Calcium entry blockade as a mechanism for chlordimeform-induced inhibition of motor activity in the isolated guinea-pig ileum.

Central and peripheral alpha 2-adrenoceptors, including those of the gastrointestinal tract, have been indicated as a toxicity target of formamidine pesticides in mammals. In this study, the inhibitory effect of chlordimeform on twitch contractions from electrically-stimulated longitudinal muscle-myenteric plexus preparations (LMMPs) of the guinea-pig ileum was found to be resistant to the action of the alpha 2-adrenoceptor antagonist idazoxan. This drug was also ineffective on chlordimeform-induced inhibition of peristalsis recorded in whole ileal segments. As expected, idazoxan antagonized the inhibitory effect of the alpha 2-adrenoceptor agonist clonidine on twitch contractions and peristaltic activity. Chlordimeform reduced the amplitude of direct mechanical responses to a variety of spasmogens such as acetylcholine, histamine and substance P, suggesting a muscular site of action. Moreover, Ca(2+)-free, K(+)-depolarized LMMPs, chlordimeform inhibited submaximal contractions caused by addition of exogenous calcium, through an action apparently similar to that of the Ca2+ entry blocker nifedipine. Both chlordimeform- and nifedipine-induced inhibition of calcium contractions were reversed by the calcium channel activator BAY K 8644. This compound also partially prevented the inhibitory action of chlordimeform on peristaltic activity. On the whole, these results indicate that chlordimeform-induced depression of motor activity in the guinea-pig ileum is, at least in part, related to inhibition of transmembrane Ca2+ fluxes responsible for smooth muscle contraction.     

Stereoselectivity of Butylidenephthalide on Voltage-dependent Calcium Channels in Guinea-pig Isolated Ileum

Two geometric isomers, the Z- and the E- forms, can be separated from synthetic mixtures of butylidenephthalide (Bdph). Z-Bdph (50–100 μm ) non-competitively inhibited Ca²⁺-induced contractions in depolarized (K⁺, 60 mm ) guinea-pig ileum longitudinal smooth muscle, with a pD₂' value of 3.88 ± 0.20 (n = 5). However, E-Bdph (20–100 μm ) competitively inhibited these contractions with a pA₂ value of 4.56 ± 0.18 (n = 5) which was significantly (P < 0.05) greater than the pD₂' value of Z-Bdph. In contrast, the two isomers had no stereoselective inhibitory action on Ca²⁺ influx through pre- or post-junctional membranes of cholinergic nerve endings from which the transmitter acetylcholine is released or on Ca²⁺ release from intracellular stores. Therefore, the trans-Z and cis-E forms of Bdph might have geometric stereoselectivity for voltage-dependent calcium channels (VDC) in guinea-pig longitudinal smooth muscle. Both isomers might inhibit more selectively the contractile twitch responses evoked by electrical stimulation than by cumulative acetylcholine-or carbachol-induced transient contractions in guinea-pig ileum longitudinal smooth muscle.     

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic α2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for α₂-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [³H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. α-Methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC₅₀ from 0.13 to 1.05 μM; E_max from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC₅₀=44.9 μM; E_max=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl]-4,5-dihydroimidazoline), whereas prazosin (10 μM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione; 10 μM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]azepi-ne; 10 μM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of α₂-adrenoceptor blockade by rauwolscine (3 μM) or RX 821002 (1 μM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the α_2A and α_2D binding sites as well as at previously classified α_2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I₁ or I₂ binding sites. When tested on the electrically induced outflow of tritium, α-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 μM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 μM), whereas idazoxan (up to 100 μM) was without effect. When RX 821002 (1 μM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic α₂-adrenoceptors belonging to the α_2D subtype. Received: 10 October 1997 / Accepted: 14 March 1998     

Prejunctional inhibitory alpha-adrenoceptors and dopaminoceptors of the rat vas deferens and the guinea-pig ileum in vitro.

The effects of α-adrenoceptor and dopaminoceptor agonists and antagonists were investigated on prejunctional receptors of the rat vas deferens and the guinea-pig ileum. The order of potency of the agonists for twitch inhibition of the rat vas deferens was clonidine > oxymetazoline > dopamine > apomorphine > noradrenaline whilst the order of potency for inhibiting the stimulated guinea-pig ileum was clonidine > oxymetazoline > noradrenaline > dopamine > apomorphine. Yohimbine readily blocked the inhibitory effects of clonidine, oxymetazoline and noradrenaline in both tissues but was less effective against dopamine and apomorphine. Pimozide selectively blocked the effects of dopamine and apomorphine on the rat vas deferens and was almost completely ineffective against clonidine, oxymetazoline and noradrenaline. However, pimozide significantly antagonized the noradrenaline-induced twitch inhibition of the stimulated guinea-pig ileum in addition to antagonising dopamine and apomorphine action. The pA₂ values for pimozide against dopamine, apomorphine and noradrenaline in both tissues were significantly different. It is concluded that the prejunctional α-adrenoceptors of the rat vas deferens are the same as those located on the terminal cholinergic neurones of the guinea-pig ileum whilst the prejunctional dopaminoceptors in these tissues appear to differ from one another.     

Endomorphin-1 and endomorphin-2 activate µ-opioid receptors in myenteric neurons of the guinea-pig small intestine

The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central μ-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM–1 μM) inhibited (–log EC₅₀ values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective μ-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), caused a concentration-dependent (30 nM–1 μM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA ₂ values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective δ-receptor antagonist) or 30 nM nor-binaltorphimine (a selective κ-receptor antagonist). These results demonstrate that endomorphins selectively activate μ-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists. Received: 6 August 1998 / Accepted: 5 October 1998     

Effects of Mercuric Chloride and Methyl Mercury on Cholinergic Neuromuscular Transmission in the Guinea-Pig Ileum

Abstract: The effects of mercuric chloride (HgCl₂) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.3-3 μM slightly enhanced the amplitude of twitch contractions in ~50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 μM mercurials increased electrically-evoked tritium outflow from [³H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl₂ IC₅₀=21.3±6.4 μM; MeHg IC₅₀=45.1±5.5 μM), as well as contractions to exogenous ACh (0.1 μM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl₂ on basal tone was antagonized by the Ca²⁺ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca²⁺ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca²⁺-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.     

Pharmacology of B-HT 920 in some Isolated Smooth Muscles of the Guinea-pig

Abstract: The effects of B-HT 920 were investigated on four isolated preparations from the guinea-pig, namely the aorta, trachea, ileum and vas deferens. The latter three preparations were studied during electrical field stimulation, which induced contractions by activating cholinergic neurones (trachea and ileum) or adrenergic neurones (vas deferens), respectively. Comparative studies were also made with clonidine and noradrenaline. In ileum and trachea B-HT 920 was almost equipotent with noradrenaline to inhibit the electrically induced contractions. In these tissues, B-HT 920 also displayed almost the same maximal effect as noradrenaline. Clonidine also inhibited the contractions in ileum and trachea; the drug was slightly more potent than noradrenaline. However, in contrast to the intrinsic activity of B-HT 920 that of clonidine was only submaximal. In vas deferens both B-HT 920 and clonidine induced inhibition of contractions on electrical field stimulation at low concentrations. In this organ, both drugs were capable of inducing complete inhibition of the contractile response. In aorta B-HT 920 as well as clonidine were only weak agonists in comparison to noradrenaline. The α₂-blocker, yohimbine, completely blocked the effect of B-HT 920 in ileum at low concentrations (1 × 10^?7 M). Remarkably, however, the inhibitory action of B-HT 920 in trachea was only marginally affected even by high concentrations of yohimbine (1 × 10^?6 M). It is suggested from the present results that B-HT 920 can induce inhibition of both cholinergic and adrenergic neurotransmission presumably by inducing selective stimulation of prejunctional α₂-receptors. In fact, the selectivity of B-HT 920 seems to be comparable to that of clonidine for the α₂-receptor. However, the mode of action of B-HT 920 in trachea may be somewhat uncertain since its effect was not inhibited by yohimbine.     

Atypical presynaptic alpha-adrenoceptor modulation of neurally-mediated cholinergic responses in guinea-pig tracheal smooth muscle

Cholinergic excitatory nerves in guinea-pig trachea are subject to inhibitory control by presynaptic α₂-adrenoceptors. Recently, the nature of these receptors has come into question insofar as the presynaptic inhibitory effects of the α₂-adrenoceptor agonist, clonidine, in the guinea-pig trachea have been shown to be antagonized by the α₂-adrenoceptor antagonist, yohimbine, as well as the α₁-adrenoceptor antagonist, prazosin. This inhibitory action of prazosin had not been described previously in the airways and may relate to the use of norepinephrine rather than clonidine as the α-adrenoceptor agonist in earlier studies. The present study evaluates the susceptibility of norepinephrine-induced inhibition of neurally-mediated cholinergic excitatory responses to antagonism by prazosin and yohimbine under conditions identical to those which showed clonidine to be sensitive to these antagonists. In tissues pretreated with guanethidine, propranolol and indomethacin, norepinephrine (1 μm) induced a 37-fold rightward shift of the frequency-response curve for neurally-mediated cholinergic contractions which was reversed partially by pretreatment of tissues with yohimbine. Norepinephrine also caused a concentration-dependent inhibition of cholinergic ‘twitch’ responses induced by intermittent (1 Hz) nerve stimulation. This action of norepinephrine was antagonized in a concentration-dependent manner by yohimbine but was unaffected by prazosin. These results indicate that in guinea-pig trachea the presynaptic inhibitory actions of norepinephrine on cholinergic nerves are mediated via classical α₂-adrenoceptors, i.e. receptors that can be blocked by yohimbine but not by prazosin. This distinguishes the action of norepinephrine from that shown previously for clonidine and provides support for the contention that these agonists do not act on the same population of α₂-adrenoceptors.     

The Adenosine Agonist NECA Inhibits Intestinal Secretion and Peristalsis

Abstract— This study aimed to determine whether the antidiarrhoeal effect of the mixed A₁/A₂ adenosine agonist NECA (5′-N-ethylcarboxamido adenosine) is due to inhibition of intestinal fluid transport or to contractility. Intestinal secretion was stimulated in anaesthetized rats by intra-arterial infusions of PGE₂ (4 μg min^?1) or vasoactive intestinal peptide (0·8 μg min^?1). NECA reversed PGE₂-induced secretion in the jejunum (ED50 16 μg kg^?1) and ileum (ED50 21 μg kg^?1, i.v.) and inhibited VIP-induced secretion in the jejunum (ED50 21·5 μg kg^?1). NECA inhibited twitch responses (0·1 Hz, 1 ms, IC50 11·2Nm ) but not tetanic contractions at 10 Hz of the transmurally stimulated guinea-pig ileum. Likewise, NECA (10 μm ) did not inhibit frequency-related contractions over the range of 2·5 to 40 Hz of rat jejunum or ileum. However, NECA was shown to be a potent inhibitor (30 Nm ) of the peristaltic reflex in the rat ileum. The results indicate that adenosine receptors are involved in modulating peristalsis as well as the secretory activity of the mucosa in the rat small intestine.     

蛇床子素对豚鼠离体回肠和结肠带的作用

以豚鼠离体回肠和结肠带为标本,观察蛇床子素(Ost)的作用与Ca²⁺)的关系。结果表明:Ost和钙拮抗剂Ver产生剂量依赖性抑制乙酰胆碱(ACh)、组胺及KCl所致回肠条或结肠带的收缩;非竞争性拮抗CaCl₂累积量—效曲线,pD₂分别为4.41±0.15,7.0±0.2。Ost 100μmol/L和Ver 1μmol/L均能对抗小剂量Ca²⁺所致结肠带收缩,但被加入较大量Ca²⁺所取消。Ost和Ver均能抑制ACh诱导的依内钙性收缩,不影响依外钙性收缩。结果提示Ost具有钙拮抗作用,其作用方式与Ver类似。     

Relaxant Effect of Spermidine on Acethylcholine and High K+-induced Gastric Contractions of Guinea-Pig

In our previous study, we found that spermine and putrescine inhibited spontaneous and acetylcholine (ACh)-induced contractions of guinea-pig stomach via inhibition of L-type voltage-dependent calcium current (VDCC_L). In this study, we also studied the effect of spermidine on mechanical contractions and calcium channel current (I_Ba), and then compared its effects to those by spermine and putrescine. Spermidine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner (IC₅₀=1.1±0.11 mM). Relationship between inhibition of contraction and calcium current by spermidine was studied using 50 mM high K⁺-induced contraction: Spermidine (5 mM) significantly reduced high K⁺ (50 mM)-induced contraction to 37±4.7% of the control (p<0.05), and inhibitory effect of spermidine on I_Ba was also observed at a wide range of test potential in current/voltage (I/V) relationship. Pre- and post-application of spermidine (5 mM) also significantly inhibited carbachol (CCh) and ACh-induced initial and phasic contractions. Finally, caffeine (10 mM)-induced contraction which is activated by Ca²⁺-induced Ca²⁺ release (CICR),'' was also inhibited by pretreatment of spermidine (5 mM). These findings suggest that spermidine inhibits spontaneous and CCh-induced contraction via inhibition of VDCC_L and Ca²⁺ releasing mechanism in guinea-pig stomach.     

CP-96,345, a non-peptide antagonist of substance P: I. Effects on the actions mediated by substance P and related tachykinins on the guinea-pig ileum and rabbit jejunum

Summary The effects of a non-peptide antagonist of substance P, CP 96,345, were investigated, in vitro, on the guinea-pig ileum and the rabbit jejunum.Contractions of the guinea-pig ileum, induced by substance P and neurokinin A, were specifically inhibited by the racemate (±)CP-96,345 (pIC₅₀ 7.8 and 7.3, respectively). The inhibition by (±)CP-96,345 of contractions evoked by neurokinin B and by bradykinin (pIC₅₀ 6.1 and 4.9, respectively) was attributed to unspecific effects of the antagonist. The inhibition of substance P-induced contractions of the rabbit jejunum required a 10 times higher concentration of (±)CP-96,345 (pIC₅₀ = 6.8) than was required with the guinea-pig ileum. The plateau phase of contraction of the guinea-pig ileum induced by high concentrations of substance P, neurokinin A or neurokinin B, which is known to be mediated through tachykinin receptors on intrinsic cholinergic neurones, was inhibited by 200 nM (±)CP-96,345 but not by the inactive enantiomer, CP-96,344. This indicates a specific inhibition of these neuronal tachykinin receptors by (±)CP-96,345 Contractions known to be mediated by the release of substance P, such as those evoked by capsaicin and by mesenteric nerve or field stimulation, were partially inhibited by (±)CP-96,345 at concentrations of 200 to 600 nM. Unspecific inhibitory effects of CP-96,345, in concentrations of 1 µM or higher, were observed on histamine-induced contractions, and on the cholinergic twitch response to electrical stimulation, of the guinea-pig ileum. Therefore, an inhibition by CP-96,345 of substance P-related effects can only be regarded as specific if the concentration of the antagonist is below 1 µM. No effect of CP-96,345 (1 µM) was seen on peristalsis in vitro. The peristaltic reflex, in situ, was also not affected by CP-96,345 (1.6 µmolkg^–1, i.v.).The present results demonstrate that, although inhibitory actions of CP-96,345 can be observed on certain motor responses in the guinea-pig small intestine, peristalsis, a physiologically relevant function, remains intact. Send offprint requests to F. Lembeck at the above address     

α-Adrenoceptor subtypes and Ca2+ mobilization the rabbit ear artery†

The mobilization of cellular and extracellular Ca²⁺ pools by selective α₁-adrenoceptor (phenylephrine) and α₂-adrenoceptor (xylazine) agonists as well as noradrenaline was evaluated in rabbit ear artery. Noradrenaline and phenylephrine possess full instrinsic activity for both types of Ca²⁺ mobilization whereas xylazine up to 1 mM had only a limited contractile effect, being more effective in inducing extracellular Ca²⁺-dependent response. However, extracellular Ca²⁺ was mobilized by xylazine in a concentration 20 times higher than that required to stimulate pre-junctional α₂-adrenoceptors. Noradrenaline (5 μM) and xylazine (1 mM) induced cellular and extracellular Ca²⁺-dependent contractions which were prazosin-sensitive and yohimbine-resistant. Xylazine-induced contractile activity, particularly that dependent upon the extracellular Ca²⁺ pool, was markedly reduced by selective adrenergic denervation with 6-hydroxydopamine, but the actions of noradrenaline were unaffected. These results suggest that: (1) rabbit ear artery contain post-junctional α¹-adrenoceptor but not α₂-adrenoceptors; (2) stimulation of these α₁-adrenoceptors can account for the overall contractile activity of exogenously added noradrenaline and (3) stimulation of α₁-adrenoceptors results in mobilization of cellular as well as extracellular Ca²⁺ pools.     

Interaction between clonidine and histamine on the guinea-pig isolated trachea

In experiments on guinea-pig isolated tracheal spirals, clonidine, in concentrations of 10^?6 to 3 times 10^?4 M. had a contracting effect which was strongly inhibited by prazosin but not significantly modified by yohimbine. Moreover, clonidine (3 times 10^?5 to 3 times 10^?4 M) potentiated histamine-induced contractions; this latter effect was inhibited specifically by α₁-adrenoceptor antagonists (e.g. prazosin, AR-C 239) but unmodified by yohimbine, nicardipine or agents acting on the arachidonic acid cascade. It would therefore appear that clonidine in high concentrations contracts the guinea-pig trachea by stimulating α₁-adrenoceptors and that, contrary to what has been reported with other animal species, notably the dog, the guinea-pig trachea is devoid of α₂-adrenoceptors that mediate contractions.     

GABA-RELATED ACTIVITIES OF AMINO PHOSPHONIC ACIDS ON GUINEA-PIG ILEUM LONGITUDINAL MUSCLE

• 1 The effects of phosphonic analogues of GABA, β-alanine and glycine on guinea-pig ileum longitudinal muscle were measured.
• 2 Aminomethylphosphonic acid (AMPh) and 2-aminoethylphosphonic acid (2-AEPh) were devoid of any effect both in non-stimulated preparations and in electrically-stimulated preparations.
• 3 The phosphonic analogue of GABA, 3-aminopropylphosphonic acid (3-APPh) possessed a GABA_B agonistic effect (relaxation and inhibition of twitch response) at doses of 10^?3 M. No agonistic effect on GABA_a receptors was observed.
• 4 3-APPh at doses tested (2 × 10^?4 M and 10^?3 M) also displayed antagonistic action on the effects of GABA_B agonists producing a parallel shift of the log dose-effect curves of GABA- and (-)-baclofeninhibition of twitch responses. In contrast 3-APPh did not antagonize the inhibitory effect of morphine and noradrenaline.
• 5 The contractile effect of GABA, mediated via GABA_A receptors, was unaffected by 3-APPh (10^?3 M).
• 6 It is concluded that 3-APPh is a partial agonist at the GABA_B site in guinea-pig ileum.

     

Role of extra- and intracellular calcium in the contractile action of agonists in the guinea-pig ileum

Summary The effects of Ca²⁺-channel blockers (nifedipine and verapamil), EGTA, caffeine or the removal of external Ca²⁺ on the contractile action of different agonists and transmural electrical stimulation were examined in isolated segments of the proximal and terminal part of the guinea-pig ileum. The effects of agonists and nerve stimulation on membrane potential were also studied by means of the sucrose gap method. Acetylcholine-elicited contractions in both parts and noradrenaline-as well as histamine-induced contractions in the terminal part of the ileum were composed of an initial phasic and a sustained tonic component. Single pulse transmural nerve stimulation elicited smooth muscle twitches, whereas addition of CaCl₂ to the tissue bath containing Ca²⁺-free and high-K⁺ medium elicited a sustained contraction. Both verapamil and nifedipine were more potent in inhibiting the tonic phase of the responses to the agonists or CaCl₂ than inhibiting the phasic contractions elicited by transmural nerve stimulation, acetylcholine or noradrenaline. The excitatory junction potentials (e j.p.s.) as well as smooth muscle twitches were reduced only by high nifedipine concentrations. The effects of acetylcholine on membrane potential and input membrane resistance were affected minimally by the omission of extracellular Ca²⁺, while the contractions gradually disappeared on repetitive agonist application in the absence of external Ca²⁺ and were blocked by caffeine preexposure. In Ca²⁺-free solution noradrenaline and histamine partially reduced each other's motor effect, while neither of them changed the contractile action of acetylcholine, yet the contraction induced by noradrenaline was prevented and that of histamine significantly reduced by preexposure to acetylcholine. These results suggest that the potency of acetylcholine to release Ca²⁺ from its caffeine-sensitive intracellular stores is much higher than that of histamine and noradrenaline. Send offprint requests to V. Bauer at the above address     

Prejunctional Control of pH 6-Induced Bronchoconstriction by NK1, NK2, μ-Opioid, α2-Adrenoceptor and Glucocorticoid Receptors in Guinea-pig Isolated Perfused Lung

This study investigated the release of calcitonin-gene related peptide-like (CGRP) immunoreactivity and bronchoconstriction induced by pH 6 buffer in guinea-pig isolated perfused lung. Both pH 6-induced CGRP-like immunoreactivity and bronchoconstriction were completely abolished after systemic pretreatment with capsaicin. Pretreatment with the NK₂ receptor antagonist SR 48968 (5 times 10^?7M) completely inhibited bronchoconstriction and significantly reduced the immunoreactivity induced by the pH 6 buffer. The NK₁ antagonist SR 140333 (5 times 10^?7M) and, to a lesser extent the NK₁ antagonist CP 96345, morphine (5 times 10^?6M), the α₂-adrenoceptor agonist UK 14304 (10^?7M) and betamethasone (10^?6M) significantly reduced both pH 6-induced bronchial response and CGRP-like immunoreactivity overflow. The effects of morphine and UK14304 were partially reversed by naloxone (5 times 10^?5M) and idazoxan (5 times 10^?5M). Therefore, NK₁, NK₂, μ-opioid, α₂-adrenoceptor and glucocorticoid receptors seemed to have a prejunctional action on pH 6 buffer-induced CGRP-like immunoreactivity and bronchoconstriction.     

Effects of Alkylxanthines on Contractility of Diaphragm Fibres Isolated from Normal and Sensitized Guinea-pigs

Abstract— This study investigates the effects of alkylxanthines on twitch tension generated by electrical stimulation (supramaximal pulses, 0·2 ms duration, 1 Hz) of diaphragm muscle fibres isolated from normal and actively-sensitized guinea-pigs. Caffeine, theophylline and theobromine increased, in a concentration-dependent manner (50–500 μm ), twitch tension in normal and sensitized diaphragm. Caffeine (500 μm ) enhanced contractility to a greater extent than theophylline or theobromine. Twitch potentiation by caffeine (500 μm ) was significantly greater in sensitized diaphragm. Verapamil (0·1–100 μm ) did not alter twitch contractions in the absence or presence of alkylxanthines in normal or sensitized strips. Dantrolene (0·01–100 μm ) depressed, in a concentration-dependent fashion, twitch contractions of normal and sensitized diaphragm. The inhibitory concentration 50% (expressed as —log IC50) was 6·78 ± 0·13 in normal tissues and 6·15 ± 0·11 in sensitized tissues (n = 6 in each group; P < 0·05). Exposure to Ca²⁺-free, EGTA (0·1 Mm )-containing medium, depressed twitch contraction of normal diaphragm to a lesser extent than that of sensitized diaphragm. Methylxanthines reversed depression of twitch contractions produced by exposure to dantrolene (5 μm ) or a Ca²⁺ -free medium. Adenosine (1–1000 μm ) was without effect whereas enprofylline (50–500 μm ) enhanced diaphragm contractility in normal tissues. This indicates that blockade of adenosine receptors is not involved in the inotropic effect of alkylxanthines in guinea-pig diaphragm. Results from this study suggest that alkylxanthines enhance diaphragm contractility in the guinea-pig by releasing intracellular Ca²⁺ and promoting extracellular Ca²⁺ entry through verapamil-insensitive pathways. An alteration of Ca²⁺ movements and stores may be present in the sensitized diaphragm.     

α1A-adrenoceptor subtype mediates contraction of the rat urethra

1 The α₁-adrenoceptor-mediated responses of the rat urethra to phenylephrine have been examined in vitro.Phenylephrine caused concentration-dependent contractions of the isolated urethra which were antagonized by WB4101 (3–30 nM) and prazosin (10–100 nM) but not idazoxan (1.5 μM). Schild plot analysis of the antagonism by prazosin and WB4101 yielded straight lines with slopes not significantly different from unity. The pA₂ value of 9.0 for WB4101 was significantly greater than the value previously obtained at the α₁₈-adrenoceptor of the rat spleen. 2 5-Methylurapidil (30 nM) and abanoquil (1 nM) caused dextral shifts of concentration-response curves yielding pK_s values of 8.3 and 9.4 respectively. Maximal responses to phenylephrine were also reduced by this concentration of abanoquil. 3 Preincubation with chloroethylclonidine (25 μM for 40 min) failed to alter responses, but removing extracellular calcium or the presence of nifedipine (1 μM) almost abolished contractions to phenylephrine. 4 These results indicate that the responses of the rat urethra to phenylephrine are mediated via the α_1A-adrenoceptor subtype and are dependent on the influx of extracellular calcium.     

Studies on the Mechanism of Spasmolytic Activity of (O-Methyl-)-N-(2,6-dihydroxybenzoyl)tyramine,a Constituent of Aniba riparia (Nees) Mez. (Lauraceae), in Rat Uterus,Rabbit Aorta and Guinea-pig Alveolar Leucocytes

Abstract— The mechanism of action of a nonspecific smooth muscle relaxant, (O-methyl-)-N-(2,6-dihydroxybenzoyl)tyramine (riparin), a constituent of Aniba riparia (Nees) Mez. (Lauraceae) was studied in relation to Ca²⁺ metabolism in smooth muscle tissues and in guinea-pig alveolar leucocytes. In rat depolarized uterus, riparin inhibited in a reversible and noncompetitive manner CaCl₂-induced contraction, a response mediated through voltage-dependent Ca²⁺ channels. The pD₂ value (mean±s.e.m.) for riparin was 4·98±006. When compared with sodium nitroprusside (IC50 2·5 μm ), an antagonist of receptor-operated Ca²⁺ channels, riparin was ineffective in suppressing noradrenaline-induced sustained contractions of rabbit aortic strips. However, in the aorta, the compound inhibited intracellular calcium-dependent transient contractions of noradrenaline and riparin (IC50 10·1 μm ), was approximately two and a half times more potent than procaine (IC50 25·5 μm ), a known inhibitor. In guinea-pig alveolar leucocytes, riparin (IC50 3·2 μm ), inhibited intracellular Ca²⁺ accumulation induced by the calcium ionophore A23187. The results suggest that the inhibition of Ca²⁺ influx and of Ca²⁺ release from intracellular stores contribute to the spasmolytic effects of riparin, which may not involve cyclic AMP generation as the levels of this nucleotide were not increased in alveolar macrophages treated with riparin (10–100 μm ).