Similarities between the relaxations induced by vasoactive intestinal peptide and by stimulation of the non-adrenergic non-cholinergic neurons in the rat stomach

Summary The characteristics of the non-adrenergic, noncholinergic inhibitory response of the rat stomach fundus to transmural nerve stimulation were compared with the relaxation induced by vasoactive intestinal polypeptide (VIP). Treatment with -chymotrypsin (5 U/ml) or VIP antiserum (1:200) significantly reduced the relaxation induced by transmural nerve stimulation at 30 Hz, indicating that the possible transmitter in the non-adrenergic, non-cholinergic nerves is a peptide and may be VIP or a closely related peptide. VIP was able to relax, fully and dose-dependently, the stomach fundus that had previously been constricted by treatment with 10^–6 M serotonin, and the IC₅₀ value for VIP was 2.4 × 10^–9 M. VIP elevated levels of cyclic AMP in a dose-dependent manner and the EC₅₀ value was 2.8 × 10^–9 M in the presence of 10^–6 M atropine and 10^–6 M guanethidine. The stomach fundus was relaxed by transmural nerve stimulation (30 Hz, 50 mA) and transmural nerve stimulation also caused production of cyclic AMP in the rat stomach in the presence of atropine and guanethidine. The basal level of cyclic AMP in the stomach was 8.7 ± 0.26 pmole/mg protein. When transmural nerve stimulation was applied for 5 min, the contraction of the stomach, induced by 10^–6 M serotonin, was inhibited by 54% in the presence of atropine and guanethidine and the level of cyclic AMP was increased to 13.0 ± 0.73 pmol/mg protein. Apamin inhibited the transmural nerve stimulation-induced relaxation and shifted the dose-response curve for VIP to the right. These results suggest that one of the putative neurotransmitter from non-adrenergic, non-cholinergic nerves in the rat stomach is VIP and that VIP-induced relaxation may be mediated by the production of cyclic AMP and by the opening of apamin-sensitive K⁺-channels.Send offprint requests to K. Kamata at the above address     

Prejunctional α-adrenoceptors regulate nitrergic neurotransmission in the rabbit urethra

We evaluated the effects of prejunctional α-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 NO₂⁻/NO₃⁻ released during electrical field stimulation was measured by an NO₂⁻/NO₃⁻ analyzer based on the Griess method. Pretreatment with phenylephrine (0.01 μM) and yohimbine (0.1–10 μM) significantly reduced the relaxation responses induced by electrical field stimulation. In contrast, pretreatment with clonidine (0.01 μM) and prazosin (0.01–1 μM) enhanced the relaxation responses. Cys-NO-induced relaxations of rabbit urethral smooth muscle were not affected by pretreatment with α-adrenoceptor agonists and antagonists. The amount of NO₂⁻/NO₃⁻ released by electrical field stimulation increased after pretreatment with clonidine (0.01 μM) and prazosin (0.01–1 μM), but decreased after pretreatment with phenylephrine (0.01 μM) and yohimbine (0.1–10 μM). The results suggest that the release of NO from nitrergic nerves in the rabbit urethra is reduced and increased by stimulation of prejunctional α₁- and α₂-adrenoceptors, respectively.     

The mechanism of action of dopamine to inhibit field stimulation-induced contractions of guinea pig stomach strips

Summary Field stimulation (0.125–10 Hz) of longitudinal smooth muscle strips taken from the fundus of guinea pig stomach caused frequency related contraction responses (atropine-sensitive) associated with relaxation at the higher frequencies (both responses tetrodotoxin-sensitive). Dopamine, noradrenaline, adrenaline and apomorphine antagonised the contraction responses at all frequencies; the concentration-response curves were steep and the use of higher concentrations was precluded by changes in base line tension per se. That noradrenaline was approximately 10 fold more potent than dopamine, that the dopamine response was not antagonised by the dopamine antagonists haloperidol, domperidone or (-)sulpiride, but was mimicked by the ₂-agonist guanfacine and partially antagonised by the ₂-adrenoceptor antagonist yohimbine (which could also antagonise the inhibitory actions of guanfacine) would indicate that a component of dopamine's action to reduce cholinergic activity is effected via ₂-adrenoceptor mechanisms. A failure of phenylephrine to mimic, or prazosin to attenuate the agonist inhibitory responses would not indicate an additional ₂-adrenoceptor involvement. The persistence of the dopamine response following disruption of noradrenergic function by reserpine or inhibition of catecholamine reuptake processes (desmethylimipramine plus corticosterone, GBR 13098) would indicate a direct action of dopamine. It is concluded that the ability of dopamine to reduce cholinergic induced contractions in longitudinal smooth muscle of the stomach does not reflect a dopamine receptor or ₁-adrenoceptor stimulation but may involve an action on ₂-adrenoceptors.     

Non-cholinergic,non-adrenergic responses to nerve stimulation of different regions of the guinea-pig small intestine

Summary Non-cholinergic, non-adrenergic responses to nerve stimulation recorded from smooth muscles of the guinea-pig duodenum, jejunum, proximal and terminal ileum were investigated in an attempt to characterize these responses.In the presence of atropine (1–2 mol · l^–1) and guanethidine (10 mol · l^–1) coaxial stimulation induced in all regions of the guinea-pig small intestine an initial relaxation (primary relaxation) upon which contraction (primary contraction) appeared, followed by rebound contraction.Noradrenaline decreased the cholinergic smooth muscle twitches, predominantly at low stimulation frequencies, and had a similar effect on the non-cholinergic, non-adrenergic primary relaxation, primary and rebound contractions.ATP decreased the smooth muscle twitches; however, this agent had only a transient influence on the non-cholinergic, non-adrenergic responses of muscle (tension and membrane potential) to single stimuli. With higher stimulus frequencies ATP increased the primary relaxation and decreased the contraction phases. ATP also inhibited the post-tetanic inhibition induced by non-cholinergic, non-adrenergic nerve stimulation.In most of the muscle cells of the guinea-pig proximal and terminal ileum the non-cholinergic, non-adrenergic nerve stimulation generated i.j.p.s., while about 15–20% of the cells responded with e.j.p.s. During long-lasting stimulation (10s) the i.j.p.s. were sometimes interrupted by action potentials or by a gradual depolarization of the membrane. The i.j.p.s. were followed by a marked rebound depolarization accompanying the action potentials. Those cells which generated i.j.p.s. in response to field stimulation, were depolarized by ATP, while those cells, which generated e.j.p.s, were hyperpolarized by ATP.A reduction in the concentration of extracellular sodium chloride decreased both the primary and rebound contractions; the primary contraction was, however, more sensitive than was the rebound contraction.Theophylline increased the primary and rebound contractions with no marked influence on the primary relaxation, lowered the action potential threshold, increased the rebound depolarization and did not markedly influence the i.j.p.s.Quinidine enhanced the primary relaxation and inhibited the primary contraction in a concentration-dependent manner. Inhibition of the rebound contraction by quinidine was slight (less than 50%).The present results demonstrate that primary relaxation, primary and rebound contractions are associated with i.j.p.s and e.j.p.s., and rebound depolarization with action potentials, respectively; they are typical responses of various regions of the guinea-pig small intestine to activation of inhibitory and excitatory non-cholinergic, non-adrenergic nerves. The P₁ and P₂ receptors, proposed by Burnstock (1975), probably do not mediate the non-cholinergic, non-adrenergic postsynaptic responses of the guinea-pig small intestine. A possible physiological function of ATP as a mediator of non-cholinergic, non-adrenergic nerves of the guinea-pig small intestine is discussed.This study was supported in part by JSPS Japan     

Effects of the potassium channel openers KRN4884 and levcromakalim on the contraction of rat aorta induced by A23187, compared with nifedipine

We examined the different vasodilatory effects of the K⁺ channel openers levcromakalim and 5-amino-N2-[2-(2-chlorophenyl)ethyl]-N-cyano-3-pyridinecarboxamidine (KRN4884), and the Ca²⁺ channel blocker nifedipine in the rat aorta. KRN4884 (10^–10-10^–5 M) and nifedipine (10^–10–10^–5 M) produced concentration-dependent relaxation in the rat aorta precontracted by 25 mM KCl. The K⁺ channel blocker glibenclamide (1 M) inhibited the relaxation induced by KRN4884 but did not influence nifedipine-induced relaxation. KRN 4884 had almost no effect on contraction induced by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl. These results indicate that KRN4884 is a K+ channel opener. We investigated the relaxant effects of KRN4884 (10^–10-10^–5 M), levcromakalim (10^–9-10^–5 M) and nifedipine (10^–9-10^–5 M) on A23187 (1 M)-induced contraction. KRN4884 and levcromakalim had a potent relaxant effect but nifedipine only a weak effect on the smooth muscle contracted by A23187. Glibenclamide (1 M) inhibited the relaxation induced by KRN4884 and levcromakalim, but did not influence the nifedipine-induced relaxation. KRN 4884 (1 M) produced a larger relaxation of A23187-induced contraction but had little effect on the increase in intracellular [Ca²⁺] induced by A23187. These results suggest that KRN4884 is a specific K⁺ channel opener and its vasodilating mechanisms involve not only deactivation of Ca²⁺ channels but also a decrease in the Ca²⁺ sensitivity of contractile elements.     

Chloroethylclonidine irreversibly activates postjunctional alpha2-adrenoceptors in the dog saphenous vein

Summary This study was aimed at analysing the contractile response of the dog saphenous vein to chloroethylclonidine. At 37°C, chloroethylclonidine (0.1–100 mol·1^–1) caused along-lasting contraction in both proximal and distal segments of the dog saphenous vein, reaching 77.6 and 52.6% of the maximal response to phenylephrine, respectively. At 18°C, and in both segments, the maximal response to chloroethylclonidine was markedly reduced, whereas that to phenylephrine was not changed and that to UK-14,304 was enhanced. The response to chloroethylclonidine was unaffected by pretreatment with cocaine. Warming to 37°C caused contraction of strips which at 18°C had remained unresponsive to chloroethylclonidine, even if these strips were repeatedly washed before warming. At 18°C, chloroethylclonidine (100 mol·1^–1) did not alter the responses to UK-14,304 and phenylephrine.At 37°C, the contractile response to chloroethylclonidine was antagonized by yohimbine, rauwolscine and prazosin, with the potency rank yohimbine = rauwolscine > prazosin. Phenoxybenzamine (30 nmol · 1^–1) displaced the concentration-response curve to chloroethylclonidine to the right and depressed its maximum. After phenoxybenzamine, yohimbine continued to be more effective than prazosin, which remained very potent.We concluded that: 1) the contractile response of the canine saphenous vein to chloroethylclonidine (both in the absence and in the presence of phenoxybenzamine) is predominantly alpha2-adrenoceptor-mediated since it is larger at the proximal than at the distal level of the vein and since it is more sensitive to yohimbine and rauwolscine than to prazosin; 2) the response to chloroethylclonidine and UK-14,304 are apparently due to activation of different alpha2-adrenoceptor subtypes, since prazosin was much more effective against chloroethylclonidine than against UK-14,304, and since at 18°C chloroethylclonidine occupies receptors without changing the response to UK-14,304; 3) there is a component of alphal-adrenoceptor stimulation in the response to chloroethylclonidine, since 30 nmol·1^–1 phenoxybenzamine partly antagonized the effect of chloroethylclonidine; 4) since the responses to UK-14,304 and chloroethylclonidine are differently affected by cooling, there is some step (or steps) in the chain of events between the receptor and the final response, which is different in the two pathways. Correspondence to S. Guimarães at the above address     

Characterization of two distinct alpha-adrenoceptor binding sites in smooth muscle cell membranes from rat and bovine aorta

Summary In order to characterize postjunctional alpha adrenoceptor binding sites of aortic smooth muscle, the specific binding of (³H)prazosin and (³H)yohimbine to membranes prepared from the medial layers of rat and bovine thoracic aorta was investigated. Binding of (¹²⁵I)-BE 2254 (2-[B-(4-hydroxyphenyl)-ethylaminomethyl] tetralone) and (³H)RX 781094 (idazoxan) was also examined in bovine membranes. Each of the ligands displayed saturable, specific binding to a single population of sites; the K _D values of the respective ligands were similar in the two animal species. The number of (³H)prazison and (¹²⁵I)BE 2254 binding sites (160–190 fmol · mg protein^–1 in the two species) was higher than the number of (³H)yohimbine and (³H)RX 781094 binding sites (110–120 fmol · mg protein^–1 in the bovine and 50 fmol · mg protein^–1 in the rat). Alpha-adrenoceptor ligands inhibited binding of the ligands with the following orders of potency: prazosin > BE 2254 > yohimbine > RX 781094 > clonidine in the case of (³H)-prazosin, and yohimbine > RX 781094 > clonidine > prazosin in the case of (³H)yohimbine. Methoxamine, in concentrations up to 10 M, was without effect on the binding of either ligand. The absence or presence of Na⁺, K⁺ or Ca²⁺ added at physiological concentrations did not change the order of potency of displacing ligands whereas Ca²⁺ reduced by 50% the numbers of (³H)prazosin and (³H)-yohimbine sites and Na⁺ increased by 3-fold the affinity of (³H)yohimbine.It is concluded that post-junctional membranes from rat and bovine aortic smooth muscle contain two distinct ₁- and ₂-adrenoceptor binding sites, the number of the latter being less than the number of the former.     

A postsynaptic effect of tyramine in ventricular muscle

Summary Tachyphylaxis and susceptibility to pretreatment with reserpine demonstrate that tyramine increases contractility of the guinea-pig papillary muscle in concentrations up to 10^–4 M by means of an indirect sympathomimetic effect.In higher concentrations (up to 3×10^–3 M) tyramine causes an increase in force of contraction which is characterized by a slowing of relaxation of the isometric contraction curve. Pretreatment with reserpine reveals that this positive inotropic effect is composed of an indirect sympathomimetic and a direct post-synaptic effect; the latter is not subject to tachyphylaxis and produces by itself (at 3×10^–3 M) an increase in force of contraction which amounts to about 50% of the maximum of the indirectly produced inotropic effect.The postsynaptically-induced positive inotropic effect of tyramine is not antagonized by propranolol (5×10^–6 M). It is unlikely, therefore, that it is brought about by stimulation of adrenergic -receptors.The height of the isometric contraction curve in reserpine-pretreated muscles is increased by tyramine as a result of an increase in the rate of force development despite a marked concentration-dependent increase in relaxation time (t ₂).The direct inotropic effect of tyramine is correlated with a prolongation of the duration of the action potential (AP) which amounts to 70% at 3×10^–3 M. In addition to its influence on the duration of the AP, tyramine slows the rate of depolarization and decreases the membrane resting potential.     

Effect of calcium antagonists (ω-conotoxin GVIA,verapamil, gallopamil,diltiazem) on bronchial smooth muscle contractions induced by soman

Summary The effect of the calcium antagonists -conotoxin GVIA, verapamil, gallopamil and diltiazem was investigated on in vitro bronchial smooth muscle contraction in the rat induced by the nerve agent soman. Soman inhibits the acetylcholinesterase activity irreversibly. The effect of the calcium channel antagonists on contractions induced by electrical field stimulation and carbachol was also investigated, in order to elucidate the mechanism by which calcium antagonists inhibit the soman induced contraction.-Conotoxin GVIA reduced the bronchial smooth muscle contraction induced by electrical field stimulation with an almost complete inhibition at approximately 1.0× 10^–6 M. The soman induced contraction was only inhibited by 15% at a concentration of 3.0× 10^–6 M -conotoxin GVIA. The organic calcium antagonists verapamil, gallopamil and diltiazem reduced both electrically and soman induced smooth muscle contraction. Complete inhibition of the contractions induced by soman was achieved at 1.4x 10^–4 M for verapamil and gallopamil, while diltiazem inhibited the contraction to 7% of control at 1.4 × 10^–4 M. Verapamil, gallopamil and diltiazem increased the EC₅₀ for carbachol significantly, while co-conotoxin GVIA had no effect. None of the calcium antagonists had any effect on the maximal contraction induced by carbachol. Verapamil, gallopamil and diltiazem blocked, however, sub-maximal contractions induced by carbachol (10^–7 -10^–5 M) resulting in a right-shift of the dose response curve.The results show that co-conotoxin GVIA inhibits the calcium-dependent release of acetylcholine which causes contraction of airway smooth muscle, while it has no effect on smooth muscle contraction induced by soman. Gallopamil, verapamil and diltiazem inhibit the contraction of bronchial smooth muscle following stimulation of postjunctional muscarinic receptors resulting from inhibition of the acetylcholinesterase activity by soman. Gallopamil and verapamil inhibit the contraction more potently than diltiazem. Correspondence to P. Aas at the above address     

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.     

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     

Biphasic effect of tricyclic antidepressants on the release of norepinephrine from the adrenergic nerves of the rabbit heart

The release of norepinephrine (NE) from the right atrium of the rabbit heart was used as a model to investigate biphasic effects due to tricyclic antidepressants, similar to those clinically observed in the treatment of depression and known as therapeutic window. Strips of the atrium were loaded with³H-NE, and then superfused by Krebs solution. The basal release and the electrical stimulation evoked release of³H-NE were measured in the presence and absence of four clinically used tricyclic antidepressants: imipramine, amitriptyline, desipramine and nortriptyline. In addition, guanethidine, an adrenergic neuron blocker, was also studied. At lower concentrations (0.5–10 µM) tricyclic antidepressants increased, whereas higher concentrations (50–100 µM), inhibited the evoked release of NE. This inhibition was not prevented by the alpha₂ adrenoceptor antagonist yohimbine, excluding the possibility of alpha₂ adrenoceptormediated inhibition of NE release. In higher concentrations the tricyclic antidepressants increased the basal release of NE in a Ca-independent way. Secondary amine derivatives were more potent inhibitors of the evoked release, and enhance the resting basal release of NE to a greater extent than the tertiary ones. Similarly, guanethidine (1–50 µM) also decreased the evoked release and increased the basal release of NE in a concentration dependent manner. Yohimbine failed to counteract the inhibition caused by guanethidine and the increment of the basal release was Ca-independent. It is concluded that the effect of tricyclic antidepressants in potentiating the release of NE is masked by their adrenergic neuron blocking properties, i.e. they inhibit the release of NE. Our findings are consistent with paradoxical clinical observations that some tricyclic antidepressants have therapeutic value only in lower plasma concentrations and they become ineffective at higher concentrations.     

Studies on alpha adrenoceptors in guinea-pig peripheral lung strips

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

Alpha2-adrenoceptor antagonism and other pharmacological antagonist properties of some substituted benzoquinolizines and yohimbine in vitro

Summary Comparison of pA₂ values for antagonism of clonidine induced inhibition of the electrically evoked contraction of the rat isolated vas deferens ( ₁-adrenoceptors) and antagonism of contractions to methoxamine on the rat isolated anococcygeus ( ₂-adrenoceptors) showed a group of substituted benzoquinolizines (Wy 25309, 26392 and 26703) to be more potent and more selective ₂-adrenoceptor antagonists than yohimbine.The benzoquinolizines and yohimbine enhanced stimulation-evoked overflow of tritium from rabbit isolated pulmonary arteries preloaded with [³H]-noradrenaline, as expected for ₂-adrenoceptor antagonists. In contrast to the results on the rat vas deferens, yohimbine was more potent than the benzoquinolizines. At higher concentrations all the -adrenoceptor antagonists reduced the stimulation-evoked contraction of the pulmonary artery.The benzoquinolizines were competitive antagonists of 5-hydroxytryptamine on the rat isolated ileum. Wy 25309 showed only weak activity (pA₂=5.21) whereas Wy 26703 was more potent (pA₂=7.25). Yohimbine was a potent antagonist of 5-hydroxytryptamine.Wy 26703 was the only compound to have histamine antagonist effects in the guinea pig isolated ileum and to antagonise the chronotropic effect of isoprenaline on the isolated atria of the guinea pig and in both instances activity was weak (pA₂ values 5.3 and 5.5 respectively). Yohimbine reduced the spontaneous beating of the atria at 3×10^–6 M. No compound at 10^–5 M antagonised acetylcholine on the guinea pig ileum. These novel substituted benzoquinolizines should be useful experimental compounds for the study of -adrenoceptors mediated responses.     

Direct and indirect actions of dopamine on tracheal smooth muscle

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

Investigation of the interaction between cholinergic and nitrergic neurotransmission in the pig gastric fundus

1. The interaction between the cholinergic and nitrergic innervation was investigated in circular muscle strips of the pig gastric fundus.
2. In physiological salt solution containing 4×10⁻⁶ M guanethidine, electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5–32 Hz, 10 s at 4 min intervals) induced small transient relaxations at 0.5–4 Hz, and large frequency-dependent contractions, sometimes followed by off-relaxations, at 8–32 Hz.
3. In the presence of L-N^G-nitroarginine methyl ester (L-NAME; 3×10⁻⁴ M) or physostigmine (10⁻⁶ M), relaxations were reversed into contractions and contractions were enhanced. Physostigmine added to L-NAME further enhanced contractions, while addition of L-NAME to physostigmine had no additional effect. Off-relaxations were enhanced in the presence of L-NAME and physostigmine. L-NAME and physostigmine consistently increased basal tone.
4. Tissues contracted by 5-hydroxytryptamine or by acetylcholine responded to EFS in a similar way as in basal conditions and L-NAME reversed the relaxations at the lower stimulation frequencies into contractions and enhanced the contractions at the higher stimulation frequencies.
5. Off-relaxations in the presence of L-NAME were partially reduced by α-chymotrypsin (10 U ml⁻¹).
6. In the absence of physostigmine, the concentration-response curve to exogenous acetylcholine was not influenced by L-NAME.
7. Contractions of the same amplitude induced by EFS at 4 Hz and by exogenous acetylcholine were either decreased or enhanced to the same extent by sodium nitroprusside (SNP; 10⁻⁵ M), depending upon the degree of relaxation by SNP.
8. These experiments suggest that endogenous nitric oxide interferes with cholinergic neurotransmission in the pig gastric fundus by functional antagonism at the postjunctional level. The interaction is independent of the degree of contraction.

     

Effect of histamine on human bronchial arteries in vitro

Summary Histamine caused a concentration-dependent relaxation at lower concentrations (1 pmol/1–1 mol/l) and contraction at higher concentrations (0.01–1 mmol/l) of isolated precontracted human bronchial arteries. In the vessels at resting tension only concentration-dependent contraction was evoked by histamine (0.01–1 mmol/l). Both the contractile and relaxant responses were significantly antagonised by mepyramine (1 gmol/l), with an estimated pK_B value of 8.4, but not by cimetidine (100 mol/l). Our results indicate that histamine induces biphasic effects on human bronchial arteries via H₁-re-ceptors. Send offprint requests to P. J. Barnes at the above address     

Functional role of sodium pump in human placental arteries

Summary Ouabain (10^–7 to 10^–4 M) elicited concentration-dependent contractile responses in human placental arteries. The contractions were reduced by 10^–4 M amiloride and Ca²⁺-free medium, but not affected by 10^–6 M nifedipine or 10^–6 M Bay-K-8644, which markedly reduced or potentiated 75 mM K⁺-induced contractions, respectively. After contracting the vessels with 10^–6 M prostaglandin F₂ in a K⁺-free medium, the subsequent addition of 7.5 mM K⁺ induced a marked relaxation, which was blocked by 10^–6 M ouabain. This glycoside (10^–8 to 10^–4 M) also produced a concentration-dependent reduction of ⁸⁶Rb⁺ uptake. Scatchard analysis of the [³H]-ouabain binding to membrane fractions from human placental arteries suggests a single class of binding sites with a KD of 88.3 nM and a B_max of 345 fmol/mg. 5-Hydroxytryptamine (5-HT; 10^–9 to 10^–5 M) caused concentration-dependent contractions. Single concentrations produced transient responses composed of an initial contraction, followed by a slow fall in tension. Ouabain (10^–8 to 10^–6 M), K⁺-free medium or the reduction of bath temperature (28°C) did not modify contractions but inhibited the relaxant phase of the response. 5-HT (10^–8 to 10^–6 M) increased both total and ouabain-insensitive ⁸⁶Rb⁺ uptake, but the difference between them was not modified. These data indicate that: (1) human placental arteries possess an important sodium pump activity, inhibition or stimulation of which markedly alters vascular tone, (2) ouabain-evoked contractions are produced by Ca²⁺ entry mainly through Na⁺-Ca²⁺ exchange, secondary to intracellular Na⁺ accumulation, (3) the relaxant component of 5-HT response is dependent on the activity of the sodium pump, (4) the activation of Na⁺,K⁺-ATPase activity by this amine is not apparently due to direct effect, and (5) the inhibition of the sodium pump can cause long lasting increases of placental vascular resistance in the presence of physiological concentrations of 5-HT. Send offprint requests to J. Marin at the above address     

Presence of muscarinic inhibitory and absence of nicotinic excitatory receptors at the terminal sympathetic nerves of chicken hearts

Summary Nicotine (2×10^–4 M) or acetylcholine (5.5×10^–4 M) in the presence of 3×10^–6 M atropine did not increase the rate or amplitude of contraction in isolated atria or ventricular strips of the chicken heart; both drugs also did not cause an output of noradrenaline or adrenaline and did not evoke antidromic discharges in the right sympathetic nerves of isolated perfused chicken hearts. In contrast, high K⁺-solutions evoked an output of noradrenaline and adrenaline and caused a burst of antidromic discharges. Dimethylphenylpiperazine (DMPP; 3.1×10^–4 M), by a tyramine-like action, elicited a small output of noradrenaline and increased rate and amplitude of contraction, but did not evoke antidromic discharges. The noradrenaline output caused by DMPP was not reduced by lowering the extracellular Ca²⁺ concentration from 1.8 to 0.1125 mM.-Acetylcholine (10^–5 and 10^–4 M) inhibited the noradrenaline and adrenaline outputs evoked by electrical stimulation of the right sympathetic nerves (3 Hz, 1 ms, 30 V); the inhibition was blocked by 3×10^–6 M atropine. —It is concluded that the terminal parts of sympathetic nerves of the chicken heart posses muscarinic inhibitory receptors but lack nicotinic excitatory receptors. Thus prejunctional nicotinic receptors are not an integral part of the terminal sympathetic neurone otherwise they would be present at this neurone in all species.This work was part of the M. D. thesis of U. Engel     

Precontraction-induced contractile response of isolated canine portal vein to alpha-2 adrenoceptor agonists

Summary The responses of isolated canine portal veins, in either transverse or longitudinal strip, to alpha-2 adrenoceptor agonists were examined. B-HT920, a selective alpha-2 adrenoceptor agonist, did not elicit an appreciable response in the transverse strips of the portal vein under resting tone (0.5 g/mm width). When the preparation was partially precontracted with phenylephrine, prostaglandin F₂, KCl, Bay K 8644, acetylcholine, 5-hydroxytryptamine, histamine, or A23187, B-HT920 evoked concentration-dependent contractile responses (3 × 10^–9-10^–6 M). Maximum contractions, which depended on the precontraction levels and the precontracting substances, ranged from 10–35% of those evoked by norepinephrine 10^–5 M. Similar precontraction-dependent contractile responses were obtained in the longitudinal strips. Concentration-response curves of B-HT920 in the transverse strips precontracted with prostaglandin F₂ were shifted by yohimbine to the right, but not by prazosin. Schild analysis yielded a slope of unity and a pA₂ of 8.79. Clonidine also showed a similar precontraction-dependent contractile response. The lower part of the concentration-response curve of clonidine was shifted by yohimbine. These results may be explained by the presence of postsynaptic alpha-2 adrenoceptors in canine portal vein, which mediate a contractile response under certain conditions. Send offprint requests to T. Furuta at the above address