Stimulation of a dopamine-sensitive adenylate cyclase in homogenates of rat striatum by a metabolite of piribedil (ET 495)

Summary The addition of dopamine (1–100M) to homogenates of rat striatum incubated with ATP evoked a 120% increase in the rate of cyclic 35 adenosine monophosphate (cyclic AMP) production. The effects of added dopamine were mimicked by the addition of the compound S 584 [1-3,4-(dihydroxybenzyl)-4-(2-pyrimidinyl) piperazine] (1–100 M), a catechol metabolite of the dopaminergic stimulant drug piribedil (ET 495). The latter substance was itself inactive in this system at these concentrations. The stimulation of cyclic AMP production by dopamine and by S 584 was potently inhibited by the dopamine antagonist dru chlorpromazine and spiroperidol (1–10 M). It is possible that the dopaminergic effects elicited by piribedil may be mediated through the active metabolite S 584.     

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     

Phorbol 12,13-dibutyrate,an activator of protein kinase C,stimulates both contraction and Ca2+ fluxes in dog saphenous vein

Summary The vascular effects of phorbol 12,13-dibutyrate (PDBu) were studied in the dog saphenous vein. PDBu (1 M) caused contraction (0.58 ± 0.22 g/mg wet wt.) and Ca uptake (74.2 ± 41.2 mol/kg wet wt.) which were unaffected by 10 M phentolamine (N = 6). The PDBu-induced contraction was greatly (60–80%) inhibited in Ca²⁺-free solution. 15 Ca efflux measurements performed in Ca²⁺-free solution showed that PDBu did not cause Ca release from intracellular storage sites. The contractile response to PDBu (1 nM-1 M) was significantly correlated with the magnitude of Ca uptake; contraction and the rise in tissue Ca²⁺ also had a similar time course. Correlation between the two measures persisted when the responses to PDBu were augmented by co-administration with 20 mM KCl. However, no synergism occurred between the two agonists. Both the contraction and Ca uptake responses to PDBu were reduced by nifedipine and verapamil, each at 1 M. In the Triton X-100 skinned saphenous vein, where the voltage-dependent Ca channel is not functional, 10 M PDBu did not cause contractions in the presence of 0.1 M Ca²⁺. Thus, contraction of the intact saphenous vein by PDBu characteristically exhibits great Ca dependence and PDBu seems to activate the voltage-dependent Ca channel, presumably through stimulation of protein kinase C; the ensuing Ca entry is primarily responsible for contraction. However, the mechanism responsible for the PDBu-induced contractions that are resistant to Ca²⁺-free PSS or Ca entry blockers remains to be defined. It appears that the dog saphenous vein differs from dog femoral artery, rabbit aorta and pig carotid artery where PDBu contractions do not display dependence on external Ca²⁺. Send offprint requests to P. J. S. Chin at the above address     

Differential effects of potassium channel blockers on neurohypophysial release of oxytocin and vasopressin. Evidence for frequency-dependent interaction with the endogenous opioid inhibition of oxytocin release

Summary Isolated rat neurohypophyses were fixed by their stalks to a platinum wire electrode and superfused with Krebs-HEPES solution. Vasopressin and oxytocin released into the medium were determined by specific radioimmunoassays. Hormone secretion was increased by electrical stimulation of the pituitary stalk at different frequencies. The effects of several potassium channel blockers, tetraethylammonium (TEA) ions, 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) were tested.The release of vasopressin and oxytocin evoked by electrical stimulation with 900 pulses at 15 Hz (about 900 and 1,000 U, respectively) was about 10 times higher than that evoked by 900 pulses at 3 Hz. Both 10 and 30 mmol/l TEA enhanced the release of vasopressin evoked by stimulation at 3 and 15 Hz, by 25- and 2-fold, respectively, to attain a maximum release of about 1,800 U per stimulation. The stimulated release of oxytocin attained a maximum of about 9,000 U at 15 Hz in the presence of 10 mmol/l TEA or at 3 Hz with 30 mmol/l TEA. Thus, in the presence of maximally effective concentrations of TEA both stimulation frequencies (3 and 15 Hz) were equieffective in evoking release of vasopressin and oxytocin. 4-AP or 3,4-DAP enhanced the release of vasopressin evoked by 15 Hz stimulation maximally to about 1,600 U and that evoked by 3 Hz stimulation to about 900 U. In the presence of 4-AP or 3,4-DAP the release of oxytocin evoked by stimulation at 15 Hz increased maximally to about 8,000 U and that evoked by stimulation at 3 Hz to about 1,500 U. Thus, in the presence of maximally effective concentrations of 4-AP or 3,4-DAP stimulation at 15 Hz induced a significantly higher release of vasopressin and oxytocin than stimulation at 3 Hz. Naloxone (1 mol/l) increased the release of oxytocin evoked by stimulation at 15 Hz to about 3,000 U and that evoked by stimulation at 3 Hz to about 700 U. The release of oxytocin evoked by stimulation at 15 Hz in the presence of 10 mmol/l TEA or 1 mmol/l 4-AP (about 8,000–9,000 U) was not further enhanced by naloxone. However, during stimulation at 1 or 3 Hz in the presence of 10 mmol/l TEA, naloxone increased the release of oxytocin from about 3,700 and 6,300 U, respectively, to the maximum of about 9,000 U. Likewise, during stimulation at 3 Hz in the presence of 1 mmol/14-AP, naloxone increased the relase of oxytocin from about 1,500 to 9,000 U. Under all condition studied, naloxone did not affect the release of vasopressin.In conclusion, neurosecretory nerve endings are endowed with different types of potassium channels. Blockade of potassium channels can oppose the opioid inhibition of oxytocin release in a complex frequency-dependent manner.Abbreviations 4-AP 4-aminopyridine - 3,4-DAP 3,4-diaminopyridine - TEA tetraethylammonium ions Send offprint requests to. K. Racké at the above address     

Differences in the prejunctional effects of methacholine and pilocarpine on the release of endogenous acetylcholine from guinea-pig trachea

We investigated the effects of the full muscarinic acetylcholine receptor agonist methacholine and the partial and putatively M₂-selective agonist pilocarpine on endogenous acetylcholine release from guinea-pig trachea by use of high-performance liquid chromatography with electrochemical detection. Atropine-induced increases in acetylcholine release were used to monitor the system.Electrical field stimulation (8 V, 30 Hz, 0.5 ms for 5 min)-induced acetylcholine release in the presence of neostigmine, with or without preincubation with choline to maximally enhance acetylcholine output, was increased to about 225% by 0.3 M atropine, indicating functional autoinhibition. However, methacholine (10 M) did not affect the acetylcholine release, whereas it was enhanced to 166% by 30 M pilocarpine. When electrical field stimulation was applied at lower intensity (8 V, 16 Hz, 0.1 ms for 5 min) and in the absence of neostigmine, an increase by 0.3 M atropine (to 177%) but a decrease of the acetylcholine release by 10 M methacholine (to 65%) and 30 M pilocarpine (to 63%) were observed. These results clearly demonstrate (i) that inhibition of evoked endogenous acetylcholine release from prejunctional nerve terminals in guinea-pig trachea can only be demonstrated under conditions of low junctional concentrations of acetylcholine, and (ii) that pilocarpine, as a partial muscarinic agonist, behaves as an antagonist under high junctional concentrations of the neurotransmitter.     

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [³H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 M and even more so by prazosin 0.3 M combined with suramin 300 M. They were also reduced by nifedipine 10 M and even more so by nifedipine 10 M combined with ryanodine 20 M (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [³H]-noradrenaline, exogenous noradrenaline 10 M and ,-methylene ATP 10 M elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 M but not suramin 300 M and were greatly reduced by nifedipine 10 M and in Ca²⁺-free medium. Responses to ,-methylene ATP were blocked by suramin 300 M but not prazosin 0.3 M were reduced by nifedipine 10 M (effect on ATP overflow not significant) and were reduced even more in Ca²⁺-free medium. Neuropeptide Y 0.3 M caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both ₁-adrenoceptor and P₂-purinoceptor activation. Ca²⁺ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A purely neural overflow of ATP was not isolated under the conditions of the experiments. Correspondence to: R. Bültmann at the above address     

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.     

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     

Methoxamine enhances the release of endogenous noradrenaline from rabbit ear artery: possible involvement of ATP

Summary The effect of methoxamine, an ₁-adrenoceptor agonist, on the electrically-evoked release of endogenous noradrenaline was examined in the isolated rabbit ear artery. Noradrenaline was quantified by high performance liquid chromatography-electrochemical detection. The release of adenine nucleotides and nucleosides by methoxamine was examined using high performance liquid chromatography-fluorescence detection.The release of noradrenaline evoked by electrical field stimulation (EFS) at 4 Hz was reduced by tetrodotoxin 0.3 mol/l and clonidine 1 mol/l by approximately 80% and 50%, respectively. On the other hand, methoxamine at 10 but not 1 mol/l enhanced the release of noradrenaline to approximately twice the control, and the enhancement was prevented by prazosin 1 mol/l. The facilitatory action of methoxamine was also abolished after desensitization of P₂-purinoceptors by ,-methylene ATP 30 mol/l as well as by the presumed P₂-purinoceptor antagonist suramin given at 10 mol/l. Exogenous ATP 10 mol/l significantly enhanced the EFS-evoked release of noradrenaline, and the enhancement was abolished by ,-methylene ATP and suramin. None of the drugs changed the spontaneous outflow of noradrenaline. These results indicate that endogenous ATP, acting at prejunctional purinoceptors, may participate in the facilitatory effect of methoxamine. Indeed, methoxamine 10 mol/l significantly enhanced the spontaneous outflow of ATP and, less so, ADP. The methoxamine evoked release of ATP and ADP was antagonized by prazosin 1 mol/l.It is concluded that methoxamine releases endogenous ATP from postjunctional sites which then, via prejunctional purinoceptors, facilitates action potential-evoked release of noradrenaline in rabbit ear artery.Supported by grants from the Mita Research Foundation, Matsue, Japan and Kanae Research Foundation, Osaka, JapanCorrespondence to K. Takeuchi at the above address     

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.     

Diethylstilbestrol potentiation of the dopamine-elicited formation of adenosine 3′,5′-monophosphate in incubated male rat hypothalamus

Summary Dopamine (DA) stimulates the cAMP-generating system in the male rat hypothalamus only to a very low extent (25% above control). Diethylstilbestrol (DES), a synthetic estrogen, was found to be extremely potent (a 4- and 16-fold stimulation at 20 M and 100 M, respectively). Addition of either one to an incubation medium containing varying concentrations of the other resulted in a synergistic response. The potentiation by 20 M DES of the effect elicited by 100 M DA was the most remarkable, namely, a 3-fold stimulation of the combined response. A 4- and 7.5-fold stimulation of cAMP accumulation was observed when adenosine (100 M) or adenosine (100 M)+DA (100 M) were present in the incubation medium. Theophylline (0.5 mM), an adenosine antagonist, could effectively reduce this effect, as did adenosine deaminase (10 g/ml). Clomiphene (50 M), an estrogen antagonist, exhibited a marked decrease in DES+DA-elicited cAMP formation. Pimozide (40 M) had the ability to significantly block the stimulatory effects of DES and DA.     

Human isolated ileum: motor responses of the circular muscle to electrical field stimulation and exogenous neuropeptides

Summary (1) Circularly-oriented muscle strips from the human ileum responded to electrical field stimulation (1–50 Hz) with frequency-related primary relaxation at low frequency and primary contractions at high frequencies of stimulation. Both responses were abolished or markedly reduced by tetrodotoxin (1 M). (2) Atropine (3 M) or omega conotoxin (0.1 M) reduced but dit not abolish contraction to electrical field stimulation and enhanced the relaxation. Omega conotoxin (0.1 M) did not affect carbachol-induced contraction nor isoprenaline-induced relaxation. (3) Neurokinin A and substance P (1 nM-1 M) produced a concentration-dependent contraction. The NK-1 receptor selective agonist, [Pro⁹]SP sulfone and the NK-2 receptor selective agonist [Ala⁸]NKA(4-10) prodneed a contraction superimposable to that of substance P and neurokinin A, respectively. On the other hand, [MePhe⁷]-neurokinin B, an NK-3 receptor selective agonist was ineffective up to 1 [M. The response to substance P or neurokinin A was unaffected by atropine (3 M). (4) Galanin, up to 0.1 M produced a weak and inconsistent contraction. (5) Vasoactive intestinal polypeptide (10 nM - 1 M) produced a concentration-dependent relaxation while human alpha calcitonin gene-related peptide exerted a weak and inconsistent relaxant effect. (6) These findings indicate that both cholinergic excitatory and non-cholinergic inhibitory nerves affect the motility of the circular muscle of the human small intestine. Transmitter release from excitatory nerves seems largely mediated by activation of omega conotoxin-sensitive (N-type) calcium channels. Tachykinins exert a potent contractile effect independently of cholinergic nerves via NK-1 and NK-2 receptors. Send offprint requests to C. A. Maggi at the above address     

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

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

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     

Role of dopamine receptors in the modulation of acetylcholine release in the rabbit hippocampus

Summary Modulation of acetylcholine release was studied in slices of the rabbit hippocampus preincubated with ³H-choline and then continuously superfused with a medium containing 10 mol/l hemicholinium-3. Electrical field stimulation of the superfused slices elicited an increase in tritium outflow, which was tetrodotoxin-sensitive and largely calcium-dependent. Stimulus-evoked acetylcholine release in the rabbit hippocampal slices was modulated by presynaptic muscarinic autoreceptors, as has been shown previously for the rat hippocampus. Drugs with affinity for - and or -adrenoceptors did not affect the evoked overflow of tritium from rabbit hippocampal slices. In contrast, the dopamine receptor agonist apomorphine (0.1 or 1 mol/l) and exogenous dopamine (1 or 10 mol/l) significantly reduced the evoked outflow by about 10 or 20%, respectively. This effect was antagonized by haloperidol (0.01 mol/l) but not by phentolamine (1 mol/l). Attempts to enhance (using nomifensine 10 mol/l) or reduce (using haloperidol, up to 1 mol/l; or bretylium, 1 mmol/l for 5 min) endogenous dopaminergic transmission in the hippocampal slices did not affect stimulation evoked acetylcholine release. In conclusion, presynaptic dopamine receptors modulating acetylcholine release are present in the rabbit hippocampus, but they seem not to be of physiological significance.     

Antagonism by γ-aminobutyric acid of the stimulant effect of angiotensin II on cardiac sympathetic ganglia in spinal dogs

Summary The effects of angiotensin II and neuro-aminoacids administered through the right subclavian artery (i. a.) to the cardiac sympathetic ganglia were investigated in spinal dogs. Angiotensin II (1–8 g) elicited a dose-dependent positive chronotropic effect which was reduced after i. a. injection of saralasin (100g). The effect of angiotensin II was not reduced after combined treatment with either hexamethonium (10 mg/kg) plus atropine (0.1 mg/kg) or hemicholinium-3 (5 mg/kg) plus preganglionic stimulation. The dosedependent response to angiotensin II of heart rate was inhibited by GABA (50, 500g), GABOB (500g) and muscimol (50, 100g). The inhibition of the response to angiotensin II by a small dose of GABA (50g), but not by a high one (500g), was antagonized by i. a. injection of picrotoxin (2 mg). The positive chronotropism induced by bethanechol (25, 50g) and a small dose of acetylcholine (25g) were significantly inhibited by a high dose (500g) but not by a low dose (50g) of GABA. These results confirm that angiotensin II stimulates cardiac chronotropism by acting on the angiotensin II receptor located at the cardiac ganglia and show that this stimulant effect is antagonized by GABA.     

Activation of β2-adrenoceptors by isoprenaline and adrenaline enhances noradrenaline release in cortical kidney slices of young spontaneously hypertensive rats

Summary The aim of the present study was to investigate -adrenoceptor modulation of noradrenaline release from sympathetic nerves in superfused cortical kidney slices of 4-week-old spontaneously hypertensive rats (SHR) and age-matched controls (WKY). After preincubation with ³H-noradrenaline the kidney slices were electrically stimulated in superfusion chambers. The stimulation induced (S-I) outflow of radioactivity was mainly composed of unmetabolized 3H-noradrenaline in both strains and thus taken as an index of noradrenaline release. There was a frequency-dependent (1.25–20 Hz) increase in the S-1 outflow of radioactivity. At all stimulation frequencies tested S-I outflow of radioactivity was similar or even slightly lower in SHR than in WKY kidney slices in either the absence or presence of cocaine (10 mol/l). The non-selective -adrenoceptor agonists isoprenaline (0.l gmol/1) and adrenaline (0.01 and 0.1 mol/l) enhanced S-I outflow of radioactivity. The facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) were blocked by the selective ₂-adrenoceptor antagonist ICI 118551 (0.1 mol/l) but not by the selective ₁-adrenoceptor antagonist atenolol (0.3 mol/l). The cell-permeable CAMP analogue 8-bromo-cAMP (300 mol/l) enhanced S-1 outflow of radioactivity to a similar extent in both SHR and WKY kidney slices. A combination of 8-bromo-cAMP (300 mol/l) and adrenaline (0.1 mol/l) did not enhance S-1 outflow of radioactivity to a greater extent than 8-bromo cAMP (300 mol/l) alone in both strains. However, the facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) but not that of adrenaline (0.01 mol/l) were significantly greater in SHR than in WKY. The results suggest that stimulation of prejunctional ₂-adrenoceptors by adrenaline even in the absence of a-adrenoceptor blockade enhances noradrenaline release in kidney cortex of young SHR and WKY. This ₂-adrenoceptor mediated effect may possibly be dependent on cAMP formation. The greater facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) in SHR as compared to WKY are in accord with receptor binding studies which show a higher density of ₂-adrenoceptors in SHR than in WKY kidney cortex.Abbreviations SHR Spontaneously hypertensive rats - WKY WistarKyoto rats - cAMP 3-5-cyclic adenosine monophosphate - S-I stimulation induced Send offprint requests to: L. C. Rump     

Effect of nicotine and tacrine on acetylcholine release from rat cerebral cortical slices

Summary The effect of nicotine (1–10 M) and tacrine (9-amino-1,2,3,4-tetrahydroacridine; THA) on stimulation evoked release of [³H]acetylcholine from the rat brain slice preparation preincubated with [³H]choline was investigated.In these preparations, nicotine enhanced while tacrine inhibited evoked [³H]acetylcholine release. These effects were blocked by (+)tubocurarine (1 M) and atropine (0.1 M) respectively. In the presence of idazoxan (0.3 M) plus atropine (0.1 M), nicotine (3 M) continued to enhance evoked [³H]acetylcholine release while the inhibitory effect of tacrine (1 M) on evoked [³H]acetylcholine release was reversed to an enhancement. Under these circumstances the effects of both nicotine and tacrine were blocked by (+)tubocurarine (1 M).These findings demonstrate that tacrine can both inhibit or enhance [³H]acetylcholine release, most likely through its activity as a cholinesterase inhibitor. Under normal circumstances following tacrine the predominant effect of the elevated levels of acetylcholine will be activation of inhibitory presynaptic muscarine receptors on cholinergic nerves and an inhibition of evoked [³H]acetylcholine release. Under conditions where both presynaptic inhibitory muscarine and ₂-adrenoceptors are blocked, the elevated levels of acetylcholine produced by tacrine will lead to the activation of facilitatory presynaptic nicotine cholinoceptors on cholinergic nerves and an enhancement of evoked [³H]acetylcholine release. Send offprint requests to R. Loiacono at the above address     

Propagation of impulses in the guinea-pig ureter and its blockade by calcitonin gene-related peptide (CGRP)

The guinea-pig ureter was placed in a three-compartment organ bath to enable the application of electrical stimuli or drugs to its renal end (R site), the middle region (M-site) or the bladder end (B-site) while recording mechanical activity at the R- and B-sites. All experiments were performed in ureters pre-exposed to capsaicin (10 M for 15 min) to prevent the release of sensory neuropeptides from afferent nerves. Electrical field stimulation (EFS, 5–25 ms pulse width, 20 V) produced a phasic contraction at the site of stimulation (direct response to EFS) which propagated to the other end of the ureter. Section of the ureter at the M-site abolished the propagated response to EFS; after section, EFS applied at the M-site induced a phasic contraction at both the R-and B-sites. Likewise, the application of KCl at the M-site produced phasic contractions at both the R- and B-sites. Tetrodotoxin (1 M), nifedipine (1 M) or Bay K 8644 (1 M) applied at the M-site had no influence on the direct or propagated responses to EFS; nifedipine (10 M) applied at the M-site abolished the propagated responses without affecting the direct responses to EFS. Bay K 8644 (1 M) applied at the R-site produced a marked enhancement of the direct response (EFS applied at R-site) while having no effect on the amplitude of the propagated response to EFS. Nifedipine (1 M), applied at the R-site, produced a graded, time-dependent, inhibition of the direct response (EFS applied at R-site) and eventually suppressed it; the propagated response was unaffected until suppression of the direct response, when an allor-none blockade of the propagated response was observed. When applied at the B-site (EFS at Rsite), 1 M nifedipine produced a graded, time-dependent, inhibition of the propagated response and eventually suppressed it, without affecting the direct response to EFS. For further pharmacological analysis of drug action on the propagated activity, EFS was applied at the R-site and drugs were applied at the M-site. Human CGRP (CGRP, 0.1 M) or cromakalim (1-3 M) were applied in superfusion at the M-site in the absence or presence of glibenclamide (1 M). Neither drug affected the direct response to EFS; both CGRP and cromakalim produced a reversible suppression of the propagated response. Glibenclamide suppressed the inhibitory activity of 1 M cromakalim and partly antagonized the effect of CGRP; the blockade by glibenclamide was partly overcome by 3 M cromakalim. The present findings are consistent with the idea that propagation of excitation occurs because of the spread of electrical activity between smooth muscle cells of the ureter and that conduction of impulses is independent of local changes in contractility. The present results also demonstrate that CGRP induced a conduction block along the ureter and that this effect involves activation of glibenclamide-sensitive K channels. Therefore, a local release of CGRP in response to pathophysiological stimuli is, in principle, capable of suppressing ureteral peristalsis and antiperistalsis.     

Possible involvement of presynaptic α1-adrenoceptors in the effects of idazoxan and prazosin on 3H-noradrenaline release from tail arteries of SHR

Summary The effects of several -adrenoceptor antagonists have been examined on tritium release elicited by electrical stimulation from isolated perfused SHR tail artery preparations prelabelled with ³H-noradrenaline (³H-NA). Phentolamine and yohimbine potently facilitated the stimulation evoked release of tritium at low frequencies of stimulation, but the ₂-adrenoceptor antagonist idazoxan was only weakly active at 1 mol/l, despite antagonising the clonidine-evoked inhibition of ³H-release at a lower concentration of 0.1 mol/l. The ₁-adrenoceptor antagonists prazosin and corynanthine also increased stimulation evoked tritium release in this preparation, suggesting the presence of prejunctional ₁-adrenoceptors. Furthermore, the ₁-adrenoceptor agonist methoxamine (3 mol/l) caused a significant inhibition of tritium-evoked release, an effect which was blocked by prazosin (10 nmol/l).When ₁-adrenoceptors were blocked in the presence of prazosin, idazoxan (0.1 mol/l) produced a significant facilitatory effect on the electrically-evoked release of ³H-transmitter. On the other hand, when ₂-adrenoceptors were blocked in the presence of yohimbine, exposure to idazoxan (0.1 mol/l) reduced significantly the stimulation-evoked release of tritium elicited by electrical stimulation.The results indicate that in the SHR tail arteries, idazoxan has a partial agonist inhibitory activity on transmitter release, which can mask the facilitatory effects due to blockade of presynaptic ₂-adrenoceptors. The inhibitory effects of idazoxan appear to involve presynaptic ₂-adrenoceptors, which when stimulated, reduce ³H-NA release in SHR tail arteries.