Lack of presynaptic modulation by isoprenaline of 3H-noradrenaline release from rabbit isolated ear artery

Summary The aim of the present investigation was to examine whether or not presynaptic facilitatory -adrenoceptors are detectable on the postganglionic nerves in the rabbit isolated ear artery. Strips of rabbit central ear artery were incubated with ³H-noradrenaline (10^–7 mol/l; 30 min or 10^–6 mol/l; 60 min). Subsequently, they were washed repeatedly with physiological salt solution. The strips were subjected to electrical-field stimulation (S₁–S₈) and the resultant ³H-overflow was determined.When the ear artery was stimulated with 150 pulses (0.5 ms; 3 Hz; 225 mA), isoprenaline (10^–9–10^–6 mol/l) either alone or in the presence of either rauwolscine (10^–6 mol/l) or phentolamine (10^–6 mol/l) did not alter the stimulation-evoked ³H-overflow. This was also the case in the presence of rauwolscine (10^–6 mol/l) plus either the selective phosphodiesterase inhibitor ICI 63 197 (3 × 10^–5 mol/l) or forskolin (10^–6 mol/l). When the ear artery was stimulated with 300 pulses (1 ms; 5 Hz; 225 mA), isoprenaline had no effect on the stimulation-evoked ³H-overflow. This was also the case when phentolamine (10^–6 mol/l) was present. Propranolol (10^–7–10^–5 mol/l) did not alter the stimulation-evoked ³H-overflow. In some experiments, the stimulation current was reduced to 175 mA in order to obtain similar reference release (S₃) values despite the presence of rauwolscine (150 pulses; 0.5 ms; 3 Hz). Even then, isoprenaline (10^–9–10^–6 mol/l) did not change stimulation-evoked ³H-overflow. The results suggest that postganglionic sympathetic nerves in rabbit central ear artery do not possess presynaptic facilitatory -adrenoceptors. Send offprint requests to J. Abrahamsen at the above address     

Effect of thyroid status on β-adrenoceptors and calcium channels in rat cardiac and vascular tissue

Summary To determine the influence of thyroid hormone on -adrenoceptors and Ca²⁺ channels, rats were treated with thyroxine (75 g/100 g sc daily for 5 days) or propylthiouracil (0.05% in drinking water for 30 days). -Adrenoceptor density in ventricular tissue, measured by [¹²⁵I]iodocyanopindolol binding, was significantly increased and decreased respectively, following thyroxine or propylthiouracil treatment to 124.7 ± 7.11 fmol/mg protein and 71.98 ± 5.37 fmol/mg protein from euthyroid (control) levels of 93.7 ± 4.58 fmol/mg protein. Ca²⁺ channel density, measured by [³H]nitrendipine binding, was altered in the opposite direction; it was significantly decreased and increased to 324 ± 24 fmol/mg protein and 691 ± 31 fmol/mg protein from 562 ± 35 fmol/mg protein after thyroxine or propylthiouracil treatment, respectively. No changes in affinity of either ligand were observed. Responses of isolated papillary muscles from propylthiouracil-treated animals accorded with changes seen in the binding studies. The geometric mean EC₅₀ of isoproterenol increased from 9.5 × 10^–9 mol/1 to 5.5 × 10^–8 mol/l, and the EC₅₀ for calcium decreasedfrom 3.16 × 10^–3 mol/1to 1.36 × 10^–3 mol/1; moreover, the responsiveness to the Ca ²⁺ channel activator Bay K 8644 was increased. The corresponding responses in thyroxine-treated animals could not be examined because of prominent arrhythmic activity. As with papillary muscles the sensitivity of left atria to isoproterenol was decreased after treatment with propylthiouracil, with geometric mean EC₅₀ values increasing from 3.21 × 10^–9 mol/1 to 89.4 × 10^–9 mol/l. This was however, associated with a decrease in the sensitivity to calcium with geometric mean EC₅₀ values increasing from 2.43 × 10^–3 mol/1 to 5.33 × 10^–3 mol/1 and a reduction in the maximal response to Bay K 8644. Treatment with thyroxine had no effect on tissue sensitivity. The responses of isolated tail artery to phenylephrine, calcium and Bay K 8644 were not significantly different in propylthiouracil- or thyroxine-treated animals. Send offprint requests to D. J. Triggle at the above address     

Cyanopindolol is a highly potent and selective antagonist at the presynaptic serotonin autoreceptor in the rat brain cortex

Summary Rat brain cortex slices preincubated with ³H-serotonin were superfused with physiological salt solution containing the serotonin uptake blocker DU 24565 (6-nitroquipazine). The effects of (±)-cyanopindolol and its enantiomers, of ICI 118,551 (erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol) and of isoprenaline on the electrically (3 Hz) evoked ³H overflow were studied.(±)-Cyanopindolol increased the evoked ³H overflow; this effect was prevented by preexposure to the previously characterized serotonin receptor antagonist metitepin. The concentration-response curve of unlabelled serotonin for its inhibitory effect on the electrically evoked ³H overflow was shifted to the right by (±)-cyanopindolol (apparent pA₂ value: 8.29), whereas that of noradrenaline (determined in the absence of DU 24565) was not affected (apparent pA₂ value: <6.0). The concentration-response curve of serotonin was also shifted to the right by (–)-cyanopindolol (apparent pA₂ value: 8.30) and (+)-cyanopindolol (6.83) but not by ICI 118,551 (<5.5). Isoprenaline (up to 10 mol/l; examined in the absence of DU 24565) did not influence the electrically evoked ³H overflow.The present results show that the presynaptic serotonin autoreceptor is blocked by cyanopindolol in a stereoselective way. This drug is 20 times more potent than metitepin as an antagonist at the presynaptic serotonin autoreceptor, and, in contrast to the latter, it does not act as an antagonist at the presynaptic ₂-adrenoceptor on the serotoninergic neurone.This study was supported by a grant of the Deutsche Forschungsgemeinschaft     

Smooth muscle of rabbit aorta contains α1- but not α2-Adrenoceptors

Summary We have investigated the residual contractile response to noradrenaline remaining after phenoxybenzamine (3×10^–7 mol/l) in rabbit aorta, since it has been reported that phenoxybenzamine at low doses completely and irreversibly blocks ₁- but not ₂-adrenoceptors. The contraction elicited by noradrenaline slowly recovered with time after it had been almost abolished by phenoxybenzamine. This residual response was blocked by the ₁-selective antagonist prazosin (3×10^–8 mol/l) but not by the ₂-selective antagonist rauwolscine (3×10^–7 mol/l). The results confirm that the smooth muscle of rabbit aorta contains ₁- not ₂-adrenoceptors.     

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     

α2-Adrenoceptor mediated inhibition of forskolin-stimulated cyclic AMP accumulation in isolated porcine palmar lateral veins

The aim of this study was to use a ³H-adenine pre-labelling technique to characterise the effect of ₂-adrenoceptor activation on forskolin-stimulated cyclic AMP accumulation in the isolated porcine palmar lateral vein. Forskolin (10^–7–10^–4 M) stimulated ³H-cyclic AMP accumulation in the isolated porcine palmar lateral vein in a biphasic and concentration-dependent manner. In the absence of the cyclic AMP-selective phosphodiesterase inhibitor rolipram, forskolin stimulated ³H-cyclic AMP accumulation approximately 7–8 fold. The response reached a peak after 5 min. In the presence of rolipram (10^–5 M), basal ³H-cyclic AMP levels were approximately 70% higher than in its absence (basal: 1823 ± 57 dpm; rolipram: 3088 ± 229, n \2 = 3) and forskolin (3 × 10^–5 M) stimulated ³H-cyclic AMP accumulation approximately 8 fold. The latter response reached a plateau 10 min after the addition of forskolin. In all subsequent studies, the tissues were incubated with forskolin (3 × 10^–5 M) for 5 min in the absence of rolipram. Noradrenaline (NA; 10^–9–10^–4 M) and UK14304 (10^–9–10^–4 M) inhibited forskolin-stimulated ³H-cyclic AMP accumulation in a concentration-dependent manner with mean pIC₅₀ values of 7.61 ± 0.37 (n \s = 4) and 7.76 ± 0.23 (n \s = 5), respectively. With either NA or UK14304, the maximal inhibition of the forskolin response obtained was approximately 75%. Neither NA (10^–4 M) nor UK14304 (10^–4 M) altered basal ³H-cyclic AMP levels. Phenylephrine (10^–4 M) had no effect on basal ³H-cyclic AMP levels and produced a 25.4 ± 7.1% inhibition of the forskolin-stimulated response, an effect that was reversed by 10^–6 M rauwolscine. Rauwolscine (10^–9–10^–6 M) produced a concentration-dependent reversal of the inhibitory effect of UK14304 10^–6 M on forskolin-stimulated ³H-cyclic AMP accumulation with a mean pK _i of 8.35 ± 0.39 (n = 3), but had no effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. Similarly, prazosin (3 × 10^–8–3 × 10^–5 M) or imiloxan (3 × 10^–8––3 × 10^–5 M) produced a concentration-dependent reversal of the UK14304 (10^–7 M)-induced inhibition of forskolin-stimulated ³H-cyclic AMP accumulation, with mean pK _i values of 6.32 ± 0.22 (n = 4) and 6.01 ± 0.30 (n = 3), respectively; neither drug had any effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. This suggests that the receptor is of the _2A-adrenoceptor subtype. It can be seen from these studies that it is possible to measure changes in cyclic AMP accumulation in porcine vascular smooth muscle using a pre-labelling technique, and it has been possible to demonstrate the presence of functional ₂-adrenoceptors, stimulation of which results in inhibition of forskolin-stimulated cyclic AMP formation.     

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     

The role of cyclic AMP in the positive inotropic effect mediated by β1- and β2-adrenoceptors in isolated human right atrium

Summary The time course of the effects of isoprenaline (3 × 10^–7 mol/l) on contractile force and on the cyclic AMP level was studied in the electrically driven isolated muscle strip of the human right atrium. Isoprenaline produced a rise in cyclic AMP content (maximum increase after 60 s) preceding the increase in contractile force. The effects of isoprenaline on contractile force and on the intracellular level of cyclic AMP were enhanced in the presence of the phosphodiesterase inhibitor papaverine (10^–5 mol/l). On the other hand, the -adrenoceptor antagonist propranolol (10^–7 mol/l) suppressed isoprenaline-induced cyclic AMP increases, but reduced the increase in force of contraction by only 35%. In addition, both the ₁-selective antagonist bisoprolol (3 × 10^–9 × 10^–8 mol/l) and the ₂-selective antagonist ICI 118,551 (3 × 10^–9 × 10^–8 mol/l) inhibited the isoprenaline-induced cyclic AMP increase concentration-dependently; ICI 118,551 produced more pronounced inhibition than bisoprolol. It is concluded that cyclic AMP is involved in the positive inotropic action of isoprenaline evoked by ₁- and ₂-adrenoceptor stimulation in isolated human right atrium; however, an additional cyclic AMP independent mechanism cannot be ruled out. Send offprint requests to O.-E. Brodde at the above address     

Celiprolol exerts microvascular dilatation by activation of β2-adrenoceptors

Summary In order to clarify the question whether the ₁-selective adrenoceptor antagonist celiprolol possesses vasodilating properties, isolated vascular networks were perfused with increasing concentrations of celiprolol (in a cumulative manner) ranging from 10^–8 to 10^–4 mol/l. The study was carried out using the isolated mesenteric vascular bed of the guinea pig mesenterium coli. Vascular diameters of four different vascular regions [vessels classified as G1 (585 ± 30 m), G2 (403 ± 25 m), G3 (282 ± 27 m) and G4 (197 ± 13 m)] were assessed by means of microscopic videoangiometry. Perfusion with celiprolol resulted in concentration dependent vasodilation which was more pronounced in G3 and G4 vessels. In addition, cumulative concentration-response curves were determined from responses obtained in the presence of 10^–8, 10^–7, 10^–6 and 10^–4 mol/l ICI 118,551 (a highly selective adrenoceptor antagonist). In the presence of ICI 118,551 at concentrations 10^–6 mol/l, no celiprolol response could be observed. Lower concentrations of ICI 118,551 shifted the celiprolol concentration-response curve to the right in a concentration-dependent manner. Therefore, it is concluded (a) that celiprolol has a vasodilating effect, (b) that this vasodilation is produced by stimulation of ₂-adrenoceptors and (c) that the vasodilating effect is more pronounced in smaller than in larger vessels (G3, G4 vs G1, G2). Send offprint requests to S. Dhein at the above address     

The depolarizing action of 5-hydroxytryptamine on rabbit isolated preganglionic cervical sympathetic nerves

Summary This study describes a depolarizing action of 5-hydroxytryptamine (5-HT) on rabbit isolated preganglionic cervical sympathetic nerves using an extracellular recording technique. From cumulative concentration-response curves for 5-HT (1 mol/1-1 mmol/1), the mean maximal depolarization was shown to be 277 ± 32 V and EC₅₀ was 9.4 mol/l(6.5–13.6 mol/l, geometric mean, 95% confidence limits, n = 42). The responses to 5-HT displayed marked tachyphylaxis. When cumulative concentration-response curves to 5-HT and 2-methyl-5-HT were determined in the same preparations (n = 4), the mean maximal response to 5-HT was 519 ± 167 V, EC₅₀ 32.2 mol/l (8.8–118 mol/l) and the mean maximal response to 2-methyl-5-HT was 317 ± 63 V, EC₅₀ 35.1 mol/l (12.9–95.5 mol/l, geometric means, 95 % confidence limits). The action of selective 5-HT antagonists was tested on repeated cumulative concentration-response curves to 5-HT. Neither methiothepin (0.1–1 mol/l, _n = 3) nor ketanserin (0.1–1 mol/l, n = 3) had an action on 5-HT responses. The selective 5-HT₃ antagonists MDL 72222, ICS 205-930 and SDZ 206–830 were all potent antagonists of the 5-HT depolarizations. The action of these antagonists was quantified by determining the apparent pA₂ from the dose ratios and a Schild plot. For MDL 72222 (1 nmol/1-0.1 mol/l), the apparent pA₂ was 9.1 ± 0.1 (n = 12), Schild plot: 9.2; for ICS 205–930 (0.1 nmol/l–3 nmol/1), the apparent pA₂ was 10.4 ± 0.1 (n = 11), Schild plot 10.3, and for SDZ 206–830 (0.03 nmol/l-1 nmol/1), the apparent pA₂ was 11.2 ± 0.1 (n = 12), Schild plot 11.2. 5-HT depolarizations were unaffected by hexamethonium (0.5 mmol/1). 5-HT depolarizations were reduced by superfusion with both Na-free (42 ± 8% of controls, n = 4) and Na/Ca-free media (35 ± 7% of controls, n = 4). It is concluded that 5-HT depolarizations of rabbit preganglionic cervical sympathetic nerve are mediated by 5-HT₃ receptors. The data with selective 5-HT₃ receptor antagonists suggest that the receptor profile may be more like that for the 5-HT₃ receptor on the terminals of sympathetic nerves than that for the 5-HT₃ receptor on the soma of superior cervical ganglion cells or on vagal afferent neurones. Send offprint requests to D. I. Wallis at the above address     

Antagonism of the effects of 5-hydroxytryptamine on the rabbit isolated vagus nerve by BRL 43694 and metoclopramide

Summary Depolarization and reduction in the C fibre compound action potential (C spike) in response to 5-HT were recorded simultaneously from rabbit isolated vagus nerve. 5-HT (0.1–100 mol/l) was applied cumulatively and EC₅₀ and IC₅₀ values measured from individual concentration-response curves. Blockade of 5-HT responses by the 3-indazole carboxamide, BRL 43694, was investigated and compared with the blocking action of metoclopramide. BRL 43694 was a selective antagonist of 5-HT responses. A concentration of 10 nmol/l BRL 43694, which nearly abolished the depolarization and reduction of the C spike evoked by 5-HT (100 mol/l), had no effect on similar responses evoked by DMPP (100 mol/l) or GABA (100 mol/l). Blockade of 5-HT responses by BRL 43694 (0.3 nmol/l) was slow in onset, a plateau blockade occurring after equilibrium of tissue with antagonist for 2 to 3 h. Metoclopramide induced a blockade of rapid onset. The maximal blockade was apparent within 30 min of application. Full recovery in the responsiveness of the tissue to 5-HT was observed within 30 min of washing out metoclopramide. BRL 43694 at concentrations of 0.3, 1, 3 and 10 nmol/l caused a progressive rightward shift of the concentration-response curves to 5-HT. At the highest concentration of antagonist, there was some depression of the maximal 5-HT response. The apparent pA₂ estimated from the Schild equation was 10.03 ± 0.09 (mean ± SEM, n = 20) against 5-HT depolarization and 10.31 ± 0.1 against C spike reduction. Schild plots had slopes not significantly different from 1.0. The slopes and extrapolated pA₂ fitted by linear regression were 0.91 (0.58 – 1.24) and pA₂ 10.16 (9.74–10.58; mean and 95% confidence levels) for the depolarizations. For reduction in C spikes the slope was 0.74 (0.39–1.08) with a pA₂ of 10.86 (10.24–11.49). There was no apparent use-dependent element to the blockade by BRL 43694. Blockade of 5-HT depolarization or C spike reduction by BRL 43694 (0.3 nmol/l) was not significantly different on repeated testing in the presence of the antagonist or without testing of 5-HT until 3 h incubation had elapsed. Metoclopramide at concentrations of 0.3, 1, 3, or 10 mmol/l progressively shifted concentration-response curves to the right. However, the response maximum, especially that for depolarization, was enhanced in the presence of the antagonist. The apparent pA₂ values from the Schilde equation were 7.04 ± 0.04 (n = 20) against 5-HT depolarization and 7.13 ± 0.06 (n = 16) against C spike reduction. Schild plots had slopes significantly less than unity. The lines fitted to the relationship gave pA₂ values of 7.41 (7.25–7.57) with a slope of 0.79 (0.69–0.9) against depolarization and 7.63 (7.28–7.97) with a slope of 0.74 (0.54–0.93) against C spike reduction. Metoclopramide at concentrations above 30 mol/l directly reduced C spike amplitude; the IC₅₀ for the local anaesthetic action was 158 ± 40 mol/l (mean ± SEM). It is concluded that BRL 43694 is a potent and selective antagonist of 5-HT₃ receptors on the rabbit vagus nerve. At concentrations below 10 nmol/l, BRL 43694 appeared to behave as a competitive antagonist while at 10 nmol/l the antagonism was unsurmountable, suggesting a pseudo-irreversible antagonism due to slow dissociation of antagonist from the receptor. Although metoclopramide behaved as a surmountable antagonist, the low slope of the Schild plots and the increase in maximal response amplitude in the presence of the antagonist are unexplained features of its blocking action.Send offprint requests to D. I. Wallis at the above address     

Agonist binding at alpha2-adrenoceptors of human platelets using 3H-UK-14,304: regulation by Gpp(NH)p and cations

Summary The agonist/₂-adrenoceptor interactions at human platelet membranes have been examined in radioligand binding studies with the full agonist ligand ³H-UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline] and the antagonist ligand ³H-yohimbine. From association kinetics of different concentrations of ³H-UK-14,304 (0.75–8.1 nmol/l) a K _D-value of 2.37 nmol/l in agreement with the high-affinity KD-value (K _DH = 1.60 ± 0.15 nmol/1) obtained from equilibrium binding studies was derived. In the presence of Gpp(NH)p about 6% of specific radioligand binding was observed in the association reaction. Addition of Gpp(NH)p at equilibrium resulted in a rapid loss (t _1/2 < 1 min) of 80% of bound radioligand. Dissociation after addition of an excess of phentolamine (10 mol/l) showed a biphasic time course independent of the radioligand concentration with the proportions of /15 of rapidly (t _/12 < 2 min) and /45 of slowly dissociating ligand (k–1 = 0.033±0.004 min^–1). Application of a sequential binding model resulted in K _D-values from this approach also in agreement with K _DH from equilibrium binding studies. The rank order of potency for different agonists and antagonists to compete for binding with ³H-UK-14,304 indicated an ₂-adrenoceptor interaction: (–)adrenaline > clonidine > (–)noradrenaline > (–)isoprenaline and yohimbine = rauwolscine > phentolamine > prazosin >- corynanthine > timolol respectively. The analysis of competition isotherms of UK-14,304 versus ³H-yohimbine (Hill-coefficient = 0.59 ± 0.03) showed that the agonist binds to two affinity states of the ₂-adrenoceptor, with high (K _DH = 1.77 ± 0.50 nmol/l) and low affinity (K _DL = 71.2 ± 11.6 nmol/l) respectively. From these experiments a fraction of 56.9%±2.1% of the total number of ₂-adrenoceptors (B _max = 198.4 ± 8.0 fmol/mg of protein) in the high-affinity state was calculated. Similar results were obtained from ³H-UK-14,304 saturation isotherms according to a two-state binding model (K _DH = 1.60±0.15 nmol/l; K _DL = 66.2±10.7 nmol/l; B _maxH = 57.6% ± 2.3%). Adrenoceptor agonists competed for specific binding of ³H-UK 14,304 and ³H-yohimbine in a manner that suggests that the ³H-UK-14,304 (3.5 nmol/l) labeled sites represent predominantly the agonist induced or stabilized high-affinity state of the ₂-adrenoceptor. Adrenoceptor antagonists had equal affinities irrespective of the receptor states labeled by the agonist or antagonist radioligand. A loss of the high-affinity binding capacity (B _maxH) of the agonist due to the presence of Gpp(NH)p was delineated from ³H-UK-14,304 saturation isotherms. An IC₅₀-value of 0.181 ± 0.007 mol/l for this Gpp(NH)p-effect was calculated. Divalent cations such as magnesium and manganese (10 mmol/l) increased specific binding of ³H-UK-14,304 by a factor of 3, without any influence on binding of the antagonist ³H-yohimbine. In contrast, sodium chloride strikingly decreased high-affinity binding of the agonist radioligand (IC₅₀ = 41.9 ± 3.7 mmol/l). Unlike Gpp(NH)p, sodium chloride (> 30 mmol/l) additionally promoted a marked decrease of the affinity of UK-14,304 at the low-affinity binding component. In contrast to the effects on agonist binding, sodium chloride concentrations of 30 to 300 mmol/l increased the binding affinity of the antagonist ³H-yohimbine about 2-fold. The sodium substitute N-methyl-D-glucamine was without effect on binding of ³H-UK-14,304 indicating that the influence of sodium chloride on binding properties was not due to changes in osmolarity. In conclusion these results suggest that ³H-UK-14,304 labels preferentially the agonist induced or stabilized high-affinity state (_2H) of the platelet ₂-adrenoceptor.Abbreviations UK 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline - Gpp(NH)p guanylyl-imidodiphosphate - EDTA ethylene dinitrilo tetraacetic acid Preliminary data were presented at the Joint Meeting of the Belgian, Dutch and German Pharmacological and Toxicological Societies at Aachen, FRG, September 23–26, 1985 (Schloos et al. 1985)This report is part of the thesis to be presented by J. Schloos in partial fulfillment of the requirements for a Doctor of Natural Science degree Send offprint requests to J. Schloos at the above address     

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.     

Direct and indirect actions of dopamine on tracheal smooth muscle

Summary The effects of dopamine (DA) were studied on guinea-pig isolated tracheal chains. At a low concentration (10^–6M) DA occasionally produced a small contraction; this was followed by a dose-dependent relaxation 3×10^–6–3×10^–3 M).On a molar basis, DA was about 40 times less potent than noradrenaline (NA) in relaxing tracheal chains, and about 2700 times less potent than isoprenaline (ISO). The maximum degree of relaxation obtained with each drug was the same.Pretreatment of the guinea-pig with reserpine (5 mg/kg) resulted in a 3-fold shift of the DA curve to the right without concomitantly affecting the ISO doseresponse curve. Reserpine completely abolished the relaxant effects of tyramine, but a small contractile response remained.Desipramine (DMI), at a concentration of 10^–5 M, caused a 4-fold shift of the DA curve to th right. The same concentration of DMI resulted in a shift to the left of the NA dose-response curve by about 8-fold. Benztropine (10^–5 M) and haloperidol (10^–5 M and 3×10^–5 M) did not affect the DA dose-response curve.The DA-induced relaxation was inhibited by propranolol (10^–8–10^–6 M) in a dose-dependent manner. The higher concentrations of propranolol (10^–7 and 10^–6 M) unmasked the contractile effect of DA. In the presence of propranolol, phentolamine (10^–5 M) abolished the contractile effect of DA.It is concluded that DA has both direct and indirect actions on guinea-pig isolated tracheal smooth muscle. The relaxant effects of DA are predominantly due to a direct action on smooth muscle -adrenoceptors, with a component due to release of NA from adrenergic nerves. The contractile effects, which under normal conditions are masked by the relaxant effect of DA, are mediated by functional -adrenoceptors. There is no evidence for either specific dopaminergic nerves, uptake mechanisms or receptors in guinea-pig trachealis muscle.     

Effects of the novel dopamine DA2-receptor agonist carmoxirole (EMD 45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney

Summary The effects of the classical dopamine DA₂-receptor agonist quinpirole (LY 171555) and the recently characterized DA₂-receptor agonist, carmoxirole (EMD 45609), on neurotransmission in rat isolated kidney were investigated. After preincubation with ³H-noradrenaline, the renal nerves were electrically stimulated. The stimulation induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. Quinpirole (0.3 mol/l) inhibited S-I outflow of radioactivity and pressor responses to renal nerve stimulation (RNS) at 1 Hz. Both effects of quinpirole were blocked by the DA₂-receptor antagonist S(–)-sulpiride (10 mol/l). The ₁, ₂-adrenoceptor antagonist phentolamine (1 mol/l) did not block the inhibitory effect of quinpirole. Carmoxirole (0.003 and 0.03 mol/l) did not alter and carmoxirole (0.3 mol/l) even enhanced S-I outflow of radioactivity, however, pressor responses to RNS were markedly reduced by carmoxirole (0.003–0.3 mol/l). Pressor responses to RNS were also markedly reduced by the ₁-adrenoceptor antagonist prazosin (0.1 mol/l). Carmoxirole (0.3 mol/l), prazosin (0.1 mol/l) and phentolamine (1 mol/l) totally abolished pressor responses to exogenous noradrenaline (0.05 mol/l). In contrast, quinpirole (0.3 mol/l) did not alter pressor responses to exogenous noradrenaline (0.05 mol/l). Furthermore, carmoxirole (0.003–0.3 mol/l) markedly reduced pressor responses induced by the ₁-adrenoceptor agonist methoxamine (1 mol/l) but even the highest concentration of carmoxirole (0.3 mol/l) had no effect on pressor responses induced by bolus injections of either neuropeptide Y (1.5 ng) or angiotensin II (1 ng). Phentolamine (1 mol/l) by itself markedly enhanced S-1 outflow of radioactivity and pressor responses to RNS were virtually unchanged. In the presence of phentolamine carmoxirole (0.03 and 0.3 mol/l) and quinpirole inhibited S-I outflow of radioactivity and pressor responses to RNS. Phentolamine resistant pressor responses to RNS were also inhibited by the P_2X-receptor desensitizing agent , -methylene adenosine triphosphate (mATP, 1 mol/l), which by itself in the presence of phentolamine did not alter S-I outflow of radioactivity. The inhibitory effects of carmoxirole (0.3 mol/l) in the presence of phentolame (1mol/l) were antagonized by S(–)-sulpiride (10 mol/l). The data suggest that activation of prejunctional DA₂-receptors by quinpirole inhibits noradrenaline release and thereby reduces pressor response to RNS at 1 Hz in rat isolated kidney. Carmoxirole activates prejunctional inhibitory DA₂-receptors, but this effect is masked by simultaneous blockade of inhibitory prejunctional -adrenoceptors. Pressor responses to RNS at 1 Hz in rat isolated kidney are largely due to neuronally released noradrenaline whereas phentolamine resistant pressor responses to RNS at 1 Hz are most likely due to ATP, which is co-released with noradrenaline. Carmoxirole inhibits pressor responses to RNS at 1 Hz as well as pressor responses induced by either exogenous noradrenaline or methoxamine by blocking postjunctional ₁-adrenoceptors. In addition carmoxirole and quinpirole seem to block phentolamine resistant pressor responses by inhibiting ATP release through activation of prejunctional DA₂-receptors. Send offprint requests to L. C. Rump at the above address     

Pharmacological characterization of the postjunctional alpha-adrenoceptors of the rat isolated seminal vesicle

Summary In an attempt to define the pharmacological characteristics of the postjunctional -adrenoceptors of the rat seminal vesicle, responses to certain phenylethanolamine and imidazoline agonists were investigated, in vitro, under experimental conditions outlined by Furchgott (1972), using ₁-selective, non-selective and ₂-selective adrenoceptor antagonists. Adrenaline (ADR), noradrenaline (NA) and phenylephrine (PE) produced concentration-dependent contractions. In many experiments the concentration-response (C-R) curves had a distinct shoulder at the level of 60–80% of the maximum response (E_max), a situation reminiscent of the rat anococcygeus muscle and the rat basilar artery. The relative potencies of ADR:NA:PE, derived from their EC₅₀ values, were 4.07:1:0.26. In contrast clonidine, oxymetazoline and naphazoline failed to contract the tissue even in concentrations up to 1 × 10^–3 M. In fact the imidazoline derivatives prevented responses to the phenylethanolamines. The antagonist action of clonidine, against phenylephrine, was studied in detail.Prazosin, phentolamine, yohimbine, corynanthine and clonidine all caused a rightward displacement of the C-R curves for NA without depressing E_max. The Arunlakshana and Schild plots of the data were linear and had slopes not significantly different from unity. The pA₂ estimates obtained were 9.17 (9.13–9.21) for prazosin, 8.58 (8.07–9.09) for phentolamine, 6.70 (6.44–6.98) for yohimbine and 7.05 (6.81–7.30) for corynanthine. Clonidine had a pA₂ value of 6.60 (6.55–6.67) against phenylephrine.On the basis of results obtained with antagonists, the postjunctional -adrenoceptors of the rat seminal vesicle could be firmly placed in the gross category of ₁. The concept of heterogeneity of ₁-adrenoceptors is discussed in the light of the profiles of the phenylethanolamine and imidazoline agonists as well as the antagonist potencies of prazosin and yohimbine. Send offprint requests to S. I. Sharif at the above address     

An inhibitory prejunctional 5-HT1-like receptor in the isolated perfused rat kidney

Summary The present study has identified a receptor for 5-hydroxytryptamine (5-HT) which functions to inhibit the stimulus-induced release of [³H] noradrenaline following sympathetic periarterial nerve stimulation to the isolated perfused rat kidney. In addition to 5-HT (IC₃₀=4.5×10^–8 mol/l), both 5-carboxamidotryptamine (IC₃₀=8×10^–9 mol/l) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl) indole (RU-24969, IC₃₀=2.5×10^–7 mol/l) acted as agonists whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was inactive. The inhibitory effect of 5-HT on the electrically-evoked release of tritium was antagonized in a concentration-dependent manner by methiothepin (IC₅₀=4×10^–9 mol/l), metergoline (IC₅₀=4×10^–8 mol/l) and methysergide (IC₅₀=1.3×10^–7 mol/l) but not by cyproheptadine, ketanserin, mesulergine, (–)-propranolol, (±)-pindolol, (±)-cyanopindolol, metoclopramide or phentolamine. It is concluded that the receptor to 5-HT conforms to general criteria defining 5-HT₁-like receptors but at the present time the receptor site cannot be fitted to the designated 5-HT_1A, 5-HT_1B or 5-HT_1C subtypes.Preliminary accounts of this work were presented to the British Pharmacological Society in London (December, 1984) and Southampton (July, 1985)     

Urapidil and some analogues with hypotensive properties show high affinities for 5-hydroxytryptamine (5-HT) binding sites of the 5-HT1A subtype and for α1-adrenoceptor binding sites

Summary The mechanism responsible for the antihypertensive effect of urapidil is not yet completely understood. Its vasodilator action has been attributed to an antagonism at vascular ₁-adrenoceptors. However, it has been suggested that a central action contributes to the hypotensive effect. Recently, three potent analogues of urapidil have been described which also lower blood pressure by a central mechanism. 5-Hydroxytryptamine (5-HT) receptors of the 5-HT_1A subtype have been implicated with the central control of cardiovascular function. In the present study, the affinities of these urapidil derivatives (5-acetyl, 5-formyl- and 5-methylurapidil) for 5-HT receptors were investigated using ³H-8-hydroxy-2-(di-n-propylamino)tetralin (³H-8-OH-DPAT), ¹²⁵I-iodocyanopindolol (¹²⁵I-ICYP) and ³H-ketanserin for labelling 5-HT_1A, 5-HT_1B and 5-HT₂ binding sites, respectively. ³H-Prazosin and ³H-clonidine were used as selective ₁- and ₂-adrenoceptor radioligands, respectively. Urapidil and its analogues produced half-maximum inhibition of ³H-8-OH-DPAT binding at concentrations of 4 × 10^–9 mol/l to 4 × 10^–7 mol/l with the following order of potency: urapidil < 5-acetyl- < 5-formyl- < 5-methyl-urapidil. Thus, 5-methyl-urapidil is one of the most potent ligands at 5-HT_1A recognition sites known to date. The IC₅₀ values of urapidil and its derivatives for ³H-prazosin binding were in the range of 5 × 10^–8 mol/l to 8 × 10^–7 mol/l (order of potency: urapidil < 5-formyl- < 5-acetyl- 5-methyl-urapidil). The affinity of these analogues for 5-HT_1B, 5-HT₂ and ₂-adrenergic recognition sites was distinctly lower. Urapidil and its congeners clearly discriminate between 5-HT- and between -receptor subtypes. It is postulated that the high affinity for 5-HT_1A receptors as well as for ₁-adrenoceptors is relevant to the hypotensive properties of these compounds. Send offprint requests to G. Groß     

The pharmacokinetics ofα-human atrial natriuretic polypeptide in healthy subjects

Summary We have analysed the pharmacokinetics of-human atrial natriuretic polypeptide (-hANP) in healthy subjects, using a two-compartment open model following bolus intravenous injection. The plasma half-times for the fast and slow components were 1.7±0.07 min and 13.3±1.69 min respectively. V₁ (the volume of the central compartment), V_z (volume of distribution) and V_ss (volume of distribution at steady-state) were 5370±855 ml (89.5±14.3 ml·kg^–1), 32000±4620 ml (533±77.0 ml·kg^–1), and 11900±1530 ml (198±25.5 ml·kg^–1) respectively. The mean plasma clearance was 1520±121 ml·min^–1 (25.4±2.0 ml·min^–1·kg^–1.     

Stereoselectivity of noradrenaline uptake into synaptic vesicles of the rat brain

Summary To investigate the stereoselectivity of the ATP-Mg²⁺-dependent uptake of noradrenaline, synaptic vesicles were isolated from the rat brain by differential centrifugation and incubated with ³H-(±)-, ³H-(–)- or ¹⁴C-(+)-noradrenaline in the absence and in the presence of ATP-Mg²⁺. The K _m values of the ATP-Mg²⁺-dependent uptake were found to be different for the two isomers (mol/l): ³H(±)-noradrenaline 14.9 ± 2.2 × 10^–1, ³H-(–)-noradrenaline 7.7 ± 0.5 × 10^–1, ¹⁴C-(+)-noradrenaline 17.3 ± 3.7 × 10^–1, whereas the V _maX of the racemate was identical with those of the two isomers (pmol/mg protein/min): ³H-(±)-noradrenaline 5.5 ± 0.4, ³H-(–)-noradrenaline 4.9 + 0.1, ¹⁴C-(+)-noradrenaline 5.1 ± 0.4. Moreover, (+)-noradrenaline inhibited competitively the ATP-Mg²⁺-dependent uptake of ³H-(±)-noradrenaline (K_i 19.2 + 1.0 × 10^–1 mol/l) and ³H-(–)-noradrenaline (Ki 17.7 ± 1.8 × 10^–1 mol/l), the K_i values being nearly identical with the K _m of the ATP-Mg²⁺-dependent uptake of ¹⁴C-(+)-noradrenaline. It is concluded that the ATP-Mg²⁺-dependent uptake of noradrenaline into synaptic vesicles of the rat brain is stereoselective and that both isomers share the same transport system.The experiments were carried out at the Institut für Pharmakologie and Toxikologie der Universitat Würzburg, FRG. This work was supported by the Deutsche Forschungsgemeinschaft Send offprint requests to A. Philippu at the above address