Prejunctional alpha-adrenoceptors regulate nitrergic neurotransmission in the rabbit urethra

We evaluated the effects of prejunctional alpha-adrenoceptors on nitric oxide (NO)-mediated urethral relaxation in rabbits using a muscle bath technique and high-performance liquid chromatography coupled with a microdialysis procedure. The amount of NO(2)(-)/NO(3)(-) released during electrical field stimulation was measured by an NO(2)(-)/NO(3)(-) analyzer based on the Griess method. Pretreatment with phenylephrine (0.01 microM) and yohimbine (0.1-10 microM) significantly reduced the relaxation responses induced by electrical field stimulation. In contrast, pretreatment with clonidine (0.01 microM) and prazosin (0.01-1 microM) enhanced the relaxation responses. Cys-NO-induced relaxations of rabbit urethral smooth muscle were not affected by pretreatment with alpha-adrenoceptor agonists and antagonists. The amount of NO(2)(-)/NO(3)(-) released by electrical field stimulation increased after pretreatment with clonidine (0.01 microM) and prazosin (0.01-1 microM), but decreased after pretreatment with phenylephrine (0.01 microM) and yohimbine (0.1-10 microM). The results suggest that the release of NO from nitrergic nerves in the rabbit urethra is reduced and increased by stimulation of prejunctional alpha(1)- and alpha(2)-adrenoceptors, respectively.     

Distribution and types of adrenoceptors in the guinea-pig ileum: the action of alpha- and beta-adrenoceptor blocking agents

1 Transmurally stimulated segments of the guinea-pig ileum have been used to analyse the different adrenoceptors in the terminal (0 to 3 cm) and the proximal (> 50 cm from the ileocaecal valve) ileum.

2 The prejunctional adrenoceptors (located on the final, cholinergic, motor nerve terminals) and postjunctional adrenoceptors (located on the smooth muscle membrane) have been characterized according to their sensitivity to α- and β-agonists and antagonists.

3 Phentolamine, phenoxybenzamine and yohimbine, in concentrations of 0.1 μM, transiently enhanced (up to 10%) the twitch response. At higher concentrations all the α- and β-antagonists studied depressed the neurogenic twitches and relaxed the smooth muscle.

4 The twitch-inhibitory effects of adrenoceptor agonists (noradrenaline, adrenaline and ephedrine) were not antagonized by phenoxybenzamine (0.1, 0.5 and 1 μM), carbidine (0.5, 1 and 5 μM) and propranolol (0.5, 1 and 5 μM); however, they were depressed by phentolamine (0.1, 0.5, 1.25 and 5 μM) and yohimbine (0.25, 0.5 and 5 μM).

5 The smooth muscle contractions induced by noradrenaline and adrenaline in the terminal ileum and by phenylephrine in both the terminal and proximal ileum were antagonized by phenoxybenzamine, carbidine and phentolamine but were not influenced by yohimbine and propranolol.

6 The smooth muscle relaxations of the proximal ileum induced by noradrenaline, adrenaline and ephedrine were inhibited by yohimbine, phentolamine, carbidine and phenoxybenzamine, and the isoprenaline-induced relaxation was antagonized by propranolol.

7 All the agonists studied, except phenylephrine, elicited relaxations of the acetylcholine-induced sustained contraction of both proximal and terminal ileum. The relaxation induced by isoprenaline was antagonized by propranolol, and the effects of noradrenaline and ephedrine by yohimbine.

8 It is concluded that in the guinea-pig ileum there are postsynaptic β-adrenoceptors and at least two types of α-adrenoceptors: α₁-excitatory postjunctional adrenoceptors activated by phenylephrine, noradrenaline and adrenaline and antagonized by phenoxybenzamine, carbidine and phentolamine; α₂-inhibitory prejunctional adrenoceptors activated by ephedrine, noradrenaline and adrenaline and inhibited by yohimbine and phentolamine. The inhibitory postjunctional α-adrenoceptors are more close to the α₂-adrenoceptors, since they were stimulated predominantly by ephedrine and noradrenaline and inhibited by yohimbine.

9 It has been shown that all α-adrenoceptor subtypes are to be found at every distance (0 to 70 cm) from the ileocaecal valve and that they can be activated in the resting or in the acetylcholine-contracted states.

     

Field stimulation-induced responses of circular smooth muscle from guinea-pig stomach

Summary Field stimulation of circular smooth muscle of guinea-pig stomach from the regions of the cardia and fundus caused contraction responses at low stimulation frequencies (0.25–1 Hz) with relaxation at higher frequencies (1–10 Hz), whilst tissues of the body and antrum responded with contraction throughout the frequency range. Atropine (10^–9–10^–8 M) antagonised the contraction responses of all tissues, with relaxation developing at higher concentrations (except for antral tissue). In contrast, metoclopramide (10^–8–10^–6 M) caused modest (cardia, fundus) or marked (body, antrum) enhancement of contractions to field stimulation, whilst domperidone (10^–8–10^–7 M), haloperidol (10^–8–10^–6 M), prazosin, propranolol and methysergide (10^–8–10^–6 M) failed to modify the contraction responses. However, whilst yohimbine and guanethidine failed to modify the contractions of the cardia, fundus and body tissues, those of the antral preparations were antagonised by nanomolar concentrations of yohimbine and by guanethidine (10^–6–5×10^–5 M). To optimise the relaxation responses for study, atropine was included in the physiological solution. Relaxation to field stimulation of preparations from the body and cardia, but not the fundus, was antagonised by reserpine pretreatment (5 mg/kg i.p., 24h), addition of guanethidine (10^–5–10^–4 M), phentolamine, prazosin or propranolol (10^–7–10^–6 M) (the effects of prazosin and propranolol being additive). Higher concentrations of haloperidol and domperidone antagonised the relaxation responses of the body preparations only. Metoclopramide, yohimbine and methysergide (10^–8–10^–6 M) were ineffective. Thus, it is concluded that the contractile effects of the 4 stomach areas to field stimulation reflects a major cholinergic involvement, with an additional ₂-adrenoceptor contractile component in antral tissue. Relaxation responses of cardia and body tissue involve ₂- and -adrenoceptors plus a further, unidentified, non-adrenergic component; the latter represents the total relaxation response of the fundic preparation.     

Role of the purinergic and noradrenergic components in the potentiation by endothelin-1 of the sympathetic contraction of the rabbit central ear artery during cooling

1. To examine the role of the purinergic and noradrenergic components in the potentiation of endothelin-1 on the vascular response to sympathetic nerve stimulation, we recorded the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1–8 Hz) under different conditions, at 37°C and during cooling (30°C).
2. Electrical field stimulation produced frequency-dependent contraction, which was reduced during cooling (about 60% for 8 Hz). Both at 37°C and 30°C, phentolamine (1 μM) or blockade of α₁-adrenoceptors with prazosin (1 μM) reduced, whereas blockade of α₂-adrenoceptors with yohimbine (1 μM) increased, the contraction to electrical field stimulation. This contraction was increased after desensitization of P2-receptors with α,β-methylene adenosine 5′-triphosphate (α,β-meATP, 3 μM) at 37°C but not at 30°C, and was not modified by blockade of P2-receptors with pyridoxalphosphate-6-azophenyl-2,4′-disulphonic acid (PPADS, 30 μM) at either temperature.
3. Endothelin-1 (1, 3 and 10 nM) at 37°C did not affect, but at 30°C it potentiated in a concentration-dependent manner the contraction to electrical field stimulation (from 28±6 to 134±22%, for 8 Hz). At 37°C, endothelin-1 in the presence of phentolamine or prazosin, but not in that of yohimbine, α,β-meATP or PPADS, potentiated the contraction to electrical stimulation. At 30°C, phentolamine or yohimbine reduced, prazosin or PPADS did not modify and α,β-meATP slightly increased the potentiation by endothelin-1 of the response to electrical stimulation.
4. The arterial contraction to ATP (2 mM) and the α₂-adrenoceptor agonist BHT-920 (10 μM), but not that to (−)-noradrenaline (1 μM), was potentiated by endothelin-1 at both 37°C and 30°C.
5. These results in the rabbit central ear artery suggest that the sympathetic response: (a) at 37°C, could be mediated mainly by activation of α₁-adrenoceptors, with low participation of P2-receptors, (b) is diminished during cooling, probably by a reduction in the participation of α₁-adrenoceptors, and in this condition the response could be mediated in part by P2-receptors, and (c) is potentiated by endothelin-1 during cooling, probably by increasing the response of both postjunctional α₂-adrenoceptors and P2-receptors.

     

Inhibitory actions of catecholamines on electrically induced contractions of the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus

1 The submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus was used to study the actions of catecholamines on the twitch responses to electrical stimulation.

2 When the preparation was stimulated coaxially (0.1 Hz, 0.5 ms, supramaximal voltage), stable twitch-like contractions were obtained. These were abolished by tetrodotoxin (0.1 μM) and atropine (0.1 μM), potentiated by physostigmine (0.1 μM), and were mediated presumably by stimulation of intramural cholinergic nerves.

3 The twitch contractions of the muscularis mucosae were inhibited by catecholamines, in a concentration-dependent manner. The order of potency was isoprenaline > adrenaline > noradrenaline > dopamine.

4 The inhibitory actions of noradrenaline (1 μM) and adrenaline (1 μM) were partly reversed by phentolamine (1 μM) or by propranolol (1 μM), and completely abolished by both antagonists together. The inhibitory effect of dopamine (300 μM) was largely reversed by phentolamine (1 μM), but not by propranolol (1 μM), while the inhibitory action of isoprenaline was competitively antagonized only by propranolol (pA₂ of 7.6).

5 The contraction of the muscularis mucosae to exogenously applied acetylcholine (ACh, 20 nM) which was comparable in magnitude with that to electrical stimulation was also inhibited by isoprenaline (0.1 μM), adrenaline (1 μM) and noradrenaline (1 μM), but not by dopamine (300 μM). In the presence of propranolol (1 μM), noradrenaline, adrenaline and dopamine potentiated the ACh-induced contraction, while the effect of isoprenaline was mainly antagonized. The potentiating effects were antagonized by further treatment with phentolamine (1 μM).

6 Adrenaline, noradrenaline and dopamine but not isoprenaline, produced a weak contraction of the longitudinal muscularis mucosae in the presence of propranolol (3 μM). The contractile responses were completely inhibited by phentolamine (3 μM). Tone in the muscularis mucosae induced by carbachol (3 μM) in the presence of phentolamine (10 μM) was inhibited by catecholamines, in a concentration-dependent manner, an effect that was competitively antagonized by propranolol.

7 In the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus there are three types of adrenoceptor, inhibitory prejunctional α-adrenoceptors, excitatory postjunctional α-adrenoceptors and inhibitory postjunctional β-adrenoceptors, and cholinergic neurotransmission is inhibited by catecholamines acting at both prejunctional α- and postjunctional β-adrenoceptors.

     

Homogeneous and non-homogeneous distribution of inhibitory and excitatory adrenoceptors in the longitudinal muscle of the guinea-pig ileum

1 The effects of adrenoceptor agonists and antagonists on spontaneous and evoked membrane activities of longitudinal muscle cells from different parts of the guinea-pig ileum were observed, using microelectrode methods.

2 Isoprenaline inhibited the generation of spikes in cells in the terminal (0-3 cm from the ileocaecal valve) and proximal (more than 50 cm from the ileocaecal valve) regions of the ileum, with no change on the membrane potential and ionic conductance of the membrane. These actions of isoprenaline were abolished by propranolol.

3 Noradrenaline and phenylephrine depolarized the membrane and increased both the spike frequency and ionic conductance of cell membranes of the terminal ileum, whereas noradrenaline and clonidine hyperpolarized the membrane, increased the ionic conductance of the membrane and inhibited the spontaneously generated spikes from cells of the proximal ileum. The excitatory effect of phenylephrine in the cells of the proximal ileum and the inhibitory effect of clonidine on cells of the terminal ileum were less pronounced.

4 The excitatory actions of noradrenaline or phenylephrine were antagonized by prazosin and phentolamine, but not by yohimbine, whereas the inhibitory actions of noradrenaline or clonidine were antagonized by yohimbine and phentolamine but not by prazosin.

5 The cholinergic e.j.ps evoked by field stimulation to the tissue were not affected by isoprenaline or phenyleprine but were inhibited by noradrenaline and clonidine, in both the terminal and proximal regions of the ileum. These actions of noradrenaline and clonidine were antagonized by yohimbine but not by prazosin.

6 The results indicate that in the myenteric plexus and longitudinal muscle tissues of the guinea-pig ileum there are prejunctional inhibitory (α₂), postjunctional inhibitory (α₂ and β) and postjunctional excitatory (α₁) adrenoceptors. The homogeneous distributions of prejunctional α₂- and postjunctional β-adrenoceptors in the ileum are responsible for inhibitions of cholinergic excitatory junction potentials (e.j.ps) and spontaneous spike activities, respectively. The density of distribution of the postjunctional α₁-adrenoceptors is higher in the terminal than in the proximal regions, and these distributions are reversed in the case of the postjunctional α₂-adrenoceptors. The postjunctional α₁-adrenoceptors are probably responsible for the membrane depolarization and α₂-adrenoceptors for the hyperpolarization induced by catecholamines.

     

Effects of the histamine H2-receptor blocking drugs burimamide and cimetidine on noradrenergic transmission in the isolated aorta of the rabbit and atria of the guinea-pig

1 In rabbit aortic strips, concentration-response curves to noradrenaline (NA) were shifted to the right in a parallel and concentration-dependent manner by the α-adrenoceptor blocking drug, phentolamine and also by the histamine H₂-receptor blocking drugs, burimamide and cimetidine. Responses to 5-hydroxytryptamine were not affected by these drugs.

2 Burimamide had the properties of a competitive antagonist of noradrenaline, possessing about one-hundredth the potency of phentolamine. Cimetidine was weaker than burimamide and did not fulfil the requirements for competitive antagonism of noradrenaline.

3 In guinea-pig isolated atria, in which noradrenergic transmitter stores were labelled with [³H]-noradrenaline, phentolamine (3 μM), burimamide (30 μM) and cimetidine (30 μM), in decreasing order of effectiveness, each enhanced stimulation-induced efflux of [³H]-noradrenaline, indicating that their blocking effects on prejunctional α-adrenoceptors in this tissue are in the same order of relative potency as on postjunctional α-adrenoceptors in rabbit aortic strips.

4 In the concentrations used (30 μM), neither burimamide nor cimetidine interfered with the neuronal uptake of noradrenaline. Burimamide, and to a much lesser extent, cimetidine, increased the resting efflux of [³H]-noradrenaline from guinea-pig atria.

5 The effect of clonidine, a partial agonist on prejunctional α-adrenoceptors in guinea-pig atria, in increasing stimulation-induced efflux of [³H]-noradrenaline when stimulated with 150 pulses at 5 Hz was blocked by cimetidine (30 μM) and reversed by phentolamine (3 μM) and burimamide (30 μM).

     

The actions of 5-hydroxytryptamine receptor agonists and antagonists at pre- and postjunctional level on the canine saphenous vein

Summary The prejunctional and postjunctional 5-HT receptors of the canine saphenous vein were studied. The release of ³H-noradrenaline (³H-NA) from incubated saphenous vein strips was inhibited by 5-hydroxytryptamine (5-HT) in a concentration-dependent way (5-HT concentrations: 0.01, 0.1 and 1.0 mol · l^–1), but not by the selective 5-HT_1A agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT; 1 and 10 mol · l^–1). The inhibitory effect of 5-HT was antagonized by metitepine and methysergide, but not by yohimbine, (–)-pindolol or ketanserin. In strips preincubated with 5-HT (1.2 mol · l^–1), the fractional release of ³H-NA was slightly reduced (paired experiments). 5-HT and 8-OH-DPAT caused concentration-dependent contractions of the saphenous smooth muscle. A parallel shift of the concentration-response curve for 8-OH-DPAT to the right was caused by metitepine and yohimbine, but not by ketanserin. The contractions caused by 5-HT were antagonized by metitepine and yohimbine (parallel displacement of the curves to the right), as well as by ketanserin and methysergide (with a depression of the upper part of the curve). Blockade of -adrenoceptors (due to prazosin plus a low concentration of yohimbine) also resulted in a weak antagonistic effect. Ketanserin and metitepine displaced the noradrenaline concentration-response curve to the right. We conclude that the saphenous vein of the dog is endowed with prejunctional receptors of the 5-HT₁ type which can not be classified as belonging either to the 1 A or 1 B subtype; and that at the postjunctional level 5-HT₁ (possibly of the 1D subtype) and 5-HT₂ receptors are present. 5-HT is able to activate all these receptors.Supported by INIC (Centro de Farmacologia e Biopatologia Química da Universidade do Porto) Send offprint requests to M. Q. Paiva at the above address     

Vasoactive responses of a human cystic artery: adrenoceptor characterization.

1. The pharmacology of various agonists and antagonists was studied in the human isolated cystic artery. 2. The estimated pA2s for the alpha 1-adrenoceptor antagonist prazosin against the alpha-adrenoceptor agonists phenylephrine, alpha-methylnoradrenaline and noradrenaline were not significantly different. Similar results were seen for estimated pA2s of the alpha 2-adrenoceptor antagonist yohimbine against these same agonists. Equivalent responses to exogenous noradrenaline and to transmural electrical stimulation were blocked to the same degree by an antagonist with alpha 1-adrenoceptor blocking properties (prazosin). Responses to transmural electrical stimulation, however, tended to be more resistant than equivalent responses to exogenous noradrenaline to blockade by antagonists with alpha 2-adrenoceptor blocking properties (phentolamine, yohimbine). 3. Relaxation to isoprenaline was observed in partially contracted arterial strips using isoprenaline concentrations of up to 10(-6) M, but cumulative addition of higher concentrations of isoprenaline sometimes then evoked a contraction from the relaxation nadir. The relaxation effect of isoprenaline was antagonized by propranolol (10(-5) M). 4. These findings suggest the human cystic artery has almost exclusively alpha 1-adrenoceptors postjunctionally, although prejunctional alpha 2-adrenoceptors may be present; and, it also has some postjunctional beta-adrenoceptors which mediate relaxation.     

Inhibitory effect of dopamine on canine gastric fundus

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

EFFECTS OF PRAZOSIN,PHENTOLAMINE AND YOHIMBINE ON NORADRENERGIC TRANSMISSION IN THE RAT RIGHT VENTRICLE IN VITRO

1 The effects of prazosin, phentolamine and yohimbine on the accumulation of radioactivity from [³H]-noradrenaline, and on the subsequent spontaneous and field stimulation-evoked outflow of radioactivity have been investigated in the rat right ventricle in vitro. In addition the effects of these agents on contractions produced by exogenous amines or by field stimulation are reported. 2 Prazosin, phentolamine and yohimbine had no effect on the accumulation of radioactivity from [³H]-noradrenaline. 3 The decline in the spontaneous outflow of radioactivity, following loading of the tissue with [³H]-noradrenaline, was reduced by prazosin (1 times 10^-8 or 1 times 10^-7M) and 1 times 10^-6M yohimbine. Phentolamine and 1 times 10^-7M yohimbine had no effect on the spontaneous outflow of radioactivity. 4 Prazosin, phentolamine and yohimbine (all at 1 times 10^-7M) reduced the decline in outflow of radioactivity evoked by field stimulation at 5Hz. In the presence of yohimbine, prazosin or phentolamine had no effect on the evoked outflow. The effect of prazosin, phentolamine and yohimbine alone probably represents antagonism at prejunctional α-adrenoreceptors. 5 The rate of beating and force of contractions evoked by 1 times 10^-6M isoprenaline were not altered by prazosin, phentolamine or 1 times 10^-7M yohimbine. The rate of beating evoked by noradrenaline (1 times 10^-6M) was reduced by prazosin and phentolamine. Yohimbine (1 times 10^-6M) reduced the rate of beating to isoprenaline and to noradrenaline. These effects of prazosin and phentolamine probably represent antagonism at postjunctional α₁-adrenoreceptors and that of yohimbine a decrease in the excitability of the postjunctional membrane. 6 In the control tissues the force of contractions evoked by field stimulation at 5Hz declined slowly with successive stimulations at 30 min intervals. In the presence of prazosin (1 times 10^-8 and 1 times 10^-7M), phentolamine (1 times 10^-7 and 1 times 10^-6M) and 1 times 10^-7M yohimbine the responses did not decline with successive stimulations. Yohimbine (1 times 10^-6M) had no effect on the stimulation-evoked responses. 7 Phentolamine or yohimbine (both at 1 times 10^-7M) had no additional effect on the stimulation-evoked responses in the presence of prazosin (1 times 10^-7M). Prazosin (1 times 10^-8M) and yohimbine (1 times 10^-7M) had no further effect in the presence of phentolamine (1 times 10^-6M). Prazosin or phentolamine (both at 1 times 10^-7M) had no effect on stimulation-evoked responses in the presence of yohimbine (1 times 10^-7M). 8 In the rat right ventricle, the ability of phentolamine and yohimbine to prevent the decline in the stimulation-evoked responses with successive stimulations is probably due to antagonism at prejunctional α-adrenoreceptors. However it is suggested that the ability of prazosin to prevent the decline in response is due to a release of endogenous noradrenaline and to antagonism at prejunctional α-adrenoreceptors.     

The central sympatho-inhibitory effect of 5,6-dihydroxy-2-dimethylaminotetralin (M7) is mediated by α2-adrenoceptors

M7 (5,6-dihydroxy-2-dimethylaminotetralin) produces in anesthetized rats a hypotensive response previously attributed to peripheral dopaminergic mechanisms. We re-examined the effects of this drug on arterial blood pressure, heart rate and sympathetic nerve activity in anesthetized rats and dogs. M7 (1–100 μg/kg i.v.) produced in the rats transient dose-dependent pressor effects, with bradycardia and sympatho-inhibition, followed by long-lasting dose-dependent hypotension, bradycardia and sympatho-inhibition. The sympatho-inhibitory and hypotensive effects were comparable in baroreceptor-denervated rats and were reversed by idazoxan (0.1 mg/kg i.v.). The sympatho-inhibitory response induced by M7 (1–100 μg/kg) was prevented by treatment with the specific α₂-adrenoceptor antagonist, 2-methoxy-idazoxan (0.03 mg/kg i.v.). This central effect of M7 was not altered by treatment with the α₁-adrenoceptor antagonist, prazosin (0.1 mg/kg i.v.) and was reduced by treatment with the α₂-adrenoceptor antagonists, yohimbine (1 mg/kg i.v.) or idazoxan (0.3 mg/kg i.v.), and the dopaminergic antagonists, haloperidol (0.5 mg/kg i.v.) or sulpiride (3 mg/kg i.v.). Bilateral microinjections of M7 (0.3–3 nmol) into the rostroventral medulla in the rat produced dose-dependent hypotension, bradycardia and sympathetic nerve inhibition which were reversed and prevented by bilateral microinjection of 2-methoxy-idazoxan (1 nmol) into the same sites. Microinjections of 2-methoxy-idazoxan into the rostroventral medulla also inhibited the central effects of M7 at 0.03 mg/kg i.v. In anesthetized dogs, M7 administered into the cisterna magna (1–10 μg/kg) reduced arterial blood pressure, heart rate and sympathetic nerve activity; these effects were reversed by administration of 2-methoxy-idazoxan (0.03 mg/kg i.v.). In conclusion, M7, a rigid catecholamine, produces a potent central sympatho-inhibitory and hypotensive effect by activation of α₂-adrenoceptors.     

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     

Evidence for two distinct types of postsynaptic α-adrenoceptor in vascular smooth muscle in vivo

1 The effects of the highly selective α₁-adrenoceptor antagonist, prazosin, and the relatively selective α₂-adrenoceptor antagonist, yohimbine, on the pressor responses to intravenous injections of phenylephrine and noradrenaline have been examined in anaesthetized cats and pithed rats in an attempt to determine whether α₁- and α₂-adrenoceptors are located postsynaptically on vascular smooth muscle.

2 In anaesthetized cats prazosin caused a much greater reduction in the pressor responses to phenylephrine than to noradrenaline or splanchnic nerve stimulation (after adrenalectomy). Yohimbine was of similar potency in reducing the pressor responses to each stimulus.

3 A differential blocking activity of prazosin against intra-arterial injections of phenylephrine and noradrenaline was also demonstrated in the blood-perfused cat hind limb. As in the whole animal, prazosin was more potent against phenylephrine than noradrenaline. A similar, though less marked, effect was seen in the mesenteric circulation, but not in the renal circulation, where prazosin was almost equipotent in reducing responses to phenylephrine and noradrenaline.

4 In pithed rats prazosin was a potent, competitive antagonist of phenylephrine, but had little effect against noradrenaline; only the responses to high doses of noradrenaline were reduced by prazosin. Yohimbine was approximately equipotent as an antagonist of phenylephrine and noradrenaline. In the anococcygeus muscle, prazosin was as potent an antagonist of noradrenaline as it was of phenylephrine on vascular smooth muscle.

5 The results suggest that there are two types of α-adrenoceptor in the vasculature of cats and rats. Phenylephrine produces pressor responses by stimulating one type of postsynaptic α-adrenoceptor that is blocked by prazosin and yohimbine; these are α₁-adrenoceptors. Noradrenaline exerts some of its effect via these receptors but most of its effect appears to be exerted through prazosin-insensitive receptors. The latter receptors appear to differ from α₂-adrenoceptors.

     

Role of α2-adrenoceptors in the regulation of intestinal water transport

1. The influence of the sympathetic nervous system on intestinal fluid transport by the jejunum and ileum of the anaesthetized rat was investigated under basal conditions and during active secretion induced by intra-arterial infusion of vasoactive intestinal peptide (VIP).
2. Intra-arterial infusion of noradrenaline (3, 10, 30 nmol min⁻¹, i.a.) and i.v. injection of the selective α₂-adrenoceptor agonist UK 14,304 (1 μmol kg⁻¹, i.v.) increased the rate of basal fluid absorption. The effect of UK 14,304 was blocked by yohimbine (10 μmol kg⁻¹, i.v). However, the selective α₁-adrenoceptor agonist phenylephrine (5 μmol kg⁻¹, i.v.) did not alter either the jejunal or ileal absorption rate.
3. The α₂-adrenoceptor antagonists yohimbine (0.3, 1.0, 3 and 10 μmol kg⁻¹, i.v.) and rauwolscine (10 μmol kg⁻¹, i.v.) decreased the basal absorption rate, while the α₁-adrenoceptor antagonist prazosin (3 μmol kg⁻¹, i.v.) was without effect. Intracerebroventricular injection of yohimbine (3 μmol kg⁻¹) caused a significant antiabsorptive effect in the jejunum but not ileum.
4. Peripheral chemical sympathectomy induced by pretreating animals with 6-hydroxydopamine (150 mg kg⁻¹, i.p., total dose) induced a trend towards impaired absorption in the jejunum and ileum.
5. The findings provide evidence that the sympathetic nervous system exerts tonic control on intestinal fluid transport and that the effect is mainly through peripheral α₂-adrenoceptors.
6. The subtype determination of α₂-adrenoceptors in modulating intestinal fluid transport was assessed by determining the effects of α₂-adrenoceptor agents on intestinal fluid secretion induced by i.a. infusion of VIP (0.8 μg min⁻¹).
7. Intravenous administration of UK 14,304 caused a dose-dependent reversal of the secretory phase of the VIP-induced response, but failed to restore fluid transport to the control level of net absorption. EC₅₀ values were 0.17 μmol kg⁻¹ in the jejunum and 0.22 μmol kg⁻¹ in the ileum.
8. The effect of UK 14,304 was blocked by the selective α_2A/D antagonist BRL 44408 and the non-selective α₂ antagonist yohimbine (each 10 μmol kg⁻¹). The selective α_2B/C antagonist ARC 239 (10 μmol kg⁻¹) did not affect the antisecretory action of UK 14,304. It is suggested that the α₂-adrenoceptors in the rat intestinal epithelium are the α_2D or α_2A-like subtype.

     

Catecholamine transport in isolated lung parenchyma of pig

1 Lung parenchyma strips of the pig incubated at 37°C with [³H]-(-)-noradrenaline ([³H]-NA) or [³H]-(±)-isoprenaline ([³H]-Iso), accumulated radioactivity via saturable, high affinity uptake processes. Apparent saturation constants (K_m) for [³H]-NA and [³H]-Iso were 1.34 × 10^-6 M and 1.63 × 10^-6 M respectively, while apparent transport maxima (V_max) were 4.86 and 1.63 × 10^-9 mol min^-1 g^-1 respectively.

2 Cellular accumulation of radioactivity from radiolabelled catecholamines was greatly reduced by lowering the temperature to 7°C, pretreatment with ouabain (100 μM), phentolamine (15 μM) or phenoxybenzamine (80 μM). However, accumulation of radioactivity derived from (³H]-NA was inhibited selectively by cocaine (10 μM) and desipramine (1 μM), while normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM) caused much greater reductions in cellular radioactivity from [³H]-Iso than from (³H]-NA. Taken together with information from kinetic studies, the results indicate that these amines are transported by separate uptake processes.

3 Cocaine (50 μM) which selectively reduced [³H]-NA transport, had no significant effect on the sensitivity (EC₅₀) of isolated parenchyma lung strips of the pig to the contractile effects of cumulative concentrations of NA. The catechol-O-methyl transferase (COMT) inhibitor, U-0521 (60 μM), also failed to alter the potency of NA, while normetanephrine (80 μM) caused a 2 fold decrease in potency.

4 Phentolamine (15 μM), which reduced the cellular accumulation of radioactivity derived from [³H]-Iso by 64%, caused a small potentiation of Iso-induced relaxations of porcine lung strips. Normetanephrine (80 μM) and 3-O-methylisoprenaline (50 μM), which also depressed the accumulation of cellular radioactivity from [³H]-Iso by > 50%, caused rightward shifts in Iso concentration-effect curves as a result of β-adrenoceptor blockade. In sharp contrast, cortisol (80 μM) and U-0521 (60 μM), which caused smaller reductions in the cellular accumulation of radioactivity derived from [³H]-Iso, both caused an approximately 9 fold potentiation of responses to Iso in isolated lung strips.

5 The results indicate that the major sites of uptake and metabolism of NA in porcine parenchyma strip are remote from α-adrenoceptors mediating NA-induced contraction. Similarly, some major sites of uptake of Iso are remote from β-adrenoceptors mediating Iso-induced relaxation. However, β-adrenoceptors are apparently in close proximity to a compartment containing COMT activity.

     

Long-term administration of 1,3-dipropyl-8-sulphophenylxanthine (DPSPX) alters α2-adrenoceptor-mediated effects at the pre- but not at the postjunctional level

The present investigation was undertaken to see whether a long-term inhibition of adenosine receptors —leading to hypertension — interferes with ₂-adrenoceptor-mediated modulation of noradrenaline release. Rat tail arteries were removed from normal and from hypertensive animals obtained by chronic treatment with intraperitoneally infused DPSPX (1,3,-dipropyl-8-sulphophenylxanthine) or orally administered L-NAME (N^G-Nitro-L-arginine methyl ester). To study prejunctional effects, the influence of UK-14,304 (5-bromo-6(imidazoline-2-ylamino)-quinoxaline) and yohimbine on the overflow of tritium evoked by electrical stimulation (100 V; 1 Hz; 2 ms; 5 min) from tissues preloaded with ³H-noradrenaline was analysed. To study postjunctional effects, concentration-response curves to UK-14,304 were determined. In DPSPX-treated rats there was an enhancement of the prejunctional effects of UK-14,304: its EC_30% was reduced from 381 (250; 579) to 85 (73; 99) nmol.l^–1 (n = 5; P<0.05) and its maximal effect — expressed as percent reduction of tritium overflow-increased from 45 ± 5% to 61 ± 5% (n = 6; P < 0.05). In L-NAME-treated rats there was no change in either of these two parameters. At the postjunctional level, there was no change in the sensitivity to UK-14,304 in tissues from either DPSPX- or L-NAME-treated rats. Yohimbine (10–1000 nmol.l^–1) caused a concentration-dependent increase of tritium overflow evoked by electrical stimulation in both control and hypertensive animals (either DPSPX- or L-NAME-treated). The EC_50%-pre-antagonist values (concentration of the antagonist that increases the evoked overflow by 50%) were not significantly different in the three situations. We conclude that long-term administration of DPSPX increases the sensitivity to the prejunctional effects of UK-14,304 without changing that to its postjunctional effects, showing a specific interaction between -adrenoceptors and adenosine receptors at a prejunctional level. The question arises whether there is any link between that alteration and the development of the hypertensive state. Correspondence to: S. Guimaraes at the above address     

Mechanism of prostaglandin E2-, F2α- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E₂-, prostaglandin F_2α- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F_2α)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E₂ caused maximum relaxation of endothelin-1 precontracted vessels (EC₅₀: 1.8×10⁻⁸ M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor,

Full-size image

-NAME (N^G-Nitro-

Full-size image

-arginine methylester). CGRP-(8–37) (calcitonin gene-related peptide fragment (8–37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK₁ receptor blocker, GR 82334 ([

Full-size image

-Pro⁹[Spiro-γ-Lactam]Leu¹⁰,Trp¹¹]physalaemin (1–11)), markedly attenuated the response. Both prostaglandin F_2α and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1α(Z),2β,3β,5α]]-(+)-7-[5-([1,1′-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), a thromboxane receptor antagonist (EC₅₀: for prostaglandin F_2α 7.9×10⁻⁹ M, and for latanoprost acid 4.9×10⁻⁹ M).

Full-size image

-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8–37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E₂ could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

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

Background and purpose:

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

Experimental approach:

Mouse isolated tail arteries were assessed using a pressure myograph.

Key results:

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

Conclusions and implications:

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

DOPAMINE RELEASES ENDOTHELIUM-DERIVED RELAXING FACTOR VIA α2-ADRENOCEPTORS IN CANINE VESSELS: COMPARISONS BETWEEN FEMORAL ARTERIES AND VEINS

1. We investigated the role of vascular smooth muscle α-adrenoceptor subtypes in the vasoconstrictor response of femoral arteries and veins to dopamine and whether the vasoconstriction is modified by endothelium-dependent relaxation mediated via the activation of α₂-adrenoceptors in ring preparations of femoral arteries and veins from mongrel dogs. 2. Dopamine contracted both arteries and veins in a dose-dependent manner and this contraction was inhibited by pre-treatment with phentolamine or prazosin. Pretreatment with yohimbine shifted the dose-response curve for dopamine to the right in femoral veins, but not in arteries. 3. Phenylephrine contracted femoral arteries and veins in a dose-dependent manner and this contraction was inhibited by pretreatment with prazosin. 4. Clonidine produced a bell-shaped dose-response curve in femoral veins and this curve was shifted upwards by pretreatment with N^G-nitro-l -arginine (l -NNA). In contrast, femoral arteries were not affected by clonidine. N^G-Nitro-l -arginine potentiated contractile responses to dopamine in both veins and arteries. This potentiation was inhibited by yohimbine or by the removal of the endothelium in both arteries and veins. 5. These results suggest that dopamine contracts femoral arteries via stimulation of α₁-adrenoceptors and contracts femoral veins via stimulation of both α₁- and α₂-adrenoceptors and that these contractions are attenuated by the vasodilator action of dopamine via α₂-adrenoceptor-mediated release of endothelium-derived relaxing factor.