Islet-activating protein (pertussis toxin) diminishes α2-adrenoceptor mediated effects on noradrenaline release

Summary The effect of islet-activating protein (IAP) on ₂-adrenoceptor mediated modulation of noradrenaline release in the rabbit hippocampus was studied. Slices of the hippocampus were incubated for 6 h with IAP, subsequently loaded with³H-noradrenaline and superfused continuously. IAP-pretreatment significantly enhanced the electrically evoked transmitter release and diminished the facilitatory effect of the ₂-adrenoceptor antagonist yohimbine. In addition, the inhibitory effect of the ₂-adrenoceptor agonist clonidine was reduced. These results provide circumstantial evidence that an inhibitory guanine-nucleotide-binding protein, most probably N₁ of a presynaptically located adenylate cyclase, is involved in the ₂-autoreceptor mediated modulation of noradrenaline release.Abbreviations IAP islet-activating protein - LPA leukocytosis-promoting activity     

Pertussis toxin does not attenuate α2-adrenoceptor mediated inhibition of noradrenaline release in mouse atria

Summary 1. The ₂-adrenoceptor agonist clonidine (0.03 and 0.1 ol/l) significantly inhibited stimulation-induced overflow of radioactivity from mouse isolated atria pre-incubated with [³H]-noradrenaline. This effect of clonidine was blocked by idazoxan (0.3 gmol/l) but not prazosin (0.3 ol/l), indicating that an ₂-adrenoceptor was involved. 2. In some experiments mice were injected with pertussis toxin (1.5 g/mouse) 4 days before their atria were removed and subsequently incubated with [³H]-noradrenaline. Alternatively, isolated atria from untreated mice were suspended in Krebs-Henseleit solution, incubated for 16 h with pertussis toxin (1.0 and 4.0 g/ml) or vehicle and subsequently incubated with [³H]-noradrenaline. The effectiveness of pertussis toxin pretreatment was assessed indirectly using carbachol. Carbachol caused a dose dependent fall in both the rate and force of contraction of isolated, spontaneously beating atria from mice pretreated with vehicle in vivo or in vitro. This effect of carbachol was not seen in atria from mice pretreated with pertussis toxin in vivo or in vitro, suggesting that active toxin penetrated the myocardium. 3. Pertussis toxin pretreatment, either in vivo or in vitro did not alter the inhibitory effect of clonidine (0.03 and 0.1 gmol/l), or the facilitatory effect of the -adrenoceptor antagonist phentolamine (1.0 mol/l), on the stimulation-induced overflow of radioactivity. These results suggest that ₂-adrenoceptor modulation of noradrenaline release from sympathetic nerve terminals is not dependent on an inhibitory guanine-nucleotide-binding protein. Send offprint requests to I. Musgrave     

Vasoconstriction mediated by postsynaptic α2-adrenoceptor stimulation

Summary The postsynaptic -adrenoceptors involved in vasoconstriction brought about by B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazolo-[5,4-d]-azepin) administered i.v. to pithed, normotensive rats were characterized. The rate of onset of the hypertensive response to i.v. B-HT 933 is slower than that induced by (–)-phenylephrine, an agonist of ₁-adrenoceptor. The antagonism of the -adrenoceptor blocking drugs rauwolscine, yohimbine and corynanthine was quantified towards B-HT 933-induced increases in diastolic pressure. Rauwolscine (pA₂=7.06) and yohimbine (pA₂=6.83) were effective antagonists, whereas corynanthine proved much less potent (pA₂=5.03). On the basis of the reported selectivity of yohimbine and its two diastereoisomers rauwolscine and corynanthine for ₁- and ₂-adrenoceptor, it is concluded that the postsynaptic -adrenoceptors triggered by B-HT 933 are of the ₂-type. B-HT 933 identifies a subclass of postsynaptic ₂-adrenoceptor in vascular smooth muscle distinct from postsynaptic ₁-adrenoceptor. Both types of -adrenoceptors are likely to be involved in the mediation of vasoconstriction.Preliminary data were presented at the 21th Spring Meeting of the German Pharmacological Soceity, Mainz, March 18–21, 1980 (Timmermans, 1980)     

Presynaptic α2-adrenoceptor,opioid κ-receptor and adenosine A1-receptor interactions on noradrenaline release in rabbit brain cortex

Summary The interaction of presynaptic, release-inhibiting ₂-adrenoceptors, opioid -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 -receptor agonist ethylketocyclazocine (EK) attenuated markedly the release-inhibiting effects of the ₂-adrenoceptor-selective agonists clonidine and -methylnoradrenaline as well as the release-facilitating effect of the ₂-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. -Adrenoceptors interact with opioid -receptors in a similar manner, independently of the chemical nature (imidazoline or phenylethylamine derivative) of the -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.Send offprint requests to N. Limberger at the above address     

Further study of prerequisites for the enhancement by α-adrenoceptor antagonists of the release of noradrenaline

Summary Segments of the rabbit ear artery were preincubated with (–)-³H-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of ³H-noradrenaline and total tritium was determined.In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 M present). Thirteen pulses (0.25 Hz) elicited an overflow of ³H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 M did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 M decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 M decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 M was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses.In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of ³H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 M did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses. TEA 100 M increased the evoked overflow at all pulse numbers. Similar results were obtained in the presence of cocaine 5 M.The results demonstrate that the enhancement by -adrenoceptor antagonists of the release of noradrenaline depends on the biophase concentration of noradrenaline. Under the present conditions, graded increases in biophase noradrenaline concentration led to graded increases in the effect of the antagonists. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train. Under the present conditions, graded increases in train length up to about 20s led to graded increases in the effect of the antagonists, even though the average biophase concentration of noradrenaline did not change with the pulse train length. This pattern of effects of the -antagonists is not shared by at least one other release-enhancing drug, namely TEA.     

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.     

Influence of neuronal uptake on pre- and postjunctional effects of α-adrenoceptor agonists in tissues with noradrenaline — ATP cotransmission

Summary This study was undertaken in an attempt to explain why in some of the tissues in which noradrenaline and ATP act as co-transmitters the noradrenergic component predominates, while in others the predominant component is purinergic. Four different tissues were used: the epididymal portion of the rat vas deferens and the rabbit ear artery, tissues where the noradrenergic component predominates, and the prostatic portion of the rat vas deferens and the rabbit jejunal artery, where the purinergic component predominates. The noradrenaline content as well as the electrically-evoked release of noradrenaline were determined in all tissues. To determine the evoked release, the tissues were pretreated with pargyline (1 mmol · 1^–1) and then exposed to ³H-noradrenaline, washed out and transmurally stimulated (1 Hz). In addition, the influence of inhibition of neuronal uptake by desipramine (40nmo1·1^–1) on pre- and postjunctional effects of adrenaline and -methylnoradrenaline (and/or noradrenaline) was compared.The noradrenaline content of the tissues averaged: 17.4, 23.2, 3.1, and 4.8 g·g^–1 for the epididymal and the prostatic portions of the rat vas deferens and for the ear and the jejunal arteries of the rabbit, respectively. The fractional electrically-evoked release of ³H-noradrenaline was 2.02 and 2.04 × 10^–5 for the epididymal and the prostatic portions of the rat vas deferens, respectively, and 3.33 and 3.26 × 10^–5 for the ear and the jejunal arteries of the rabbit, respectively. Desipramine enhanced much more the postjunctional effect of noradrenaline, adrenaline, and -methylnoradrenaline in the epididymal than in the prostatic portion of the rat vas deferens. Moreover, desipramine similarly enhanced the effects of noradrenaline and adrenaline in the ear artery, while it did not modify the responses to these amines in the jejunal artery. The influence of inhibition of neuronal uptake on the prejunctional effect of sympathomimetic agonists was studied in the rat vas deferens only. Desipramine caused very marked and similar enhancements of the inhibitory effect of the amines in the epididymal and the prostatic portions (72.5 and 93.5 times, respectively, for adrenaline, and 34.7 and 41.4 times, respectively, for -methylnoradrenaline).These results are compatible with the hypothesis that postjunctional adrenoceptors are situated closer to the varicosities in the epididymal than in the prostatic portion of the rat vas deferens and also closer in the rabbit ear than in the rabbit jejunal artery. Therefore, the variation in the relative importance of the noradrenergic and the purinergic components may depend on the distance between the sites from where the transmitters are released and the sites where they act. Send offprint requests to J. Gonçalves at the above address     

The effects of the α2-adrenoceptor antagonist idazoxan on sleep in normal volunteers

The effects of idazoxan, a novel antidepressant that is a potent and selective α(2)-adrenoceptor antagonist, were studied on sleep. Twelve normal male volunteers had sleep recordings made using the Oxford ambulatory system (Medilog 9000) with subsequent analysis by automatic sleep staging and visual inspection. Idazoxan was given in a dose of 40 mg three times a day for 21 days. Sleep recording on day 3 demonstrated markedly reduced indices of rapid eye movement (REM) sleep, although other sleep parameters were only minimally affected. The effect on REM persisted unchanged at day 18, however on the second day after withdrawal a rebound in REM measures was observed. These data show that idazoxan has an effect on sleep which is very similar to that of other antidepressants, and emphasize a role for noradrenaline in the regulation of REM sleep.     

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     

Discriminative stimulus effects of the α2-adrenoceptor antagonist idazoxan

Rats were trained to discriminate a dose of the ₂-adrenoceptor antagonist idazoxan (10 mg/kg IP) from saline. The discriminative stimulus produced by idazoxan was dose related and generalised to yohimbine. However, generalisation did not occur with a variety of compounds from other pharmacological categories including the ₁-adrenoceptor agonist cirazoline, the ₂-adrenoceptor antagonist prazosin, and the ₂-adrenoceptor agonist clonidine. The idazoxan stimulus was not antagonised by either prazosin or clonidine, although it was clear that idazoxan antagonised the reductions in response rate produced by clonidine. Dose-related responding on the idazoxan-associated lever was produced by the anxiolytics buspirone and ipsapirone and by their metabolite MJ 13653 (1-PP), which has previously been shown to be an ₂-adrenoceptor antagonist. In general, however, high levels of generalisation occurred with these three compounds only at doses which substantially reduced response rates. These results demonstrate that idazoxan can give rise to a discriminative stimulus which is probably mediated through antagonism at ₂-adrenoceptors although the failure of clonidine to block the idazoxan stimulus is difficult to explain.     

Effects of α2-adrenoceptor agonists on locus coeruleus firing rate and brain noradrenaline turnover in N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-treated rats

Summary Previous studies have shown that a low dose of the alkylating compound N-ethoxycarbonyl-2-ethoxy1,2-dihydroquinoline (EEDQ) reduces the density of ₂-adrenoceptors in rat cerebral cortex and antagonizes the effects of an ₂-adrenoceptor agonist on noradrenaline release in rat cortical slices. In the present study, a corresponding dose of EEDQ (1 mg/kg, s. c., 24 h) was shown to reduce the effect of the ₂-adrenoceptor agonists clonidine and guanfacine on noradrenaline turnover in rat brain while not affecting the inhibitory effect of clonidine on locus coeruleus (LC) cell firing. When considerably higher doses of EEDQ were administered (10 and 20 mg/kg, s. c., 24 h) not only the biochemical but also the electrophysiological effects of clonidine were markedly reduced (or even reversed). The data support the notion that EEDQ decreases the responsiveness of brain ₂-adrenergic receptors; moreover, they indicate that ₂-adrenoceptors regulating LC activity are characterized by a larger receptor reserve or are less sensitive to the influence of alkylation than are the population of ₂-adrenoceptors regulating noradrenaline utilization. Send offprint requests to G. Engberg, at the above address     

In vivo potentiation of reboxetine and citalopram effect on extracellular noradrenaline in rat brain by α2-adrenoceptor antagonism

The therapeutic activity of noradrenaline reuptake inhibitors (NaRIs) and serotonin reuptake inhibitors (SSRIs) as antidepressant is based on their ability to increase monoamine concentrations in the synaptic cleft. α₂-Adrenoceptors inhibit noradrenaline (NA) release, which modulates antidepressant neurochemical activity. The present study assesses the influence of the addition of the selective α₂-adrenoceptor antagonist RS79948 to the NaRI reboxetine and the SSRI citalopram on brain extracellular NA. Dual-probe microdialysis technique in the locus coeruleus (LC) and prefrontal cortex (PFC) was performed in freely moving rats. Acute reboxetine (3 and 5 mg/kg i.p.) promoted a dose-dependent increase of NA in LC (164 ± 15%; 243 ± 24%) and PFC (140 ± 7%; 181 ± 30%). Acute citalopram (5 mg/kg i.p.) did not change NA in LC or PFC, but at 10 mg/kg i.p. increased NA in LC (144 ± 14%) and decreased it in PFC (− 42 ± 7%). An inactive dose of RS79948 (0.1 mg/kg i.p.) in rats pretreated with reboxetine (3 mg/kg i.p.) or citalopram (5 mg/kg i.p.) induced a significant enhancement of NA in LC (reboxetine: 462 ± 137%; citalopram: 142 ± 11%) and PFC (reboxetine: 281 ± 56%; citalopram: 130 ± 16%). The results indicate that co-administration of selective α₂-adrenoceptor antagonist drugs might improve the effects of NaRI or SSRI antidepressants by enhancing extracellular NA concentrations in the brain.     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address     

Acute dilation to α2-adrenoceptor antagonists uncovers dual constriction and dilation mediated by arterial α2-adrenoceptors

Background and purpose:

In mouse tail arteries, selective α₂-adrenoceptor antagonism with rauwolscine caused powerful dilation during constriction to the α₁-adrenoceptor agonist phenylephrine. This study therefore assessed phenylephrine''s selectivity at vascular α-adrenoceptors and the mechanism(s) underlying dilation to rauwolscine.

Experimental approach:

Mouse isolated tail arteries were assessed using a pressure myograph.

Key results:

The α₂-adrenoceptor agonist UK14,304 caused low-maximum constriction that was inhibited by rauwolscine (3 × 10⁻⁸ M) but not by the selective α₁-adrenoceptor antagonist prazosin (10⁻⁷ M). Concentration–effect curves to phenylephrine, cirazoline or noradrenaline were unaffected by rauwolscine but were inhibited by prazosin, which was more effective at high compared with low levels of constriction. In the presence of prazosin, rauwolscine inhibited the curves and was more effective at low compared with high levels of constriction. Although rauwolscine alone did not affect concentration–effect curves to phenylephrine, noradrenaline or cirazoline, it caused marked transient dilation when administered during constriction to these agonists. Dilation was mimicked by another α₂-adrenoceptor antagonist (RX821002, 3 × 10⁻⁸ M), was dependent on agonist selectivity, and did not occur during adrenoceptor-independent constriction (U46619). During constriction to UK14,304 plus U46619, rauwolscine or rapid removal of UK14,304 caused transient dilation that virtually abolished the combined constriction. Endothelial denudation reduced these dilator responses.

Conclusions and implications:

Inhibition of α₂-adrenoceptors caused transient dilation that was substantially greater than the contribution of α₂-adrenoceptors to the constriction. This reflects a slowly reversing α₂-adrenoceptor-mediated endothelium-dependent dilation and provides a rapid, sensitive test of α₂-adrenoceptor activity. This approach also clearly emphasizes the poor selectivity of phenylephrine at vascular α-adrenoceptors.     

Vasopressor nerve responses in the pithed rat, previously identified as α2-adrenoceptor mediated, may be α1D-adrenoceptor mediated

Responses to pressor nerve stimulation in the pithed rat have been variously described as mediated, at least in part, by α(2)-adrenoceptors and by α(1A) and α(1D)-adrenoceptors. We have examined the subtypes of α-adrenoceptor involved in rises in diastolic blood pressure in the pithed rat preparation produced by vasopressor nerve stimulation with 10 pulses at 1 Hz or 20 pulses at 5 Hz. Vasopressor nerve responses to 1 Hz stimulation were markedly inhibited by the α(1A)-adrenoceptor antagonist RS 100329 (0.1mg/kg) and by the α(1D-)adrenoceptor antagonist BMY 7378 (0.1mg/kg). The α(2)-adrenoceptor antagonist yohimbine (0.1mg/kg) significantly increased pressor nerve responses to 1 Hz stimulation, but yohimbine (1mg/kg) significantly reduced pressor nerve responses. However, following BMY 7378 (0.1mg/kg), yohimbine (1mg/kg) did not produce any further inhibition of pressor nerve responses to 1 Hz stimulation. The α(2A)-adrenoceptor antagonist BRL 44408 (1mg/kg) did not reduce pressor responses to 1 Hz stimulation. BMY 7378 produced much less inhibition of pressor nerve responses to 5 Hz stimulation, whereas RS 100329 produced similar inhibition of 1 Hz and 5 Hz responses. Yohimbine (0.1 and 1mg/kg) did not significantly affect pressor nerve responses to 5 Hz stimulation. In conclusion, pressor nerve responses in the pithed rat involve both α(1A) and α(1D)-adrenoceptor, but there is no clear evidence for the involvement of α(2)-adrenoceptors.     

The effects of ethanol in combination with the α2-adrenoceptor agonist dexmedetomidine and the α2-adrenoceptor antagonist atipamezole on brain monoamine metabolites and motor performance of mice

The time course of the effects of ethanol alone and in combination with the selective α₂-adrenoceptor agonist dexmedetomidine and the α-adrenoceptor antagonist atipamezole was studied in NIH-Swiss mice. Core body temperature, rotarod performance, motility and changes in the noradrenaline, dopamine, and 5-hydroxytryptamine (5-HT) metabolite contents of different brain parts (limbic forebrain, striatum, lower brainstem, the rest of the forebrain + midbrain and hypothalamus) were measured. Atipamezole (3 mg/kg) attenuated the hypothermia induced by either ethanol (3 g/kg) alone or ethanol in combination with dexmedetomidine (0.3 mg/kg). Atipamezole shortened the duration of the ethanol-impaired and ethanol + dexmedetomidine-impaired rotarod performance. Further, atipamezole prevented the decreased motility due to the combined treatment with ethanol and dexmedetomidine. Ethanol increased 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) values. Dexmedetomidine alone decreased MHPG and 5-hydroxyindoleacetic acid (5-HIAA) concentrations and increased DOPAC and HVA values. Dexmedetomidine combined with ethanol resulted in a further increase in DOPAC and HVA values. Pharmacokinetic parameters did not contribute to this antagonism of ethanol's effects by atipamezole, nor did the antagonism observed in rotarod performance or hypothermia seem to correlate with the changes seen in the brain noradrenaline and dopamine or 5-HT metabolism. In conclusion, these findings suggest that several ethanol effects are not mediated via direct activation of α₂-adrenoceptors, even though some of ethanol's behavioral and physiological effects may be antagonized by coadministration of α₂-adrenoceptor antagonists.     

The effects and after effects of the α2-adrenoceptor antagonist idazoxan on mood, memory and attention in normal volunteers

Idazoxan, an α( 2)-adrenoceptor antagonist, is an effective antidepressant with a mode of action different from that of conventional antidepressants. As it is used as an antidepressant it is important to know whether there are any unwanted CNS side effects. Study of its effects will also provide information on the relationship between noradrenergic function and mood and performance. Twelve normal male volunteers who were given the drug (40 mg orally three times daily for 21 days) were compared with 12 matched controls. A computerized test battery was used to assess mood and various aspects of memory and attention. Many of the tests of memory and attention in the battery have been widely used over the last 20 years, and in addition two new selective attention tasks were included. The subjects were tested 3 days before starting the drug, on days 3 and 17 while on the drug, and after they had stopped taking the drug (4 days after and 24 days after). Control subjects followed a similar testing schedule. The results showed that the drug had no effect on mood, logical reasoning, retrieval from semantic memory or sustained attention. However, the drug did improve one aspect of selective attention (the place repetition effect), although this effect was only observed on the third day on the drug. Overall, the results suggest that idazoxan produces selective performance improvements, and that the measures of selective attention used here may be more sensitive indicators of drug effects than some of the traditional tasks currently in use.     

Down-regulation of α2-adrenoceptor subtypes

To characterize further the α₂-adrenoceptor subtypes in terms of their regulation, monolayers of cells expressing either the α_2A (CHO-A2AR cells) or α_2C (OK cells) subtype were preincubated with norepinephrine for various times and the extent of receptor down-regulation was assessed. Exposure to 30 μM norepinephrine caused a similar time course and extent of down-regulation (approximately 50%) in both cells lines. The extent of down-regulation caused by 0.3 μM norepinephrine in OK cells was similar to that with 30 μM norepinephrine in CHO-A2AR cells, although the time course was somewhat slower. Reversal of the down-regulation of the α₂-adrenoceptor caused by 30 μM norepinephrine was more rapid in the CHO-A2AR than in the OK cell. With 0.3 μM norepinephrine, reversal of down-regulation of the α₂-adrenoceptor in the OK cell was slightly faster than that of the CHO-A2AR cell with 30 μM norepinephrine. These data indicate that although norepinephrine is more potent in causing down-regulation of the α_2C (OK cells) as compared to the α_2A subtype (CHO-A2AR cells), the time courses for down-regulation and its reversal are similar for the two subtypes.     

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.     

Protein kinase C activation increases noradrenaline release from the rat hippocampus and modifies the inhibitory effect of α2-adrenoceptor and adenosine A1-receptor agonists

Summary We have studied the effect of stimulating protein kinase C with phorbol esters on the release of [³H]-noradrenaline (NA) in the absence or presence of presynaptic ₂-adrenoceptor blocking agents and compared that to the elevation of cyclic AMP levels more than 10-fold by a combination of rolipram and forskolin. 4--Phorbol 12,13-dibutyrate (PDiBu) increased stimulated (3 Hz) [³H]-NA release markedly and in a concentration dependent manner. 4--Phorbol-12,13-didecanoate was ineffective. The effect of PDiBu was not significantly reduced by nifedipine (1 M), but was proportionally less in the presence of an ₂-adrenoceptor antagonist, yohimbine. PDiBu inhibited the presynaptic effect of ₂-adrenoceptor agonists clonidine and UK 14304. By contrast, the presynaptic effect of the adenosine analogue R-PIA was not reduced by PDiBu. PDiBu caused an increase in cyclic AMP that depended on adenosine receptor stimulation. Elevation of cyclic AMP had a limited effect on NA release from rat hippocampus, and did not significantly decrease the presynaptic inhibitory effect of UK 14304 (0.1 M), of morphine (1 M) or of the adenosine A₁-receptor agonist CHA (1 M). The effect of phorbol esters and several presynaptic inhibitors of NA-release in the rat hippocampus cannot be explained by changes in cyclic AMP levels in the tissue. Phorbol esters that stimulate protein kinase C appear to interact with a target that is the site of action ₂-adrenoceptors in this tissue. This site is not a dihydropyridine sensitive Ca-channel and is also different from the target of presynaptic adenosine receptors. Thus, activation of protein kinase C discriminates between apparently similar presynaptic mechanisms. Send offprint requests to B. Fredholm at the above address