (1 M) to 170 ± 10% and 199 ± 26%, and attenuated by the alpha₂-adrenoceptor agonist B-HT 920 (1 M) to 70 ± 9% and 68 ± 9%, respectively. The adenosine analogue cyclohexyladenosine (1 M) significantly reduced the stimulated overflow of both noradrenaline (to 57 ± 5%) and NPY (to 73 ± 8%). Exogenous NPY (100 nM) attenuated the stimulated overflow of noradrenaline by 30 ± 6%. Uptake₁ blockade with desipramine (100 nM) or nisoxetine (100 nM) prior to the second stimulation significantly increased noradrenaline overflow and attenuated that of NPY; the attenuation of the stimulation-evoked overflow of NPY was abolished by yohimbine (1 M).Our results indicate that electrical stimulation induces a calcium-dependent, exocytotic co-release of noradrenaline and NPY. The co-release of both transmitters is regulated by presynaptic receptors in a parallel manner; furthermore, both transmitters, noradrenaline and possibly NPY, modulate their own release by a presynaptic negative feedback mechanism via presynaptic alpha₂-adrenoceptors and NPY-receptors.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 320 - Herzfunktion und ihre Regulation). Presented in part at the 61st Scientific Sessions of the American Heart Association. Washington DC, November 1988 Send offprint requests to M. Haass at the above address     

Inhibition by interleukin-1\ of noradrenaline release in rat spleen: involvement of lymphocytes,NO and opioid receptors

Effects of indomethacin, N-nitro-L-arginine (NNA) and naloxone, and of pretreatment with cyclophosphamide (CY), on the interleukin (IL)-I\ induced inhibition of exocytotic noradrenaline release were investigated in the isolated, vascularly perfused spleen of the rat. Neurotransmitter release was evoked by perivascular electrical stimulation (4 Hz) and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection.Perfusion of the spleen with Tyrode's solution containing IL-1\ (100 pg/ml) for 90 min caused an inhibition of the stimulation-evoked noradrenaline overflow which persisted for at least 20 min after washout of the IL. The evoked overflow was reduced in the presence of NNA 30 mol/l, but remained unaffected by indomethacin 3 mol/l, naloxone 0.1 mol/l or treatment of the rats with CY (250 mg/kg). The opioid agonist etorphine 10 mol/1 inhibited the evoked overflow of noradrenaline and this effect was prevented by naloxone 0.1 mol/1. The inhibition of evoked overflow by IL-1\ was not affected by indomethacin but was reduced or even prevented in the presence of NNA or naloxone, or after lymphocyte depletion of spleens by CY.The results are compatible with the idea that in the rat spleen exocytotic noradrenaline release is accompanied by a concomitant secretion of a nitric oxide (NO)-like compound which, in turn, reinforces noradrenaline release, and that the release can be inhibited via prejunctional opioid receptors. The IL-1\ induced inhibition of evoked release appears to be a complex process which involves as one of many steps a decrease of the facilitatory NO-like compound and the release of endogenous opioids probably from spleen lymphocytes.     

Inhibition by the putative potassium channel opener pinacidil of the electrically-evoked release of endogenous dopamine and noradrenaline in the rat vas deferens

Summary The effect of pinacidil on the release of endogenous noradrenaline and dopamine from the sympathetic innervation of the rat vas deferens was examined. Amine release was evoked by electrical stimulation (1, 2, 5 and 10 Hz) or by depolarization with high potassium (75 mmol/l) in the medium. Dopamine and noradrenaline were measured by means of high pressure liquid chromatography with electrochemical detection.Pinacidil (1, 5, 10 and 50 mol/l) produced a concentration-dependent inhibition of the electrically stimulated (2 Hz) overflow of noradrenaline and dopamine. Only pinacidil 50 mol/l increased the spontaneous loss of dopamine and noradrenaline. The inhibitory effects of pinacidil (5 mol/l) on amine overflow were also observed at other frequencies of stimulation (1, 5 and 10 Hz). The magnitude of the inhibitory effect on noradrenaline release was approximately the same at all frequencies (63% to 56% reduction); for dopamine, the higher the frequency of stimulation, the greater the inhibitory effect of pinacidil (up to 73% reduction). When the preparations were continuously stimulated for 70 min at 2 Hz, pinacidil (5 mol/l) reduced the overflow of dopamine and noradrenaline during the first 40 or 30 min of stimulation only. The addition of phentolamine (1 mol/l) to the perifusion medium slightly reduced the inhibitory effect of pinacidil on amine overflow, but the inhibition by pinacidil remained statistically significant. Tetraethylammonium (10 mmol/l) completely abolished the inhibitory effect of pinacidil (10 mol/l). Pinacidil (5 mol/l) did not reduce the potassium-evoked release of the amines.The results demonstrate that pinacidil impairs transmitter release from the sympathetic innervation of the rat vas deferens, probably as a consequence of the opening of potassium channels. Send offprint request to P. Soares-da-Silva at the above adress     

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     

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     

Metabolism of endogenous and exogenous noradrenaline in the rabbit perfused heart

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from rabbit perfused hearts was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs and without nerve stimulation, the outflow of endogenous noradrenaline over a period of 108 min averaged 0.17 pmol×g^–1×min^–1 and the outflow of DOPEG 2.1 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.13 pmol×g^–1×min^–1). The effect of perfusion with (–)-noradrenaline 0.1, 1 or 10 mol/l for 18 min was then investigated. As the concentration of noradrenaline increased so did the outflow of DOPEG. Moreover, DOMA was found in the venous effluent during and after perfusion with noradrenaline 1 or 10 mol/l. The increase in the outflow of DOPEG and DOMA was almost abolished when cocaine 10 mol/l was present during the perfusion with noradrenaline 1 mol/l. The release of endogenous noradrenaline by sympathetic nerve stimulation or tyramine 10 mol/l, but not the release evoked by nicotine 30 mol/l, was accompanied by an increase in the outflow of DOPEG; an outflow of DOMA was not observed.It is concluded that, in the rabbit perfused heart, DOPEG is an important metabolite of endogenous noradrenaline. DOMA is at best a minor product, either when the neurones are at rest or when noradrenaline is released by sympathetic nerve stimulation, nicotine or tyramine. DOMA is formed in detectable amounts when the tissue is exposed to a high concentration of exogenous noradrenaline. Like DOPEG, it is formed intraneuronally. The results confirm and extend those obtained previously on guinea-pig incubated atria. They make it unlikely that, in these tissues at least, DOMA formation is one of the physiological pathways of noradrenaline catabolism.     

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.     

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.     

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     

Kainate receptors are involved in the glutamate-induced indirect,purinergic inhibition of [3H]-noradrenaline release in rabbit brain cortex

Activation of ionotropic but not of metabotropic glutamate receptors causes an indirect inhibition of the release of noradrenaline in slices of rabbit brain cortex. The inhibition is mediated by adenosine which activates presynaptic adenosine A₁-receptors. The present study characterizes the ionotropic receptor types through which glutamate itself produces this indirect inhibition. Rabbit brain cortex slices were preincubated with [³H]-noradrenaline, superfused with medium containing desipramine (1 M) and stimulated electrically by trains of 6 pulses at 100 Hz.Glutamate (100–3000 M) reduced the electrically evoked overflow of tritium by up to 58 %. The effect did not differ 20 min and 60 min after addition of glutamate. Adenosine deaminase (1 U ml^-1) as well as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 30 M) and d--glutamylamino-methanesulfonate (GAMS; 30 M), both of which block kainate receptors, attenuated the glutamate-induced inhibition. The NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5; 100 M) and the AMPA receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX; 30 M) did not change the effect of glutamate. Given alone, CNQX and GAMS, but not AP5 and NBQX, slightly increased the evoked overflow of tritium; the increases were abolished in the presence of adenosine deaminase.The results indicate that activation of kainate but not NMDA and AMPA receptors is involved in the indirect, adenosine-mediated inhibition by exogenous glutamate of the release of noradrenaline in rabbit brain cortex slices. Moreover, as shown by the increase caused by CNQX and GAMS, endogenous excitatory amino acids inhibit the release of noradrenaline through the kainate receptor-adenosine mechanism and thus contribute to the purinergic inhibitory control of noradrenaline release in the brain.     

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.     

Cocaine-sensitive O-methylation of noradrenaline in dental pulp of the rabbit: Comparison with the rabbit ear artery

Summary Incisor pulp from the rabbit metabolises exogenous noradrenaline in concentrations between 0.12 and 1.2 mol/l mainly to NMN.Effects of chronic sympathetic denervation indicated that in incisor pulp the NMN is extraneuronal in origin, and that DOPEG and DOMA formation, as well as a major part of the noradrenaline which accumulates in the tissue, are associated with the sympathetic nerves.NMN formation was unaffected by hydrocortisone 210 mol/l, but was strongly inhibited by cocaine 30 mol/l. These effects contrasted with those in the rabbit ear artery, where NMN formation was increased by cocaine 30 mol/l and decreased by hydrocortisone 210 mol/l.In COMT-inhibited denervated pulp, cocaine inhibited the accumulation of noradrenaline.Monoamine fluorescence histochemistry of pulp exposed to noradrenaline 50 mol/l indicated that cocaine-sensitive uptake occurred in fibroblasts.It is concluded that O-methylation of noradrenaline in dental pulp involves prior uptake of the amine by a process resembling uptake, but which is distinguished from uptake₁ by its extraneuronal location.Abbreviations DOMA 3,4-dihydroxy mandelic acid - DOPEG 3,4-dihydroxyphenylethyleneglycol - NMN normetanephrine - OMDA O-methyl deaminated metabolite fraction, comprising vanillyl-mandelic acid (VMA) plus the 3-methoxy derivative of DOPEG (MOPEG) - MAO monoamine oxidase - COMT catecholO-methyl transferase Send offprint requests to I. S. de la Lande at the above address     

Depression by neuropeptide Y of noradrenergic inhibitory postsynaptic potentials of locus coeruleus neurones

Summary Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus (LC). The spontaneous firing of action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode. Focal electrical stimulation evoked a synaptic depolarization (PSP) followed by a hyperpolarization (IPSP). Neuropeptide Y (NPY; 0.1 mol/l) inhibited the IPSP only. Pressure ejection of noradrenaline produced hyperpolarization which was potentiated in the presence of NPY (0.1 mol/l). Hence, NPY appears to inhibit the release of noradrenaline from dendrites or recurrent axon collaterals of LC neurones. Correspondence to: P. Illes at the above address     

Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [³H]-noradrenaline was investigated.Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline (0.05 M), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 LM-3 mM), 2-methylthio-ATP (3–100 M), as well as DMPP (10 and 100 M) induced a significant overflow of tritium. The EC₅₀-value of ATP was 20 M. Both the ATP-induced and the DMPP-induced tritium overflow was Ca²⁺-dependent and sensitive to tetrodotoxin (0.3 M) and -conotoxin (0.1 M); in addition, it was inhibited by the ₂-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 M). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P₂-purinoceptor antagonists suramin (300 M) and reactive blue 2 (3 M); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 M) and markedly inhibited by hexamethonium (100 M). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [³H]-choline.The results suggest that ATP at molar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.     

The effects of quinpirole and fenoldopam on the potassium-evoked overflow of endogenous dopamine and noradrenaline in dog mesenteric arteries

Summary (1) Dopamine and noradrenaline overflow from the main trunk of the dog mesenteric artery and its proximal branches, elicited by K⁺ (52 mmol/l), was measured by high pressure liquid chromatography with electrochemical detection. (2) Quinpirole (0.1, 1 and 10 nmol/l) produced a concentration dependent reduction of dopamine and noradrenaline overflow in both segments of the mesenteric artery. The inhibitory effect of quinpirole (10 nmol/l) on amine overflow was antagonized by sulpiride (1 mol/l) but not by phentolamine (0.2 mol/l) or the selective dopamine (DA₁), antagonist SK&F 83566 (1 ol/l). (3) Fenoldopam (0.1 and 1 mol/l) did not alter dopamine and noradrenaline overflow from both segments of the mesenteric artery; only 10 mol/l fenoldopam was found to increase the overflow of dopamine and noradrenaline in both segments of the mesenteric artery. This effect of fenoldopam on amine overflow was not altered by the addition to the perifusion fluid of SK&F 83566 (1 ol/l). (4) Clonidine (100 nmol/l) significantly reduced amine overflow from both segments of the mesenteric artery and this effect was antagonized by fenoldopam (10 mol/l) (5) These results suggest that quinpirole inhibits sympathetic neurotransmission through the activation of prejunctional dopamine receptors of the DA₂ subtype. The facilitatory effect of fenoldopam (10 mol/l) on amine release appears to be mediated through the blockade of prejunctional ₂-adrenoceptors. Send offprint requests to P. Soares-da-Silva 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     

Releasing activities of d-fenfluramine and fluoxetine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin

Summary Rat hippocampal synaptosomes preloaded with [³H]serotonin and maintained in a superfusion apparatus were exposed for 3 min to d-fenfluramine or fluoxetine. Both drugs evoked a tritium overflow which was reserpine-sensitive requiring the presence of intact synaptic vesicles. However the two drugs displayed different characteristics: 1) the overflow was immediate with dfenfluramine whereas the releasing activity of fluoxetine showed a delay of about 2 min; 2) d-fenfluramine-induced overflow was already apparent at 0.15 mol/l whereas the minimal effective concentration of fluoxetine was 2.5 mol/l. Their concentration-effect curves were differently shaped, the effect of d-fenfluramine being saturable at 5–20 mol/l (EC₅₀ about 1 gmol/l) while no saturation was observed with fluoxetine up to 10 mol/l; 3) only 1907o of the tritium overflow evoked by fluoxetine (2.5–10 mol/l) consisted of true [³H]serotonin, compared with 7001o when 0.5 mol/l d-fenfluramine was used; 4) the releasing action of 0.5 mol/l d-fenfluramine was completely Ca⁺⁺-dependent, while at higher dfenfluramine concentrations the Ca⁺⁺-independent overflow became more important. The fluoxetine induced overflow was mainly. (70010) Ca⁺⁺-independent; 5) the releasing acitvity of d-fenfluramine was mainly (80%) blocked by the serotonin uptake blockers indalpine, midalcipram and also fluoxetine whereas fluoxetine-induced overflow was insensitive to inhibition of the serotonin carrier.In conclusion, the releasing activity of d-fenfluramine is already present at a very low concentration (0.5 mol/l) and at this concentration its mechanism of action was Ca⁺⁺-dependent, together with the requirement of a functional serotonin carrier. These data therefore do not support the hypothesis of a simple. displacement of 5-HT from its storage vesicles but suggest an exocytotic release possibly triggered by interaction of d-fenfluramine with intracellular receptors. A direct releasing activity is also shown for fluoxetine, very marked at 5–10 mol/l; such effect is different from that of d-fenfluramine and is probably due to the overflow of 5-hydroxyindoleacetic acid, formed in the synaptosomes after the fluoxetine-induced displacement of serotonin from its storage vesicles. The active concentrations of fluoxetine on serotonin release are compatible with those found in rat brain at doses inducing an anorectic activity. Send offprint requests to M. Gobbi at the above address     

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 calcium channel activator,bay K 8644, enhances K+-evoked efflux of acetylcholine and noradrenaline from rat brain slices

Summary Slices of rat brain were incubated with either (³H) choline (hippocampus) or (³) noradrenaline (hypothalamus) and superfused with Krebs buffer. The release of (³H) acetylcholine and (³H) noradrenaline after inhibition of monoamine oxidase by pargyline was induced by a short exposure to Krebs buffer containing elevated K⁺ ions (25 mmol/l). Nifedepine (1 mol/l) caused only a slight inhibition of noradrenaline efflux and was without effect on acetylcholine overflow. The calcium channel activator, Bay K 8644 (0.1–1 mol/l), increased the K⁺-evoked efflux of both neurotransmitters. The additional efflux evoked by Bay K 8644 (0.3 mol/l) was blocked by nifedipine (1 mol/l). The results from the present study thus extend the earlier findings with the neurotransmitter 5-hydroxytryptamine to include noradrenaline and acetylcholine. The functional correlates for voltage operated calcium channels concerned with transmitter release are clearly a widespread phenomenon in the CNS.     

Differences in the metabolic fate of noradrenaline released by electrical stimulation or by tyramine

Summary Strips of canine saphenous vein were loaded with ³H-noradrenaline (1.4 M) and perifused with Krebs solution and either subjected to field stimulation or exposed to tyramine 40 M. ³H, noradrenaline and its metabolites were determined in the perifusion fluid. Stimulation caused an increase predominantly in noradrenaline, followed by DOPEG, whereas tyramine released DOPEG in larger amounts than noradrenaline. Tyramine had more sustained effects than stimulation. Cocaine (1.6 M) drastically reduced DOPEG efflux due to stimulation, but had no effects on the pattern of release by tyramine. It is concluded that tyramine releases noradrenaline which is deaminated before it reaches the synaptic gap, whereas after stimulation deamination of the transmitter occurs after re-uptake.Results presented in part to the 8th Annual Meeting of the Portuguese Pharmacological Society (Coimbra, December 1977) and to the 3rd International Symposium on Vascular Neuroeffector Mechanisms (Louvain-Antwerpen, July 24–26, 1978). This work was supported by a grant from Instituto Nacional de Investigação Científica (FmPl)