Facilitatory and inhibitory modulation by endogenous adenosine of noradrenaline release in the epididymal portion of rat vas deferens

Summary The present study aimed at determining the modulation by adenosine of the release of noradrenaline in the epididymal portion of the rat vas deferens. The tissues were treated with pargyline and perifused in the presence of desipramine and yohimbine. Up to four periods of electrical stimulation were applied (5 Hz, 9 min).The A₁-adenosine receptor selective agonist R-N⁶-phenylisopropyladenosine (R-PIA; 100–900 nmol·l^–1) reduced, whereas the A_2A-receptor selective agonist 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine (CGS21680; 3–30nmol·l^–1) increased the electrically-evoked noradrenaline overflow in a concentration-dependent manner. The nonselective agonist 5-N-ethy1carboxamidoadenosine (NECA; 30–300 nmol·l^–1) reduced noradrenaline overflow, but the effect did not depend on the concentration. Adenosine deaminase at the concentration of 0.5 ·ml^–1 decreased but at that of 2.0 ·ml^–1 increased noradrenaline overflow. The inhibitors of adenosine uptake, S-(4-nitrobenzyl)-6-thioinosine (NBTI; 50 nmol·l^–1) and dipyridamole (3 mol·l^–1), increased the electrically-evoked noradrenaline overflow. The A₁-adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 20 nmol·l^–1) caused an increase whereas the A₂-adenosine receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX; 0.1 mol·l^–1) caused a decrease. NBTI (50 nmol·l^–1), partially antagonized the effect of both DPCPX (20 nmol·l^–1) and DMPX (0.1 mol·l^–1).It is concluded that, in the epididymal portion of the rat vas deferens, endogenous adenosine tonically modulates the release of noradrenaline evoked by electrical stimulation, through activation of both inhibitory (A₁) and facilitatory (A_2A) adenosine receptors.Abbreviations CGS 21680 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine - DMPX 3,7-dimethyl-l-(2-propynyl)xanthine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - NBTI S-(4-nitrobenzyl)-6-thioinosine - NECA 5-N-ethylcarboxamidoadenosine - R-PIA R-N⁶-phenylisopropyladenosine Correspondence to J. Gongalves at the above address     

Ca2+-dependent release of endogenous GABA from rat cortical slices from different pools by different stimulation conditions

Summary The previously reported inhibitory effect of (–)-baclofen on the electrically evoked release of endogenous GABA from rat brain slices indicated the possibility of existence of GABA_B autoreceptors. In this study, we have tested an alternative explanation, i. e. the possibility that (–)-baclofen reduced an excitatory glutamatergic input to GABAergic neurons by inhibiting glutamate release, by investigating the interaction of 10 mmol/1 l-glutamate with the inhibitory effect of 10 mol/1(–)-baclofen. l-Glutamate did not affect the electrically evoked release of GABA on its own and did not abolish the effect of (–)-baclofen, suggesting that the latter was not secondary to a reduction of glutamate release. On the other hand, it greatly increased the basal release of GABA and more than doubled the GABA content of the slices at the end of the perfusion, indicating a marked enhancement of GABA synthesis. This additional GABA, apparently formed from exogenous l-glutamate, was not releasable by electrical stimulation at 0.5 or 24 Hz, but at least in part by stimulation with 30 mmol/l K⁺. The previously reported increase of GABA release at 12 Hz as compared to 4 Hz was studied in more detail. GABA released by electrical stimulation at 8–48 Hz was Ca²⁺-dependent and tetrodotoxin-sensitive. No evidence was obtained for a decrease of the amount of GABA released per impulse with increasing frequency in this range. Moreover, neither (–)-baclofen nor muscimol at 10 mol/l altered the release of the amino acid at 24 Hz; the former was also tested at a low Ca²⁺ concentration (0.3 mmol/l) and found to be inactive under these conditions. Thirty mmol/l K⁺ released about 30% higher amounts of GABA than electrical stimulation at 24 Hz under comparable conditions, in a Ca²⁺-dependent manner. K⁺-induced release was not modified by 10 mol/l (–)-baclofen or muscimol. Our results suggest the existence of at least 2 different, presumably neuronally located, releasable pools of GABA. One is sensitive to electrical stimulation at 0.25–4 Hz and responds to (–)-baclofen, suggesting control by GABA_B-type autoreceptors. The existence of a 2nd pool is indicated by the fact that K⁺ releases substantially more GABA than electrical stimulation and by the exclusive sensitivity of K⁺-evoked GABA release to exogenous l-glutamate. GABA released by electrical stimulation at frequencies above 4 Hz may come from a 3rd pool. Both the 2nd and the 3rd pool seem to be insensitive to (–)-baclofen and muscimol. Send offprint requests to P. C. Waldmeier at the above address     

Postnatal development of vascular β-adrenoceptor-mediated responses and the increase in the adrenaline content of the adrenal gland have a parallel time course

The present study was undertaken to analyse the relationship between postnatal development of vascular ₂-adrenoceptor-mediated responses and the content of adrenaline in the adrenal gland and its concentration in plasma. Dog saphenous vein tissue from newborn, two-weeks old and adult animals were either preloaded with ³H-noradrenaline (or ³H-adrenaline) to study prejunctional -adrenoceptor-mediated effects or mounted in organ baths to determine isoprenaline-induced relaxation of preparations contracted by phenylephrine to about 65010 of the maximum. The adrenal glands and samples of blood from the same animals were taken for estimation of adrenaline and noradrenaline.At birth, there were no -adrenoceptor-mediated effects pre- or postjunctionally. At two weeks, while the results at the prejunctional level were not significantly different from those obtained in newborns, at the postjunctional level there was a relaxant response to isoprenaline, which antagonised about 35010 of the previous contraction to 1.75 mol·l^–1 phenylephrine. In adults, isoprenaline (50 nmol·l^–1) increased by 24% tritium overflow evoked by electrical stimulation of tissues preloaded with ³H-noradrenaline but not that of tissues preloaded with ³H-adrenaline. On the other hand, propranolol (1 mol·l^–1) reduced by 21% the overflow of tritium evoked by electrical stimulation of tissues preloaded with ³H-adrenaline but not that of tissues preloaded with ³H-noradrenaline; postjunctionally, the maximal response to isoprenaline antagonised 70% of the previous contraction to 1.75 mol·l^–1 phenylephrine.At birth the catecholamine content of the adrenals was relatively low (2.9 ol·g^–1) and the adrenaline/noradrenaline ratio was 0.26; two weeks later, the catecholamine content was 14.5 mol·g-1and the adrenaline/noradrenaline ratio was 0.74; in adults, the catecholamine content was 24.5 mol·g^–1 and the adrenaline/noradrenaline ratio was 2.3. In plasma, the highest concentration of adrenaline was observed at birth (11.8 nmol·l^–1); two weeks later it was 5.5 nmol·l^–1 and in adulthood it fell to 3.1 nmol·l^–1.On the basis of these results, it is concluded that some link between the postnatal increase in adrenaline adrenal content and the development of ₂-adrenoceptor-mediated pre- and postjunctional effects may exist. Additionally it is suggested that circulating adrenaline may trigger the development of ₂-adrenoceptor-mediated responses as well as some hypertensive states occurring as a consequence of an overreactivity of the sympathoadrenal system. Correspondence to: S. Guimarães at the above address     

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     

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address     

Increase by NO synthase inhibitors of acetylcholine release from guinea-pig myenteric plexus

The effects of nitric oxide (NO) synthase inhibitors on the electrically evoked release of [³H]acetylcho-line were studied in guinea-pig myenteric plexus preparations preincubated with [³H]choline. N^G-monomethyl-l-arginine (EC₅₀ 5.3 mol l^–1) and N^G-nitro-l-arginine (EC₅₀ 1.3 mol l^–1) concentration-dependently increased the evoked release of [³H]acetylcholine without affecting the basal outflow. The facilitatory effect of N^G-monomethyl-l-arginine was prevented by l-arginine but not by d-arginine. The results suggest that endogenous NO inhibits the depolarisation-evoked release of acetylcholine. Correspondence to: H. Kilbinger at the above address     

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.     

Role of nitric oxide formation in the regulation of haemodynamics and the release of noradrenaline and adrenaline

Summary This study in the anaesthetized rabbit aimed at determining the role of nitric oxide (NO), the putative endothelium-derived relaxing factor, in the regulation of haemodynamics and the release into plasma of noradrenaline and adrenaline. Specific inhibition of NO formation was achieved by i.v. bolus injection of l-N^G-monomethyl-arginine (l-NMMA; 3–100 mg kg^–1). Phenylephrine was infused i.v. at constant rates (2.5–20 g kg^–1 min^–1) in order to assess baroreflex-mediated changes in release due to direct peripheral vasoconstriction. Rates of noradrenaline and adrenaline release into plasma were determined by the radio-tracer technique. l-NMMA, but not d-NMMA, dose-dependently increased mean arterial pressure and total peripheral vasular resistance, whereas both heart rate and cardiac output decreased concomitantly. The corresponding ED₅₀ values for l-NMMA ranged from 11.2 to 18.5 mg kg^–1. Inhibition of NO formation by l-NMMA as well as phenylephrine infusion caused decreases in the plasma clearance of noradrenaline and adrenaline which were correlated with the drug-induced decreases in cardiac output. Both l-NMMA and phenylephrine reduced the rate of noradrenaline release into plasma as they increased total peripheral resistance. Moreover, the curvilinear relationship between these two parameters obtained for l-NMMA was virtually identical to that produced by phenylephrine, indicating that the reduction in noradrenaline release by l-NMMA is mediated solely by the baroreflex. From the l-NMMA-induced maximum inhibition of noradrenaline release, it is concluded that the counter-regulation against peripheral vasodilation by NO accounts for 69% of basal noradrenaline release. The baroreflex-sensitive component of noradrenaline release, as determined by the maximum inhibition of release induced by phenylephrine, amounted to 83% of basal release. l-NMMA also reduced the release into plasma of adrenaline; the maximum inhibition of release was 52%. However, when related to total peripheral resistance, this inhibition of adrenaline release was more pronounced than that induced by phenylephrine, suggesting that the formation of endogenous NO facilitates the release of adrenaline.This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5-3). A preliminary account of the present results was presented to the German Pharmacological Society (Halbrügge and Lütsch 1991) Send offprint requests to T. Halbrügge at the above address     

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     

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.     

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     

Differential effects of electrical stimulation,blockade of neuronal amine uptake and activation of α2-adrenoceptors on the release of endogenous noradrenaline and 5-hydroxytryptamine from the isolated rat pineal gland

Summary Isolated rat pineal glands were incubated in vitro and the release of endogenous noradrenaline or 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was determined by HPLC with electrochemical detection. In the absence of test drugs, the spontaneous outflow of noradrenaline was about 10 fmol/10 min and electrical stimulation (5 Hz, 1500 pulses) evoked the release of about 70 fmol noradrenaline. Nomifensine enhanced the spontaneous outflow of noradrenaline about threefold and the electrically evoked release of noradrenaline about sixfold. In the presence of nomifensine, the ₂-adrenoceptor antagonist yohimbine markedly increased the electrically evoked release of noradrenaline, whereas the ₁-adrenoceptor antagonist prazosin had no effect. Clonidine inhibited the electrically evoked release of noradrenaline by about 65%, and this was antagonized by yohimbine in a competitive manner. In the absence of drugs, the initial spontaneous outflow of 5-HT was (compared with noradrenaline) very high 64 mol/10 min. It declined by 80% within 1 h of incubation in vitro. The outflow of 5-HIAA amounted initially to 38 mol/10 min and declined by 40% within 1 h of incubation. Addition of l-tryptophan (10 mol/1) after 1 h of incubation in vitro largely enhanced the outflow of 5-HT and 5-HIAA within 30 min of incubation (about ten- and fourfold, respectively). When l-tryptophan was present from the onset of incubation the initial outflow of 5-HT and 5-HIAA was only slightly elevated, but the decline was largely attenuated. Neither omission of calcium nor addition of nomifensine, clonidine or yohimbine significantly affected the spontaneous outflow of 5-HT or 5-HIAA. Likewise, neither electrical stimulation in the absence or presence of nomifensine and yohimbine nor stimulation by high potassium (45 mmol/1) significantly affected the outflow of 5-HT or 5-HIAA.In conclusion, the release of endogenous noradrenaline from the sympathetic nerves terminating in the pin eal gland is inhibited by presynaptic ₂-adrenoceptors. The outflow of 5-HT from the pineal gland originates almost exclusively from non-neuronal cells, most probably the pinealocytes, and depends largely on a continuous de novo synthesis. Catabolism of 5-HT to 5-HIAA in the pineal gland occurs mainly in an extraneuronal compartment, probably the pinealocytes and/or the interstitial cells of the pineal gland. Send offprint requests to K. Racké at the above address     

Functional relation between nitric oxide and noradrenaline for the modulation of vascular tone in rat mesenteric vasculature

As previously reported, N-nitro-l-arginine (l-NNA), an inhibitor of nitric oxide (NO) synthesis, decreased transmural field stimulation (TFS)-induced noradrenaline overflow from the isolated perfused rat mesenteric vasculature attached to the intestine. The decrease was attenuated by l-arginine. This suggests that NO may increase noradrenaline release (Yamamoto et al. 1993).The present experiments with this preparation were done in order to monitor changes in vascular perfusion pressure caused by TFS or by noradrenaline infusion in parallel with those in the noradrenaline outflow caused by TFS in the presence of atropine (0.1 mol/l) (to block acetylcholine-induced release of endothelial NO) and of indomethacin (3 mol/l) (to inhibit l-NNA-induced production of vasoconstrictor prostanoids). (1) TFS (2–10 Hz) caused a frequency-dependent increase in noradrenaline overflow and perfusion pressure. (2) l-NNA (10 and 30 mol/l) caused a concentration-dependent inhibition of TFS-induced noradrenaline overflow, whereas the TFS-induced pressure increase was augmented by l-NNA in a concentration-dependent manner. At any given concentration of l-NNA, the potentiation of vasoconstriction by l-NNA became greater in magnitude as the frequency of the TFS was raised. (3) Infusion of noradrenaline (0.38–6 nmol) caused a dose-dependent increase in perfusion pressure up to a value comparable with that caused by TITS. The pressure increase in response to noradrenaline infusion was also enhanced by l-NNA, relatively, to a greater extent than the enhancement, by l-NNA, of the pressure response to TFS. (4) These effects of l-NNA were significantly attenuated by l-arginine (0.3 mmol/l) or sodium nitroprusside (1 mol/l). Our results suggest that NO, presumably originating from several sites, may stimulate the release of noradrenaline in the mesenteric vasculature and that the consequent rise in circulating noradrenaline, in turn, causes the liberation of endothelial NO. Correspondence to: R. Yamamoto at the above address     

Functional characterization of substance P receptors in the rabbit ear artery

Summary Rabbit isolated ear arteries were perfused at a constant flow and stimulated with field pulses (5 Hz, 5 impulses). Different tachykinins and capsaicin depressed stimulation-induced vasoconstriction, substance P (SP) being the most potent inhibitor. The rank order of potency of the tachykinins was, SP physalaemin eledoisin>SP-methylester; that of SP and its C-terminal fragments, SPSP-(2–11)SP-(4–11)>SP-(6–11). SP-(1–9) was inactive. The SP antagonist (Arg⁵,d-Trp^7,9,Nle¹¹)SP-(5–11) 10 mol/l shifted the concentration-response curve of SP to the right (pA₂=5.43), whereas it did not reduce the action of capsaicin. Another SP antagonist (d-Pro⁴,d-Trp^7,9,10)SP-(4–11) 10 mol/l failed to affect the SP depression. Neither antagonist changed vasoconstriction by itself. Pretreatment of the arteries with a mixture of yohimbine, propranol, atropine, diphenhydramine, burimamide, methysergide and indomethacin, all 1 mol/l, did not influence the effect of SP or capsaicin. Only the inhibition by SP, but not that by capsaicin was abolished after mechanical destruction of the endothelium. SP, physalaemin and eledoisin, all 3 mol/l, reduced vasoconstriction by noradrenaline or histamine; capsaicin 30 mol/l depressed noradrenaline-induced vasoconstriction. In arteries preincubated with³H-noradrenaline, electrical stimulation (1 Hz, 120 pulses) triggered an increase in the outflow of tritium and evoked vasoconstriction. SP 1 mol/l did not change either basal or stimulation-evoked tritium outflow, whereas it reduced vasoconstriction. In conclusion, SP depresses nerve stimulation-induced vasoconstriction solely by changing smooth muscle contractility; the receptor activated seems to belong to the SP-P type. Under the conditions of these experiments there is no indication for a similar effect of endogenous SP.     

Inhibitory effects of apomorphine and pergolide on neurogenic vasoconstriction in the hindquarters of the rat

Summary The effects of locally administered apomorphine and pergolide were studied in the isolated autoperfused hindquarters of the rat, in an attempt to assess the possible role of presynaptic dopamine receptors at the level in the hypotensive effect of these dopamine agonists.Local infusion of apomorphine (1g·kg^–1·min^–1 for 5 min) or pergolide (1g·kg^–1·min^–1 for 5 min) [into the hindquarters] did not alter perfusion pressure per se, but reduced the pressor response to electrical stimulation of the lumbar sympathetic chains for the whole frequency range used during a cumulative frequency-response curve (0.25–16 Hz, 1 ms, supramaximal voltage). Apomorphine and pergolide reduced the pressor response elicited by 4 Hz electrical stimulation (applied until maximum response was reached) to 54.8±7.1% and 53.9±1.7% respectively, but they did not modify similar increases of perfusion pressure produced by locally administered noradrenaline.The inhibition by apomorphine and pergolide of the 4 Hz stimulation-evoked pressor response was completely antagonized by local administration of the dopamine antagonist haloperidol (1g·kg^–1), but was not influenced by the ₂-antagonist rauwolscine (100g·kg^–1). This dose of rauwolscine antagonized the inhibitory effect of the ₂-agonist UK-14,304, which was not influenced by haloperidol.Local administration of rauwolscine increased the pressor response to stimulation at 4 Hz by 37.4–46.2%. In contrast, local administration of haloperidol did not influence the 4 Hz stimulation-evoked pressor response.These results indicate that dopamine receptors are pressent on the sympathetic innervation of the vascular bed in the rat hindquarters but do not provide evidence for a physiological role of these receptors in modulating peripheral sympathetic neurotransmission. Stimulation of these receptors, leading to a decrease of noradrenaline release and thus of vasomotor tone, might—at least in part—explain the blood pressure lowering effects of intravenous apomorphine and pergolide in the rat.     

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.     

Cultured chick sympathetic neurons: prostanoid EP1 receptor-mediated facilitation of noradrenaline release

Prostanoid EP receptor-mediated modulation of noradrenaline release from cultured chick sympathetic neurons was investigated. Transmitter release from dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline, was elicited either by electrical field stimulation (36 pulses/3 Hz) or by elevating the extracellular concentration of K⁺ (to 30 mM; for 2 min).Prostaglandin E₂ (PGE₂; 0.01–3 M) enhanced electrically evolved [³H]-noradrenaline release in a concentration-dependent manner with a maximal increase by about 50% at 1 M. Also iloprost (0.1–3 M) increased transmitter release concentration-dependently, whereas misoprostol (0.1–3 M) had no effect. Indometacin (10 M) influenced neither evoked release per se nor the enhancement caused by PGE₂. AH6809 (3 M), a selective EP₁ receptor antagonist, blocked the enhancement caused by both PGE₂ and iloprost. K⁺-evoked noradrenaline release, which was virtually insensitive to tetrodotoxin (0.3 M), was increased by PGE₂ to an extent comparable to that observed after electrical stimulation.In summary, the present data indicate that PGE₂ facilitates noradrenaline release from cultured chick sympathetic neurons by a receptor which shows the pharmacological profile of the EP₁ subtype and is probably located at the processes of the neuron.     

Stimulus-evoked release of tritiated monoamines from rat periaqueductal gray slices in vitro and its receptor-mediated modulation

Summary The periaqueductal gray is a brain region of considerable interest. It is innervated by monoamine-containing neurons as well as by a variety of peptidergic fiber systems, and it participates in the regulation of various functions. Virtually nothing is known about monoamine release in the periaqueductal gray and its receptor-mediated modulation. We therefore studied the release of radioactivity from periaqueductal gray slices preloaded with tritriated monoamines, using an in vitro superfusion method.The release of radioactivity from superfused periaqueductal gray slices after preloading of the tissue with [³H]noradrenaline increased upon electrical stimulation in a frequency-dependent manner. The stimulus-evoked release of radioactivity was Ca²⁺-dependent. Clonidine reduced and yohimbine enhanced the release. The inhibition curve for the effect of clonidine was shifted to the right in the presence of 10^–6 M yohimbine. While phenylephrine, isoprenaline, SK&F 38393, quinpirole, carbachol, [Arg⁸]vasopressin, -MSH and ACTH-(1-24), at a concentration of 10^–6 M, did not influence the electrically evoked release of radioactivity, [Leu⁵]enkephalin reduced it. The selective -opioid receptor agonists [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin and [d-Arg²,Lys⁴]-dermorphin-(1–4)-amide reduced the release of radioactivity, whereas the selective opioid receptor agonist [d-Pen²,d-Pen⁵]enkephalin and the selective K opioid receptor agonist U-69593 had no effect. In the presence of naloxone, which by itself had no effect on the release of radioactivity, the effect of [d-Arg²,Lys⁴]dermorphin-(1–4)-amide was abolished. These results show that the release of noradrenaline from periaqueductal gray slices is via a Ca²⁺-dependent. exocytotic process, and that it is modulated through ₂-adrenoceptors as well as via -opioid receptors. Though the overflow of radioactivity from slices preloaded with [3H]dopamine in the presence of desipramine was measurable, there are reasons to assume that we are dealing here with the release of tritiated catecholamines from a population of nerve endings consisting of noradrenergic and dopaminergic terminals.The release of radioactivity from periaqueductal gray slices preloaded with [³H]5-hydroxytryptamine upon elevation of the K⁺ concentration in the superfusion medium was much more pronounced than that induced by electrical stimulation. The K⁺-evoked release of radioactivity was almost completely abolished in the absence of Cat²⁺; showing that the release is via a Ca²⁺-dependent process. 5-Hydrotryptamine reduced the K⁺-evoked release of radioactivity in a concentration-dependent manner.Some of these data were presented at the XIth International Congress of Pharmacology, 1–6 July 1990, Amsterdam, The Netherlands (Eur J Pharmacol 183:408) Send offprint requests to D. H. G. Versteeg 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.     

Long-term administration of 1,3-dipropyl-8-sulphophenylxanthine (DPSPX) alters α2-adrenoceptor-mediated effects at the pre- but not at the postjunctional level

The present investigation was undertaken to see whether a long-term inhibition of adenosine receptors —leading to hypertension — interferes with ₂-adrenoceptor-mediated modulation of noradrenaline release. Rat tail arteries were removed from normal and from hypertensive animals obtained by chronic treatment with intraperitoneally infused DPSPX (1,3,-dipropyl-8-sulphophenylxanthine) or orally administered L-NAME (N^G-Nitro-L-arginine methyl ester). To study prejunctional effects, the influence of UK-14,304 (5-bromo-6(imidazoline-2-ylamino)-quinoxaline) and yohimbine on the overflow of tritium evoked by electrical stimulation (100 V; 1 Hz; 2 ms; 5 min) from tissues preloaded with ³H-noradrenaline was analysed. To study postjunctional effects, concentration-response curves to UK-14,304 were determined. In DPSPX-treated rats there was an enhancement of the prejunctional effects of UK-14,304: its EC_30% was reduced from 381 (250; 579) to 85 (73; 99) nmol.l^–1 (n = 5; P<0.05) and its maximal effect — expressed as percent reduction of tritium overflow-increased from 45 ± 5% to 61 ± 5% (n = 6; P < 0.05). In L-NAME-treated rats there was no change in either of these two parameters. At the postjunctional level, there was no change in the sensitivity to UK-14,304 in tissues from either DPSPX- or L-NAME-treated rats. Yohimbine (10–1000 nmol.l^–1) caused a concentration-dependent increase of tritium overflow evoked by electrical stimulation in both control and hypertensive animals (either DPSPX- or L-NAME-treated). The EC_50%-pre-antagonist values (concentration of the antagonist that increases the evoked overflow by 50%) were not significantly different in the three situations. We conclude that long-term administration of DPSPX increases the sensitivity to the prejunctional effects of UK-14,304 without changing that to its postjunctional effects, showing a specific interaction between -adrenoceptors and adenosine receptors at a prejunctional level. The question arises whether there is any link between that alteration and the development of the hypertensive state. Correspondence to: S. Guimaraes at the above address