Comparison of pre-junctional α-adrenoceptors at the neuromuscular junction with vascular post-junctional α-receptors in cat skeletal muscle

1 Activation of pre-junctional α-adrenoceptors at the skeletal neuromuscular junction enhances acetylcholine release whereas activation of such receptors at autonomic nerve endings inhibits transmitter output. In the present study the characteristics of pre-junctional α-adrenoceptors at motor nerve terminals have been compared with post-junctional (vascular) α-adrenoceptors in the cat hind limb.

2 Reversal of partial (+)-tubocurarine blockade of contractions of the tibialis anterior muscle was used to monitor pre-junctional activity and increases in hindlimb vascular resistance to assess post-junctional actions at α-adrenoceptors.

3 Responses to intra-arterial injections of noradrenaline, adrenaline, phenylephrine, oxymetazoline, methoxamine and clonidine were monitored. Dose-response lines for all the compounds except clonidine were parallel. The latter agent produced only weak and inconsistent effects.

4 Ratios of the doses of the agents required to produce pre- and post-junctional effects indicated that oxymetazoline and adrenaline possessed some preferential activity at post-junctional sites, whereas the remaining agents were non-selective in their actions. If dose-ratios with respect to noradrenaline were compared at the two sites none of the compounds possessed a marked degree of selectivity.

5 In the presence of phentolamine or tolazoline, dose-response curves to the pre- and post-junctional effects of phenylephrine were shifted to a similar extent. Thymoxamine showed preferential activity as a pre-junctional α-receptor antagonist.

6 In comparing the results of this study with those of other authors, it is apparent that there are marked differences in the characteristics of pre-junctional α-receptors at the skeletal neuromuscular junction and at autonomic nerve endings. The pre- and post-junctional α-receptors in skeletal muscle show less divergence.

     

Inhibition of rabbit intestine mediated by α- and β-adrenoceptors

1. The effects of some alpha- and beta-adrenoceptor agonists and antagonists were studied on isolated segments of rabbit intestine in an attempt to characterize the two types of inhibitory response produced by sympathomimetic amines.2. Phenylephrine, an alpha-adrenoceptor agonist, produced an inhibition of rapid onset, from which recovery occurred despite the continued presence of the drug. On washout there was an overshoot in contraction height. Isoprenaline, a beta-adrenoceptor agonist, produced an inhibition of slow onset which was maintained throughout the presence of the drug and there was no overshoot on washout.3. Adrenaline resembled phenylephrine more closely than it resembled isoprenaline, in that it showed more affinity for alpha-adrenoceptors, whereas noradrenaline, and the transmitter released on periarterial nerve stimulation, behaved more like isoprenaline, although both types of receptor were affected.4. Adenosine-5'-triphosphate produced an inhibition resembling that produced by an alpha-adrenoceptor agonist, whereas the dibutyryl analogue of cyclic adenosine 3',5'-monophosphate (cyclic 3',5'-AMP) produced an inhibition resembling that produced by a beta-adrenoceptor agonist.5. In critical concentrations theophylline augmented and imidazole inhibited beta-adrenoceptor mediated responses, as well as responses to dibutyryl cyclic AMP. However, additional actions of theophylline and imidazole were also demonstrated.6. Responses mediated by alpha-adrenoceptors, but not those mediated by beta-adrenoceptors, were blocked by membrane stabilizers, quinidine being the most potent of those studied.7. The results are discussed in relation to the possible mechanisms of action of alpha- and beta-adrenoceptor agonists.     

Distribution of α- and β-adrenoceptors in human urinary bladder

1 The distribution of alpha- and beta-adrenoceptors in isolated preparations of human bladder neck and detrusor muscle has been studied.2 Adrenaline caused contraction of the bladder neck which was blocked by phenoxybenzamine but unaltered by propranolol.3 Isoprenaline caused relaxation of the bladder neck which was blocked by propranolol. High concentrations caused contraction which was enhanced by propranolol but blocked by phenoxybenzamine.4 Detrusor muscle was relaxed by isoprenaline and this effect was blocked by propranolol. Phenylephrine caused relaxation of detrusor which was unaffected by phenoxybenzamine; in some cases contraction was produced in the presence of propranolol.5 It is concluded that the bladder neck contains mainly alpha-receptors and the detrusor mainly beta-receptors but both regions posses both types of adrenoceptor.     

Characterization of α-adrenoceptors in the myocardium

1. In the driven rabbit left atrium alpha-adrenoceptors mediate the inotropic effect of phenylephrine and the prolongation of functional refractory period by sympathomimetic amines.2. These receptors are highly sensitive to blockade by phentolamine and phenoxybenzamine.3. Prolongation of functional refractory period is greater with the secondary amines, phenylephrine, adrenaline and epinine, than with the primary amines, norphenylephrine, noradrenaline and dopamine.     

Assessment of selective β-adrenoceptor blockade in man

1. Selective antagonism of the cardiac beta(1)-adrenoceptors has been studied in normal human volunteers.2. Practolol and UK 6558 produced greater antagonism of the chronotropic and inotropic responses to i.v. isoprenaline than of the vasodilator response to either i.v. or intra-arterial isoprenaline. A third drug, M&B 17,803A, produced non-selective beta-adrenoceptor blockade in 2 of 3 subjects studied.3. Practolol, UK 6558 and M&B 17,803A, produced an attenuation of the responses to Valsalva's manoeuvre.4. A substantial reduction in blood pressure was seen in 3 of 4 normotensive subjects given UK 6558.     

α- and β-Adrenoceptors in the detrusor muscle and bladder base of the pig and β-adrenoceptors in the detrusor muscle of man

1 The presence and type of adrenoceptors in the smooth muscle of the pig and human urinary bladder was assessed on the basis of the relative potency of α- and β-adrenoceptor agonists and antagonists.

2 In isolated, carbachol-contracted bladder strips from the pig detrusor muscle the relaxing potency of isoprenaline was four times that of salbutamol and ritodrine and thirty times that of noradrenaline.

3 Propranolol caused a parallel shift to the right of the noradrenaline dose-response curve which was not changed by phentolamine.

4 Propranolol and butoxamine showed, in contrast to practolol, a dose-dependent antagonism of the response to isoprenaline. A pA₂ value of 9.2 ± 0.2 and 6.8 ± 0.2 (mean ± s.e. mean) for the first two antagonists was calculated.

5 In the bladder base of the pig, propranolol caused a parallel shift to the right and phentolamine a shift to the left of the dose-response curve to noradrenaline.

6 In the human detrusor muscle the potency and maximum effect of isoprenaline and salbutamol were less than those in the pig detrusor muscle. The potency of isoprenaline was sixty times that of salbutamol.

7 Whereas a parallel shift to the right of the dose-response curve to isoprenaline was obtained with propranolol, no antagonism was obtained with butoxamine or practolol.

8 The results are interpreted as indicating the presence of β₂-adrenoceptors in the detrusor muscle of the pig and β-adrenoceptors with neither β₁- nor β₂-characteristics in the detrusor muscle of man. An indication of the presence of α-adrenoceptors in the bladder base but not in the detrusor muscle of the pig was obtained.

     

Comparison of the effects of propranolol and MJ 1999 on cardiac β-adrenoceptors in man

1. Propranolol, a beta-adrenoceptor blocking drug with local anaesthetic and a direct myocardial depressant action, and MJ 1999, a beta-adrenoceptor blocking drug which has no local anaesthetic or intrinsic sympathomimetic action, were compared for beta-adrenoceptor blocking activity in man.2. Propranolol was 2.67 times more active than MJ 1999 in reducing by 50% the tachycardia produced by the intravenous infusion of isoprenaline in healthy volunteers.3. Propranolol and MJ 1999 intravenously both reduced resting heart rate in the standing position and an exercise tachycardia, but there was no qualitative or quantitative difference between them.4. On oral administration, both propranolol and MJ 1999 reduced resting heart rate and an exercise induced tachycardia; propranolol was only slightly more active than MJ 1999.5. In patients with thyrotoxicosis propranolol was about twice as active as MJ 1999 in reducing the heart rate.     

Temperature modulation of α- and β-adrenoceptors in the isolated frog heart

1. The effects of adrenaline on the isolated frog's heart at 27 degrees C are not antagonized by phentolamine (1.5 x 10(-6)M) but are abolished at 7 degrees C.2. At 27 degrees C isoprenaline was more potent than noradrenaline, but at 7 degrees C noradrenaline was more potent than isoprenaline.3. Phenoxybenzamine (1.5 x 10(-5)M) or dibenamine (1.5 x 10(-5)M) at 7 degrees C abolished the work output induced by adrenaline. When the temperature was raised to 24 degrees C, adrenaline caused an increase in work output.4. It is concluded that in the isolated frog heart there are at least two pools of adrenoceptors, the availability of which can be governed by temperature.     

Pharmacology of AH 5158; a drug which blocks both α- and β-adrenoceptors

1. AH 5158 differs from conventional adrenoceptor blocking drugs in producing competitive blockade of both alpha- and beta-adrenoceptors.2. AH 5158 is 5-18 times less potent than propranolol in blocking beta-adrenoceptors. It resembles propranolol in its non-selective blockade of beta(1)-cardiac and beta(2)-vascular and tracheal adrenoceptors and in its lack of intrinsic sympathomimetic activity.3. AH 5158 is 2-7 times less potent than phentolamine in blocking alpha-adrenoceptors. AH 5158 itself is more active on beta- than alpha-adrenoceptors.4. Blockade of noradrenaline vasopressor responses by AH 5158 in anaesthetized dogs was dose-dependent up to 1 mg/kg but no further blockade was obtained with larger doses of AH 5158. ;Self-limiting' blockade was not observed in dogs pretreated with cocaine, or in untreated dogs if the vasopressor agent was oxymetazoline instead of noradrenaline. A possible cause of ;self-limiting' blockade is discussed.5. In doses higher than those required for either alpha- or beta-adrenoceptor blockade, AH 5158 produced effects on cardiac muscle that are attributable to membrane-stabilizing activity. This was manifested as a negative inotropic action in spinal dogs and in guinea-pig left atrial strips, as a negative chronotropic action in syrosingopine pre-treated dogs, and as an increase in the effective refractory period of guinea-pig left atrial strips. AH 5158 was 3-11 times less potent than propranolol in these tests.6. In open chest dogs AH 5158 resembled propranolol in reducing cardiac output, rate and contractility, effects which are attributable to beta-adrenoceptor blockade. The drug differed from propranolol in decreasing rather than increasing total peripheral resistance and in causing larger decreases in arterial blood pressure at equipotent beta-adrenoceptor blocking doses. These differences are attributable to the alpha-adrenoceptor blocking actions of AH 5158.7. In anaesthetized dogs, intravenously administered AH 5158 antagonized both catecholamine and ouabain-induced arrhythmias. Orally administered AH 5158 lowered systolic arterial pressure in conscious renal hypertensive dogs.8. These results show AH 5158 to possess a novel profile of activity. Possible uses of the drug in cardiovascular disorders such as hypertension, angina pectoris and cardiac arrhythmias are discussed.     

Comparison of mianserin with desipramine, maprotiline and phentolamine on cardiac presynaptic and vascular postsynaptic α-adrenoceptors and noradrenaline reuptake in pithed normotensive rats

1 The cardiovascular effects of intravenous desipramine (0.03 and 0.1 mg/kg), maprotiline (0.5 mg/kg), mianserin (1.0 and 3.0 mg/kg) and phentolamine (0.25 mg/kg) were examined and compared in pithed rats. Several experimental procedures were used in order to distinguish between the effects of the compounds on cardiac presynaptic alpha-adrenoceptors and on neuronal noradrenaline reuptake, as inhibition of either mechanism produces an increase of neurotransmitter concentration within the sympathetic synapse and therefore results in a greater end organ response.2 Pressor responses elicited by noradrenaline were potentiated by desipramine and maprotiline, reduced by phentolamine and not significantly modified by mianserin. However, all four compounds inhibited the pressor action of tyramine. Furthermore, mianserin reduced the pressor response to adrenaline.3 Desipramine, maprotiline and mianserin, but not phentolamine enhanced the positive chronotropic effects of noradrenaline, without affecting those of isoprenaline.4 All four compounds abolished the clonidine-induced inhibition of heart rate responses to short term electrical stimulation of the spinal cord. Moreover, in rats with a persistent tachycardia (induced by continuous stimulation of the thoracic spinal cord) desipramine, maprotiline and mianserin further increased heart rate. This effect was also observed in animals pretreated with phentolamine, administered in order to inhibit cardiac presynaptic alpha-adrenoceptors.5 In rats with a sustained tachycardia (100 beats/min produced by electrical stimulation of the spinal cord) both mianserin and phentolamine, in contrast to desipramine, shifted the clonidine heart rate dose-response curve to the right. Phentolamine was about 34 times more potent than mianserin in this respect.6 In pithed, reserpine-treated rats, the pressor responses to clonidine were not significantly modified by desipramine. The dose-response curves were shifted to the right by phentolamine (0.25 mg/kg) and mianserin (3.0 mg/kg).7 These results indicate that mianserin is an antagonist of both cardiac presynaptic and vascular postsynaptic alpha-adrenoceptors and also inhibits the neuronal reuptake of noradrenaline.     

Central α- and β-adrenoceptors modifying arterial blood pressure and heart rate in conscious cats

1 In conscious unrestrained cats noradrenaline, alpha-methylnoradrenaline and clonidine, infused into the lateral cerebral ventricles (i.c.v.) caused dose-related falls in blood pressure and heart rate; both effects were abolished after i.c.v. phentolamine.2 In 12 out of 20 cats, i.c.v. isoprenaline and salbutamol when given caused dose-related pressor responses and tachycardias. These effects were abolished after i.c.v. beta-adrenoceptor blocking drugs but were unaffected by alpha-adrenoceptor blocking agents.3 In 5 out of 20 cats, i.c.v. isoprenaline regularly produced dose-related falls in blood pressure with associated tachycardias; both effects were abolished after i.c.v. beta-adrenoceptor blocking agents.4 Intracerebroventricular dopamine produced cardiovascular responses which were qualitatively similar to those produced by i.c.v. isoprenaline.5 Intracerebroventricular adrenaline produced complex responses in untreated animals but typical alpha-effects were obtained after prior i.c.v. treatment with a beta-adrenoceptor blocking agent and typical beta-effects after i.c.v. pretreatment with an alpha-adrenoceptor blocking agent.6 The cardiovascular changes produced by i.c.v. beta-adrenoceptor agonists were abolished after systemic administration of hexamethonium or bethanidine.7 The results are discussed in the light of the mode of action of beta-adrenoceptor stimulants and beta-adrenoceptor blocking agents in the treatment of hypertension.     

Roles for α1-adrenoceptors during contractions by electrical field stimulation in mouse vas deferens

We have investigated the relative roles of α₁-adrenoceptors and purinoceptors in contractions to low and high frequency stimulation of the mouse vas deferens, in terms of the time course of responses. In separate experiments, isometric contractile responses were obtained to 10 pulses at 1 Hz and 40 pulses at 10 Hz. Responses to 1 Hz stimulation consisted of a series of discrete peaks. The α_1A-adrenoceptor antagonist RS100329 (10^–9M–10^–7M) significantly reduced the response to the first pulse, the α_1D-adrenoceptor antagonist BMY7378 (10^–7M–10^–6M) significantly reduced the response to the first two pulses, and the non-selective α₁-adrenoceptor antagonist prazosin (10^–8M) reduced the response to the first 4 pulses at 1 Hz. Responses to 10 Hz stimulation consisted of an early peak response and a maintained plateau response. RS100329 significantly reduced the peak response but did not significantly affect the plateau response. Prazosin, significantly reduced both the peak and plateau responses. The α_1A-adrenoceptor antagonist RS17053 in high concentrations reduced mainly the plateau response leaving a clear early peak response. The plateau response of contraction was almost abolished by the purinoceptor antagonist suramin. These results suggest that there is a relatively minor early α_1D-adrenoceptor and a larger early α_1A-adrenoceptor component to stimulation-evoked contractions of mouse vas deferens, but the major α₁-adrenoceptor component is revealed by prazosin to be α_1B-adrenoceptor mediated. α_1B-Adrenoceptor activation probably facilitates contractions mediated by other α₁-adrenoceptors and by purinoceptors. These results suggest that combined non-selective α₁-adrenoceptor blockade, particularly α_1B-adrenoceptor blockade, in addition to P2X1-purinoceptor blockade is useful in reducing male fertility.     

Effect of cocaine on the affinity of α-adrenoceptors for noradrenaline

1. Doses of cocaine which cause specific or unspecific supersensitivity in cat spleen did not alter the blocking effect of phenoxybenzamine on the responses of isolated cat spleen strips to noradrenaline.2. The same doses of cocaine did not increase the protection of alpha-adrenoceptors given by noradrenaline during a standard exposure to phenoxybenzamine.3. It is concluded that cocaine does not change the affinity of the alpha-adrenoceptor for noradrenaline, and therefore changes in affinity are not responsible for the potentiating action of cocaine.     

Mechanism of potentiation by amines of non-equilibrium blockade of the α-adrenoceptor

1. The mechanism by which sympathomimetic and certain other amines enhance blockade of the alpha-adrenoceptors by the non-equilibrium antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in strips of rabbit aorta was examined.2. Non-equilibrium blockade of the 5-hydroxytryptamine receptors by EEDQ was not increased by sympathomimetic amines and was decreased by 5-hydroxytryptamine.3. Low concentrations of reversible competitive antagonists appeared to protect selectively against the additional blockade by EEDQ which develops in the presence of an amine.4. Phenoxybenzamine potentiated EEDQ blockade of the alpha-receptors but not of the 5-hydroxytryptamine receptors.5. Augmentation of EEDQ blockade was also detected in a variety of other tissues, but not in segments of rabbit intestine where alpha-adrenoceptors mediate an inhibitory response.6. It was concluded that EEDQ acts at two sites in antagonizing alpha-receptor mediated responses, and that one of these sites (site II) is separate from the site of action of agonists and phenoxybenzamine (site I). Amines which enhance blockade appear to exert their action by combining with a third site (site III), which may induce a conformational alteration at site II.7. It appears that the alpha-adrenoceptor may have multiple sites for drug interaction.     

Actions of phenylephrine on β-adrenoceptors in guinea-pig trachea

1. Phenylephrine produced relaxation of the isolated guinea-pig tracheal chain preparation, its potency being 1/5 that of noradrenaline on normal tissues.2. The potentiation of phenylephrine by cocaine (10(-5)M) was only slight. Thus on cocaine-treated tissues phenylephrine was 1/45 as potent as noradrenaline.3. The dose-response lines to phenylephrine were shifted in a parallel manner by propranolol 10(-8)M and 10(-7)M, suggesting that the relaxations were mediated through beta-adrenoceptors.4. Phenylephrine had a lower intrinsic activity than the catecholamines and produced multiphasic dose-response lines at the higher doses used in the presence of propranolol (10(-6)M). These observations have been explained by the evidence obtained that phenylephrine is a partial agonist with beta-adrenoceptor blocking activity.5. From experiments using alpha-adrenoceptor blocking drugs, it has been concluded that stimulation of alpha-adrenoceptors has little influence on the beta-adrenoceptor relaxation to phenylephrine on the guinea-pig tracheal chain preparation.     

Clinical investigation of an antagonist at α- and β-adrenoceptors-AH 5158A

1. The alpha- and beta-adrenoceptor blocking action of AH 5158A was investigated in man using the veins of the hand, the arterial bed of the forearm, and certain responses of the circulation as a whole.2. In the veins, locally infused AH 5158A resulted in specific and competitive antagonism of the constrictor response to locally infused noradrenaline and of the dilator response to isoprenaline.3. Brachial artery infusions of AH 5158A resulted in competitive antagonism of the arterial blood flow changes produced by local infusions of noradrenaline and isoprenaline.4. Systemic infusion of AH 5158A (0.5-0.9 mg/kg) produced clear blockade of the heart rate response to systemic infusion of isoprenaline. It also attenuated the response to exercise at 80 watts for 4 min; mean arterial pressure during exercise was reduced by 16% and heart rate by 18%. Blockade lasted at least 1 hour.5. AH 5158A caused small changes in arterial pressure and heart rate at rest supine, but had no effect on the response of pressure and rate to tilting.     

The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site

α-conotoxins are 13–19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. α-conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets α7 nAChR. Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site.     

The presence of β-adrenoceptors in the guinea-pig seminal vesicle

1. The preparation of longitudinal smooth muscle strips from guinea-pig seminal vesicles is described.2. Isoprenaline and salbutamol inhibited contractions produced by parasympathomimetic agents.3. The inhibitory action of isoprenaline was blocked by low concentrations of propranolol and butoxamine. It was concluded that beta(2)-adrenoceptors were present in the tissue.4. The inhibitory action of isoprenaline was not apparent when adrenaline, noradrenaline, hypogastric nerve or transmural stimulation were used to contract the tissue.     

Effect of catecholamines and angiotensin on extracellular water and cation movements and the effect of α and β-adrenoceptor blockade

1. The extracellular fluid volume (ECFV), as raffinose space, and its content of Na, K and Ca were measured in anaesthetized dogs in acute experiments before and during the vascular response to intravenous injections and infusions of noradrenaline, adrenaline, isoprenaline and angiotensin.2. In male dogs the effect of noradrenaline was unpredictable, the ECFV might increase or decrease. In female dogs noradrenaline caused an increase in the ECFV. The difference between the responses of the two sexes was statistically significant (P<0.001). After phentolamine, noradrenaline exerted no effect at all in either sex. After bretylium the results were like those in normal animals.3. In both males and females adrenaline generally induced an increase in the ECFV. After phentolamine, adrenaline decreased the ECFV in males and caused little change in females. The differences before and after blockade were statistically significant (P<0.001). After bretylium the results in both sexes were like those in normal animals.4. In both male and female dogs isoprenaline induced an increase in the ECFV and the results were the same as in the normal animals after both phentolamine and bretylium.5. In male dogs there was no change in the ECFV as a result of administering angiotensin, either alone or in the presence of phentolamine or bretylium. In normal females angiotensin induced a decrease in ECFV and the difference between the responses of the males and females was statistically significant (P<0.005). In females which had received either phentolamine or bretylium the results were indistinguishable from those in the males.6. Blockade of the beta-adrenoceptors with pronethalol in a few animals did not change the response to the drugs from those seen in normal animals.7. The cation content of the ECF changed in the same direction and to about the same extent as the water, except after noradrenaline when in some experiments the proportionate change in potassium concentration was considerably greater than that of the other substances.8. The inulin space and its Na and K content were measured in several dioestrous, oestrous and pro-oestrous rats and in normal and stilboestrol treated males before and after giving an intravenous injection of angiotensin. There was little difference between the results of control injections of 0.9% saline solution and of angiotensin in dioestrous and oestrous females and normal males. On the other hand, pro-oestrous females and stilboestrol treated males responded alike to angiotensin in the form of a decrease in ECFV which was statistically different from the responses in the other three groups (P<0.0005).9. It is suggested that the various results depend on two factors: the site of action of the drug-for example, whether it increases or decreases capillary pressure and therefore, fluid transfer-and also the sex of the animal. The ground substance of the small blood vessels is probably important in taking up and releasing fluid, and its capacity for so doing may well vary with the amount of available oestrogen. It appears that the effect of oestrogens and events at the alpha-adrenoceptor site are connected in some way.