Effects of clonidine on the stimulation-evoked release of3H-noradrenaline from superfused rat brain slices as a function of the biophase concentration

The influence of clonidine on the stimulation-evoked overflow of tritium was studied in brain slices preincubated with 3H-noradrenaline. The slices were prepared from parietal cortex (Cx), nucleus anterior hypothalami (nah) and nucleus tractus solitarii (nts). After preincubation, the tissues were superfused at 23 degrees C or 37 degrees C with a medium containing the noradrenaline uptake inhibitor desipramine. Electrical field stimulation was applied using stimulation frequencies of 0.3-10 Hz. At 23 degrees C/0.3 Hz, clonidine concentration-dependently inhibited the evoked overflow of tritium in all three brain regions. In contrast, at 23 degrees C/3 Hz the inhibitory effect of the drug in the Cx was abolished and a facilitation was observed in the nah and nts. When tested at increasing frequencies of stimulation in the nts at 23 degrees C, clonidine exerted a dual action, characterized by a reduction of electrically evoked responses at frequencies below 1 Hz and a facilitation at frequencies above 1 Hz. At 37 degrees C, clonidine concentration-dependently decreased the evoked overflow in all brain regions studied, this effect being more pronounced at 0.3 Hz than at 3 Hz. The apparent lack of an effect of clonidine on the stimulation-evoked overflow of tritium in the Cx at 23 degrees C/3 Hz was turned to a facilitation when noradrenaline (0.01 mumol/l) was included in the superfusion medium. Conversely, an inhibitory effect of clonidine was seen when the uptake blocker desipramine (as well as noradrenaline) was omitted from the superfusion medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester,an activator of protein kinase C,enhances calcium-dependent release of sympathetic neurotransmitter

Summary The effect of phorbol ester, phorbol 12,13-dibutyrate, was investigated on the overflow of tritium from ³H-noradrenaline-loaded sympathetic neurons of the isolated perfused salivary gland of the rat. Stimulation (1 Hz for 60 s)-evoked overflow of tritium was enhanced by phorbol ester. A significant enhancement was seen at 1 nmol/l, which increased to a maximum level (over 4-fold) at 30 nmol/l. The spontaneous overflow of radioactivity, however, was not affected by any concentration of phorbol ester. The facilitatory effect of phorbol ester on stimulation-evoked overflow was observed in the presence of inhibitors of neuronal and extraneuronal uptake as well as after removal of negative feedback inhibition of release by presynaptic alpha-adrenoceptors. Tyramine (7 mol/l for 10 min) caused a marked increase in the overflow of tritium in either the presence or absence of calcium. However, tyramine-induced overflow was not enhanced by phorbol ester. It is concluded that protein kinase C of sympathetic neurons is involved in an exocytotic release of the transmitter.     

Role of cyclic AMP in the prejunctional α2-adrenoceptor modulation of noradrenaline release from the rat tail artery

Summary Experiments were designed to evaluate the effect of cyclic AMP on the electrically-induced release of noradrenaline from vascular sympathetic nerve terminals. The possible implication of the inhibition of adenylate cyclase in the negative feed-back control by prejunctional α₂-adrenoceptors of neurotransmitter release was also investigated. Rat isolated tail arteries were preincubated with [³H]-noradrenaline; the preparations were subsequently perfused/superfused with [³H]-noradrenaline-free medium and their perivascular nerves were field stimulated with 24 pulses at 0.4 Hz (0.3 ms, 200 mA). 2 compounds known to enhance the intracellular concentration of cyclic AMP, namely the membrane permeant analogue 8-Br-cAMP (10–300 μmol/l) and forskolin (0.3–10 μmol/l), an activator of adenylate cyclase, concentration-dependently enhanced the stimulation-evoked tritium overflow. The 1,9-dideoxy derivative of forskolin, which does not stimulate adenylate cyclase, was ineffective. Exposure to the cyclic AMP phosphodiesterase inhibitor rolipram 30 μmol/l produced a moderate increase (about 20%) in tritium overflow. However, in the presence of rolipram the facilitatory effect of forskolin was significantly more pronounced than in its absence. Whereas 8-Br-cAMP produced a slight concentration-dependent enhancement of the stimulation-induced vasoconstriction, forskolin and rolipram depressed it. The α₂-adrenoceptor agonist B-HT 933 (3–30 μmol/l) concentration-dependently inhibited the tritium overflow. The effect of B-HT 933 30 μmol/l was slightly, but significantly reduced in the presence of 8-Br-cAMP 100 and 300 μmol/l, but was not changed in the presence of forskolin 3 μmol/l The facilitatory effect of rauwolscine 1 μmol/l was enhanced in the presence of 8-Br-cAMP 100 μmol/l. During perfusion with 8-Br-cAMP 100 μmol/l, the current strength and frequency were decreased to 150 mA and 0.2 Hz, respectively in order to obtain similar amounts of tritium overflow to those observed in the absence of the cyclic AMP analogue with the initial stimulation parameters. Under these conditions, the inhibition of the overflow by B-HT 933 30 μmol/l and the facilitation by the α₂-adrenoceptor antagonist rauwolscine 1 μmol/l were unaltered as compared to controls under initial stimulation conditions. It is concluded that, in the rat tail artery, the terminals of perivascular sympathetic nerves are endowed with an adenylate cyclase system. Cyclic AMP is able to modulate noradrenaline release, but does not appear to play a role in the initiation of the release process itself. In addition, the results do not support the hypothesis that prejunctional α₂-adrenoceptors depress noradrenaline release through the inhibition of adenylate cyclase. Send offprint requests to B. Bucher at the above address     

Interaction between presynaptic facilitatory angiotensin II receptors and inhibitory muscarinic cholinoceptors on3H-noradrenaline release in the rabbit heart

Summary The existence of a functional interaction between presynaptic receptors modulating the release of noradrenaline was studied in the rabbit heart. Isolated right atria were prelabelled with³H-noradrenaline and the overflow of tritium was induced by field stimulation (2 Hz, 0.1 ms duration, supramaximal voltage for a total of 180 pulses). In atria superfused with Krebs' solution containing 10 mol/l cocaine and 30 mol/l corticosterone, angiotensin II (10 nmol/l) increased the stimulation-evoked overflow of³H-transmitter by 2.8-fold. The addition of atropine (0.3 mol/l) to the perfusion medium, either in the presence or in the absence of uptake inhibitors, further enhanced the facilitatory effect of angiotension II (³H-transmitter release increased by 3.5-fold). Exposure to 1 mol/l carbachol decreased by 65% the stimulation-evoked release of³H-transmitter while the facilitatory effect of angiotensin II determined in the presence of the muscarinic cholinoceptor agonist was enhanced (³H-transmitter release increased by 6.6-fold). Conversely, during sustained activation of presynaptic angiotensin receptors producing a 2.5-fold increase in the release of³H-transmitter, the inhibitory effect of carbachol remained unchanged. These results suggest a functional interaction between presynaptic inhibitory muscarinic cholinoceptors and the presynaptic facilitatory angiotensin receptor which modulate the release of noradrenaline from cardiac noradrenergic nerves.     

Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery

1. Interactions between A(2A)-adenosine receptors and alpha(2)-, A(1)- and P2- release-inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [(3)H]-noradrenaline, superfused with desipramine-containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz). 2. Blockade of alpha(2)-autoreceptors with yohimbine (1 microM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz. 3. The selective A(2A)-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 1-100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA; 100 nM) or by the P2-receptor agonist 2-methylthioadenosine triphosphate (2-MeSATP; 80 microM). 4. CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The alpha(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304; 30 nM), the A(1)-receptor agonist CPA (100 nM) or the P2-receptor agonist 2-MeSATP (80 microM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow. 5. Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow. 6. It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A(2A)-adenosine receptors is favoured by activation of release inhibitory receptors.     

Evidence for beta 2-adrenoceptor-mediated facilitation of 3H-noradrenaline release from isolated guinea pig papillary muscles

The effects of beta-adrenoceptor agonists and antagonists on field-stimulated release of radioactivity from superfused guinea pig papillary muscles preincubated with 3H-noradrenaline were studied. Stimulation-evoked overflow of tritium was abolished in the absence of Ca2+ or the presence of tetrodotoxin. Isoprenaline (1 mumol/L) caused a slight facilitation of evoked overflow, whereas phentolamine (1 mumol/L) exerted a strong facilitatory action. However, when phentolamine (1 mumol/L) was present throughout superfusion, isoprenaline and the selective beta 2-adrenoceptor agonist, zinterol, caused concentration-dependent increases (half-maximal effects at 1 nmol/L). The effects of the agonists were inversely related to stimulation frequency. Furthermore, the concentration-response curve of isoprenaline was shifted to the right by the selective beta 2-adrenoceptor antagonist, ICI 118,551, but not by the selective beta 1-adrenoceptor antagonist, ICI 89,406. Schild-plot analysis revealed competitive antagonism and a pA2 value of 9.04 for ICI 118,551. Both ICI 118,551 and ICI 89,406, as well as beta-adrenoceptor antagonists with intrinsic sympathomimetic activity (pindolol and celiprolol; 1 mumol/L), had no effect on stimulation-evoked overflow of tritium (phentolamine present). It is concluded that guinea pig papillary muscles are endowed with prejunctional beta 2 adrenoceptors facilitating impulse-evoked noradrenaline release. The facilitation is markedly promoted by blockade of prejunctional alpha adrenoceptors.     

Effects of prostaglandin E1 and indomethacin on the stimulation- evoked overflow of 3H-noradrenaline from guinea-pig taenia caecum

Prostaglandin E₁ (5.8 × 10^?8 M) markedly and reversibly reduced the stimulation-evoked overflow of total tritium, while it had no effects on basal outflow. Indomethacin (8.4 × 10^?6 M) increased the stimulation-evoked overflow of total tritium at low frequencies (2–5 Hz), while it had no effect at high frequencies of stimulation (more than 10 Hz). It was concluded that endogenous prostaglandin E₁ also plays a regulatory role in adrenergic inhibitory neurotransmission by inhibiting the noradrenaline release from adrenergic nerve terminals of the guinea-pig taenia caecum.     

Chloroethylclonidine: an irreversible agonist at prejunctional alpha 2-adrenoceptors in rat vas deferens.

1. The possibility that chloroethylclonidine (CEC) activates prejunctional alpha 2-adrenoceptors was studied in the isolated vas deferens of the rat. Tissues were stimulated electrically and both the stimulation-evoked overflow of tritium (after preincubation with [3H]-noradrenaline) and the purinergic contraction component (isolated by prazosin 0.3 microM) were measured. 2. CEC (0.1-3 microM) concentration-dependently reduced the overflow of tritium evoked by trains of 6 pulses/100 Hz. The inhibition by CEC was not altered by prazosin (0.3 microM) but was prevented by pre-exposure to rauwolscine (0.3 microM). The inhibition, once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 3. CEC (0.1-3 microM) concentration-dependently reduced the purinergic component of contractions elicited by single pulses. The inhibition, again, was prevented by pre-exposure to rauwolscine (0.3 microM) and once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 4. CEC (3 microM) reduced the overflow of tritium evoked by 20 pulses/10 Hz, did not alter the overflow evoked by 100 pulses/10 Hz and increased the overflow evoked by 500 pulses/10 Hz. 5. CEC (3 microM) reduced the early peak, but increased the late plateau phase, of purinergic contractions elicited by 100 pulses/10 Hz. 6. It is concluded that CEC reduces the release of noradrenaline and a purinergic co-transmitter by irreversible activation of prejunctional alpha 2-adrenoceptors. CEC seems to be a partial alpha 2-agonist with an efficacy lower than that of noradrenaline. The prejunctional inhibitory effect limits the suitability of CEC for the characterization of postjunctional alpha 1-adrenoceptors mediating responses to sympathetic nerve stimulation.     

Alpha-adrenoceptor antagonists and the release of noradrenaline in rabbit cerebral cortex slices: support for the alpha-autoreceptor hypothesis.

Slices of rabbit cerebral cortex were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S1, S2) at various frequencies (0.2-3 Hz). The stimulation-evoked overflow of tritium (S1) increased with increasing frequency and was higher when cocaine (10 microM) was present. In the absence of cocaine, tetraethylammonium (TEA; 100 and 300 microM), added before S2, increased the stimulation-evoked overflow of tritium to about the same extent, irrespective of the frequency. In contrast, rauwolscine (0.1 and 1 microM) and idazoxan (0.1-10 microM) increased the evoked overflow much more, the higher the frequency of stimulation. Phentolamine (0.1 and 1 microM) reduced the overflow elicited at 0.3 and 1 Hz, and (1 microM) caused an increase only at 3 Hz. In slices superfused throughout with cocaine 10 microM, rauwolscine (1 microM) and idazoxan (1 and 10 microM) again increased the evoked overflow of tritium more, the higher the frequency of stimulation. For a given frequency, rauwolscine and idazoxan enhanced the evoked overflow to a greater extent in the presence than in the absence of cocaine. Idazoxan (1 and 10 microM) and rauwolscine (1 microM) counteracted the inhibition that phentolamine (0.1 microM) produced at low frequency. The increases caused by rauwolscine (1 microM) and TEA (300 microM) were approximately additive, but those caused by rauwolscine (1 microM) and idazoxan (10 microM) were not. The effects of rauwolscine, idazoxan and phentolamine depend on the experimental conditions (frequency, cocaine) in a manner compatible with the operation of a presynaptic alpha 2-adrenoceptor-mediated autoinhibition of noradrenaline release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Facilitation of noradrenaline release from rat brain slices by \-adrenoceptors

The present study examined whether stimulation of \-adrenoceptors facilitated noradrenaline release in the rat brain. Electrical stimulation-evoked overflow of tritium from rat cerebral cortical, hypothalamic and hippocampal slices labelled with ³H-noradrenaline was measured during superfusion for 100 min. Tissue slices were electrically simulated (1 Hz, 20 mA, 2 ms, 2 min), at 20(S1) and 70(S2) min after the onset of superfusion. The nonselective \-adrenoceptor agonist isoproterenol (0.1 – 10 nM) enhanced stimulation-evoked overflow of tritium from slices of cerebral cortex, hypothalamus and hippocampus in a concentration-dependent manner; mean S2/S1 ratios with 10 nM isoproterenol were 161 +- 11%, 142 +- 15% and 143 - 12% of control, respectively, in the three brain regions. The facilitatory effect of isoproterenol in cerebral cortical slices was antagonized by propranolol (50 nM), a nonselective \sb-adrenoceptor antagonist, and by the \sb₁- and \sb₂-selective adrenoceptor antagonists ICI 89,406 (1 nM) and ICI 118,551(1 nM), respectively. The \sb₁- and \sb₂-selective adrenoceptor agonists prenalterol and albuterol (0.1 \2- 10 nM), respectively, also increased stimulation-evoked overflow of tritium from cerebral cortical slices; these effects were antagonized by \sb-adrenoceptor antagonists. These findings suggest that stimulation of \sb-adrenoceptors enhance noradrenaline release from rat cerebral cortical, hypothalamic and hippocampal slices; this release mechanism appears to involve both \sb₁- and \sb₂-adrenoceptor subtypes. These facilitating presynaptic receptors may be involved in mediating the antidepressant-like behavioral effects of \sb₂-adrenoceptor agonists.     

Transmitter release patterns of noradrenergic,dopaminergic and cholinergic axons in rabbit brain slices during short pulse trains,and the operation of presynaptic autoreceptors

Summary Slices of rabbit brain were field-stimulated either by single electrical pulses or by trains of 4 or 8 pulses at 1 or 100 Hz in order to study transmitter release patterns and the autoinhibition of transmitter release. The slices were preincubated with ³H-noradrenaline (cortex), ³H-dopamine (caudate nucleus) or ³H-choline (caudate nucleus).Slices preincubated with ³ H-noradrenaline were superfused with medium containing desipramine 1 gmol/l. The overflow of tritium elicited by single pulses amounted to 0 .19% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was similar, whereas that elicited by 4 pulses/100 Hz was 5.1-fold higher. Yohimbine 101000 nmol/l increased up to 2.5-fold the overflow evoked by 4 pulses/1 Hz but did not change the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-dopamine were superfused with medium containing nomifensine 1 mol/l. The overflow of tritium elicited by single pulses was 0.39% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was 1.3-fold and the overflow elicited by 4 pulses/100 Hz 1.4-fold higher. Domperidone 1–100 nmol/l and sulpiride 10–1000 nmol/1 increased up to 2.4-fold the overflow evoked by 4 pulses/ 1 Hz but increased only slightly the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 mol/l. In physostigmine-free medium, atropine did not increase the evoked overflow of tritium at any stimulation condition. In physostigmine-containing medium, the overflow elicited by single pulses was 0.18% of the tritium content of the tissue. The overflow elicited by 8 pulses/1 Hz was 2.0-fold and the overflow elicited by 8 pulses/100 Hz 2.2-fold higher. Atropine 2–200 nmol/1 increased up to 2.4-fold the overflow evoked by 8 pulses/1 Hz but increased only slightly the overflow evoked bysingle pulses or 8 pulses/100 Hz. In physostigmine-free medium, sulpiride 10–1000 nmol/1 did not change the single-pulse-evoked overflow of tritium in the absence but increased it in the presence of nomifensine 1 mol/l.Single pulses elicit a large release of ³H-noradrenaline, ³H-dopamine and ³H-acetylcholine under the conditions of these experiments. Release elicited by single pulses is not subject to autoinhibition except for a small inhibition by spontaneously released transmitter in the case of dopaminergic and cholinergic axons. When 3 or 7 further pulses follow the first one at intervals of 1 s, they elicit much smaller release. At least a great part of the fall is due to autoreceptor mediated inhibition (for 3H-acetylcholine release in the presence of physostigmine only). When 3 or 7 further pulses follow at intervals of 10 ms, they elicit release that is either similar to that evoked by the first pulse (³H-noradrenaline) or much smaller (³H-dopamine, ³H-acetylcholine). However, the fall is not due to stimulation-dependent, auto-receptor-mediated inhibition; autoinhibition does not develop in these short high-frequency trains. Overall, the results are in accord with the autoreceptor theory. They demonstrate the role of autoinhibition in determining the transmitter release patterns of central noradrenergic, dopaminergic and cholinergic neurones. Send offprint requests to N. Limberger at the above address     

Effects of prostaglandin E2, prostaglandin I2 and 6-keto-prostaglandin F1 alpha on adrenergic neurotransmission in the pulmonary artery of the rabbit.

Strips of the rabbit pulmonary artery were preincubated with 3H-noradrenaline and then superfused and stimulated electrically at 2 or 4 Hz. PGE2 and PGI2 reduced the stimulation-evoked overflow of total tritium and 3H-noradrenaline. PGI2 was about 10 times less potent than PGE2. High concentrations of 6-keto-PGF 1alpha also diminished the evoked overflow, but the effect was small. It is concluded that PGI2, in comparison with PGs of the E series, is a relatively weak inhibitor of noradrenaline release.     

Role of the L-arginine-NO pathway and of cyclic GMP in electrical field-induced noradrenaline release and vasoconstriction in the rat tail artery.

1. The possible roles of the L-arginine-NO pathway and of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in regulating the prejunctional release of noradrenaline and neurogenic vasoconstriction were investigated in the perfused rat tail artery. 2. In the presence of N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of NO formation, the vasoconstrictor responses to perivascular nerve stimulation (24 pulses at 0.4 Hz, 0.3 ms, 200 mA) and to exogenous noradrenaline (1 microM) were significantly enhanced, whereas the stimulation-evoked tritium overflow from [3H]-noradrenaline preloaded arteries was not modified. The vasoconstriction enhancing effect of L-NAME was prevented by L-arginine (1 mM) but not D-arginine (1 mM) and was abolished by removal of the endothelium. 3. The NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1; 0.1-30 microM), and the cyclic GMP phosphodiesterase inhibitor, zaprinast (0.1-30 microM) both induced a concentration-dependent inhibition of the electrical field stimulation-induced vasoconstriction, while atrial natriuretic peptide (ANP; 100 nM) produced only a slight decrease of the vasoconstrictor response. Methylene blue (3 microM), a known inhibitor of soluble guanylate cyclase increased the electrical field stimulation-induced vasoconstriction. SIN-1 and methylene blue when administered simultaneously, antagonized each others effect. None of the compounds tested (SIN-1, zaprinast, ANP or methylene blue) had any significant effect on the stimulation-evoked [3H]-noradrenaline overflow. 4. 8-Bromo-cyclic GMP, a potent activator of cyclic GMP-dependent protein kinase, markedly and concentration-dependently (3-300 microM) increased [3H]-noradrenaline overflow but decreased field stimulation-induced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain

The effect of histamine and related drugs on the tritium overflow evoked electrically (0.3 Hz) or by introduction of Ca²⁺ ions into Ca²⁺-free K⁺-rich (25 mmol/l) medium containing tetrodotoxin was studied in superfused guinea-pig brain cortex, cerebellum, hippocampus or hypothalamus slices and in mouse brain cortex slices preincubated with ³H-noradrenaline. The electrically evoked tritium overflow in guinea-pig cortex slices was inhibited by histamine; the H₃ receptor antagonist clobenpropit reversed the effect of histamine to a slight facilitation. The facilitatory effect of histamine (obtained in the presence of clobenpropit) was not affected by the H₁ receptor antagonist mepyramine but abolished by the H₂ receptor antagonist ranitidine. In the absence of clobenpropit, ranitidine augmented the inhibitory effect of histamine. In slices superfused in the presence of ranitidine, the evoked overflow was inhibited by histamine and, more potently, by the H₃ receptor agonist R-α-methylhistamine in a concentration-dependent manner (maximum inhibitory effect obtained for both agonists 30–35%). The concentration-response curve of histamine was shifted to the right by the H₃ receptor antagonist thioperamide. R-α-Methylhistamine inhibited the electrically evoked tritium overflow also in guinea-pig cerebellar, hippocampal and hypothalamic slices. In cortex slices superfused in the presence of clobenpropit, the H₂ receptor agonists impromidine and, less potently, R-sopromidine facilitated the evoked overflow in a concentration-dependent manner. S-Sopromidine only tended to increase the evoked overflow. The effect of impromidine was counteracted by the H₂ receptor antagonists ranitidine and cimetidine. The extent of the maximum facilitatory effect of impromidine (by 15–20%) was about the same when (i) the Ca²⁺ concentration in the medium was reduced from 1.3 to 0.98 mmol/l, (ii) the time of exposure to impromidine was reduced from 28 to 8 min or (iii) cerebellar, hippocampal or hypothalamic slices were used instead of cortical slices. The Ca²⁺-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca²⁺-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H₃ receptors and facilitated via H₂ receptors not located presynaptically. In the mouse brain cortex, only inhibitory H₃ receptors occur. The extent of the H₃ receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex. Received: 25 September 1997 / Accepted: 17 November 1997     

Leucine-enkephalin- and neuropeptide Y-modulation of [3H]noradrenaline release in the oviduct of mature and juvenile rabbits

1. The effects of leucine-enkephalin and neuropeptide Y (NPY) on [3H]noradrenaline release induced by electrical field stimulation were studied in the isthmic part of the oviduct of juvenile and mature rabbits. 2. [3H]noradrenaline and total tritium overflow in the presence of cocaine, corticosterone and hyoscine were determined by liquid scintillation spectrometry. 3. Tritium overflow evoked by electrical stimulation (1 or 4 Hz, 1 msec) was calcium dependent. [3H]noradrenaline content (measured by ion exchange chromatography) accounted for 85% of the total tritium overflow. 4. Leucine-enkephalin (1 microM) in the presence of the peptidase inhibitor bacitracin reduced the stimulation-evoked tritium overflow in mature rabbits by 26.1 +/- 1.6% and in juvenile rabbits by 11.9 +/- 1.9%. Naloxone (1 microM) antagonized the effect of leucine-enkephalin. 5. NPY (0.2 microM) reduced the evoked tritium overflow in mature rabbits by 23.4 +/- 2.4% and in juvenile rabbits by 17.2 +/- 4.3%. 6. It is concluded that leucine-enkephalin and NPY inhibited [3H]noradrenaline release in rabbit oviduct and the modulatory effect of leucine-enkephalin depends on maturity while NPY modulation is a more independent system.     

Differential effects of D600, nifedipine and dantrolene sodium on excitation-secretion coupling and presynaptic beta-adrenoceptor responses in rat atria.

The stimulation-evoked release of tritium was measured from rat atria labelled with [3H]-noradrenaline. The calcium dependence of evoked release and the facilitation of this release via activation of presynaptic beta-adrenoceptors were examined using D600 (methoxyverapamil), nifedipine and dantrolene sodium. Both D600 and nifedipine at dose levels of 20 and 100 microM inhibited evoked release. Dantrolene (20, 100 microM) reduced release by 25%, the effect being maximal at 20 microM. In the presence of 20 nM isoprenaline, a facilitation of evoked release occurred, which was blocked by 0.1 microM (-)-propranolol. The facilitatory action of isoprenaline was abolished by omission of calcium from the buffer, or by D600 or nifedipine, (100 microM). In contrast, the response to isoprenaline was not modified by dantrolene (20, 100 microM). It is concluded that the evoked release of noradrenaline (NA) utilizes Ca from both intra- and extracellular sources and that isoprenaline increases NA secretion by promoting the depolarization-induced influx of Ca.     

The influence of uptake2 on the inhibition by unlabelled noradrenaline of the stimulation-evoked overflow of 3H-noradrenaline in rabbit aorta with regard to surface of amine entry

The release by electrical field stimulation of 3H-noradrenaline from the adrenergic nerve endings in the rabbit aorta was studied in a special double-chambered organ bath. Independently of the frequency (1-10 Hz) and the number of pulses used (300-3,000 pulses), only 10-20% of the stimulation-evoked overflow of tritium (total evoked overflow) left the aortic wall via the intimal surface (intimal evoked overflow). The corresponding percentage value for the basal efflux was twice that for the evoked overflow, thus indicating that part of the radioactivity in the basal efflux originated from an extraneuronal compartment. The radioactivity in the total evoked overflow (in response to stimulation at 10 Hz) originated from at least two compartments (compartment I and II) with half times for efflux of about 2 and 6 min, respectively. In the intimal evoked overflow, compartment II (but not compartment I) was involved. When uptake1 was inhibited, 70% of the radioactivity in the intimal evoked overflow (stimulation at 1 Hz) consisted of metabolites, while unchanged amine prevailed by far in the total and adventitial evoked overflow, respectively. Additional inhibition of uptake2 thus had a striking effect only on the composition of the radioactivity in the intimal evoked overflow. The intimal surface was exposed to unlabelled noradrenaline in order to inhibit the evoked overflow of tritium (stimulation at 1 Hz; uptake1 inhibited). When uptake2 was inhibited additionally, the dose-response curve for the inhibitory effect of noradrenaline was shifted to the left by a factor of 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of fentanyl,levorphanol and pethidine on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of fentanyl, levorphanol and pethidine on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by low, and was accelerated by high concentrations of the drugs. The overflow of tritium evoked by electrical stimulation at 3 Hz was diminished by 10^–8–10^–7 M fentanyl and by 10^–7–10^–6 M levorphanol, but was augmented by 10^–5 M levorphanol. Naloxone prevented the inhibitory effect of fentanyl and levorphanol. In contrast to fentanyl and levorphanol, pethidine did not decrease, but at concentrations of 10^–6–10^–5 M greatly increased the stimulation-induced overflow of tritium. However, the increase was abolished, and the stimulation-evoked overflow slightly reduced, after the re-uptake of noradrenaline had been blocked by cocaine. It is concluded that fentanyl, levorphanol and pethidine share with morphine the ability to inhibit the release of transmitter from cerebrocortical noradrenaline neurones evoked by nerve impulses.     

Autoreceptors and α2-adrenoceptors at the serotonergic axons of rabbit brain cortex

Summary Slices of the rabbit occipito-parietal cortex were preincubated with ³H-serotonin and then superfused and stimulated electrically (2 min at 3 Hz). In the absence of drugs, the stimulation-evoked overflow of tritium was approximately 3% of the tritium content of the tissue. Unlabelled serotonin and 5-carboxamido-tryptamine, when administered in the presence of 6-nitroquipazine, reduced the evoked overflow of tritium. Their effects were antagonized by metitepin (apparent pA₂ value 8.1) and (±)-cyanopindolol (apparent pA₂ value 6.4). Metitepin, but not cyanopindolol, increased evoked tritium overflow; the effect of metitepin was greater in the presence than in the absence of nitroquipazine. The evoked overflow of tritium was also depressed by clonidine, an effect antagonized by idazoxan (apparent pA₂ value 7.0) but not by prazosin. Phenylephrine caused a decrease only at high concentrations that simultaneously accelerated basal tritium efflux. Prazosin and idazoxan did not change evoked tritium overflow, and phentolamine increased it significantly only when administered in the presence of (+)-oxaprotiline. Rauwolscine produced an inhibition that was prevented by metitepin. It is concluded that the serotonergic axons of the rabbit occipitoparietal cortex possess presynaptic, release-inhibiting serotonin autoreceptors and ₂-adrenoceptors. The receptors appear to receive an input of endogenous serotonin and, to a lesser extent, noradrenaline, under the conditions of these in vitro experiments.     

An examination of the pre- and postsynaptic alpha-adrenoceptors involved in neuroeffector transmission in rabbit aorta and portal vein.

1 The alpha-adrenoceptor agonists, clonidine and xylazine, reduced and the alpha-antagonists, yohimbine an rauwolscine, increased the stimulation-evoked tritium overflow from rabbit aorta and portal vein pre-incubated with [3H]-noradrenaline. 2 Based on an order of agonist potency of clonidine greater than xylazine greater than phenylephrine and antagonist potency of rauwolscine = yohimbine greater than prazosin, the presynaptic receptor mediating these effects is of the alpha 2 type. 3 In the aorta, stimulation-evoked contractions were abolished by prazosin (0.1 micrometers) and potentiated by rauwolscine and yohimbine in concentrations that increased the stimulation-evoked overflow tritium. 4 In the portal vein, prazosin was less potent in reducing, and rauwolscine and yohimbine failed to potentiate, the stimulation-evoked contraction. 5 In experiments in which tissues were pre-exposed to phenoxybenzamine (30 nM) to block some of the postsynaptic alpha-receptors, rauwolscine in concentrations that increased stimulation-evoked tritium overflow, reduced the evoked contraction in the portal vein but not in the aorta. 6 It is concluded that presynaptic alpha 2-autoreceptors are present in both tissues and that the postsynaptic alpha-receptors which mediate nerve stimulation-evoked contractions are alpha 1 in the aorta but a mixture of alpha 1 and alpha 2 in the portal vein.