Impulse interval-dependent effect of sympathetic nerve stimulation on evoked acetylcholine release from the rabbit perfused atria preparation

The aim of the present study was to explore possible prejunctional effects mediated by impulse activity of sympathetic terminals on evoked acetylcholine release in an organ innervated by the autonomic ground plexus. Rabbit atria were isolated with the extrinsic right vagus and sympathetic nerves intact and perfused with Tyrode solution. Acetylcholine overflow was determined after labelling of the transmitter stores with [¹⁴C]choline and fractionation of the radioactivity on cation exchange columns. The overflow of endogenous noradrenaline was measured by HPLC and electrochemical detection.The vagus nerve was stimulated at 2 Hz for 3 min four times at intervals of 10 min. During the second stimulation the postganglionic sympathetic nerves were stimulated (2 Hz, 3 min) in such a way that the impulses preceded the vagus stimuli by a fixed time interval which was varied in different experiments (0, 7, 19, 50, 132, and 350 ms). Evoked acetylcholine release was significantly enhanced when the vagus was excited 7, 19 and 50 ms after the sympathetic nerves but it was unaltered at the 132 or 350 ms intervals, and when both nerves were stimulated simultaneously. Noradrenaline release was similar (about 6 ng per stimulation period) in all experimental groups. When sympathetic nerve stimulation had little effect in releasing noradrenaline (<2.0 ng per stimulation period), facilitation of acetylcholine release at the 19 ms pulse interval was absent. The resting outflow of acetylcholine was unaffected by sympathetic nerve stimulation.The experiments show a facilitation of evoked acetylcholine release by sympathetic activity. As revealed by the pulse-to-pulse method this effect is confined to a relatively brief interval immediately following the excitation of the noradrenergic terminal, and is unlikely to be mimicked by exogenous drug application.     

Role of cyclic AMP in the release of noradrenaline from isolated rat atria

Summary The possible role of cyclic AMP (cAMP) on tritium overflow evoked by stimulation of the cardioaccelerant nerves was studied in rat atria preincubated with [³H]-noradrenaline. Addition of the activator of adenylate cyclase forskolin (1 µmol/l), or of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMx, 100 µmol/l), did not affect both basal and evoked overflow. However, in the presence of the ₂-adrenoceptor antagonist yohimbine (0.03 µmol/l) both forskolin and IBMx increased the stimulation-induced transmitter overflow by 49% and 141%, respectively (compared to yohimbine 0.03 µmol/l). Thus, in rat atria the cAMP-dependent facilitation of noradrenaline release is only present when the autoinhibition exerted by activation of prejunctional ₂-adrenoceptors is blocked. Propranolol (0.1 µmol/l) that did not produce any effect on noradrenaline release markedly reduced the facilitatory response induced by forskolin in the presence of yohimbine. When rats were pretreated with the ₂-adrenoceptor agonist clenbuterol (0.3 mg · kg^–1, s. c., twice daily, 14 days), a treatment which desensitizes -adrenoceptor-me-diated facilitation of noradrenaline release (Kazanietz and Enero 1989), the facilitatory effect of forskolin and IBMx in the presence of yohimbine was abolished. The results indicate that in rat atria the effect of forskolin and IBMx on noradrenaline release are only to be observed after blockade of presynaptic ₂-adrenoceptor autoinhibition. -adrenoceptor blockade or clenbuterol pre-treatment decreases the facilitatory response to forskolin and hence prejunctional -adrenoceptor-mediated enhancement of noradrenaline release is linked to the stimulation of adenylate cyclase. Correspondence to M. A. Enero at the above address     

Modulation of noradrenaline release in rat isolated stomach by prostanoids,but not by histaminergic mechanisms

Several gastric functions are modulated by the sympathetic nervous system, but local mechanisms involved in the control of noradrenaline release are largely unknown.Overflow of endogenous noradrenaline was studied from isolated rat stomach incubated in Ussing chambers allowing the separate determination of mucosal and serosal overflow. Spontaneous noradrenaline overflow was similar at the mucosal and serosal side, but electrical field stimulation caused a frequency-dependent increase in noradrenaline overflow selectively at the serosal side. Evoked noradrenaline overflow was blocked by tetrodotoxin, not affected by indometacin and markedly enhanced (by about 250%) by yohimbine. In the presence of indometacin and yohimbine, sulprostone (an agonist at EP₁/EP₃ receptors) and misoprostol (an agonist at EP₂/EP₃ receptors) reduced the noradrenaline overflow evoked by stimulation at 3 Hz maximally by about 80% (EC₅₀: 6 nmol/l and 11 nmol/l, respectively). The EP₁ receptor selective antagonist AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) did not antagonize the inhibition by sulprostone. Noradrenaline overflow evoked by stimulation at 1 Hz and 3 Hz was increased by scopolamine by about 50% and almost completely inhibited by oxotremorine. Neither, histamine nor the H₃ receptor selective agonist (R)--methyl-histamine, nor the H₁, H₂ and H₃ selective receptor antagonists mepyramine, cimetidine and thioperamide significantly affected noradrenaline overflow evoked by stimulation at 1 Hz or 3 Hz.In conclusion, impulse-induced noradrenaline release in the rat stomach is controlled by multiple presynaptic mechanisms involving ₂-adrenergic autoreceptors, EP₃ prostanoid and muscarine heteroreceptors, whereas histaminergic mechanisms do not appear to be significant.     

A presynaptic excitatory M1 muscarine receptor at postganglionic cardiac noradrenergic nerve fibres that is activated by endogenous acetylcholine

Summary Rabbit atria were isolated with the extrinsic right vagus and sympathetic nerves intact and perfused with Tyrode solution. Noradrenaline overflow evoked by sympathetic nerve stimulation (SNS) at 3 Hz for 3 min was determined before, during, and after vagus nerve stimulation (VNS), also at 3 Hz and for 3 min. The VNS pulses preceded the SNS pulses by 3, 100 and 233 ms. Acetylcholine overflow was determined after labelling of the transmitter stores with [¹⁴C]choline.Pirenzepine 80 nmol/l failed to alter the muscarinic inhibition of noradrenaline overflow when the vago-sympathetic impulse intervals were 3 and 233 ms. At an interval of 100 ms VNS did not significantly inhibit noradrenaline overflow in the absence of pirenzepine but produced an inhibition in the presence of the drug. When the pirenzepine concentration was varied (0.4–300 nmol/l) the largest inhibition of noradrenaline overflow was observed at 5.7 nmol/l whereas 300 nmol/l fully antagonized the inhibition. Acetylcholine overflow evoked by VNS was not altered by pirenzepine 0.4–300 nmol/l.AF-DX 116 (11-[{2[oi(diethylamino)methyl]-1-piperidinyl}-acetyl]-5,11-dihydro-6H-pyrido-[2,3-b]-[1,4]benzodiazepine-6-one), an M₂ receptor selective antagonist, concentration-dependently (100–800 nmol/l) inhibited the decrease of tension development elicited by VNS. At the 100 ms vago-sympathetic impulse interval noradrenaline overflow was enhanced in the presence of AF-DX 116 400 and 800 nmol/l. However, already 100 nmol/l of the drug caused a maximum (fourfold) increase of acetylcholine overflow.It is concluded that acetylcholine released onto noradrenergic nerve fibres causes a small facilitation of noradrenaline overflow at a vago-sympathetic impulse interval of 100 ms. This response is mediated by an M₁ receptor and is superimposed on the well-known M₂ receptor mediated inhibition of noradrenaline release which is obtained at vago-sympathetic impulse intervals ranging between 3 and 233 ms. The M₂ autoreceptor regulating acetylcholine release is activated by lower synaptic concentrations of the transmitter than the M₂ heteroreceptor regulating noradrenaline release.Abbreviations SNS sympathetic nerve stimulation - VNS vagus nerve stimulation Send offprint requests to: E. Muscholl 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     

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.     

Effect of desipramine-induced blockade of neuronal uptake mechanisms on adrenoceptor-mediated responses in the guinea-pig colon

In order to clarify whether adrenoceptors in the guinea-pig distal colon are under sympathetic control, we assessed possible variations in the sensitivity to adrenoceptor agonists after blockade of neuronal catecholamine uptake mechanisms by desipramine (DMI). First, experiments were carried out to investigate the effects of DMI added in the organ bath on propulsion velocity, endogenous and [³H] prelabelled acetylcholine overflow, electrically evoked noradrenaline overflow and longitudinal smooth muscle tone. Secondly, we studied the effects of adrenoceptor agonists on the above parameters in untreated animals and in animals chronically treated with DMI.DMI added in the organ bath at concentrations equal to or higher than 30 nM inhibited all the parameters under study. Thus, when evaluating the effect of DMI on concentration-response curves to adrenoceptor agonists, concentrations which were per se inactive were used. DMI added in the organ bath at concentrations up to 30 nM potentiated the inhibitory effects of exogenous noradrenaline on propulsion velocity and acetylcholine overflow, but it did not affect the concentration-response curve to exogenous noradrenaline on longitudinal smooth muscle tone. Furthermore, 30 nM DMI inhibited propulsion velocity during sympathetic nerve stimulation. In preparations obtained from animals chronically treated with DMI, no significant change of propulsion velocity, endogenous and [³H] prelabelled acetylcholine overflow was found with respect to untreated animals. Nevertheless, in such preparations subsensitivity to isoprenaline (acting mainly on muscular -adrenoceptors) and clonidine (acting on neuronal ₂-adrenoceptors) and supersensitivity to phenylephrine were observed. Electrically evoked noradrenaline overflow was enhanced, in a frequency-dependent way, by yohimbine and inhibited by clonidine.We conclude that in the guinea-pig colon: 1) - and -adrenoceptors are under tonic neuronal control, as indicated by the sensitivity changes to - and -adrenoceptor agonists after chronic DMI treatment; 2) exogenous NA reaching neuronal, but not muscular adrenoceptors, is affected by neuronal uptake mechanisms; 3) NA released by adrenergic terminals undergoes neuronal uptake and is controlled by ₂-autoreceptors. Correspondence to: G. M. Frigo at the above address     

Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein by presynaptic histamine H3 receptors

Summary The human saphenous vein was used to examine whether presynaptic histamine receptors can modulate noradrenaline release and, if so, to determine their pharmacological characteristics. Strips of this blood vessel were incubated with [³H]noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. Electrically (2 Hz) evoked ³H overflow was inhibited by histamine and the H₃ receptor agonist R-(–)--methylhistamine. Histamine-induced inhibition of electrically evoked tritium overflow was not affected by ₂-adrenoceptor blockade by rauwolscine. S-(+)--methylhistamine (up to 10 mol/l) as well as the histamine H₁ and H₂ receptor agonists 2-(2-thiazolyl)ethylamine (up to 3 mol/l) and dimaprit (up to 30 mol/l), respectively, were ineffective. The selective histamine H₃ receptor antagonist thioperamide abolished the inhibitory effect of histamine. The histamine H₂ and H₁ receptor antagonists ranitidine and pheniramine, respectively, did not affect the histamine-induced inhibition of evoked tritium overflow. The present results are compatible with the suggestion that the sympathetic nerves of the human saphenous vein are endowed with inhibitory presynaptic histamine receptors of the H₃ class. Send offprint requests to M. Gothert 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.     

AF-DX 116 differentiates between prejunctional muscarine receptors located on noradrenergic and cholinergic nerves

Summary Prejunctional affinity constants of the cardioselective muscarine receptor antagonist AF-DX 116 (11-[(2[(diethyl-amino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro6 H-pyrido [2,3-b] [1,4] benzodiazepine-6-one) were determined for muscarine autoreceptors on cholinergic nerves of the guinea-pig ileum and for heteroreceptors on noradrenergic nerves of the rat heart and guinea-pig iris. AF-DX 116 antagonized with low affinity the muscarinic inhibition induced by arecaidine propargyl ester of the stimulation-evoked [³H]acetylcholine overflow (pA₂ 6.74) from the guinea-pig ileum. In contrast, AF-DX 116 was more potent in antagonizing the methacholine-induced inhibition of the stimulation-evoked [³H]noradrenaline overflow from rat heart (pA₂ 7.29) or guinea-pig iris (pA₂ 7.57). The data confirm previously reported differences between prejunctional muscarine heteroreceptors in the rat heart which belong to the cardiac subtype (M₂ or M₂) and autoreceptors in the guinea-pig ileum that cannot be distinguished from the ileal subtype (M₂) or (M₃). Send offprint requests to H. Fuder at the above address     

Desensitization of inhibitory prejunctional α2-adrenoceptors and putative imidazoline receptors on rabbit heart sympathetic nerves

Summary To find out whether sympathetic nerves of the rabbit heart possess pharmacologically relevant prejunctional imidazoline receptors different from -autoreceptors, the inhibition by oxymetazoline, aganodine and BDF 6143 (4-chloro-2-[2-imidazoline-2-ylamino]-isoindoline hydrochloride) of endogenous noradrenaline overflow evoked by stimulation of extrinsic postganglionic sympathetic nerves (0.66 Hz, 80 pulses) was investigated. In addition we wanted to find out whether either type of these prejunctional receptors undergoes desensitization upon pre-exposure to respective agonists.The ₂-adrenoceptor agonist oxymetazoline inhibited the evoked noradrenaline overflow (2.9 nmol/l, IC₅₀; about 90010, maximum inhibition). The inhibition was antagonized by rauwolscine (–log K_B 8.20). This confirms the presence of ₂-autoreceptors. Endogenous noradrenaline activated autoinhibition to a small extent as indicated by a rauwolscine-induced increase in evoked overflow by less than 2-fold.The ₂- and imidazoline receptor agonist aganodine inhibited the evoked noradrenaline overflow (2.4 nmol/l, IC₅₀; about 80%, maximum inhibition). The inhibition was antagonized by rauwolscine with a potency (–log K_B 6.75), about 1/30 of that found at the ₂-autoreceptor. Neither an ₂-selective low concentration of rauwolscine nor the ₁-adrenoceptor antagonist prazosin, nor SKF 104078, a mixed _1/2-antagonist, reduced the aganodine effect. The ₂-adrenoceptor antagonist and imidazoline receptor agonist BDF 6143 inhibited the evoked noradrenaline overflow (18 nmol/l, IC₅₀; about 70% maximum inhibition). The inhibition was insensitive to a low rauwolscine concentration.In hearts pre-exposed for 30 min (followed by washout; rauwolscine 0.1 mol/l added later on to minimize presynaptic ₂-adrenoceptor activation or blockade by drugs persisting in the biophase) to oxymetazoline 10 mol/l, aganodine 2 mol/l or BDF 6143 10 mol/l, the inhibitory effects of oxymetazoline 30 nmol/l and aganodine 10 nmol/l were concomitantly reduced. No significant reduction of the agonist effect was seen after pre-exposure to BDF 6143 2 mol/l. Pre-exposure to BDF 6143 10 mol/l shifted the concentration for half-maximum inhibition to the right and depressed the maximum effect of both, oxymetazoline and aganodine, but did not affect the inhibitory action of the muscarinic agonist methacholine.It is concluded that inhibitory prejunctional ₂-autoreceptors and putative imidazoline receptors coexist on postganglionic sympathetic nerves of the rabbit heart. They are both subject to desensitization upon exposure to a high agonist concentration. The findings are compatible with a mutual cross-desensitization under the conditions investigated.This study was supported by the Deutsche Forschungsgemeinschaft (FU163/3) Correspondence to H. Fuder 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     

Adrenergic mechanisms during hypertension induced by sucrose and/or salt in the spontaneously hypertensive rat

Summary Spontaneously hypertensive rats received tap water, 1% NaCl, 5% sucrose or NaCl and sucrose in combination for 4 weeks. The blood pressure (tail plethysmography) and renal excretions of sodium and catecholamines were followed. After 4 weeks the noradrenaline turnover (disappearance after -methyltyrosine) was assessed in the heart and brain. In pithed rats the pressor responses to intravenous noradrenaline and to electrical stimulation of the spinal sympathetic nerves (SNS) were determined together with the rise in plasma noradrenaline concentrations during the SNS. Salt alone caused an increase in peripheral sympathetic activity, measured as turnover of noradrenaline in the heart and spillover of noradrenaline in the urine, a modest enhancement of vascular responsiveness to noradrenaline and a blood pressure elevation. Sucrose alone increased the peripheral sympathetic activity but influenced neither the vascular responsiveness to noradrenaline nor the basal blood pressure. The largest increase in sympathetic activity and in blood pressure was observed with sucrose and salt in combination. The release of noradrenaline from the sympathetic nerve endings was not significantly influenced by any diet regime. The changes in noradrenaline turnover in the heart was accompanied by reciprocal changes in brain stem noradrenaline turnover. Send offprint requests to K. Gradin     

Pharmacological characterization of the imidazoline receptor which mediates inhibition of noradrenaline release in the rabbit pulmonary artery

Summary Imidazoline receptors involved in modulation of noradrenaline release were characterized in the rabbit pulmonary artery preincubated with [³H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Tritium overflow was evoked by transmural electrical stimulation.The ₂-adrenoceptor blocking imidazolines tolazoline, BDF 6100 [2-(2-imidazoline-2-ylamino)-isoindoline] and BDF 7572 (4,7-dichloro-derivative of BDF 6100) increased the electrically evoked ³H overflow; the concentration-response curves were bell-shaped. In contrast, two other imidazolines, i. e. moxonidine and clonidine, two guanidine derivatives structurally related to BDF 6100, i. e. aganodine and BDF 7579 [4-chloro(2-isoindolinel)-guanidine], as well as the catecholamine noradrenaline concentration-dependently inhibited the evoked ³H overflow. The concentration-response curves for moxonidine, clonidine, aganodine, BDF 7579 and noradrenaline were shifted to the right by rauwolscine. The apparent pA₂ value of rauwolscine against moxonidine was 8.22, whereas those against clonidine, aganodine, BDF 7579 and noradrenaline were in the range of 6.37–6.77 and, hence, considerably lower than reported for ₂-adrenoceptors. In the presence of rauwolscine an inhibitory effect was also observed with the ₂-adrenoceptor blocking imidazolines tolazoline, BDF 6100, BDF 7572, and the imidazolineST 587 [2-(2-chloro-5-trifluoromethylphenylimino)-imidazoline]; the rank order of potency of all guanidines and imidazolines investigated was: aganodine > BDF 7579 > BDF 7572 > BDF 6100 > clonidine > ST 587 > moxonidine > tolazoline. Amiloride, 1-benzylimidazole and histamine were ineffective. After irreversible blockade of -adrenoceptors by preexposure to phenoxybenzamine, evoked ³H overflow was still inhibited by aganodine, BDF 7579 and noradrenaline. Under this condition the maximal effects obtainable with the guanidines and in particular with noradrenaline were lower than in the presence of rauwolscine.These findings are compatible with our previous suggestion that imidazoline receptors mediating inhibition of noradrenaline release exist on the sympathetic nerve terminals of the rabbit pulmonary artery. Comparison of the present data with those obtained in other preparations containing imidazoline recognition sites revealed that those sites are different from the present ones. It is conceivable that the receptor characterized here represents an allosteric site of the ₂-adrenoceptor or a so far undescribed -adrenoceptor subtype since it can be activated not only by imidazolines and guanidines but also by noradrenaline and can be blocked by rauwolscine. Comparison of the properties of isoindolines substituted with either aminoimidazoline or guanidine reveals that the imidazolines (e.g. BDF 7572) possess both ₂-adrenoceptor antagonistic and imidazoline receptor agonistic properties, whereas the analogous guanidines (e. g. aganodine) are imidazoline receptor agonists as well as ₂-adrenoceptor agonists. Send offprint requests to M. Göthert 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 N-methyl-d-aspartate (NMDA)-stimulated noradrenaline release in rat brain cortex by presynaptic α2-adrenoceptors

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

Prejunctional inhibition of sympathetically evoked pupillary dilation in cats by activation of histamine H3 receptors

Summary Frequency-dependent pupillary dilations were evoked by electrical stimulation of the pre- or post-ganglionic cervical sympathetic nerve (sympatho-excitation) or the hypothalamus (parasympatho-inhibition) in sympathectomized anesthetized cats. Systemic administration of the selective histamine H₃ receptor agonist (R)--methylhistamine (RMeHA) produced a dose-dependent depression of mydriasis due to direct neural sympathetic activation but had no effect on responses elicited by parasympathetic withdrawal. The histamine H₂ receptor agonist, dimaprit, was inactive. RMeHA was much more effective in depressing sympathetic responses obtained at lower frequencies when compared to higher frequencies of stimulation.Responses evoked both pre- and postganglionically were inhibited by RMeHA. This peripheral sympathoinhibitory action of RMeHA was antagonized by the histamine H₃ receptor blocker thioperamide but not by intravenous pretreatment with the histamine H₁ receptor antagonist chlorpheniramine. Histamine H₂ receptor blockers cimetidine and ranitidine were also without effect. RMeHA did not depress pupillary responses elicited by i.v. (-)-adrenaline.The results demonstrate that histamine H₃ receptors modulate sympathetic activation of the iris at a site proximal to the iris dilator muscle. The predominant mechanism of action appears to the prejunctional inhibition of noradrenaline release from postganglionic sympathetic nerve endings. However, a concomitant ganglionic inhibitory action cannot be excluded. Correspondence to M. C. Koss 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     

Inhibition of uptake1 by (+)-oxaprotiline reveals a differential central regulation of noradrenaline and adrenaline release

Summary Inhibition of uptake, in the central nervous system leads to a decrease of sympathetic outflow to many tissues; central a2-adrenoceptors are involved in this decrease. The aim of the present study was to compare the effects of the selective uptake, inhibitor (+)-oxaprotiline on the plasma kinetics of noradrenaline and adrenaline in anaesthetized and in conscious rabbits. [³H]Noradrenaline and [^3H]adrenaline were infused iv. The arterial plasma concentrations of endogenous and radiolabelled noradrenaline and adrenaline were measured, and the clearance from and spillover into the plasma of noradrenaline and adrenaline were calculated.Results obtained in conscious and anaesthetized rabbits were similar. (+)-Oxaprotiline 0.2, 0.6 and 1.8 mg kg^–1 iv. dose-dependently reduced the clearance of [³H]noradrenaline from the plasma. The clearance of [³H]adrenaline was reduced less. The spillover of endogenous noradrenaline was decreased by up to 35%. In contrast, the spillover of adrenaline tended to be enhanced. Prazosin 0.1 and 1 mg kg^–1 was injected iv. in a second part of each experiment. It lowered the blood pressure and caused a marked increase in noradrenaline spillover but no increase or even a decrease in adrenaline spillover.The results are compatible with the following hypothesis. The sympathetic outflow from the central nervous system is subject to a twofold a-adrenoceptor-mediated modulation: -adrenoceptor-mediated inhibition and ₁-adrenoceptor-mediated excitation. In the control of the sympathetic outflow to many extra-adrenal tissues, the ₂-adrenergic inhibition prevails. Uptake₁ inhibitors depress sympathetic outflow to such tissues by enhancing the ₂-adrenergic inhibition. In the regulation of the sympathetic outflow to the adrenal medulla, in contrast, ₂-adrenergic inhibition and ₁-adrenergic excitation have a similar impact. Uptake, inhibitors, hence, cause little change in adrenaline release: the two opposing influences cancel out. Prazosin produces an increase in noradrenaline but not adrenaline release because the loss of the central ₁ sympathoexcitation attenuates at best slightly the baroreflex to most extra-adrenal tissues but dampens markedly the baroreflex to the adrenal medulla. Correspondence to B. Szabo at the above address     

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