THE CHRONIC EFFECTS OF β-ADRENORECEPTOR BLOCKING AGENTS ON TRANSMITTER OVERFLOW IN THE ANOCOCCYGEUS MUSCLE OF THE RAT

1 The effect of chronic administration of three β-adrenoreceptor blocking agents, propranolol, timolol and atenolol on transmitter release from sympathetic nerves has been investigated in the isolated anococcygeus muscle of the rat. 2 Oral treatment for 11 weeks with either propranolol (12 mg/kg/day), or timolol (1.2 mg/kg/day) but not atenolol (12 mg/kg/day) significantly increased the stimulation-induced overflow of radioactivity following preincubation of the tissue with ³H-NA. 3 No increase in end-organ sensitivity, as measured in terms of responsiveness to exogenous acetylcholine, tyramine or NA, was observed. 4 The ineffectiveness of atenolol compared to the other β-adrenoreceptor blocking drugs may be related to the finding that, in acute experiments, atenolol inhibited tissue uptake of ³H-NA. 5 It is postulated that increased release of transmitter may be related to prolonged blockade of presynaptic β-receptors and a resultant increase in presynaptic receptor sensitivity.     

POSITIVE CHRONOTROPIC REPONSES TO CARDIAC α1-ADRENORECEPTOR ACTIVATION IN THE PITHED RAT

• 1 Adrenaline (0.1–10 μg/kg), noradrenaline (0.1–10 μg/kg) and phenylephrine (1–100 μg/kg) acted on both cardiac α₁- and β-adrenoreceptors to induce positive chronotropic responses in the pithed rat.
• 2 When β-adrenoreceptors were blocked by propranolol (1 mg/kg), the residual chronotropic responses were due to activation of α₁-adrenoreceptors since they were significantly reduced by prazosin (10–100 μg/kg).
• 3 Methoxamine (10–300 μg/kg) acted solely on cardiac α₁-adrenoreceptors to induce positive chronotropic responses which were abolished by prazosin (10–100 μg/kg) alone, as has been demonstrated previously for amidephrine.
• 4 The rank order of potency for eliciting the positive chronotropic response to α₁-adrenoreceptor activation was adrenaline > noradrenaline > phenylephrine > methoxamine.
• 5 The positive chronotropic responses to adrenaline (3–10 μg/kg), noradrenaline (3–10 μg/kg) and phenylephrine (30–100 μg/kg) produced by activating α₁-adrenoceptors had a slower time course than did the chronotropic responses produced by activation of β-adrenoreceptors.

     

β-ADRENORECEPTOR ANTAGONISTS WITH MULTIPLE PHARMACOPHORES: PERSISTENT INHIBITION OF RAT HEART ADENYLATE CYCLASE

• 1 β-Adrenoreceptor antagonists containing several pharmacophores, so called alprenolol-Jeffamines, were studied. These compounds are derived from Jeffamine^R NH₂-CH-(CH₃)-CH₂-[-O-CH₂-CH(CH₃)-]_n- NH₂₉n = 2.6 ave. by substitution of nitrogen with 3-(2-allylphenoxy)-2-hydroxypropyl groups, which are part of the Alprenolol^R pharmacophore.
• 2 Alprenolol-Jeffamines inhibited (-)isoprenaline stimulated adenylate cyclase activity; the derivative with one pharmacophore was about 4-fold and the derivative with two pharmacophores was about 17-fold less potent than (+)alprenolol; the trisubstituted derivative which has one complete and two partial pharmacophores was ineffective.
• 3 The ratio of K_i's for inhibition of (-)³H-DHA binding and for inhibition of adenylate cyclase were approximately one for each derivative.
• 4 When (+)alprenolol-Jeffamine derivatives were injected intraperitoneally into rats and heart membranes or homogenates were prepared 18–20 h afterwards, the mono, di, and trisubstituted derivatives, but not (+)alprenolol, inhibited bindng of (-)³H-DHA. This persistency pattern is different from that observed in vitro, where only di and trisubstituted derivatives are persistent. Slow metabolism/slow excretion of the monosubstituted derivative may be a source of the increased persistency in vivo.
• 5 In similarly prepared animals, the dose-response curve for (-)isoprenaiine stimulated adenylate cyclase was shifted to the right 3-to-4 fold for mono and disubstituted derivatives but was unaffected by (+) alprenolol and the trisubstituted derivative.
• 6 The results suggest that these derivatives interact with physiologically important β-adrenoreceptors in vitro, and that, in vivo, they persistently block β-adrenoreceptors and inhibit isoprenaline stimulated adenylate cyclase.

     

THE EFFECT OF β-ADRENORECEPTOR ANTAGONISTS ON THE INHIBITION OF PENDULAR MOVEMENTS OF RABBIT ILEUM PRODUCED BY PERIARTERIAL SYMPATHETIC NERVE STIMULATION AND SOME SYMPATHOMIMETIC AMINES

• 1 The effects of propranolol and of the selective β₁ and β₂-adrenoreceptor blocking drugs atenolol and ICI 118,551 were determined on the inhibitory responses of isolated segments of rabbit ileum to noradrenaline, isoprenaline and salbutamol and to periarterial sympathetic nerve stimulation.
• 2 Responses to isoprenaline (0.04–10.24 μM) and salbutamol (1.4–89.6 μM) were blocked by propranolol in concentrations up to 5.0 and 12.8 μM, respectively. Responses to sympathetic nerve stimulation were reduced but responses to noradrenaline (0.03-1.92 μM) were unaffected by propranolol in concentrations up to 10.0 and 5.0 μM, respectively.
• 3 Atenolol in concentrations up to 30.0 μM blocked responses to isoprenaline (0.04-2.56 μM) but did not affect responses to noradrenaline, salbutamol or sympathetic nerve stimulation in concentrations up to 3.0,3.0 and 1.0 μM, respectively. However, when responses to noradrenaline and sympathetic nerve stimulation were reduced by phentolamine (1.0 μM), atenolol then produced further reductions.
• 4 Responses to isoprenaline (0.04-2.56 μM) and salbutamol (1.4–89.6 μM) were blocked by ICI 118,551 in concentrations up to 0.5 μM. Responses to sympathetic nerve stimulation were reduced but responses to noradrenaline were unaffected by ICI 118,551 in concentrations up to 0.01 and 0.3 μM, respectively.
• 5 Salbutamol (0.1 μM.) increased the inhibitory response to sympathetic nerve stimulation and this effect was blocked by ICI 118,551 (0.01 μM).
• 6 It was concluded that blockade of β₂-adrenoreceptors, presumably located on sympathetic nerve terminals, decreases the release of transmitter noradrenaline and that blockade of β₁-adrenoreceptors, presumably located in longitudinal smooth muscle cells, reduces the response to transmitter noradrenaline when α-adrenoreceptors are also blocked.

     

EFFECT OF 8-SULFOPHENYL THEOPHYLLINE ON ENDOGENOUS NORADRENALINE RELEASE FROM SYMPATHETIC NERVES OF THE RABBIT EAR ARTERY

1. The release of endogenous noradrenaline (NA) and adenyl purine (ATP, ADP, AMP and adenosine) from the rabbit ear artery, evoked by electrical stimulation (ES; 16Hz), was examined. 2. ES evoked a significant release of NA and adenyl purine; the ratio of the amount of total purine released to NA released was approximately 180 on a molar base. 3. ES-evoked purine release was significantly reduced by the denudation of the endothelium and abolished by the α₁-adrenoceptor antagonist, prazosin (1 μmol/L). 4. ES-evoked NA release was significantly reduced by a P₁-purinoceptor antagonist, 8-sulfophenyl theophylline (8SPT). Purine release was slightly reduced by 8SPT. 5. These results suggest that endogenous NA released by ES results in the release of a large amount of purine, which may, in turn, increase the release of NA by acting on prejunctional purinoceptors on sympathetic nerve terminals.     

INHIBITION BY COCAINE OF NORADRENALINE RELEASE FROM SYMPATHETIC NERVES IN THE RABBIT EAR ARTERY

1. Effects of cocaine on release of noradrenaline (NA) from sympathetic nerves were studied in the isolated perfused central artery of the rabbit ear. Indices of release were the vasoconstrictor response to nerve stimulation and stimulation-induced overflow of radioactivity after the nerves had been loaded with [3H]-NA. The overflow studies were carried out on phenoxybenzamine-treated arteries to eliminate the effect of cocaine on neuronal uptake. 2. Cocaine enhanced the constrictor responses of the artery to stimulation in concentrations of 3 and 30 mumols/l, but in higher concentrations (tested up to 300 mumols/l) the enhancement declined and was replaced by inhibition. Responses to extraluminal NA remained enhanced throughout the concentration range (tested up to 150 mumols/l). 3. In contrast, cocaine depressed the overflow of radioactivity, the effect being detectable in a concentration of 3 mumols/l (a decrease of 15%); the decrease was 40% at the highest concentration tested (90 mumols/l). 4. It is suggested that when assessed in terms of the vasoconstrictor response, inhibition of transmitter release by cocaine is masked by inhibition of neuronal uptake except in high concentrations of cocaine.     

STUDIES ON THE PRE- AND POSTJUNCTIONAL ACTIVITIES OF α-ADRENORECEPTOR AGONISTS AND THEIR CARDIOVASCULAR EFFECTS IN THE ANAESTHETIZED RAT

• 1 The selectivity of a number of agonists for peripheral α₁- and α₂- adrenoreceptors was determined and related to their cardiovascular effects in pentobarbitone-anaesthetized rats.
• 2 In isolated tissues, presynaptic α₂- and postsynaptic α₁-adrenoreceptor activity was determined on the rat vas deferens and anococcygeus muscle respectively. Xylazine, guanabenz, guanoxabenz and guanfacin were more selective, whilst lofexidine, tiamenidine, naphazoline, oxymetazoline and St 91 were less selective than clonidine for presynaptic α₂-adrenoreceptors.
• 3 The anococcygeus muscle of the pithed rat was used to determine the α-adrenoreceptor selectivity of the compounds in vivo. The selectivities were similar to those obtained in vitro.
• 4 Following intravenous administration to pentobarbitone-anaesthetized rats guanabenz and guanfacin caused small transient pressor responses and a prolonged secondary hypotension. In contrast oxymetazoline and St 91 produced marked initial pressor responses and the secondary reduction in blood pressure was absent. Clonidine and tiamenidine produced marked initial pressor responses followed by secondary hypotensive effects. All compounds reduced heart rate.
• 5 Intracerebroventricular (i.c.v.) administration of clonidine, guanabenz and guanfacin elicited falls in blood pressure and heart rate. In contrast blood pressure was either unaffected or elevated following i.c.v. administration of St 91, oxymetazoline and tiamenidine.
• 6 It is concluded that in the normotensive rat, central α₂-adrenoreceptors play a major role in the hypotensive effects of clonidine and related compounds.

     

THE EFFECTS OF KF-4317, A NOVEL COMBINED α- and β1-ADRENORECEPTOR ANTAGONIST,ON THE RAT ISOLATED RIGHT VENTRICLE AND AORTA

• 1 The effects of KF-4317 on the accumulation of radioactivity from [³H]-noradrenaline, and on the subsequent spontaneous and noradrenergic nerve-evoked outflow of radioactivity have been investigated in the rat isolated right ventricle. In addition the effects of KF-4317 on the contractions of the electrically-driven directly muscle stimulated rat right ventricle to isoprenaline and of the rat isolated aorta to phenylephrine and 5-hydroxytryptamine are reported.
• 2 KF-4317 at 1μM had no effect on the ability of the rat right ventricle to accumulate radioactivity from [³H]-noradrenaline.
• 3 The spontaneous outflow of radioactivity, following loading of the ventricle with [³H]-noradrenaline, was increased by KF-4317 at 1μM by a cocaine-insensitive mechanism.
• 4 KF-4317 at 1μM had no effect on the noradrenergic nerve-evoked outflow or radioactivity, and therefore is not an α₂-adrenoreceptor antagonist, but reduced the associated contractile response probably mainly by acting as an antagonist at postjunctional β₁-adrenoreceptors.
• 5 KF-4317 caused a parallel rightward shift of the concentration-response curve of the electrically-driven directly muscle stimulated rat right ventricle to isoprenaline. However the inhibitory effect, × 9.0 and × 237.2 in the presence of 0.1 and 1μM KF-4317, was not closely concentration-related. At 1μM, KF-4317 also depressed the maximum responses to isoprenaline. This suggests that in addition to β₁-adrenoreceptor antagonism, KF-4317 probably exerts membrane stabilizing activity.
• 6 The responses of the rat isolated aorta to phenylephrine were inhibited in a non-concentration related manner by KF-4317. Thus the responses were inhibited × 1.8, × 2.7 and × 4.7 in the presence of KF-4317 at 0.1, 1 and 1μM, respectively. The responses of the aorta to 5-hydroxytryptamine were inhibited to a small extent by KF-4317 at 1μM but potentiated to a small degree by KF-4317 at 10μM. We suggest that KF-4317 is an antagonist at α₁-adrenoreceptors and 5-hydroxytryptamine receptors but that it also has an additional postjunctional action, in the rat aorta, to increase the sensitivity to both phenylephrine and 5-hydroxytryptamine.

     

RESISTANCE OF SYMPATHETIC CARDIAC NERVE FUNCTION IN THE PITHED RAT TO PREJUNCTIONAL EFFECTS OF ANGIOTENSIN II

1. The spinal sympathetic outflow (C7-T2) of pithed male Wistar rats was electrically stimulated (30-50 V, 0.5 ms, 0.1-1.0 Hz) and heart rate and arterial blood pressure were recorded. D-Tubocurarine (1 mg/kg) and atropine (1 mg/kg) were administered intravenously (i.v.) to reduce voluntary and parasympathetic nerve activity. 2. Angiotensin II (0.39-3.4 micrograms/kg per min) was infused at a rate which caused a sustained rise in diastolic blood pressure of at least 25 mmHg but which did not alter basal heart rate. 3. Chronotropic responses to sympathetic nerve stimulation were not affected by infusion of angiotensin II, and were also unaltered by pretreatment of rats with indomethacin (5 mg/kg, i.v.) 10 min prior to commencement of stimulation. 4. These results suggest that positive chronotropic responses to cardiac sympathetic nerve stimulation in pithed rats are not affected by increased angiotensin II levels. Indomethacin had no effect, which indicates that cyclo-oxygenase products were not involved in modulation of chronotropic responses to cardiac sympathetic nerve stimulation.     

COMPARISON OF THE CARDIAC EFFECTS OF β-ADRENORECEPTOR AGONISTS IN ANAESTHETISED AND CONSCIOUS DOGS

• 1 The cardiovascular effects of some β-adrenoreceptor agonists on heart rate, blood pressure and myocardial contractility (maximum rate of change of left ventricular pressure/integrated isometric tension) were measured in pentobarbitone-anaesthetised and conscious, instrumented greyhounds.
• 2 In anaesthetised dogs isoprenaline increased heart rate and myocardial contractility and reduced blood pressure. Prenalterol and RO 363, in equiactive inotropic doses, induced greater increases in heart rate than isoprenaline if blood pressure fell by less than 25 mmHg. Salbutamol had hypotensive activity at all doses and appeared to be a relatively selective inotrope.
• 3 None of the agonists caused blood pressure to fall in the conscious dogs. Prenalterol and RO 363 were more effective inotropic stimulants, producing smaller increases in heart rate and more pronounced increases in myocardial contractility. Salbutamol, however, elicited greater increases in heart rate in the conscious animals and the inotropic selectivity demonstrated in the anaesthetised animals was lost.
• 4 The direct effects of the β-adrenoreceptor agonists, without modification by reflexes could be observed in the anaesthetised animals. The differences in the actions of the agonists in the conscious animals appear to be attributable to the state of the baroreceptor reflex control system and the relatively enhanced responsiveness of the heart.

     

A STUDY ON THE POSTJUNCTIONAL EXCITATORY α-ADRENORECEPTOR SUBTYPES IN THE MESENTERIC ARTERIAL BED OF THE RAT

• 1 The nature of the postsynaptic adrenoreceptor subtypes which mediate vasoconstriction in the mesenteric arterial bed of the rat was investigated using mixed and selective α₁, α₂ and β-agonists and antagonists.
• 2 Phenylephrine (PE) an α₁selective agonist and noradrenaline (NA) a mixed α₁and α₂-agonist, produced a rise in perfusion pressure (vasoconstriction). The responses to NA remained stable with time whereas responses to PE considerably increased.
• 3 UK14304 an α₂-selective agonist at low doses (10^?8 ?10^?7 moles), caused small, slow contractions in most preparations. Repeated administration of these doses or slightly higher ones, desensitized the tissue to this compound but not to NA or PE. Finally, UK14304 given simultaneously with NA or PE, at doses higher than 5 × 10^?7 moles, reduced contractions to the latter compounds and this effect was not altered by 10^?7 M rauwolscine, an α₂-selective antagonist.
• 4 Prazosin, an α₁-selective antagonist, as expected, reduced contractions to NA considerably at 10^?10 ?10^?8 M and abolished contractions to UK14304 at 2 × 10^?9 M.
• 5 Rauwolscine, at 10^?8 M, potentiated contractions to NA and at 10^?6 M reduced contractions to both NA and PE (when compared to time controls).
• 6 When propranolol (10^?6 M), a β-antagonist was included in the perfusion fluid, rauwolscine no longer potentiated responses to NA but reduced them at all concentrations. Under the same conditions rauwolscine affected the responses to PE in a similar direction to that observed in the absence of propranolol.
• 7 These results suggest that in the rat mesenteric arterial bed:

• a. rauwolscine exerts an effect additional to α₂-adrenoreceptor antagonism. Modification of this effect by propranolol indicates an interaction between this effect of rauwolscine and the β-adrenoreceptor.
• b. vasoconstriction in the mesenteric arterial bed of the rat is mainly mediated postsynaptically by α₁-adrenoreceptors although the contribution of an α₂-mediated component cannot be excluded.
• c. UK14304 is an α₁-partial agonist as well as an α₂-agonist.

     

RELATIVE CONTRIBUTION OF DIFFERENT VASCULAR BEDS TO THE PRESSOR EFFECTS OF α-ADRENORECEPTOR AGONISTS AND VASOPRESSIN IN PITHED RATS: RADIOACTIVE MICROSPHERE DETERMINATION

• 1 The relative fractional distribution of SICr-labelled microspheres was evaluated in pithed rats during equieffective vascoconstrictor responses evoked by infusions of the α-adrenoreceptor agonists methoxamine (α₁-selective), UK- 14,304 (α₂-selective) or vasopressin. The proportion of injected radioactive microspheres trapped in each tissue during a sustained pressor response relative to saline treated controls is considered a reflection of the degree of local vascoconstriction in the tissue analysed.
• 2 All three agonists (methoxamine, UK-14,304 and vasopressin) decreased the number of micro-spheres trapped in the mesentery and tail. Only methoxamine reduced the blood flow to the kidney and spleen. UK-14,304 did not modify the number of microspheres in the sample of skeletal muscle, however, both vasopressin and methoxamine reduced the blood flow to this tissue. Vasopressin increased the counts in the lungs and particularly in the liver but decreased the number of spheres trapped in the stomach and skin. In contrast to the a-adrenoreceptor agonists, vasopressin did not increase the number of microspheres trapped in the heart.
• 3 Since a reduction in the number of microspheres trapped in the tissue reflects a decrease in blood flow, to that organ it is reasonable to conclude that al-adrenoreceptor stimulation increases kidney, spleen, mesentery, caudal and skeletal muscle vascular resistance, whereas a2-adrenoreceptors appear to preferentially vasoconstrict the mesenteric and the caudal vascular beds.

     

DIFFERENTIAL BLOCKING EFFECTS OF PRAZOSIN AND YOHIMBINE ON PRESSOR RESPONSES TO NEURONALLY RELEASED AND EXOGENOUSLY ADMINISTERED NORADRENALINE IN THE PITHED RAT

The effects of prazosin and yohimbine on pressor responses to sympathetic nerve stimulation and intravenous injections of noradrenaline, phenylephrine and clonidine were examined in pithed rats to determine the postjunctional location of alpha 2-adrenoceptors in the vascular smooth muscle. Prazosin antagonized the pressor responses to phenylephrine and to sympathetic nerve stimulation more effectively than the responses to noradrenaline and to clonidine. Yohimbine antagonized the pressor responses to noradrenaline and to clonidine more effectively than the responses to sympathetic nerve stimulation and to phenylephrine. These results suggest that alpha 2-adrenoceptors as well as alpha 1-adrenoceptors produce vasoconstriction in the rat vasculature and support the hypothesis that alpha 1-adrenoceptors are predominantly located within the neuroeffector junction in contrast to an extrajunctional location of alpha 2-adrenoceptors.     

HETEROGENEITY OF β-ADRENORECEPTOR SUBTYPES IN THE HUMAN PLACENTA

• 1 The human placenta is a rich source of β-adrenoreceptors. However, previous work has provided conflicting evidence as to the predominant receptor subtype present. This study was designed to clarify this situation.
• 2 The radioligand ³H-dihydroalprenolol (³H-DHA) was used to identify β-adrenoreceptor binding sites in membranes prepared from human placentae obtained immediately post partum from normal full term pregnancies.
• 3 ³H-DHA binding to human placental membranes was saturable, of high affinity and exhibited the pharmacological characteristics of a β-adrenoreceptor. Displacement by β-adrenoreceptor agonists suggested a predominant β₁-subtype with a hierarchy of potency isoprenaline > adrenaline > noradrenaline. Analysis of the displacement of the specific binding of ³H-DHA by β-adrenoreceptor antagonists yielded Hill plots with an apparent slope (nH) of one for the non selective antagonist (?)propranolol, and of less than one for the highly selective antagonists practolol (β₁), and ICI 118,551 (β₂) indicating a possible heterogeneity of binding sites.
• 4 A direct comparison of the displacement of ³H-DHA binding by selective antagonists in the placenta with that occurring in rat and rabbit lung, further established that a mixture of β₁- and β₂-adrenoreceptor binding sites was present.
• 5 Graphical analysis by Hofstee plot and computer assisted iterative curve fitting was used to further evaluate the displacement by the selective agents and established the presence of an heterogeneous population of β-adrenoreceptor subtypes in the human placenta with approximately 65%β₁ and 35%β₂.