Notes: 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 μmol/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 μmol/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 μmol/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.

Notes: Summary 1. Receptor protection experiments were carried out in order to study the site of action of α-adrenoceptor agonists and antagonists on the release of noradrenaline. Cerebrocortical slices from rabbits were preincubated with 3H-noradrenaline. They were then superfused with medium containing cocaine 30 μmol/l and stimulated electrically (3 Hz) three times, after 60, 250 and 295 min of superfusion (S1, S2, S3). Phenoxybenzamine 10 μmol/1 when used, was added between S1 and S2 for 30 min; putative protecting drugs (clonidine 100 μmol/1 or yohimbine 10 μmol/1) were present 5 min before and during the exposure to phenoxybenzamine and then washed out together with the latter. Either the voltage drop between the electrodes at S2 and S3 or the Ca2+-concentration of the superfusion medium at S2 and S3 was diminished, if necessary, in order to bring the overflow evoked by S2 close to the overflow at S1. Blockade by phenoxybenzamine, or protection against the blockade, was examined by addition of the test compounds noradrenaline 0.1 μmol/1 or yohimbine 1 μmol/1 before S3. 2. In slices not exposed previously to α-adrenoceptor ligands, noradrenaline 0.1μmol/1 greatly reduced, whereas yohimbine 1 μmol/1 greatly increased the evoked overflow of tritium. Both effects were abolished in slices treated with phenoxybenzamine 10 pmol/1 alone between S1 and S2. 3. In contrast to phenoxybenzamine alone, exposure to phenoxybenzamine 10 μmol/1 in the presence of either clonidine 100 pmol/1 or yohimbine 10 μmol/1 failed to abolish the effects of the test compounds noradrenaline 0.1 μmol/1 and yohimbine 1 μmol/1, although the effects were reduced. 4. It is concluded that the irreversible antagonist phenoxybenzamine, the protecting agents clonidine and yohimbine, the test compounds noradrenaline and yohimbine, and by inference endogenous noradrenaline as well, all act at the same site, namely the presynaptic α-autoreceptor.

Notes: Summary Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic α2-autoreceptors of effects mediated by presynaptic opioid κ- and adenosine A1-receptors requires activation of the α2-receptors. The slices were preincubated with 3H-noradrenaline and then superfused with medium containing desipramine 1 μmol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz. The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca2+-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 μmol/l or yohimbine 0.3 μmol/l. Ethylketocyclazocine (EK; 0.1–10 nmol/l) and R-(−)-N6-phenylisopropyladenosine (PIA; 1–1,000 nmol/1) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. — The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 μmol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca2+ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine. The results demonstrate that there is no α2-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic α2-adrenoceptor does not interfere with presynaptic opioid κ- and adenosine A1-receptor mechanisms. It is only when the autoreceptor is activated by released noradrenaline that it attenuates neighbouring presynaptic receptor mechanisms, and this attenuation is removed by α2-adrenoceptor antagonists.

Notes: Summary The interaction of presynaptic, release-inhibiting α2-adrenoceptors, opioid κ-receptors and adenosine A1-receptors was studied in slices of the occipito-parietal cortex of the rabbit. The slices were preincubated with 3H-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S1, S2). The stimulation-evoked overflow of tritium was taken to reflect action potential-evoked release of noradrenaline. One of two release-modulating compounds to be examined for interaction was kept in the medium throughout superfusion, the other one was added before S2. In many experiments, the stimulation parameters were adjusted (frequency 0.5–7 Hz; voltage drop 2–5 V/cm) in order to obtain similar reference release (S1) values despite the presence of the first release-modulating compound. The selective κ-receptor agonist ethylketocyclazocine (EK) attenuated markedly the release-inhibiting effects of the α2-adrenoceptor-selective agonists clonidine and α-methylnoradrenaline as well as the release-facilitating effect of the α2-adrenoceptor-selective antagonist yohimbine. The attenuation occurred both when the parameters of electrical stimulation were kept constant and when they were adjusted to obtain similar S1 release values. The selective A1-receptor agonist R-N6-phenylisopropyladenosine (PIA) also attenuated the effects of clonidine and yohimbine. Conversely, clonidine attenuated and yohimbine enhanced the release-inhibiting effect of PIA. Yohimbine also enhanced the release-facilitating effect of the adenosine receptor antagonist 8-phenyltheophylline. Again, these changes occurred both at constant stimulation parameters and when stimulation parameters were adjusted. EK attenuated the release-inhibiting effect of PIA, and conversely PIA attenuated the effect of EK, both at constant and at adjusted parameters of electrical stimulation. The release-inhibiting effects of tetrodotoxin and Cd2+ remained unchanged in the presence of clonidine or EK. These results demonstrate mutual interactions between presynaptic a2-, opioid K- and adenosine A1-receptors. As soon as any one of the three systems is activated, the inhibition due to activation of either of the two remaining systems is blunted. The interactions are not a consequence of the change in release per se that the first receptor ligand inevitably produces. α-Adrenoceptors interact with opioid κ-receptors in a similar manner, independently of the chemical nature (imidazoline or phenylethylamine derivative) of the α-agonist used. The interaction is specific for release-modulating receptors and does not extend to Na+ ar Ca2+ channel blockers. It may occur at the level of the receptors themselves or at the post-receptor transduction mechanisms.

Notes: Summary Ear arteries from rabbits were preincubated with3H-noradrenaline and then perfused. 2-[2-(1,4-Benzodioxanyl)]-2-imidazoline (RX 781094) 0.1 or 1μM reduced the overflow of3H-noradrenaline and total tritium elicited by 13 electrical pulses at 0.25 Hz or 26 pulses at 0.5 Hz. RX 781094 1 μM increased the overflow elicited by 52 pulses at 1 Hz. The inhibitory effects were blocked by yohimbine 10 μM but not by prazosin 1 μM. The alleged antagonist RX 781094 possesses intrinsic activity at the presynaptic α2-autoreceptor of the ear artery.

Notes: Summary An attempt was made to determinepA2 values of antagonists at the presynaptic, release-inhibiting α2-autoreceptorsof rabbit and rat brain cortex under conditions when there was very little released noradrenaline in the autoreceptor biophase and, hence,pA2 values were not distorted by endogenous autoinhibition. Cortex slices were preincubated with3H-noradrenaline and then superfused and stimulated by trains of 4 pulses delivered at 100 Hz or, in a few cases, by trains of 36 pulses at 3 Hz. The α-adrenoceptor agonists clonidine, noradrenaline, and α-methylnoradren-aline concentration-dependently decreased the stimulation-evoked overflow of tritium. The a-adrenoceptor antagonists yohimbine, rauwolscine and idazoxan did not increase the overflow of tritium elicited by 4 pulses/100 Hz in rabbit brain slices and increased it only slightly in rat brain slices. In contrast, the antagonists increased markedly the overflow at 36 pulses/3 Hz. All antagonists caused parallel shifts to the right of the concentration-response curves of clonidine, noradrenaline, and α-methylnoradrenaline.pA2 values were calculated either from linear regression of log [agonist concentration ratio − 1] on log [antagonist concentration] or from sigmoid curve fitting. The slopes of the linear regression lines were close to unity, and thepA2 values calculated by the two methods agreed well. There was no consistent preferential antagonism of any antagonist to any agonist.pA2 values determined with stimulation by 4 pulses/100 Hz were by 0.53–0.80 log units higher than those determined with stimulation by 36 pulses/3 Hz. ThepA2 values (4 pulses/100 Hz) of yohimbine and rauwolscine in rabbit brain slices (approximately 7.9 and 8.2, respectively), were slightly higher than in rat brain slices (approximately 7.6 and 7.7, respectively), whereas thepA2 value of idazoxan in the rabbit. (about 7.1) was lower than itspA2 value in the rat (about 8.0). The experiments confirm thatpA2 values determined under conditions of autoinhibition are too low. Stimulation with short (30 ms) bursts of pulses permits the estimation ofpA2 values at presynaptic a2-autoreceptors without (rabbit) or almost without (rat) the complication of autoinhibition. The values suggest that α2-adrenoceptors in rabbit brain cortex differ slightly from those in rat brain cortex.

Notes: Abstract The effect of neuropeptide Y [NPY(1–36)] and related peptides on the voltage-dependent currents and the nicotinic acetylcholine receptor (nAChR) currents (IACh) of bovine adrenal chromafptn cells was investigated using the whole-cell patch clamp technique. Catecholamine release from single chromaffin cells was measured by means of fast cyclic voltammetry. The potency order of these peptides in inhibiting IACh evoked by nicotine was NPY(1–36), NPY (16–36) 〉 peptide YY(PYY) 〉 [Leu31, Pro34] NPY. NPY(16–36) produced a similar degree of inhibition, irrespective of whether nicotine or an equipotent concentration of acetylcholine was used to evoke IACh. NPY(16–36) failed to alter voltage-dependent inward or outward currents. Intracellular cAMP, and extracellular dibutyryl-cAMP, produced a slowly developing increase in IACh. Intracellular cAMP, extracellular 8-Br-cAMP or dibutyryl-cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases 3-isobutyl-l-methylxanthine (IBMX), decreased the inhibitory effect of NPY(16–36) on lACh. Although the intracellular application of the cAMP-dependent protein kinase A inhibitor [PKI(14–24)amide] alone did not alter IACh, it potentiated the effect of NPY(16–36) in interaction experiments. While the NPY(16–36)-induced inhibition of IACh was reversed on washout of the peptide, the slightly shorter C-terminal fragment NPY(18–36) caused a long-lasting depression of both IAch and catecholamine secretion evoked by nicotine. This depression was smaller in the presence of extracellular 8-Br-cAMP than in its absence. NPY(18–36) did not alter the secretory activity induced by a high concentration of potassium. It appears that, by activating Y3-receptors, NPY inhibits nAChR-current and the resulting secretion of catecholamines from bovine chromaffin cells. This process may involve a G protein-mediated decrease in intracellular cAMP with a subsequent decrease in the degree of phosphorylation of the nAChR-channel.