Pertussis toxin abolishes the inhibition of Ca2+ currents and of noradrenaline release via α2-adrenoceptors in chick sympathetic neurons

Summary Effects of ₂-adrenoceptor agonists on whole-cell Ca²⁺ currents and ³H-noradrenaline release were investigated by applying the patch-clamp technique and electrical field stimulation to cultured embryonic chick sympathetic neurons. A 24-h exposure of the sympathetic neurons to pertussis toxin (100 ng/ml) abolished both the 2-adrenoceptor-mediated inhibition of Ca ²⁺ currents and the modulation of noradrenaline release caused by noradrenaline (1 mol/l; in the presence of 10 mol/l cocaine) or the ₂-adrenoceptor agonists 5-bromo-6-(2imidazolin-2-ylamino)quinoxaline (UK 14,304, 10 mol/ l) and clonidine (10 mol/l). These results suggest that the ₂-autoreceptor-mediated inhibition of noradrenaline release from chick sympathetic neurons operates through the modulation of Ca²⁺ channels via pertussistoxin-sensitive GTP-binding-proteins. Send offprint requests to S. Boehm at the above address     

Modulation of calcium currents via α2-adrenoceptors in embryonic chick sympathetic neurons

Summary In order to gain insight into the mechanism of the autoinhibition of noradrenaline release, the present study explores the effects of substances acting at various adrenoceptor-subtypes on voltage-activated Ca ²⁺ currents. Experiments were carried out on cultured embryonic chick sympathetic neurons using the patch clamp technique.Ca²⁺ currents associated with a (fully activating) depolarizing 150 ms voltage step to 0 mV were reduced by noradrenaline and the two a2-adrenoceptor agonists UK 14,304 and clonidine, predominantly during the early phase of activation. We quantified these effects by measuring Ca²⁺ current amplitudes in the absence and presence of substances 10 ms after the beginning of the depolarization. Noradrenaline effects were maximal at 5 µmol/l, causing a 28% depression of the current. Half-maximal effects (IC₅₀) were apparent at 0.7 µmol/l. UK 14,304 was equipotent to noradrenaline (IC₅₀: 0.5 µmol/l; maximal effect: 26% depression). Clonidine, while active in the same range of concentration (IC₅₀: 0.6 µmol/l), had a smaller maximal effect (20% depression). Methoxamine and isoprenaline, on the other hand, did not significantly reduce the Ca ²⁺ current at 10 µmol/l. The noradrenaline-induced inhibition was attenuated by yohimbine (1 µmol/I). Neither prazosin (1 µmol/l) nor propranolol (1 µmol/l) interfered with the effect of noradrenaline.These results indicate a reduction of Ca ²⁺ influx via ₂-adrenoceptors and suggest that the autoreceptor-mediated inhibition of transmitter release in embryonic chick sympathetic neurons operates through the modulation of Ca²⁺ channels. Send offprint requests to S. Boehm at the above address     

Influence of neuronal uptake on the presynaptic α-adrenergic modulation of noradrenaline release

Summary Conditions required for the inhibitory feedback modulation of noradrenergic neurotransmission were studied in isolated atria of the rat.The alpha adrenergic antagonist, yohimbine, 0.8 M, or phentolamine, 1 M, did not affect the chronotropic response to 4 or 8 shocks at 0.8 Hz but increased it when a higher number of shocks was applied. When neuronal uptake was inhibited by cocaine, 2.9 M, or desipramine, 0.1 M, the enhancement of neurotransmission by yohimbine or phentolamine was higher than that observed in the presence of -adrenergic antagonists alone.In atria preincubated with ³H-noradrenaline, the effect of the drugs on the ³H-overflow evoked by 240 shocks at 2.0 Hz was studied. Cocaine 2.9 M, did not increase the evoked overflow but yohimbine, 0.8 M, did. The ³H-overflow obtained in the group of yohimbine plus cocaine was significantly higher than was expected from the effects of both drugs alone.It is concluded that yohimbine or phentolamine enhance the chronotropic response in rat atria only when the concentration of noradrenaline in the biophase is sufficiently high to activate presynaptic receptors. In this tissue, the efficiency of the neuronal uptake influences the degree of -adrenergic autoinhibition.     

A kinetic study of the release of noradrenaline by electrical stimulation: influence of presynaptic α-adrenoceptors

Summary Dog saphenous vein strips were incubated with 1.4 mol/l ³H-(-)-noradrenaline for 60 min, after inhibition of the noradrenaline-metabolizing enzymes and of extraneuronal uptake. At the end of the incubation period the strips were perifused for 150 min; cocaine (10 mol/l) was added to the perifusion fluid from t=75 min onwards. In some experiments either phentolamine (10 mol/l) or clonidine (0.1 mol/l) was also added at this time. Some strips were subjected to electrical stimulation from t=100 to 150 min of perifusion (t=0 being the start of perifusion), with frequencies ranging from 0.5 to 13.5 Hz. A compartmental analysis of spontaneous or electrically-induced efflux of ³H-noradrenaline was made. The spontaneous efflux had a long half time (t/2=124 min) and most of the ³H-noradrenaline which had accumulated in the strips did not participate in the efflux (bound fraction, representing 90% of tissue activity at t=100 min of perifusion). Neither phentolamine nor clonidine modified the half time or the bound fraction observed for spontaneous efflux. Electrical stimulation (>0.5 Hz) mobilized only one compartment of noradrenaline, which represented about 50% of the noradrenaline accumulated in the strips. The half time of ³H-efflux induced by electrical stimulation decreased when the frequency increased from 0.5 Hz up to 13.5 Hz. Phentolamine increased the rate of efflux for all frequencies of stimulation and decreased the half time of efflux. However, the releasable pool of noradrenaline was only increased by phentolamine at 0.5 Hz, but not at higher frequencies. Clonidine was used only at two frequencies of stimulation, 1.5 and 4.5 Hz. For the low frequency clonidine decreased the releasable pool, but no change was observed at 4.5 Hz.The results support the view that there is a norarenaline pool which is resistant to electrical stimulation and that its magnitude is not dependent on the activity of presynaptic -adrenoceptors.Results presented in part to the 13th Annual Meeting of the Portuguese Pharmacological Society (Porto, December 1982) and to the 5th Meeting on Adrenergic Mechanisms (Porto, October 1983)Work supported by a grant from Instituto Nacional de Investigação Científica (FmPl)     

Activation of presynaptic α2-adrenoceptors attenuates the inhibitory effect of μ-opioid receptor agonists on noradrenaline release from brain slices

Summary ³H-noradrenaline release from rat neocortical slices induced by 15 mM K⁺ was concentration-dependently inhibited by morphine, [D-Ala²-D-Leu⁵] enkephalin (DADLE) and the calcium entry blocker Cd²⁺. Blockade of presynaptic ₂-adrenoceptors with phentolamine, almost doubling K⁺-induced ³H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd²⁺ remained unaffected. In contrast, activation of presynaptic ₂-adrenoceptors with clonidine (1 M) or TL-99 (1 M), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd²⁺. When in the presence of 1 M clonidine adenylate cyclase was activated with forskolin (10 M), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, -opioid receptor efficacy appears to be dependent on the degree of activation of ₂-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane.     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Modulation of N-methyl-d-aspartate (NMDA)-stimulated noradrenaline release in rat brain cortex by presynaptic α2-adrenoceptors

Summary Rat brain cortex slices and synaptosomes preincubated with [³H]noradrenaline were used to investigate whether the NMDA-evoked noradrenaline release is modulated by agonists or antagonists at presynaptic ₂-adrenoceptors.In experiments on slices, noradrenaline and the preferential ₂-adrenoceptor agonists talipexole (former B-HT 920) and clonidine inhibited the NMDA evoked tritium overflow whereas the selective ₁-adrenoceptor agonists cirazoline and methoxamine were ineffective. The ₂-adrenoceptor antagonists rauwolscine and idazoxan facilitated the NMDA-evoked tritium overflow whereas the preferential ₁-adrenoceptor antagonist prazosin was ineffective. The concentration-response curve of talipexole for its inhibitory effect on NMDA-evoked overflow was shifted to the right by idazoxan (apparent pA₂ = 7.5). The EC₅₀ of NMDA (97 mol/l) for its stimulating effect on tritium overflow was not substantially changed by blockade of ₂-autoreceptors with 1 mol/l rauwolscine (EC₅₀ of NMDA in the presence of the ₂-adrenoceptor antagonist, 155 mol/l), but the maximal overflow of tritium was increased 2.5 fold by this rauwolscine concentration. In experiments on synaptosomes, talipexole and noradrenaline inhibited the NMDA-evoked tritium overflow. The inhibitory effect of talipexole was abolished by idazoxan which, given alone, was ineffective, as was prazosin. Talipexole did also not produce an inhibition when tritium overflow was evoked by NMDA in the presence of -conotoxin GVIA 0.1 mol/l; the latter, by itself, decreased the response to NMDA by about 55%. It is concluded that the NMDA-evoked noradrenaline release in the cerebral cortex is modulated via presynaptic ₂-adrenoceptors on the noradrenergic neurones. Stimulation of these autoreceptors in slices by endogenous noradrenaline does not result in a decreased potency of NMDA, but in a decreased maximum effect, in stimulating noradrenaline release. The inhibitory effect of ₂-adrenoceptor agonists on the NMDA-evoked release is at least partially due to a functional interaction between the NMDA receptors and ₂-autoreceptors at the level of the same varicosities. The results obtained with -conotoxin GVIA suggest that Ca²⁺ influx via the N-type voltage-sensitive calcium channel (VSCC) occurs in response to NMDA receptor stimulation and contributes substantially to the induction of NMDA-evoked noradrenaline release. The inhibitory effect of ₂-adrenoceptor stimulation on this release appears to be ultimately due to an inhibition of the influx of Ca²⁺ via the N-type VSCC. Correspondence to: M. Göthert at the above address     

Estimation of the biophase concentration of noradrenaline at presynaptic α2-adrenoceptors in brain slices

Summary The aim of the present study was to determine the local concentrations of noradrenaline existing at presynaptic ₂-adrenoceptors during electrical pulse train stimulation of brain slices at different frequencies. The experiments are based on the assumption that the concentration of released noradrenaline at the ₂-adrenoceptors exerting a certain autoinhibition should be equal to the concentration of exogenous noradrenaline causing the same inhibition under conditions in which any influence of the released transmitter is excluded. In order to avoid autoinhibition, hippocampus and cortex slices of the rabbit and the rat, prelabelled with [³H]noradrenaline and superfused in presence of an uptake inhibitor, were electrically stimulated using 4 pulses delivered at 100 Hz (POP stimulation). Exogenous noradrenaline diminished the overflow of tritium elicited by POP stimulation in a concentration-dependent manner. In rabbit brain tissues the EC₅₀ value and maximum inhibition of noradrenaline release were found to be approximately 6 nmol/l and more than 95%, respectively, whereas in rat tissues the corresponding values were between 20 and 30 nmol/l and approximately 90%. When electrical stimulation was performed with trains of 36 pulses delivered at 0.1, 0.3 or 3 Hz in absence or presence of an uptake inhibitor, the ₂-adrenoceptor antagonist yohimbine (1 or 10 mol/l) enhanced the evoked tritium overflow in a manner which was dependent on the frequency of stimulation and on blockade of the re-uptake mechanism. The facilitatory effects of yohimbine reflected an extent of autoinhibition which was between 53% (36 pulses/0.1 Hz, no uptake inhibitor) and 85% (36 pulses/3 Hz, uptake inhibitor present) in rabbit and between 16% (36 pulses/0.3 Hz, no uptake inhibitor) and 71% (36 pulses/3 Hz, uptake inhibitor present) in rat brain slices. Accordingly, the corresponding estimated biophase concentrations of noradrenaline were generally higher in rat than in rabbit tissues (they were between 32.5 and 74.5 or 5.1 and 51.6 nmol/l in the presence or absence of an uptake inhibitor, respectively, in the rat, and between 15 and 23.1 or 6.1 and 18.6 nmol/l in the rabbit). The observed frequency dependence of the effect of re-uptake blockade on the calculated biophase concentrations of noradrenaline would be compatible with the idea of a dependence of the effectiveness of the re-uptake mechanism on the firing rate of the neurone in being more effective at lower frequencies. Moreover, the stikingly low biophase concentrations of noradrenaline suggest that also in brain tissue noradrenaline causes lateral inhibition of release as has recently been shown for guinea-pig vas deferens. Send offprint requests to C. Allgaier at the above address     

Subclassification of presynaptic α2-adrenoceptors: α2A-autoreceptors in rabbit atria and kidney

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors of rabbit atria and kidney in terms of _2A, _2B, _2C and _2D-A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with ³H-Noadrenaline and then superfused and stimulated electrically.In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to ₂-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC_30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to ₂-autoinhibition, and concentration-inhibition curves of the ₂-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pEC_30% values correlated with pK_d values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC_30% values in atria correlated with pEC_30% values in the kidney (r = 0.988) and pK_d values in atria correlated with pK_d values in kidney (r = 0.923).It is concluded that the ₂-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both autoreceptors are _2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in guinea-pig atria and brain

The study was devised to classify, by means of antagonist and agonist affinities, the presynaptic ₂-autoreceptors in guinea-pig heart atria and brain cortex in terms of _2A, _2B, _2C and _2D. A set of antagonists and agonists was chosen that was able to discriminate between the four subtypes. Small pieces of the atria and slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically.In one series of experiments (atria only), tissue pieces were stimulated by relatively long pulse trains (1 min) leading to marked ₂-autoinhibition. All 10 antagonists increased the evoked overflow of tritium. pEC_30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments (atria and brain slices), tissue pieces were stimulated by brief pulse trains (0.4 s or 40 ms) that led to little (atria) or no (brain slices) ₂-autoinhibition, and antagonist effects against the ₂-selective agonist 5-bromo-6(2-imidazolin-2-ylamino)-quinoxaline UK 14,304 were examined. All 10 (atria) or 8 (brain) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. In a third series of experiments (brain slices only), also with brief pulse trains (40 ms), pK_a values (negative logarithms of dissociation constants of agonist-₂-adrenoceptor complexes) were determined by comparison of concentration-inhibition curves of UK 14,304, guanoxabenz and oxymetazoline in normal tissue and in tissue in which a fraction of the receptors had been blocked by phenoxybenzamine. pEC_30% values in atria correlated with pK_d values (r = 0.942). pK_d values in atria correlated with pK_d values in the brain cortex (r = 0.970).It is concluded that the ₂-autoreceptors in atria and the brain cortex are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature - indicates that both autoreceptors are _2D. In atria, this identification is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d, and in the brain cortex it is supported by the agonist pK_a values. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2AD branch of the ₂-adrenoceptor tree.     

NANC transmitters in the female pig urethra–localization and modulation of release via α2-adrenoceptors and potassium channels

1. To investigate further the release, localization and identity of a non-nitrergic mediator of smooth muscle relaxation in the female pig urethra, we studied the effects of drugs acting at α₂-adrenoceptors or K⁺ channels, the effects of capsaicin and chemical sympathectomy, and the actions of several transmitter candidates.
2. Electrical field stimulation (EFS; frequencies above 12 Hz) of spontaneously contracted smooth muscle strips from the female pig urethra evoked long-lasting, frequency-dependent relaxations in the presence of prazosin, scopolamine, and N^G-nitro-L-arginine. Treatment with the selective α₂-adrenoceptor agonist UK-14 304 markedly reduced the relaxations evoked by EFS at all frequencies tested (16–30 Hz). The inhibitory effect of UK-14 304 was completely antagonized by the α₂-adrenoceptor antagonist rauwolscine. The muscarinic M₁ receptor antagonist, pirenzepine, or exogenously administered carbachol, did not have any effects on the electrically evoked relaxations.
3. Inhibition of high conductance Ca²⁺ activated K⁺ channels by iberiotoxin or charybdotoxin significantly enhanced the relaxations evoked by EFS at all frequencies. However, inhibition of voltage-sensitive K⁺ channels with 4-aminopyridine or dendrotoxin-1, treatment with the ATP-sensitive K⁺ channel blocker, glibenclamide, or treatment with the high and low conductance Ca²⁺ activated K⁺ channel blockers, tetraethylammonium chloride and apamin, had no effect on the relaxations evoked by EFS.
4. Electrically evoked relaxations were not affected by adrenergic denervation with 6-hydroxydopamine (6-OHDA) at any frequency. However, treatment with 6-OHDA abolished prazosin-sensitive electrically induced contractions, and a long-lasting relaxation was revealed. Treatment with capsaicin, believed to damage selectively a subpopulation of primary afferent fibres, did not affect basal tone or relaxations evoked by EFS.
5. Exogenously applied vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP)-27, PACAP-38, adenosine, ATP and 5-hydroxy-tryptamine caused relaxations of the urethral preparations, whereas prostaglandin E₂ and calcitonin gene-related peptide had no effects. VIP 10-28, α, β-methylene-ATP, reactive blue-2, suramin or indomethacin did not reduce the electrically-evoked relaxations at any frequency. However, the relaxations were slightly reduced by trypsin or α-chymotrypsin.
6. The present results suggest that the release of the unknown mediator in the female pig urethra can be modulated via α₂-adrenoceptors and high conductance Ca²⁺ activated K⁺ channels. Furthermore, the mediator does not appear to be localized to or released from adrenergic or capsaicin-sensitive sensory nerve-endings. The identity of the transmitter remains to be established.

     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in mouse brain

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors in mouse cerebral cortex in terms of _2A, _2B, _2C and _2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Slices of the cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically.The stimulation periods used (4 pulses, 100 Hz) did not lead to ₂-autoinhibition as shown by the lack of an increase by rauwolscine of the evoked overflow of tritium. The ₂-selective agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and -methylnoradrenaline reduced the evoked overflow. All 10 antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. Rauwolscine also shifted the concentration-inhibition curve of -methylnoradrenaline to the right. pK_d values of the antagonists were calculated from the shifts. The pK_d values of rauwolscine against UK 14,304 and -methylnoradrenaline were very similar (8.0 and 7.9, respectively).Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that the ₂-autoreceptors in mouse brain cortex are _2D. This is the first subtype determination of ₂-autoreceptors in the mouse. It supports the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree.     

Evidence supporting the existence of presynaptic α-adrenoceptors in the regulation of endogenous noradrenaline release upon hepatic sympathetic nerve stimulation in the dog liver in vivo

Summary The existence and functional signficance of presynaptic -adrenoceptors within the liver was investigated in anesthetized dogs. The stimulation-evoked endogenous catecholamine overflow was determined in hepatic venous blood upon hepatic nerve stimulation (12 V, 1–8 Hz, 1 min). Under resting conditions, average plasma catecholamine levels in hepatic venous and aortic blood were 0.064 ng/ml and 0.334 ng/ml, respectively. A frequency-dependent increase (P<0.001) was found in the hepatic venous catecholamine overflow, while aortic catechlamine levels did not change significantly at any stimulation frequency tested. The increases in catecholamine overflow were associated with decreases in hepatic arterial vascular conductance (35–66%, P<0.001) at all frequencies tested. Yohimbine (0.3 mg/kg, i. v.) potentiated the catecholamine overflow by 110–140% (P<0.05) upon stimulation at low frequencies (1–4 Hz). Clonidine (20g/kg, i. v.) inhibited the catecholamine overflow by 55–76% (P<0.05) at frequencies of 1 and 2 Hz, and reduced the hepatic arterial response by 46% (P<0.01) at 1 Hz. The pretreatment with yohimbine (0.1 mg/kg, i. v.) abolished the inhibitory effects of clonidine both on the catecholamine overflow and the hepatic arterial responses. The results support the existence of a negative feedback mechanism mediated by presynaptic -adrenoceptors in the local regulation of noradrenaline release from hepatic sympathetic fibers. A functional significance of this process was suggested by an improved correlation found in the presence of clonidine between the catecholamine overflow and the hepatic arterial vascular conductance.     

Pronounced facilitation of endogenous noradrenaline release by presynaptic β2-adrenoceptors in the vasculature of freely moving rats

Summary The facilitation of the noradrenaline (NA) over-flow by stimulation of the presynaptic -adrenoceptor of the rat portal vein was investigated, using the freely moving unanesthetized permanently cannulated rat as a model. The ₂-selective agonist fenoterol caused a maximal enhancement of about 300% of the basal NA level at a dose of 0.5 mg/kg. Following administration of cocaine (2.5 mg/kg plus 0.05 mg/kg/min) basal NA levels increased to 150% whereas combination of cocaine and fenoterol results in a dose dependant rise up to over 560% of the basal level (at a fenoterol dosage of 0.5 mg/kg). Blockade of the ₂-adrenoceptors with yohimbine (0.5 mg/kg) which enhances the NA level to 486%, followed by 0.125 mg/kg fenoterol results in a further 2.53-fold rise to more than 1,200% of the basal level, indicating the pronounced counterregulatory role of the presynaptic ₂-adrenoceptor. After ganglionic blockade with hexamethonium (3 mg/kg plus 6 mg/kg/h) the effect of yohimbine (0.5 mg/kg) alone was diminished to 162%, but the additional facilitatory effect of 0.125 mg/kg fenoterol still was 1.82-fold, to 294% of the basal level. Combination of cocaine (2.5 mg/kg plus 0.05 mg/kg/min), yohimbine (0.5 mg/kg) and fenoterol (0.125 mg/kg) induced a rise to over 9,000 pg/ml NA (about 40-fold of the basal NA level). During electrical stimulation (2 Hz, 3 ms, 5 mA) of the local portal vein nervous plexus, the role of the inhibitory ₂-adrenoceptor becomes even more pronounced. Both in absence and in presence of cocaine, at the highest dose of fenoterol (0.5 mg/kg), the levels of electrically evoked release reverted to control values. However, after yohimbine (0.5 mg/kg) the evoked release was further enhanced by a much lower dosage of 0.125 mg/kg fenoterol to 450% of the control evoked release. This value was not changed significantly after ganglionic transmission blockade (hexamethonium 3 mg/kg plus 6 mg/kg/h). The results demonstrate that presynaptic -adrenoceptors in the vasculature of the unanesthetized freely moving rat actually possess a pronounced capacity to facilitate NA release from sympathetic varicosities. Send offprint requests to R. Remie at the above address     

Opposite modulation of cotransmitter release in guinea-pig vas deferens: increase of noradrenaline and decrease of ATP release by activation of prejunctional β-adrenoceptors

Effects of isoprenaline on contraction, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) as well as on contractions elicited by exogenous noradrenaline and ATP were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique.In [³H]-noradrenaline-pretreated tissues, electrical stimulation elicited an overflow of tritium and ATP and a biphasic contraction. Isoprenaline (1–100 nM) reduced the contraction, mainly phase I, and enhanced the evoked overflow of tritium; evoked overflow of ATP was not changed significantly. No, or almost no, contraction remained in [³H]-noradrenaline-pre-treated tissues exposed to both prazosin (0.3 M) and suramin (300 M), and the evoked overflow of ATP was reduced by about 82%. Under these conditions, isoprenaline (1–100 nM) again enhanced the evoked overflow of tritium, but it now decreased the evoked overflow of ATP. Propranolol (1 M), when added on top of prazosin and suramin, prevented the effects of isoprenaline (1–100 nM). In some tissues not pretreated with [³H]-noradrenaline, purinergic and adrenergic components of the neurogenic contraction (again to 210 pulses, 7 Hz) were isolated by exposure to prazosin (0.3 M) and suramin (300 M), respectively. Isoprenaline (1–100 nM) decreased the isolated purinergic component but did not change significantly the isolated adrenergic component. Contractions elicited by ATP (1000 M) were not changed and contractions elicited by noradrenaline (100 M) were slightly increased by isoprenaline (1–100 nM). Isoprenaline (100 nM) did not change the degradation of ATP (100 M) by pieces of the vas deferens.It is concluded that, in the guinea-pig vas deferens, activation of prejunctional -adrenoceptors modulates the neural release of noradrenaline and ATP in opposite directions: release of noradrenaline is enhanced, whereas release of ATP is decreased.     

Methoxamine inhibits noradrenaline release through activation of α1- and α2-adrenoceptors in rat isolated kidney: involvement of purines and prostaglandins

Summary The effects of the at-adrenoceptor agonist methoxamine and the ₂-adrenoceptor agonist bromoxidine (UK 14034) on the stimulation induced (S-1) outflow of radioactivity at 100 Hz/6 pulses from rat isolated kidney preincubated with ³H-noradrenaline were investigated. Methoxamine (0.3 – 30 mol/l) inhibited S-1 outflow of radioactivity to a maximum of 83% with a pEC₅₀ of 5.85 (5.71–5.94). UK 14304 (0.0003-0.3 mol/l) inhibited S-I outflow of radioactivity to a maximum of 99% with a pEC₅₀ of 8.35 (8.26–8.47). a Adrenoceptor antagonist affinities (pK_D) against methoxamine and UK 14304 at prejunctional -adrenoceptors were determined. The concentration response curve of methoxamine was shifted to the right by the 1/_2B-adrenoceptor antagonist prazosin (0.1 mol/l) with a pK_D of 7.41 and that of UK 14304 by prazosin (0.3 mol/l) with a pK_D of 6.24. The ₂-adrenoceptor antagonist rauwolscine (0.1 mol/l) shifted the concentration response curve of UK 14304 potently to the right with a p_KD of 8.34. The concentration response curve of methoxamine was shifted also to the right by rauwolscine (0.1 mol/l) and the ₂-adrenoceptor antagonist idazoxan (0.1 mol/l), however, both antagonists suppressed the maximum response of methoxamine to 466% and 56%, respectively. A ten times lower concentration of rauwolscine (0.01 mol/l) did not shift the concentration response curve of methoxamine but the inhibitory effect of methoxamine still reached only a maximum of 59%. The concentration response curve of methoxamine obtained in the presence of rauwolscine (0.01 mol/l) was shifted to the right by further addition of prazosin (0.1 mol/l) with a pK_D of 8.80 but was also shifted to the right by either the purinoceptor antagonist 8-(p-sulfophenyl) theophylline (8-SPT; 100 mol/l) or the prostaglandin synthesis inhibitor indomethacin (20 mol/l). These results suggest that methoxamine inhibits S-1 outflow of radioactivity in rat isolated kidney probably through three different mechanisms. 1. Activation of postjunctional 1-adrenoceptors and prostaglandin mediated transjunctional inhibition. 2. Activation of postjunctional ₂-adrenoceptors and purine mediated transjunctional inhibition. 3. Activation of prejunctional inhibitory ₂-adrenoceptors at which methoxamine seems to be a partial agonist. Correspondence to L. C. Rump at the above address     

α2-Adrenoceptor-mediated inhibition of cultured sympathetic neurons: changes in α2A/D-adrenoceptor-deficient mice

Alpha2-Adrenoceptor-mediated inhibition of [3H]noradrenaline release and alpha2-adrenoceptor-mediated inhibition of voltage-activated Ca2+ currents were compared in cultured thoracolumbar postganglionic sympathetic neurons from newborn wildtype (WT) mice and mice in which the alpha2A/D-adrenoceptor gene had been disrupted (alpha2A/DKO). In cultures prepared from WT mice and preincubated with [3H]noradrenaline, the alpha2-adrenoceptor agonist 5-bromo-6-(2-imidazolidinylidenamino)quinoxaline (UK 14,304) reduced the (autoinhibition-free) release of [3H]noradrenaline elicited by single electrical pulses or trains of 8 pulses at 100 Hz. The maximal inhibition by UK 14,304 amounted to 70%-85%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) and, to a smaller extent, rauwolscine (0.3 microM). Pretreatment of the cultures with pertussis toxin abolished the effect of UK 14,304. Phentolamine and rauwolscine increased the (alpha2-autoinhibited) release of [3H]noradrenaline elicited by 18, 36 or 72 pulses at 3 Hz. In cultures from alpha2A/DKO mice, UK 14,304 failed to reduce the release of [3H]noradrenaline elicited by single pulses and phentolamine and rauwolscine failed to increase the release of [3H]noradrenaline elicited by 18-72 pulses at 3 Hz. In neurons from WT mice examined with the amphotericin B-perforated configuration of the patch clamp method, UK 14,304 reduced depolarisation-evoked Ca2+ currents. The inhibition was voltage-dependent as shown by a decline at strong depolarisation during ramp-like voltage commands and by an attenuation briefly after a conditioning depolarising pulse. The maximal inhibition by UK 14,304 was 39%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) but not significantly changed by rauwolscine (0.3 microM) and prazosin (1 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304. In neurons from alpha2A/DKO mice, UK 14,304 also reduced depolarisation-evoked Ca2+ currents, but with a smaller maximal effect, namely 18% inhibition. Its concentration-response curve was shifted to the right by rauwolscine (0.3 microM) and prazosin (1 microM) but not significantly changed by phentolamine (0.3 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304 also in cultures from alpha2A/DKO mice. It is concluded that the only presynaptic alpha2-autoreceptors that detectably depress transmitter release from cultured thoracolumbar sympathetic neurons taken from newborn mice are alpha2A/D. In contrast, the soma-dendritic alpha2-autoreceptors that inhibit voltage-gated Ca2+ channels are both alpha2A/D and non-alpha2A/D (i.e. alpha2B or alpha2c). Both presynaptic alpha2A/D- and soma-dendritic alpha2A/D- and non-alpha2A/D-autoreceptors operate through pertussis toxin-sensitive G proteins in these neurons.     

Protection of presynaptic α-adrenoceptors against irreversible blockade by phenoxybenzamine: preservation of the modulatory effects of exogenous noradrenaline and yohimbine

Summary 1. Receptor protection experiments were carried out in order to study the site of action of -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 mol/l and stimulated electrically (3 Hz) three times, after 60, 250 and 295 min of superfusion (S₁, S₂, S₃). Phenoxybenzamine 10 mol/1 when used, was added between S₁ and S₂ for 30 min; putative protecting drugs (clonidine 100 mol/1 or yohimbine 10 mol/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 mol/1 or yohimbine 1 mol/1 before S₃. 2. In slices not exposed previously to -adrenoceptor ligands, noradrenaline 0.1mol/1 greatly reduced, whereas yohimbine 1 mol/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 mol/1 in the presence of either clonidine 100 pmol/1 or yohimbine 10 mol/1 failed to abolish the effects of the test compounds noradrenaline 0.1 mol/1 and yohimbine 1 mol/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 -autoreceptor. Send offprint requests to K. Starke at the above address     

Involvement of presynaptic imidazoline receptors in the α2-adrenoceptor-independent inhibition of noradrenaline release by imidazoline derivatives

Summary An involvement of imidazoline recognition sites in the modulation of transmitter release was investigated in the rabbit pulmonary artery and aorta preincubated with [³H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Electrical impulses were applied transmurally at 0.66 or 2 Hz. In the absence of further drugs, rauwolscine as well as the imidazoline derivatives BDF 6143 [4-chloro-2-(2-imidazoline-2-ylamino)-isoindoline], idazoxan and phentolamine increased the ³H overflow from the pulmonary artery, evoked by electrical stimulation at 2 Hz; the effect was due to the ₂-autoreceptor blocking property of these drugs. The maximum increase in overflow obtainable with the imidazolines was considerably lower than with rauwolscine. The concentration-response curves of the imidazolines were bell-shaped. At 0.66 Hz, BDF 6143 did not facilitate, but concentration-dependently inhibited, whereas idazoxan failed to change the evoked ³H overflow. When, at the stimulation frequency of 2 Hz, presynaptic ₂-adrenoceptors were blocked by rauwolscine and/or pre-exposure to phenoxybenzamine, the electrically evoked ³H overflow from the pulmonary artery and/or aorta was inhibited by the following imidazoline derivatives: the ₂-adrenoceptor antagonists BDF 6143, idazoxan and phentolamine, the ₁-adrenoceptor agonist with ₂-blocking property cirazoline as well as the ₂-adrenoceptor agonists clonidine and moxonidine. The maximum inhibition caused by BDF 6143 was greater than that due to clonidine and moxonidine; the latter two, hence, behaved as partial agonists. At the stimulation frequency of 0.66 Hz, the imidazolines exhibited a higher potency than, but a similar intrinsic activity to that at 2 Hz. Noradrenaline did not affect the evoked ³H overflow. The BDF 6143-induced inhibition of evoked ³H overflow was not modified by metitepine, atropine, theophylline, dipyridamole and indometacin, but was counteracted by the partial agonists clonidine and moxonidine.The results exclude the possibility that ₁- and ₂-adrenoceptors, 5-HT₁ receptors, muscarine receptors, P₁ purinoceptors and prostaglandin receptors are involved in the imidazoline-induced inhibition of noradrenaline release. They provide evidence indicating that the inhibitory effect is mediated by imidazoline receptors on the postganglionic sympathetic nerve terminals of the rabbit pulmonary artery and aorta.     

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional β-adrenoceptors: evidence for the β2 subtype

Activation of prejunctional -adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of -adrenoceptor involved.In [³H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1–100 nM), salbutamol (0.01–1 M) and terbutaline (0.1–10 M) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the ₂-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 M). Isoprenaline (1–100 nM), salbutamol (0.01–1 M) and terbutaline (0.1–10 M) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin-and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained contraction elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the ₁-selective antagonist 1-[2-((3carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM).The results confirm the opposite -adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional -adrenoceptor is of the ₂ subtype.