Inhibition of neuronal uptake reduces the presynaptic effects of clonidine but not of alpha-methylnoradrenaline on the stimulation-evoked release of 3H-noradrenaline from rat occipital cortex slices

The iminoimidazolidine clonidine reduced concentration-dependently the release of 3H-noradrenaline evoked by electrical stimulation from the rate cerebral cortex. Exposure to the neuronal uptake inhibitors cocaine (10 micro M), desipramine (0.1 to 1 micro M) and amphetamine (1 micro M) significantly increased the stimulation-evoked overflow of tritium. These uptake inhibitors antagonized the effects of clonidine on stimulation evoked 3H-noradrenaline release but failed to modify the inhibition induced by the catecholamine alpha-methylnoradrenaline. Inhibition of monoamine oxidase by preincubation of cerebral cortex slices with 0.5 mM pargyline significantly increased the stimulation-evoked overflow of tritium, but clonidine was as effective as in the controls in inhibiting 3H-noradrenaline overflow. The antagonism by desipramine of the clonidine-induced inhibition of neurotransmission could not be attributed to a blockade of presynaptic alpha-adrenoceptors because: (1) the facilitating effect of phentolamine on 3H-noradrenaline overflow was not modified in the presence of desipramine; (2) the magnitude of the inhibition of the stimulation-evoked 3H-noradrenaline release elicited by alpha-methylnoradrenaline was the same in the presence of cocaine or desipramine; (3) exposure to desipramine in the presence of cocaine did not further increase the stimulation-evoked release of 3H-transmitter. Since the catecholamine alpha-methylnoradrenaline inhibited neurotransmission in the presence of desipramine or cocaine, we can conclude that inhibition of neuronal uptake of noradrenaline antagonized selectively the presynaptic inhibitory effects of imidazolines on alpha 2-adrenoceptors. The influence of the inhibition of neuronal uptake on the presynaptic effects of imidazolines and catecholamines should be taken into account when the relative order of potencies of various alpha 2-adrenoceptors agonists is determined.     

B-HT 920, B-HT 933, and B-HT 958: presynaptic effects on electrically evoked 3H-noradrenaline release from slices of rat brain cortex and hypothalamus

Summary The effects of the three azepine compounds, B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]azepine), B-HT 933 [2-amino-6-ethyl-5, 6, 7, 8-tetrahydro-4H-oxazolo [4,5-d]azepine; azepexole] and B-HT 958 (2-amino-6-(p-chloro-benzyl)-4H-5, 6, 7, 8-tetrahydrothiazolo[5,4-d]azepine) on electrically evoked overflow of ³H-noradrenaline were studied. Slices from parietal cortex (C) or nucleus anterior hypothalami (nah) were incubated with ³H-noradrenaline, superfused at 23°C or 37°C in the presence of the noradrenaline uptake inhibitor desipramine (3 mol/l) and field stimulated at frequencies of 0.3 or 3 Hz. 1. At 37°C/0.3 Hz, B-HT 920 and B-HT 933 concentration-dependently decreased the evoked overflow of tritium in both regions studied, whereas B-HT 958 had no effect. 2. In a second set of experiments each drug was tested under three additional experimental conditions, i. e. 37°C/3 Hz, 23°C/ 0.3 Hz and 23°C/3 Hz. Increasing the stimulation frequency to 3 Hz or lowering the superfusion temperature to 23°C reduced the effects of B-HT 920 (1 mol/l) and B-HT 933 (10 mol/l) as compared to the effects at 37°C/0.3 Hz. When tested at 23°C/3 Hz, both drugs did not significantly affect the evoked overflow of tritium in the Cx or the nah. In contrast, B-HT 958 (10 mol/l), caused a facilitation of evoked overflow in both regions when the higher stimulation frequency or the lower superfusion temperature was used. Its release-enhancing action was most pronounced at 23°C/3 Hz. 3. Idazoxan (0.1 mol/l) antagonized the effects of BHT 920 (10 mol/l) and B-HT 933 (10 gmmol/l) in the Cx at 37°C/0.3 Hz. The ineffectiveness of B-HT 958 under these experimental conditions was not changed in the presence of idazoxan. Sulpiride (10 mol/l) did not affect the action of any of the three compounds. 4. From the patterns of effects obtained under the different experimental conditions it is concluded that B-HT 920 and B-HT 933 act as agonists at prejunctional ₂-adrenoceptors, whereas B-HT 958 acts as a weak antagonist at these sites. Send offprint requests to E. A. Singer at the above addressPreliminary results were presented at the 27th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, March 11–14, 1986, Mainz, Federal Republic of Germany     

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.     

Modulation of noradrenaline release in human saphenous vein via presynaptic alpha 2-adrenoceptors

Strips of human saphenous veins were incubated with [3H]noradrenaline and subsequently superfused with physiological salt solution containing cocaine, corticosterone and propranolol. The electrically (6 Hz) evoked overflow of tritium (78% of which was accounted for by unmetabolized [3H]noradrenaline) was abolished by tetrodotoxin or omission of Ca2+ from the superfusion fluid. Unlabelled noradrenaline, alpha-methylnoradrenaline, B-HT 920 and clonidine inhibited the evoked overflow (maximum effect of clonidine lower than that of the other compounds) whereas methoxamine was ineffective. The alpha 2-adrenoceptor antagonists, BDF 6143 and rauwolscine, facilitated the evoked overflow but no effect was obtained with prazosin. Rauwolscine produced a shift to the right of the concentration-response curve of B-HT 920 for its inhibitory effect on evoked outflow and BDF 6143 caused a shift to the right of the corresponding curve of clonidine. It is concluded that the stimulation-evoked release of noradrenaline from the sympathetic nerve fibres of the human saphenous vein is modulated via presynaptic alpha 2-adrenoceptors.     

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 by gallamine in the rat salivary gland

Summary The effect of gallamine on spontaneous and stimulation-evoked overflow of tritium was studied in the submandibular gland of the rat. The gland was perfused retrogradely and labeled with³H-noradrenaline. The stimulation-evoked (1 Hz for 60 s) overflow of tritium was facilitated by increasing concentrations of gallamine (0.3–20 mM). None of the concentrations of gallamine increased the spontaneous overflow of the tritium. The facilitatory effect of gallamine was observed in 0.3 to 5 mM calcium medium; the maximum facilitation was observed at the normal concentration of calcium (2.5 mM). The facilitatory effect of gallamine was inversely related to the frequency of stimulation (10-fold facilitation at 1 Hz and 3-fold at 10 Hz).Stimulation of the salivary gland by a single pulse (1 ms duration) in the normal medium did not evoke an overflow of tritium; however, the same stimulus produced a marked increase in the overflow in the presence of gallamine.The facilitatory action of gallamine on the release of sympathetic transmitter is ascribed to the enhanced availability of calcium ions to the secretory process resulting from blockade of potassium conductance during nerve activity.     

B-HT 920 and B-HT 958: Presynaptic effects on electrically evoked 3H-dopamine release from slices of rat nucleus accumbens

The effects of two thiazoloazepine derivatives, B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepine) and B-HT 958 (2-amino-6-(p-chloro-benzyl)-4H-5,6,7,8-tetrahydrothiazolo[5,4-d]a zepine) on electrically evoked overflow of 3H-dopamine were studied. Slices from nucleus accumbens of the rat were preincubated with 3H-dopamine and superfused at 23 degrees C or 37 degrees C. Electrical field stimulation was applied using frequencies of 0.5 or 5 Hz. At 37 degrees C/5 Hz, B-HT 920 markedly and dose-dependently (0.01-0.1 mumol/l) reduced the stimulation evoked overflow of tritium. Its dose-response curve was shifted to the right at 23 degrees C/0.5 Hz and 23 degrees C/5 Hz, respectively. A similar result was obtained with the dopamine receptor agonist, apomorphine (1 mumol/l). B-HT 958 (0.1-10 mumol/l) also reduced electrically induced overflow of tritium at 37 degrees C/5 Hz, had no effect at 23 degrees C/0.5 Hz, and facilitated tritium overflow at 23 degrees C/5 Hz. Sulpiride (10 mumol/l) completely prevented the effects of B-HT 920 (1 mumol/l) or B-HT 958 (1 mumol/l) at 37 degrees C/5 Hz, whereas phentolamine (1 mumol/l) had no effect on the actions of the two drugs under these experimental conditions. From the patterns of effects obtained under the different experimental conditions it is concluded that B-HT 920 acts as full agonist at presynaptic dopamine autoreceptors whereas B-HT 958 acts as partial agonist.     

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)     

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.     

α-Adrenoceptor-mediated inhibition of noradrenaline release in rabbit brain cortex slices

Summary Brain cortex slices from rabbits were preincubated with [³H]noradrenaline and then superfused and stimulated electrically at 3Hz. In the presence of cocaine 30 M, unlabelled noradrenaline, -methylnoradrenaline, clonidine, oxymetazoline, xylazine and guanabenz decreased, whereas yohimbine, corynanthine, phentolamine, tolazoline and azapetine increased the stimulation-evoked overflow of tritium. Phenylephrine and prazosin had no effect on the evoked overflow except at concentrations that greatly accelerated the basal outflow of tritium. The results indicate that the noradrenergic axons of rabbit brain cortex are endowed with presynaptic -adrenoceptors which are exclusively of the ₂-type. Addition of various concentrations of cocaine, addition of pargyline, or stimulation at different current strengths was used to obtain either a high or a low stimulation-evoked overflow of tritium. Independently of the method used, a low evoked overflow coincided with a large percentage inhibition produced by 0.1M clonidine, whereas a high evoked overflow coincided with a smaller percentage inhibition produced by clonidine. The results indicate that drugs which block the re-uptake of noradrenaline diminish the presynaptic inhibitory effect of -adrenergic agonists by increasing the biophase concentration of released noradrenaline.     

Presynaptic alpha 2-adrenoceptors modulate the release of [3H]noradrenaline from rat spinal cord dorsal horn neurones

Slices of the dorsal half of the rat spinal cord were used to investigate the existence of a noradrenergic feedback modulation of noradrenaline release. After crude preparation of the vertebral column, the spinal cord was ejected by hydraulic pressure and transverse slices were cut. These were preincubated with [3H]noradrenaline during 0.1 Hz electrical stimulation and then superfused and stimulated electrically for two periods. The stimulation-evoked release of [3H]noradrenaline was Ca2+-dependent and tetrodotoxin-sensitive. Pretreatment of the animals with the noradrenergic neurotoxin, DSP-4, reduced the tritium content in the slices and the stimulation-evoked release to less than 10% of the controls. Clonidine (0.01-1 microM) inhibited the evoked overflow by 60% maximally and yohimbine (0.1-1 microM) enhanced it by 160% maximally. The effects of clonidine were antagonized by yohimbine. These results provide evidence that noradrenaline release from spinal cord slices is controlled by an alpha 2-adrenoceptor-mediated, negative feedback mechanism.     

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     

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.     

Noradrenaline release in the human pulmonary artery is modulated by presynaptic alpha 2-adrenoceptors

We used strips of human pulmonary arteries from patients undergoing surgery for lung tumor to investigate whether or not this human tissue (like that of the rabbit) is endowed with inhibitory presynaptic alpha 2-adrenoceptors. The strips were incubated with [3H]noradrenaline and subsequently superfused with physiological salt solution containing cocaine, corticosterone, and propranolol. The electrically (2 Hz) evoked overflow of tritium consisted of 89% unmetabolized [3H]noradrenaline and was abolished by tetrodotoxin or omission of Ca2+ from the superfusion fluid. Unlabeled noradrenaline, adrenaline, and the preferential alpha 2-adrenoceptor agonists B-HT 920, alpha-methylnoradrenaline, and clonidine concentration dependently inhibited the evoked overflow (maximum effect of clonidine lower than that of the other compounds): the alpha 1-selective agonist methoxamine was ineffective. The alpha 2-adrenoceptor antagonists BDF 6143 and rauwolscine facilitated the evoked overflow; the alpha 1-selective antagonist prazosin was ineffective. The concentration-response curve of B-HT 920 for its inhibitory effect on evoked overflow was shifted to the right by rauwolscine. It is concluded that the sympathetic nerve fibres of the human pulmonary artery possess presynaptic alpha 2-adrenoceptors. Stimulation-evoked release of noradrenaline is inhibited by activation and facilitated by blockade of these receptors.     

A study of ATP as a sympathetic cotransmitter in human saphenous vein.

1. Strips of human saphenous veins were superfused with Krebs-Henseleit solution at either 25 degrees C or 37 degrees C. Constrictor responses to electrical stimulation (10 Hz, 40 s) but not to exogenous noradrenaline (0.1, 1 microM) were abolished by guanethidine (10 microM) and tetrodotoxin (1 microM). Hence, responses to electrical stimulation are due to action potential-induced release of sympathetic neurotransmitters. 2. Constrictor responses to electrical stimulation and noradrenaline were reduced by the alpha 1-adrenoceptor antagonist, prazosin (0.3 microM) as well as by the alpha 2-adrenoceptor antagonist, rauwolscine (1 microM). The combination of prazosin and rauwolscine abolished constrictor responses to noradrenaline at 25 degrees C and 37 degrees C. However, constrictor responses to electrical stimulation were partly resistant to alpha-adrenoceptor blockade by prazosin and rauwolscine (at 25 degrees C about 30%). Residual constrictor responses to electrical stimulation were also observed in the presence of the combination of prazosin (3 microM) and rauwolscine (10 microM) as well as in the presence of phenoxybenzamine (10 microM). 3. Veins, incubated with [3H]-noradrenaline, released tritium upon electrical stimulation (10 Hz, 40 s). Moreover, electrical stimulation also induced an overflow of ATP amounting to 4.8 +/- 1.5 pmol g-1 at 25 degrees C and 2.0 +/- 0.5 pmol g-1 at 37 degrees C. Both tritium and ATP overflow were abolished by tetrodotoxin (0.5 microM). The combination of prazosin (0.3 microM) and rauwolscine (1 microM) increased tritium overflow at either 25 degrees C or 37 degrees C by about 120%, but reduced ATP overflow by about 70%.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Antagonistic properties of quipazine at presynaptic serotonin receptors and α-adrenoceptors in rat brain cortex slices

Rat brain cortex slices preincubated with 3H-serotonin or 3H-noradrenaline were superfused with physiological salt solution, and the effect of quipazine on the 3H overflow evoked by electrical field stimulation was examined. 1. The electrically evoked tritium overflow from brain slices preloaded with 3H-serotonin was increased by quipazine in a concentration-dependent manner. The concentration-response curves of unlabelled serotonin and noradrenaline for their inhibitory effects on impulse-evoked 3H overflow were shifted to the right by quipazine. 2. In brain slices preincubated with tritiated noradrenaline, quipazine caused an increase in electrically stimulated 3H overflow which was less marked than the effect on brain slices preloaded with 3H-serotonin. The concentration-response curve of unlabelled noradrenaline for its decreasing effect on stimulation-evoked 3H overflow was shifted to the right by quipazine. We conclude that quipazine blocks presynaptic inhibitory serotonin receptors and alpha-adrenoceptors on monoaminergic neurones of the rat brain cortex.     

Receptor protection experiments confirm the identity of presynaptic α2-autoreceptors

Receptor protection experiments were carried out in cerebrocortical slices from rabbits in order to study the sites at which drugs with alpha-adrenoceptor affinity modulate the release of noradrenaline. The slices were preincubated with 3H-noradrenaline. They were then superfused with 3H-noradrenaline-free medium and stimulated electrically (3 Hz) twice for 2 min each, after 60 and 250 min of superfusion (S1, S2). Phenoxybenzamine was added from 85 to 95 min of superfusion. Potential protecting drugs were present for 5 min before and during the exposure to phenoxybenzamine and then washed out together with the latter. Phenoxybenzamine 0.1 and 1 mumol/l increased the evoked overflow of tritium by 77 and 287%, respectively, as indicated by the S2/S1 overflow ratio. When cocaine was present throughout superfusion, phenoxybenzamine 0.1 and 1 mumol/l increased the evoked overflow by 97 and 353%, respectively. Clonidine 0.1-100 mumol/l, when added before and during the contact with phenoxybenzamine, reduced or even abolished the increase caused by the latter. This interaction was not changed when cocaine was included in the superfusion fluid. The increase caused by phenoxybenzamine was also reduced or abolished by noradrenaline 1-100 mumol/l (tested in the presence of cocaine), yohimbine 0.01-1 mumol/l and phentolamine 0.1-10 mumol/l. Only high concentrations of clonidine, noradrenaline, yohimbine and phentolamine changed the evoked overflow when given alone (and subsequently washed out). The effect of phenoxybenzamine was not modified by prazosin 1 mumol/l, morphine 1 mumol/l and naloxone 10 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subclassification of release-regulating alpha 2-autoreceptors in human brain cortex.

1. Release-regulating alpha 2-autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [3H]-noradrenaline ([3H]-NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions. 2. The stimulus-evoked tritium overflow was almost totally Ca(2+)-dependent and tetrodotoxin-sensitive. 3. Clonidine and oxymetazoline 0.01 to 1 microM inhibited in a concentration-dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC50 = 0.03 microM) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 microM, did not affect tritium overflow. 4. Yohimbine (0.01-0.1 microM) shifted the concentration-response curve of clonidine to the right. The calculated pA2 value was 8.29. 5. Prazosin and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]ethyl]-4,4- dimethyl-1,3(2H,4H)-isoquinolinedione (AR-C 239), tested at 0.3 microM, did not modify the concentration-response curve of clonidine. 6. The effect of clonidine was antagonized by (+)-mianserin (pA2 = 7.74), but not by up to 0.3 microM of the (-)-enantiomer. The concentration-response curve of clonidine was shifted to the right by the novel alpha 2-adrenoceptor antagonist, 5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-ami ne (Z)-2-butenedioate (1:1) salt (ORG 20350) (pA2 = 7.55). 7. Yohimbine, (+)-mianserin and ORG 20350, but not prazosin and (-)-mianserin, increased the electrically-evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA. 8. Desipramine (1 microM) increased evoked tritium overflow from human cortex slices.(ABSTRACT TRUNCATED AT 250 WORDS)