Effects of hypoxia on noradrenaline release and neuronal reuptake in isolated rabbit thoracic aortic strips

Summary To clarify the effects of hypoxia on stimulus-evoked noradrenaline release and on neuronal reuptake of the released noradrenaline, we examined the effects of hypoxia on contraction responses of rabbit thoracic aortic strips to transmural electrical stimulation and on the stimulation-evoked overflow of total [³H] and [³H]noradrenaline from the strips prelabelled with [³H]noradrenaline. This was done in the presence or absence of an inhibitor of neuronal uptake (cocaine). In a medium equilibrated with a gas mixture of 95% O₂/5% CO₂ (control), cocaine doubled the stimulation-evoked overflow of total [³H] and [³H]noradrenaline; there was a concomitant increase (130%) in contractions to electrical stimulation. At 0% O₂ (95% N₂/5% CO₂, hypoxia), cocaine had no significant effects on either the stimulation-evoked overflow of total [³H] and [³H]noradrenaline or contractions. In the absence of the drug, hypoxia decreased the stimulation-evoked overflow of total [³H] and [³H]noradrenaline to 47% and 43%, respectively, of the control values, whereas these values were 31% and 28%, respectively, after exposure to cocaine. The inhibition by hypoxia of contraction responses to electrical stimulation was greater in the presence of cocaine than in its absence. These results show that hypoxia inhibits both noradrenaline release evoked by a given stimulus and neuronal uptake.This work was supported by a Grant-in-Aid for New Drug Development from the Ministry of Health and Welfare of Japan and by a Grant from Smoking Research Foundation, Japan Send offprint requests to S. Miwa at the above address     

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

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

Pharmacological characterization of the imidazoline receptor which mediates inhibition of noradrenaline release in the rabbit pulmonary artery

Summary Imidazoline receptors involved in modulation of noradrenaline release were characterized in the rabbit pulmonary artery preincubated with [³H]noradrenaline and superfused with physiological salt solution containing cocaine, corticosterone and propranolol. Tritium overflow was evoked by transmural electrical stimulation.The ₂-adrenoceptor blocking imidazolines tolazoline, BDF 6100 [2-(2-imidazoline-2-ylamino)-isoindoline] and BDF 7572 (4,7-dichloro-derivative of BDF 6100) increased the electrically evoked ³H overflow; the concentration-response curves were bell-shaped. In contrast, two other imidazolines, i. e. moxonidine and clonidine, two guanidine derivatives structurally related to BDF 6100, i. e. aganodine and BDF 7579 [4-chloro(2-isoindolinel)-guanidine], as well as the catecholamine noradrenaline concentration-dependently inhibited the evoked ³H overflow. The concentration-response curves for moxonidine, clonidine, aganodine, BDF 7579 and noradrenaline were shifted to the right by rauwolscine. The apparent pA₂ value of rauwolscine against moxonidine was 8.22, whereas those against clonidine, aganodine, BDF 7579 and noradrenaline were in the range of 6.37–6.77 and, hence, considerably lower than reported for ₂-adrenoceptors. In the presence of rauwolscine an inhibitory effect was also observed with the ₂-adrenoceptor blocking imidazolines tolazoline, BDF 6100, BDF 7572, and the imidazolineST 587 [2-(2-chloro-5-trifluoromethylphenylimino)-imidazoline]; the rank order of potency of all guanidines and imidazolines investigated was: aganodine > BDF 7579 > BDF 7572 > BDF 6100 > clonidine > ST 587 > moxonidine > tolazoline. Amiloride, 1-benzylimidazole and histamine were ineffective. After irreversible blockade of -adrenoceptors by preexposure to phenoxybenzamine, evoked ³H overflow was still inhibited by aganodine, BDF 7579 and noradrenaline. Under this condition the maximal effects obtainable with the guanidines and in particular with noradrenaline were lower than in the presence of rauwolscine.These findings are compatible with our previous suggestion that imidazoline receptors mediating inhibition of noradrenaline release exist on the sympathetic nerve terminals of the rabbit pulmonary artery. Comparison of the present data with those obtained in other preparations containing imidazoline recognition sites revealed that those sites are different from the present ones. It is conceivable that the receptor characterized here represents an allosteric site of the ₂-adrenoceptor or a so far undescribed -adrenoceptor subtype since it can be activated not only by imidazolines and guanidines but also by noradrenaline and can be blocked by rauwolscine. Comparison of the properties of isoindolines substituted with either aminoimidazoline or guanidine reveals that the imidazolines (e.g. BDF 7572) possess both ₂-adrenoceptor antagonistic and imidazoline receptor agonistic properties, whereas the analogous guanidines (e. g. aganodine) are imidazoline receptor agonists as well as ₂-adrenoceptor agonists. Send offprint requests to M. Göthert at the above address     

Involvement of α1- and α2-adrenoceptors in the responses of proximal and distal segments of the canine saphenous vein to exogenous and endogenous noradrenaline

Summary In the present study the relationship between adrenergic nerve terminals and postjunctional -adrenoceptors mediating the responses to the endogenous transmitter was compared at proximal and distal levels of the canine saphenous vein.Concentration-response curves to noradrenaline and to tyramine as well as frequency-response curves to electrical stimulation were compared at both levels of the vessel, in the absence and presence of either prazosin (100nmol·l^–1) or yohimbine (100nmol·l^–1) The influence of inhibition of neuronal uptake by cocaine (12 µmol·l^–1) on the responses to noradrenaline in the presence of prazosin (56 nmol·l^–1) or yohimbine (20 nmol·l^–1) was compared at the proximal level. The results show that, at the proximal level, the maximal responses to electrical stimulation and tyramine reached 80.1±2.2 (n = 18) and 74.2±1.9 (n = 18)%, respectively, of the maximal responses to noradrenaline, and 70.3±0.8 (n = 15) and 53.1 ± 1.2 (n = 14) %, respectively, at the distal level. Furthermore, the proximal strips were more sensitive to electrical stimulation than the distal ones. Prazosin had a much greater inhibitory effect on the contractile responses to noradrenaline than on those to electrical stimulation, at both levels. At proximal level, the shifts (to the right) of the concentration (frequency)-response curves (at EC₅₀) amounted to 0.58±0.02 (n = 16) and 0.18±0.02 (n = 8) log units, respectively (P<0.05), but, at the distal level, to 1.12±0.03 (n = 16) and 0.28±0.08 (n = 8) log units, respectively (P< 0.05). At the proximal level, yohimbine antagonizes about equally the responses to noradrenaline and the responses to electrical stimulation. However, at the distal level, the shift of the concentration-response curve to noradrenaline was much larger than that of the frequency-response curve to electrical stimulation [1.12±0.07 and 0.80±0.10 (n = 6) log units at EC₅₀, respectively (P<0.05)]. The leftward shift of the concentration-response curve to noradrenaline caused by cocaine was more pronounced in the presence of prazosin than in the presence of yohimbine: 0.95±0.15 and 0.69±0.12 (n = 12) log units, respectively (P<0.05).We conclude that, in the canine saphenous vein: 1) noradrenaline released from the adrenergic nerve terminals by electrical stimulation and by tyramine preferentially activates ₂-adrenoceptors at both proximal and distal levels; 2) the effectiveness of ₂-adrenoceptor stimulation is greater at the proximal than at the distal level; 3) ₁-adrenoceptors at the distal level seem to be different from those at the proximal one. Send offprint requests to S. Guimarães at the above address     

Presynaptic imidazoline receptors and α2-adrenoceptors in the human heart: discrimination by clonidine and moxonidine

The involvement of presynaptic ₂-autoreceptors and imidazoline receptors in the modulation of noradrenaline release was investigated in strips from human atrial appendages preincubated with [³H]noradrenaline and superfused with medium containing desipramine and corticosterone. Electrical impulses were applied transmurally at 2 Hz. The imidazoline derivatives moxonidine and clonidine reduced the evoked tritium overflow in a concentration-dependent manner. Moxonidine was 18-fold more potent than clonidine. The concentration-response curve for moxonidine, but not for clonidine was shifted to the right by the ₂-adrenoceptor antagonist rauwolscine. The apparent pA₂ value of rauwolscine against moxonidine was 8.41. An inhibitory effect was also observed for the imidazoline derivative BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), a mixed ₂-adrenoceptor antagonist/imidazoline receptor agonist; BDF 6143 was about 2-fold more potent than clonidine. Rauwolscine (1 M) did not substantially shift the concentration-response curve of BDF 6143.It is concluded that noradrenaline release in the human atrium is inhibited not only via presynaptic ₂-autoreceptors but also via presynaptic non-I₁, non-I₂ imidazoline receptors. The failure of rauwolscine to antagonize the inhibitory effect of clonidine suggests that clonidine preferentially stimulates the presynaptic imidazoline receptors; the ₂-adrenoceptor component of its action is probably suppressed by an inhibitory interaction between the two receptors. In contrast, moxonidine acts via presynaptic ₂-autoreceptors, leaving the presynaptic imidazoline receptor unaffected.     

Alterations in β-adrenoceptor number and catecholamine content of chick atria after reversible sympathetic denervation with 6-hydroxydopamine

Summary 1. The characteristics of [³H]-dihydroalprenolol (DHA) binding were determined in atria from untreated chicks. [³H]-DHA binding to atrium homogenates was rapid (k1 = 8.52 × 10⁸ 1 mol^–1 min^–1), reversible (k _–1 = 0.47 min^–1), saturable, and of high affinity (K _D = 0.61.–nmol/l). Isoprenaline competed for specific [³H]-DHA binding in a stereoselective manner; IC₅₀ values (mol/l) were: (–)isoprenaline 0.12, (+)isoprenaline 4.7. 2. The number of [³H]-DHA binding sites and catecholamine content of left and right atria were examined after injection of chicks with a single dose of 6-hydroxydopamine hydrobromide (100 mg/kg). There were transient increases in the number of [³H]-DHA binding sites in both the left and the right atrium after 6-OHDA treatment. These increases were quicker in onset and in offset in the right atrium than in the left atrium. [³H]-DHA binding was significantly increased- in the left atrium at 5 and 7 days, and in the right atrium at 3 and 5 days after 6-OHDA injection. 3. Saturation binding isotherms indicated that the increase in [³H]-DHA binding was due to an increase in -adrenoceptor number with no change in affinity for [³H]-DHA. 4. Twenty four hours after 6-OHDA treatment there was a significant (80%) decrease in noradrenaline content of left and of right atria. 5. The decrease in noradrenaline content was reversible, noradrenaline levels returning to 55% of control in left atrium and to 71% of control in right atrium by 21 days after 6-OHDA treatment. These changes are consistent with reversible sympathetic denervation of the atria. 6. Adrenaline levels in the atria were 5 to 18% of total catecholamine (noradrenaline+adrenaline) content and were not significantly altered by 6-OHDA treatment.     

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     

Actions of alpha-adrenoceptor blocking agents on two types of intracellular calcium stores mobilized by noradrenaline in rat aorta

Summary In isolated rat aortic strips noradrenaline induces a biphasic contractile response in Ca-free medium, associated with two different intracellular calcium pools, one of which is common to caffeine. We analyzed the mechanisms involved in the depletion and repletion of both intracellular Ca pools sensitive to noradrenaline in different experimental procedures in presence of prazosin, phentolamine and yohimbine.At 37°C the -adrenergic blocking agents inhibited contractile responses to noradrenaline in Ca-free medium, with prazosin being highly selective. ₂-adrenoceptors probably do not participate in the release of Ca from internal stores, as no contractile response was observed after addition of clonidine in Ca-free medium. This indicates that noradrenaline-induced Ca-release from internal stores is mainly due to activation of ₁-adrenoceptors. At 25°C, these compounds failed to inhibit caffeine-induced contraction in Ca-free medium, but abolished the release of Ca from an intracellular store only sensitive to noradrenaline. This effect is attributale to a blockade of ₁-adrenoceptors and/or inhibition of receptor-mediated signal transduction. Correspondence to: M.P. D'Ocon at the above address     

Inhibition of uptake1 by (+)-oxaprotiline reveals a differential central regulation of noradrenaline and adrenaline release

Summary Inhibition of uptake, in the central nervous system leads to a decrease of sympathetic outflow to many tissues; central a2-adrenoceptors are involved in this decrease. The aim of the present study was to compare the effects of the selective uptake, inhibitor (+)-oxaprotiline on the plasma kinetics of noradrenaline and adrenaline in anaesthetized and in conscious rabbits. [³H]Noradrenaline and [^3H]adrenaline were infused iv. The arterial plasma concentrations of endogenous and radiolabelled noradrenaline and adrenaline were measured, and the clearance from and spillover into the plasma of noradrenaline and adrenaline were calculated.Results obtained in conscious and anaesthetized rabbits were similar. (+)-Oxaprotiline 0.2, 0.6 and 1.8 mg kg^–1 iv. dose-dependently reduced the clearance of [³H]noradrenaline from the plasma. The clearance of [³H]adrenaline was reduced less. The spillover of endogenous noradrenaline was decreased by up to 35%. In contrast, the spillover of adrenaline tended to be enhanced. Prazosin 0.1 and 1 mg kg^–1 was injected iv. in a second part of each experiment. It lowered the blood pressure and caused a marked increase in noradrenaline spillover but no increase or even a decrease in adrenaline spillover.The results are compatible with the following hypothesis. The sympathetic outflow from the central nervous system is subject to a twofold a-adrenoceptor-mediated modulation: -adrenoceptor-mediated inhibition and ₁-adrenoceptor-mediated excitation. In the control of the sympathetic outflow to many extra-adrenal tissues, the ₂-adrenergic inhibition prevails. Uptake₁ inhibitors depress sympathetic outflow to such tissues by enhancing the ₂-adrenergic inhibition. In the regulation of the sympathetic outflow to the adrenal medulla, in contrast, ₂-adrenergic inhibition and ₁-adrenergic excitation have a similar impact. Uptake, inhibitors, hence, cause little change in adrenaline release: the two opposing influences cancel out. Prazosin produces an increase in noradrenaline but not adrenaline release because the loss of the central ₁ sympathoexcitation attenuates at best slightly the baroreflex to most extra-adrenal tissues but dampens markedly the baroreflex to the adrenal medulla. Correspondence to B. Szabo at the above address     

Evidence against a functional link between noradrenaline uptake mechanisms and presynaptic alpha-2 adrenoceptors

Summary Slices prepared from rat cerebral cortex were labelled with ³H-noradrenaline and superfused. Electrical field stimulation was carried out 15 min (S₁) and 45 min (S₂) after the start of collection of 5-min samples using 4 pulses delivered at 100 Hz. Drugs acting at ₂-adrenoceptors were added 20 min before S₂, and their effects were evaluated using the S₂/S₁-ratio. The ₂-adrenoceptor antagonists idazoxan (1 mol/l) and rauwolscine (1 mol/l) failed to increase stimulation-evoked overflow of radioactivity in the absence or presence of the noradrenaline reuptake inhibitor desipramine (1 gmol/l). This indicates that the duration of electrical stimulation was too short to allow development of ₂-adrenoceptor-mediated auto-inhibition by released noradrenaline. The effect of clonidine (3–1000 nmol/l) on stimulation-evoked overflow of radioactivity was tested in the absence and presence of three different reuptake inhibitors (desipramine, 1 ol/l; maprotiline, 1 ol/l; cocaine, 10 mol/l). The analysis yielded identical concentration-response curves under all conditions. These results argue against an action of inhibitors of neuronal reuptake of noradrenaline at the presynaptic ₂-adrenoceptor and against the concept of a functional link between uptake site and receptor. Send offprint requests to E. A. Singer     

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.     

α2-Adrenoceptor mediated inhibition of the release of radiolabelled 5-hydroxytryptamine and noradrenaline from slices of the dorsal region of the rat brain

Summary Possible local interactions between noradrenergic and serotonergic systems in the dorsal raphe region of the rat were investigated by studying the effects of various drugs on depolarization (20 mmol/l K⁺)-induced release of [³H]5-hydroxytryptamine (5-HT) and [³H]noradrenaline (NA) in vitro using a superfusion method. Exogenous 5-HT did not influence the release of [³H]NA. However, NA (in the presence of 10 mol/l desipramine) as well as the selective ₂-adrenoceptor agonists clonidine and oxymetazoline strongly inhibited [³H]5-HT release. The selective ₁-adrenoceptor agonists phenylephrine and methoxamine did not affect the release of either [³H]5-HT or [⁵H]NA. The inhibition by NA of both [³H]5-HT and [⁵H]NA release was not affected by the -adrenoceptor antagonist sotalol nor by the selective ₁-adrenoceptor antagonist prazosin. However, phentolamine and the selective ₂-adrenoceptor antagonists yohimbine and rauwolscine competitively antagonized the inhibitory effect of NA on [³H]NA release (respective pA₂-values 7.5 and 8.3) and on [³H]5-HT release (respective pA₂-values 7.7 and 8.2). Moreover, the release of [³H]NA and also, but to a lesser extent, that of [³H]5-HT were increased by the antagonists. It is concluded that the release of both 5-HT and NA in the dorsal raphe region may be subject to presynaptic inhibition by NA via activation of ₂-adrenoceptors.Send offprint requests to A. L. Frankhuijzen     

Studies on alpha adrenoceptors in guinea-pig peripheral lung strips

Summary Contractile responses of guinea-pig peripheral lung strips to noradrenaline were determined in the presence of propranolol (2.5 × 10^–6 mol/l). All strips (n = 44) contracted to noradrenaline and a geometric mean EC₅₀ of 1.4 × 10^–6 mol/l (1.1 × 10^–6 mol/l, 1.8 x 10^–6 mol/l 95% confidence limits) was obtained. Contractions were antagonised by phentolamine (5 × 10^–7–10^–5 mol/l) and by prazosin (10–10^–7 mol/l). Dose-ratios (DR) were calculated and log (DR-1) was plotted against log concentration of antagonist to yield slopes (± SE) of 0.84 ± 0.14 and 0.73 ± 0.22 respectively which were not significantly different from unity. A pA₂ value (± SE) of 6.7 ± 0.2 was obtained for phentolamine and 7.5 ± 0.1 for prazosin. Yohimbine (10^–7–10^–5 mol/l) did not significantly affect the maximal tension generated or the EC₅₀ values for noradrenaline. These results suggest that ₁ adrenoceptors are mediating the contractile responses to noradrenaline. In the presence of cocaine (10^–5 mol/l, n=18), normetanephrine (2 × 10^–5 mol/l, n = 15), hydrocortisone (2.5 × 10^–5 mol/l, n = 15) and normetanephrine (2 × 10^–5-5 mol/l) plus cocaine (10^–6 5 mol/l, n=15) pA₂ values for phentolamine of 6.9, 6.7, 6.6, and 6.3 respectively were obtained. Since these pA₂ values are not significantly different from 6.7, it is unlikely that this original pA₂ value, which is lower than values obtained with phentolamine at -adrenoceptors in other tissues, may be explained by neuronal or extraneuronal uptake of noradrenaline. A possible explanation may be that more than one population of -adrenoceptors contribute to changes in tension in peripheral lung strips. Send offprint requests to J. P. Seale at the above address     

Differences between full and partial α-adrenoceptor agonists in eliciting phasic and tonic types of responses in the longitudinal smooth muscle of the rat portal vein

Summary The aim of the present investigation was to study, taking into account both quantitative and qualitative differences, the influence of full and partial -adrenoceptor agonists on spontaneous myogenic activity in the rat portal vein.We found that the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, phenylephrine, St 587, Sgd 101/75, B-HT 920 and UK-14,304 could increase the amplitude of the phasic myogenic contractions in the rat portal vein with apparent differences in EC₅₀ and E_max values. In addition to an increase in phasic myogenic activity, the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, and phenylephrine were also able (in higher concentrations) to increase the basal tone of the rat portal vein preparation, again with apparent differences in EC₅₀ and E_max values. Changing the extracellular Ca²⁺ concentration from 0.9 mmol/l to 2.5 mmol/1 had no influence on the phasic character and the concentration range in which St 587 and UK-14,304 increased spontaneous myogenic activity, although changes in amplitude and frequency of the spontaneous myogenic contractions were less pronounced at a higher extracellular Ca²⁺ concentration (2.5 mmol/1). By the use of Schild analysis with the competitive a-adrenoceptor antagonists prazosin (pA₂ = 8.74) and 5-methyl-urapidil (pA₂ = 8.37), it was established that the contractile responses to St 587 were mediated by the same ₁-adrenoceptor subtype as the phasic and tonic type of contraction elicited by phenylephrine as described in a previous study. The concentration-response curve of UK-14,304 was significantly shifted to the right by low concentrations of prazosin (3 nmol/1–30 nmol/1), indicating stimulation of ₁-adrenoceptors by UK-14,304 in the rat portal vein. The -adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine irreversibly blocked the contractile responses to St 587. Based on the method of receptor alkylation with phenoxybenzamine an affinity constant was calculated for St 587 (pK_a = 5.91). Phenoxybenzamine was approximately 1000-fold more potent in inactivating ₁-adrenoceptors than chloroethylclonidine.In conclusion there appeared to be a divergence in the excitation-contraction coupling of ₁-adrenoceptors in the rat portal vein, which is reflected by two types of contraction (phasic versus tonic). The extent to which both the phasic and tonic types of contraction are stimulated by agonists depends on the affinity and intrinsic efficacy for each of the receptor-coupled effector pathways. Thus, partial and full agonism can only meaningfully be discussed if confined to one particular effector pathway. Send offprint requests to H. R. Schwietert at the above address     

Inhibitory effect of dopamine on canine gastric fundus

Summary The mechanism of the inhibitory effect of dopamine on canine stomach fundus was studied in longitudinal and circular muscle fundus strips, contracted by transmural electrical stimulation or by methacholine.Results obtained for longitudinal and circular strips were similar. Dopamine (1 · 10^–6–1 · 10^–4 M) concentration-dependently inhibited frequency-response curves to electrical stimulation; these concentrations did not change the resting tone of the strips. Dopamine (1 · 10^–4 M), tested on contractions of similar amplitude induced in the same strips by electrical stimulation at 0.5 Hz and by methacholine, inhibited the electrically induced contractions but had little influence on the contractions induced by methacholine. The inhibition of the electrically induced contractions by dopamine 1 · 10^–4 M was not influenced by the presence of cocaine 3 · 10^–5 M or hydrocortisone 3 · 10^–5 M.The ₁- and ₂-adrenoceptor antagonist phentolamine and the ₂-adrenoceptor antagonist rauwolscine markedly antagonized the inhibitory effect of dopamine on the response to electrical stimulation at 0.5 Hz. The ₁-adrenoceptor antagonist prazosin and the dopamine receptor antagonists haloperidol and domperidone had no effect. The dopamine receptor antagonist metoclopramide decreased the inhibitory effect of dopamine but had a similar effect on the inhibition caused by noradrenaline.These results indicate that the inhibitory effect of dopamine in the dog gastric fundus is mainly mediated by an interaction with ₂-adrenoceptors on the intramural cholinergic neurons; this effect is largely direct since it was not influenced by cocaine. These results are different from those obtained in the rat gastric fundus, where the inhibitory effect of dopamine is mainly indirect, and due to an interaction with -adrenoceptors on the intramural cholinergic neurons and with -adrenoceptors on the smooth muscle cells. Dedicated to Prof. Dr. E. J. Ariëns (Department of Pharmacology, University of Nijmegen, The Netherlands) on the occasion of his retirement     

Influence of the renin-angiotensin system on sympathetic neurotransmission in canine skeletal muscle in vivo

Summary The physiological importance of interactions between angiotensin II and sympathetic neurotransmission was studied in an in vivo model with constant flow blood perfused gracilis muscle in situ in dogs pretreated with desipramine and atropine. Sympathetic nerve stimulation- (2 and 8 Hz, 480 pulses) evoked overflow of endogenous noradrenaline and vasoconstriction, and vasoconstrictor responses to exogenous noradrenaline (0.5 nmol, locally i. a.) were evaluated.Angiotensin converting enzyme inhibition by benazeprilat (10 mg i. v.; n = 8) reduced arterial angiotensin II levels from 26 ± 8 to 2 +- 1 pM and reduced mean arterial and basal muscle perfusion pressures. Subsequent resubstitution of angiotensin II (3, 30 and 90 ng kg^–1 min^–1 i.v.) elevated arterial angiotensin II dose-dependently (to 67 ± 14, 622 ± 63 and 1940 ± 251 pM, respectively), as well as mean arterial and muscle perfusion pressures. Nerve stimulation-evoked noradrenaline overflow was unchanged following benazeprilat (–4 ± 4 and + 1 ± 8% at 2 and 8 Hz, respectively) and during subsequent infusions of angiotensin II. Vasoconstrictor responses to nerve stimulation and exogenous noradrenaline were also uninfluenced by these treatments. Thus, angiotensin II did not enhance sympathetic neurotransmission at the postjunctional level.Another group of animals was pretreated with noncompetitive -adrenoceptor blockade locally by phenoxybenzamine and benextramine (0.5 mg kg^–1 i. a. of each; n = 7), which abolished vasoconstrictor responses to nerve stimulation. The effects of benazeprilat and subsequent angiotensin II infusions (3 and 30 ng kg^–1 min^–1 i.v.) on circulating angiotensin II levels, mean arterial and muscle perfusion pressures were similar in this group. Following -adrenoceptor blockade, however, inhibition of angiotensin converting enzyme reduced sympathetic nerve stimulation-evoked noradrenaline overflow by 23 ± 4 and 21 ± 5% at 2 and 8 Hz, respectively (P < 0.01 for both). Angiotensin II infusions failed to enhance evoked noradrenaline overflow (–5 ± 10 and –18 ± 10% at 2 Hz; +6 ± 13% and –3 ± 14% at 8 Hz) also under these conditions.It is concluded that circulating angiotensin II does not influence sympathetic vascular control in canine skeletal muscle even at very high levels in arterial plasma. Angiotensin converting enzyme inhibition reduces nerve stimulation-evoked noradrenaline overflow only in the presence of -adrenoceptor blockade, suggesting that prejunctional -adrenoceptors have an overriding importance over prejunctional angiotensin II-receptors in the modulation of noradrenaline release in this model. The effect of converting enzyme inhibition may be related to merely local changes in angiotensin II concentration or — unrelated to the renin-angiotensin system — to other consequences of the blockade of this unspecific enzyme. Send offprint requests to J. Schwieler at the above address     

Effect of prostaglandin E2 on ACTH and β-endorphin release from rat adenohypophysis in vitro after secretagogues which can mimic various first or second messengers

Summary The modulation of the depolarization induced release of [³H]-acetylcholine by agonists acting on -adrenoceptors was studied in superfused rat atrial slices. In this model, noradrenaline and methoxamine, but not UK 14304 reduced the potassium evoked release of [³H]-acetylcholine. The inhibitory action of these drugs was antagonized by the ₁ selective adrenoceptor antagonist prazosin. Propranolol, idazoxan and sulpiride did not antagonize the inhibition by noradrenaline of the potassium-evoked release of [³H]-acetylcholine. Exposure to amphetamine, -phenylethylamine, m- or p-tyramine, increased in a concentration-dependent manner the spontaneous outflow of [³H]-noradrenaline from atrial slices. Yet, these concentrations of the indirectly acting sympathomimetic amines, tested in the presence of an inhibitor of monoamine oxidase (MAO), failed to modify the potassium evoked release of [³H]-acetylcholine. Desipramine 3 mol/l or cocaine 10 mol/l did not affect the release of [³H]-acetylcholine evoked by potassium stimulation. Under similar experimental conditions, -phenylethylamine facilitated the spontaneous outflow of [³H]-noradrenaline, and inhibited the electrically-evoked release of [³H]-serotonin from the hippocampus by activation of ₂-adrenoceptors. It is concluded that the release of acetylcholine from atrial cholinergic neurons can be modulated through inhibitory ₁-adrenoceptors, which are not activated when the release of noradrenaline is induced by indirectly acting sympathomimetic amines. In addition, amphetamine or structurally related amines do not activate directly recognition sites in the cholinergic postganglionic parasympathetic neuron to modify the release of [³H-acetylcholine.     

On the relation between release of prostaglandins and contractility of rabbit splenic capsular strips

Summary Rabbit splenic capsular strips release prostaglandins E and F when contracted by noradrenaline or methoxamine. Contractions and prostaglandin release are dose-dependent. Cocaine increases significantly the effect of noradrenaline, but not that of methoxamine, on contraction of the strips and release of prostaglandin E. Release of prostaglandin F was increased by the addition of cocaine not only when noradrenaline was used as an agonist but also at two of three dose levels of methoxamine.When indometacin is added to the bath fluid, it inhibits prostaglandin release and at the same time potentiates the contractile effects of noradrenaline and methoxamine on the rabbit splenic capsular strips. The prostaglandin-synthetase blocker 5,8,11,14-eicosatetraynoic acid also potentiates the contractions induced by noradrenaline and methoxamine. Both the effects on prostaglandin synthesis and on contraction exerted by indometacin can be reversed, when indometacin is washed out.Exogenous prostaglandins E₁, E₂ and F₂ in concentrations up to 150 ng/ml do not influence contractions of the strips induced by either noradrenaline or methoxamine. At higher concentrations prostaglandin E₁ decreases, but prostaglandins E₂ and F₂ increase the contractions induced by both agonists.The potentiation of the effects of noradrenaline and methoxamine on rabbit splenic strips by indometacin and 5,8,11,14-eicosatetraynoic acid cannot be explained by inhibition of uptake₁ or uptake₂, release of endogenous noradrenaline or inhibition of metabolism of the agonists. It is suggested that the potentiation is caused by inhibition of synthesis of endogenous prostaglandins, although an undefined sensitizing effect of indometacin and 5,8,11,14-eicosatetraynoic acid cannot be completely excluded.Supported by the Deutsche Forschungsgemeinschaft (SFB 70).A preliminary report was given at the Pharmacology Meeting at Mainz, West Germany, March 17–20, 1974 (Jobke, A., and Hertting, G., 1974).     

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     

An increase in cardiac alpha1-adrenoceptors following chronic clonidine treatment

Summary Chronic treatment (22 days) of rats with clonidine (0.5 mg/kg s.c. twice a day followed by 20 h of withdrawal) resulted in a significant increase in the specific [³H]WB4101 binding to ventricular and intraventricular septal ₁-adrenoceptors but no alteration of the atrial ₁-adrenoceptors. Scatchard analysis indicated that the increase in the [³H]WB4101 binding to the clonidine-treated cardiac tissues was due to an enhancement of the ₁-adrenoceptor density since there was a significant increase in the B _max value for the [³H]WB4101 binding to the treated ventricles without a change in the K _d value. The specific [³H]WB4101 binding to cardiac ₁-adrenoceptors was not altered by the acute (1 day) or 7 days treatment with clonidine. Chronic treatment with clonidine had no significant effect on the specific [³H](–)DHA binding to the atrial and ventricular -adrenoceptors. The noradrenaline (NA) concentrations in the clonidine-treated ventricles and intraventricular septae were decreased by 16–20%. These data provide biochemical evidence compatible with a significant reduction of sympathetic outflow to the ventricular myocardium by clonidine.