Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

α-Bungarotoxin, κ-bungarotoxin, α-cobratoxin and erabutoxin-b do not affect [3H]acetylcholine release from the rat isolated left hemidiaphragm

Endplate preparations of the rat left hemidiaphragm were incubated with [³H]choline to label neuronal transmitter stores. Nerve evoked release of newly-synthesized [³H]acetylcholine was measured in the absence of cholinesterase inhibitors to investigate whether snake venom neurotoxins by blocking presynaptic nicotinic autoreceptors affect evoked transmitter release. Contractions of the indirectly stimulated hemidiaphragm were recorded to characterize the blocking effect of -neurotoxins at the postsynaptic nicotinic receptors.Neither the long chain neurotoxins -cobratoxin (1 g ml^–1) and -bungarotoxin (5 g ml^–1) nor the short chain neurotoxin erabutoxin-b (0.1, 1 and 10 gml^–1) affected the nerve-evoked release of [³H]acetylcholine. -Bungarotoxin (1 and 5 g ml^–1), a toxin preferentially blocking neuronal nicotinic receptors, did also not affect evoked [³H]acetylcholine release, whereas (+)-tubocurarine (1 M) under identical conditions reduced the release by about 50%. -Bungarotoxin, -cobratoxin and erabutoxin-b concentration-dependently (0.01–0.6 g ml^–1)inhibited nerve-evoked contractions of the hemidiaphragm. All neurotoxins except erabutoxin-b enhanced the basal tritium efflux immediately when applied to the endplate preparation or to a non-innervated muscle strip labelled with [³H]choline. This effect was attributed to an enhanced efflux of [³H]phosphorylcholine, whereas the efflux of [³H]choline and [³H]acetylcholine was not affected.It is concluded that the -neurotoxins and -bungarotoxin do not block presynaptic nicotinic receptors of motor nerves. These nicotinic autoreceptors differ from nicotinic receptors localized at the muscle membrane and at autonomic ganglia.     

Effects of the novel dopamine DA2-receptor agonist carmoxirole (EMD 45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney

Summary The effects of the classical dopamine DA₂-receptor agonist quinpirole (LY 171555) and the recently characterized DA₂-receptor agonist, carmoxirole (EMD 45609), on neurotransmission in rat isolated kidney were investigated. After preincubation with ³H-noradrenaline, the renal nerves were electrically stimulated. The stimulation induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. Quinpirole (0.3 mol/l) inhibited S-I outflow of radioactivity and pressor responses to renal nerve stimulation (RNS) at 1 Hz. Both effects of quinpirole were blocked by the DA₂-receptor antagonist S(–)-sulpiride (10 mol/l). The ₁, ₂-adrenoceptor antagonist phentolamine (1 mol/l) did not block the inhibitory effect of quinpirole. Carmoxirole (0.003 and 0.03 mol/l) did not alter and carmoxirole (0.3 mol/l) even enhanced S-I outflow of radioactivity, however, pressor responses to RNS were markedly reduced by carmoxirole (0.003–0.3 mol/l). Pressor responses to RNS were also markedly reduced by the ₁-adrenoceptor antagonist prazosin (0.1 mol/l). Carmoxirole (0.3 mol/l), prazosin (0.1 mol/l) and phentolamine (1 mol/l) totally abolished pressor responses to exogenous noradrenaline (0.05 mol/l). In contrast, quinpirole (0.3 mol/l) did not alter pressor responses to exogenous noradrenaline (0.05 mol/l). Furthermore, carmoxirole (0.003–0.3 mol/l) markedly reduced pressor responses induced by the ₁-adrenoceptor agonist methoxamine (1 mol/l) but even the highest concentration of carmoxirole (0.3 mol/l) had no effect on pressor responses induced by bolus injections of either neuropeptide Y (1.5 ng) or angiotensin II (1 ng). Phentolamine (1 mol/l) by itself markedly enhanced S-1 outflow of radioactivity and pressor responses to RNS were virtually unchanged. In the presence of phentolamine carmoxirole (0.03 and 0.3 mol/l) and quinpirole inhibited S-I outflow of radioactivity and pressor responses to RNS. Phentolamine resistant pressor responses to RNS were also inhibited by the P_2X-receptor desensitizing agent , -methylene adenosine triphosphate (mATP, 1 mol/l), which by itself in the presence of phentolamine did not alter S-I outflow of radioactivity. The inhibitory effects of carmoxirole (0.3 mol/l) in the presence of phentolame (1mol/l) were antagonized by S(–)-sulpiride (10 mol/l). The data suggest that activation of prejunctional DA₂-receptors by quinpirole inhibits noradrenaline release and thereby reduces pressor response to RNS at 1 Hz in rat isolated kidney. Carmoxirole activates prejunctional inhibitory DA₂-receptors, but this effect is masked by simultaneous blockade of inhibitory prejunctional -adrenoceptors. Pressor responses to RNS at 1 Hz in rat isolated kidney are largely due to neuronally released noradrenaline whereas phentolamine resistant pressor responses to RNS at 1 Hz are most likely due to ATP, which is co-released with noradrenaline. Carmoxirole inhibits pressor responses to RNS at 1 Hz as well as pressor responses induced by either exogenous noradrenaline or methoxamine by blocking postjunctional ₁-adrenoceptors. In addition carmoxirole and quinpirole seem to block phentolamine resistant pressor responses by inhibiting ATP release through activation of prejunctional DA₂-receptors. Send offprint requests to L. C. Rump at the above address     

Phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex

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

Dopamine inhibits prostaglandin F2α-induced depolarization of rabbit jejunal arteries via activation of DA1-receptors

Summary In rabbit jejunal arteries, the membrane potential of single smooth muscle cells decreased on the application of noradrenaline 3 mol/1. LY 171555 1 mol/1 did not change, whereas SKF 38393 10 mol/1 reversed the effect of noradrenaline. When prostaglandin F₂ (PGF₂) was used to evoke depolarization in the presence of prazosin 0.1 mol/1, rauwolscine 1 mol/1 and propranolol 1 mol/1, both SKF 38393 10 mol/1 and dopamine 10 mol/1 repolarized the membrane. SCH 23390 1 mol/1 antagonized the effects of SKF 38393 10 mol/1 and dopamine 10 mol/1. Thus, the change in membrane potential is mediated by a DA₁-recep-tor.     

Blockade of α2-adrenoceptors increases opioid μ-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones

Summary In pontine slices of the rat brain, the frequency of spontaneous action potentials of locus coeruleus (LC) neurones was recorded extracellularly. Noradrenaline 0.1–100 mol/l, UK 14,304 0.01–100 nmol/l, [Met⁵]-enkephalin 1–10,000 nmol/l and [D-Ala², D-Leu⁵]enkephalin 0.1–1,000 nmol/l, all depressed the firing rate. Rauwolscine 1 mol/l antagonized the effects of both noradrenaline and UK 14,304, but potentiated the effects of [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin. Idazoxan 1 mol/l acted in a similar manner. Prazosin 1 mol/l did not change the effects of either noradrenaline or [Met⁵]enkephalin. Naloxone 0.1 mol/l antagonized both [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin, but failed to alter the effects of either noradrenaline or UK 14,304. Rauwolscine, idazoxan and prazosin, all 1 mol/l, as well as naloxone 0.1 mol/l, did not influence the firing rate when given alone. Desipramine 1 mol/l inhibited the discharge of action potentials in a rauwolscine-antagonizable manner. Noradrenaline 10 mol/l produced the same depression of firing, both in the presence of noradrenaline 1 mol/l and [Met⁵]enkephalin 0.03 mol/l. Likewise, the effect of [Met⁵]enkephalin 0.3 mol/l was the same, irrespective of whether it was added to a medium containing [Met⁵]enkephalin 0.03 mol/l or noradrenaline 1 mol/l. The spontaneous activity of LC neurones is inhibited by somatic ₂-adrenoceptors and opioid -receptors. We suggest that the two receptors interact with each other at a site located between themselves and not in the subsequent common signal transduction system.Send offprint requests to: P. Illes at the above address     

The involvement of central cholinergic system in the pressor effect of intracerebroventricularly injected U-46619, a thromboxane A2 analog, in conscious normotensive rats

The aim of this study was to determine the involvement of the central cholinergic system in the rise in blood pressure evoked by the thromboxane A2 (TxA2) analog, U-46619, given centrally. Intracerebroventricular (i.c.v.) injections of U-46619 (0.5, 1.0 and 2.0 g) caused dose- and time-related increases in blood pressure and decreased heart rate in awake rats. U-46619 (1 g; i.c.v.) also produced an approximately 65% increase in posterior hypothalamic extracellular acetylcholine and choline levels. Pretreatment with SQ-29548 (8 g; i.c.v.), selective TxA2 receptor antagonist, completely inhibited both the cardiovascular responses and the increase in acetylcholine and choline levels to subsequent injection of U-46619 (1 g; i.c.v.). Atropine (10 g; i.c.v.), nonselective muscarinic receptor antagonist, pretreatment did not affect the cardiovascular responses observed after U-46619 (1 g; i.c.v.). Pretreatment with the nonselective nicotinic receptor antagonist, mecamylamine (50 g; i.c.v.) attenuated the pressor effect of U-46619 (1 g; i.c.v.). Higher doses of mecamylamine (75 and 100 g; i.c.v.) pretreatments did not change the magnitude of the blockade of pressor response to U-46619; however, they abolished the bradycardic effect of U-46619 dose-dependently. Interestingly, pretreatment of rats with methyllycaconitine (10 g; i.c.v.) or -bungarotoxin (10 g; i.c.v.), selective antagonists of 7 subtype of nicotinic acetylcholine receptors (7nAChRs), partially abolished the pressor response to i.c.v. injection of U-46619 (1 g). Similar to the mecamylamine data, the use of higher doses of methyllycaconitine (25 and 50 g; i.c.v.) produced the same magnitude of blockade that was observed after the 10 g methyllycaconitine pretreatment, but it completely abolished the bradycardic effect of U-46619 (1 g; i.c.v.) at the dose of 25 g. The present results show that central administration of U-46619 produces pressor and bradycardic effect and increase in hypothalamic acetylcholine and choline levels by activating central TxA2 receptors. The activation of central nicotinic receptors, predominantly 7nAChRs, partially mediates the cardiovascular responses to i.c.v. injection of U-46619.     

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.     

Influence of N-allyl-normetazocine on acetylcholine release from brain slices: involvement of muscarinic receptors

Summary The effects of (±)N-allyl-normetazocine on the release of acetylcholine from different areas of guinea-pig and rat brain were investigated. 1. The drug did not modify the electrically (2 Hz) evoked tritium efflux from guinea-pig cerebral cortex, thalamus and caudate nucleus slices, preloaded with ³H-choline 0.1 mol/l and superfused with Krebs solution containing hemicholinium-3 10 mol/l. 2. (±)N-allyl-normetazocine 10 mol/l. enhanced the evoked ³H efflux from guinea-pig brain slices superfused with Krebs solution containing physostigmine 30 mol/l or oxotremorine 0.3 -1 gmol/l; the effect was naloxone-insensitive and was abolished by atropine 0.15 mol/l, but not by pirenzepine 1 mol/l. 3. (±)N-allyl-normetazocine 5 mol/l enhanced the electrically evoked release of endogenous acetylcholine as well, in a naloxone-insensitive way. 4. Both (±) and (+)N-allyl-normetazocine were without effect on ³H efflux from rat caudate nucleus slices electrically stimulated at 0.2 Hz frequency, after preloading with ³H-choline and during superfusion with hemicholinium-3. 5. The results are discussed in view of the antimuscarinic properties of the drug. Send offprint requests to A. Siniscalchi     

Multiple inhibitory mechanisms mediate non-adrenergic non-cholinergic relaxation in the circular muscle of the guinea-pig colon

Summary The mechanisms responsible for nerve-mediated, non-adrenergic, non-cholinergic (NANC) relaxation in mucosa-free circular muscle strips from the proximal colon of the guinea-pig were investigated. Electrical field stimulation (EFS, 1–20 Hz, trains of 5 s duration, 100 V, 0.25 ms pulse width) in the presence of atropine (1 mol/l) and guanethidine (3 mol/l) evoked a triphasic motor response consisting of. (a) a primary relaxation, (b) a rebound contraction and (c) a secondary relaxation. These three responses were abolished by tetrodotoxin (1 mol/l). Both apamin (0.01–0.3 mol/l), a known blocker of low conductance, calcium-activated potassium channels in smooth muscles, and L-nitroarginine (L-NOARG) (1–100 mol/l), a known blocker of nitric oxide (NO) synthase, increased the tone of the strips. Maximum effects on tone were observed with 0.1 mol/l apamin (21 ± 3% of KCl-induced contraction) and 30 mol/l L-NOARG (26 ± 4% of KCl response). The combined administration of 0.1 mol/l apamin and 30 mol/l L-NOARG produced an increase in tone (47 ± 5% of KCl response) that was larger than that produced by either compound alone. Neither apamin (0.1 mol/l) nor L-NOARG (30 mol/l) affected the isoprenaline-induced relaxation.Apamin (0.1 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies without affecting the secondary relaxation. Apamin also enhanced the rebound contraction at a frequency of 1 Hz. L-NOARG (30 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies, had no effect on the rebound contraction and inhibited the secondary relaxation evoked at frequencies of 1–5 Hz, but not 10–20 Hz. L-arginine (300 mol/l) reversed the effect of L-NOARG on tone and the inhibitory effect on the EFS-evoked relaxation. In the presence of apamin and L-NOARG, the primary relaxation was suppressed at all frequencies; the secondary relaxation was inhibited at 1–5 Hz and unchanged at 10–20 Hz, as observed with L-NOARG alone. We conclude that three distinct mechanisms mediate the NANC relaxation of the circular muscle of the proximal colon of the guinea-pig in response to EFS. One mechanism can be operationally defined as apamin-sensitive and a second as L-NOARG-sensitive, the latter implying a possible role of NO as an inhibitory transmitter. A third NANC inhibitory mechanism, which is apamin- and L-NOARG-resistant, is also suggested.Correspondence to: C. A. Maggi at the above address     

Further studies on the action of 5-hydroxytryptamine on lumbar motoneurones in the rat isolated spinal cord

Summary Using the hemisected spinal cord of the neonate rat, the effects of altered external Ca, thyrotrophin-releasing hormone (TRH) and a number of antagonists were tested on depolarizations evoked by 5-hydroxytryptamine (5-HT). Responses of populations of motoneurones were recorded via a ventral root. 5-Hydroxytryptamine depolarizations were not Ca-dependent but were enhanced in amplitude in Ca-free solutions. Raised Mg reversed this enhancement. 5-Hydroxytryptamine depolarizations persisted in the presence of Mn (1.53 mmol/l). TRH depolarized motoneurones; there was no evidence of modulation of 5-HT responses on concurrent application of TRH. Ritanserin (0.1 mol/l) had a modest blocking action on 5-hydroxytryptamine depolarizations reducing the maximum; 1mol/l ritanserin caused a greater antagonism which was unsurmountable (pIC₅₀ 5.2). Ritanserin (0.1 or 1 mol/l) did not depress responses to noradrenaline (NA). Ketanserin (0.1 mol/l) caused a blockade of slow onset, equilibrium with the receptors requiring 1 h. Blockade by 0.01, 0.1 and 1 mol/l ketanserin was concentration-dependent (pIC₅₀ 6.2). Ketanserin 1 mol/l, but not at lower concentrations, depressed noradrenaline responses. Mianserin (0.1 mol/l) also caused a blockade of slow onset; 0.1 or 1 mol/l produced a flattening of the 5-hydroxytryptamine concentration-response curve but did not depress noradrenaline responses (pIC₅₀ 4.7). The pIC₅₀ for spiperone was 8.0. DOI (10–100 mol/l) had no detectable agonist action but at concentrations of 0.01 and 0.1 mol/l it acted as an antagonist. Equilibration with the receptors occurred over 2 h. DOI (0.01 mol/l) depressed 5-hydroxytryptamine but not noradrenaline responses; higher concentrations of DOI also depressed noradrenaline responses. The pharmacological profile of the 5-hydroxytryptamine receptor mediating depolarization of spinal and facial motoneurones suggests that it belongs to the 5-HT_1C-5-HT₂, group of 5-hydroxytryptamine receptors but is not identical to 5-HT_1C or the 5-HT₂ CNS binding sites. Alternatively, the response might arise from a mixed population of 5-HT₁-like and 5-HT₂ receptors. Send offprint requests to D. I. Wallis at the above address     

Identification of the neuroeffector transmitter in jejunal branches of the rabbit mesenteric artery

Summary Vasoconstriction or excitatory junction potentials (e.j.ps) evoked by nerve stimulation (15 field pulses at 2 Hz every 3 min) were recorded in rabbit isolated jejunal arteries. The resting diameter of the arteries and its decrease in response to stimulation was measured by a photoelectric method. Vasoconstriction was insensitive to prazosin 0.1 or 1 mol/l. Yohimbine 1 mol/l considerably enhanced, whereas ,-methylene ATP (,-meATP) 1 mol/l abolished the contractile response. In order to test the effect of exogenously applied transmitter candidates, noradrenaline (0.1–1 mol/l) and ATP (10–30 mol/l) were added in concentrations which evoked a vasoconstriction comparable to that induced by electrical stimulation. The action of noradrenaline was prevented by prazosin 0.1 mol/l, but was unaffected by both yohimbine 1 mol/l and ,-meATP 1 mol/l. ,-meATP 1 mol/l depressed the effect of ATP. The e.j.ps evoked by a train of 15 pulses showed facilitation up to the third response and thereafter depression; a partial summation was also observed. Prazosin 0.1 mol/l did not change the e j.p. amplitudes. By contrast, when yohimbine 0.1 or 1 mol/l was added to the prazosin-containing medium, both the late e j.ps in the train and the summation were enhanced in a concentration-dependent manner. ,-meATP 1 mol/l almost abolished the e.j.ps. In conclusion, in rabbit jejunal arteries, stimulation of postganglionic sympathetic nerves may release noradrenaline together with ATP which is probably the sole neuroeffector transmitter under our conditions. Transmitter release seems to be modulated by the activation of presynaptic ₂-adrenoceptors. Under the stimulation conditions of the present experiments the released transmitter does not activate postsynaptic ₁-adrenoceptors. Send offprint requests to P. Illes     

Changes in the excretion of endogenous glycine conjugate as a possible artifact in toxicological experiments

Changes in the urinary excretion of hippuric acid (HIA) and phenaceturic acid (PUA) as well as their metabolic precursors, i.e. benzoic (BA) and phenylacetic acid (PAA), in rats housed in glass metabolic cages for 4 days were monitored using gas-liquid chromatography. The amount of HIA excreted was 128±63 mol/kg for female and 79±43 mol/kg for male rats in the first 24 h and decreased to 11±7 mol/kg (p< 0.01) for female and 3.2±2.4 mol/kg (p< 0.001) for male rats on the 2nd day. These values remained nearly at the same level until the end of the experiment. The amount of PUA decreased from 48±12 mol/kg on the 1st day to 22±9 mol/kg (p< 0.05) on the 2nd day by male rats, whereas by the females the decrease from 30±9 mol/kg to 21±8 mol/kg was not significant. The decrease in the excretion of glycine conjugates was compensated by a parallel increase in the level of unconjugated BA and PAA.     

Effect of phorbol esters and polymyxin B on modulation of noradrenaline release in mouse atria

Summary Phorbol 12-myristate 13-acetate (PMA; 0.03, 0.1 and 1.0 mol/l), a protein kinase C activating phorbol ester, significantly enhanced the stimulation-induced (S-I) outflow of radioactivity at 5 Hz stimulation in mouse atria preincubated with [³H]-noradrenaline, whereas a phorbol ester which does not activate protein kinase C, phorbol 13-acetate (0.1 mol/l), had no effect. This suggests that protein kinase C may have a role in modulating sympathetic neurotransmission.Polymyxin B (7 and 21 mol/l), an inhibitor of protein kinase C, had no effect on the S-I outflow of radioactivity. However, it had a significant inhibitory effect in a concentration of 70 mol/l. Polymyxin B (21 mol/l) reduced the facilitation of the S-I outflow of radioactivity produced by PMA (0.03 mol/l), 8-bromo-cyclic AMP (90 mol/l), tetraethylammonium chloride (300 mol/l), and idazoxan (0.1 mol/l). Furthermore, when a higher frequency of stimulation was applied (10 Hz rather than 5 Hz), polymyxin B (21 pmol/1) by itself inhibited the S-I outflow of radioactivity.In the presence of a concentration of PMA (0.1 mol/l) that was maximally effective in enhancing the S-I outflow of radioactivity, both idazoxan (0.1 mol/l) and 8-bromocyclic AMP (90 mol/l) still enhanced the S-I outflow. This suggests that these agents are not operating through protein kinase C and further suggests that the inhibitory effect of polymyxin B on these agents cannot be due to inhibition of protein kinase C. The effects of clonidine on the S-I outflow were not affected by a maximally effective concentration of PMA (0.1 mol/l). These results suggest that protein kinase C is not involved in a ₂-adrenoceptor mediated modulation of noradrenaline release. Send offprint requests to I. F. Musgrave at the above address     

Evidence for M1 muscarinic cholinoceptors mediating facilitation of noradrenaline release in guinea-pig carotid artery

Summary The muscarinic agonists acetylcholine (150 mol/l), carbachol (1–10 mol/l) and McN-A-343 (1–50 mol/l, selective for M₁ receptors) increased, in a concentration-dependent manner, the electrically-evoked tritium overflow from guinea-pig carotid arteries preincubated with [³H]-noradrenaline. The increase caused by acetylcholine was not modified by hexamethonium (300 mol/l) but was reduced by the muscarinic receptor antagonists methylatropinium (0.5 and 1 nmol/l, nonselective), pirenzepine (1 and 5 mol/l, M₁-selective), methoctramine (1 and 5 mol/l, M₂-selective) and pfluoro-hexahydro-sila-difenidol (0.1–1 mol/l, M₃-selective). The order of potencies (expressed as negative logarithms of concentrations that reduced by 50% the facilitatory effect of acetylcholine) was: methylatropinium (9.93) > pirenzepine (8.83) > p-fluoro-hexahydro-siladifenidol (6.81) methoctramine (6.20). These results demonstrate the existence of facilitatory M₁ receptors modulating noradrenaline release in blood vessels. Correspondence to M. Salaices at the above address     

Target sites for the inhibition of prostacyclin effect in guinea-pig ileum

Summary In the guinea-pig terminal ileum a maximally effective concentration of prostacyclin (PGI₂) (1 ol/l) induced contractions that were partially resistant to tetrodotoxin (TTX) 0.1 mol/l, to low temperature (20°C) and to atropine (30 nmol/l). Half maximum contractions evoked by PGI₂ (20 nmol/l) were abolished by TTX and by low temperature, which did not modify the response to exogenous acetylcholine (ACh), as well as by atropine. Procaine (5–500 ol/l) caused a concentration-dependent inhibition of contractions induced by PGI₂ (20 nmol/l and 1 mol/l) and by equieffective concentrations of ACh (20 nmol/l and 0.4 ol/l, respectively). The order of magnitude for this inhibition was ACh 20 nmol/l = PGI₂ 20 nmol/l > PGI₂1 mol/l > ACh 0.4 mol/l. In preparations exposed to TTX or to low temperature procaine (50 mol/l) did not affect the residual response to PGI₂ (1 mol/l). Quercetin (1 and 5 ol/l) inhibited the effect of PGI₂ and, at higher concentrations, it also caused partial depression of the responses to ACh. Quercetin did not alter TTX-resistant and low temperature-resistant contractions induced by PGI₂ 1 mol/l. Carbonyl cyanide-trifluoromethoxyphenyl hydrazone (FCCP) (0.1–1 ol/l) reduced the effect of PGI₂ and of ACh to approximately the same extent and inhibited the residual response to PGI₂ 1 mol/l in preparations treated with TTX or expressed to low temperature. The present results show that PGI₂, besides acting on cholinergic neurons, also exerts a direct effect on smooth muscle cells and FCCP can be used to block this effect. In contrast procaine and quercetin selectively inhibit the ACh-mediated component of PGI₂ action. Send offprint requests to R. M. Gaion     

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

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     

Pharmacological characterization of central α-adrenoceptors which mediate clonidine-induced locomotor hypoactivity in the developing rat

Summary The present experiment was designed to pharmacologically characterize receptors which mediate the clonidine-induced locomotor change in the developing rat. A subcutaneous injection of clonidine (0.78 mol/kg) produced locomotor hyperactivity in 7-day-old rats but hypoactivity in 20-day-old rats. Phenoxybenzamine (1.5 mol/kg, 5.9 mol/kg and 15 mol/kg) decreased spontaneous activity in a dosedependent manner but did not antagonize clonidineinduced hypoactivity in 20-day-old rats. By contrast, the significant reversal of the clonidine-induced hypoactivity by pretreatment with phentolamine (1.6 mol/kg and 6.3 mol/kg), yohimbine (1.3 mol/kg and 5.1 mol/kg) and piperoxan (7.4 mol/kg) was observed at such doses when the blockers did not cause and hypoactivity by themselves. It is suggested that clonidine could induce locomotor hypoactivity by activating presynaptic (₁-type) -adrenoceptors in the CNS of 20-day-old rat.     

Different muscarinic receptors mediate autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat isolated perfused heart

Summary Experiments were carried out on rat isolated perfused hearts with both vagus nerves attached. The acetylcholine stores were labelled with [¹⁴C]-choline. The effects of muscarinic receptor antagonists on the [14C]overflow and increase in perfusion pressure evoked by vagus nerve stimulation (10 Hz, 4–10 mA) were studied in order to determine the muscarinic receptor type involved in autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat heart.Stimulation of the vagus nerves (1200 pulses) caused an increase in [¹⁴C]-overflow and in perfusion pressure which was significantly reduced by hexamethonium 500 mol/l and abolished by tetrodotoxin 0.3 mol/l or perfusion with Ca²⁺-free solution. The fractional rate of evoked [¹⁴C]-overflow per pulse upon stimulation at 10 Hz (720 pulses) was doubled in the presence of the non-selective antagonist atropine (0.01–1 mol/l) as well as in that of the M₂-selective compounds methoctramine (0.1 mol/l) and AF-DX 116 (0.1–1 mol/l), but remained unaffected by the M3-selective hexahydrosiladifenidol (0.1 mol/l). The increase in perfusion pressure upon nerve stimulation was reduced by atropine (0.01 mol/l) or hexahydrosiladifenidol (0.1 mol/l) to approximately 50% and increased by about 50% in the presence of AF-DX 116 (0.1 mol/l).The results show that the autoinhibition of acetylcholine release in the rat heart is mediated by M₂ receptors. On the other hand, the increase in perfusion pressure upon vagus nerve stimulation is caused by a different muscarinic receptor, more sensitive to hexahydrosiladifenidol than to M₂-selective antagonists. Send offprint requests to I. T. Bognar at the above address