Tachykinin receptors mediate atropine-resistant rat duodenal reflex contractions in vivo

The study aimed to establish the possible role of tachykinins as mediators of atropine-resistant reflex contractions evoked by balloon distension in the proximal duodenum of urethane-anesthetized, guanethidine (34 mol/kg s.c.)-pretreated rats. Distension of the balloon with a small amount (0.2–0.3 ml) of saline induced the appearance of phasic rhythmic contractions (about 11 mmHg in amplitude) which were promptly suppressed by either atropine (3 mol/kg i.v.) or hexamethonium (28 mol/kg i.v.). Despite the continuous i.v. infusion of atropine (2 mol/h), low-amplitude rhythmic phasic contractions recovered, which were promptly suppressed by hexamethonium, to indicate the involvement of an atropine-resistant excitatory reflex. The amplitude of these atropine-resistant contractions was increased to about 4–5 mmHg by further distension of the balloon (0.4–0.6 ml) : under these conditions, the atropine-resistant contractions undergo a progressive fading. The fading was prevented by i.v. administration of the nitric oxide (NO) synthase inhibitor, L-nitroarginine methyl ester (L-NAME, 55 mol/h), to provide a suitable baseline (amplitude of contractions was 7–8 mmHg) for studying the effect of tachykinin receptor antagonists.I.v. administration of the selective tachykinin NK₂ receptor antagonists, MEN 10,627 (10–100 nmol/kg) and SR 48968 (100–300 nmol/kg) or of the selective NK₁ antagonist SR 140333 (100 nmol/kg), at doses which do not affect the duodenal contractions induced by acetylcholine (5.5 µmol/kg i.v.), produced a prompt and long lasting suppression of the atropine-resistant reflex duodenal contractions produced by balloon distension in urethane-anesthetized rats, whilst SR 48965 (300 nmol/kg), the enantiomer of SR 48968 devoid of NK₂ receptor blocking activity, was without effect.I.v. administration of the selective NK_i receptor agonists [Sar⁹] substance P sulfone and septide or of the NK₂ receptor selective agonist, Ala⁸] neurokinin A(4–10) produced dose-dependent contractions of the duodenum. SR 140333 (100 nmol/kg i.v.) selectively antagonized the duodenal contractions produced by [Sar⁹] substance P sulfone and septide without affecting those produced by [Ala⁸] neurokinin A(4–10). On the other hand, MEN 10,627 (30–100 nmol/kg i.v.) and SR 48968 (100–300 nmol/kg i.v.) but not SR 48965 (300 nmol/kg i.v.) antagonized, at a comparable extent, duodenal contractions induced by both the selective NK₂ and NK₁ receptor agonists.We conclude that endogenous tachykinins are involved in mediating atropine-resistant reflex contractions evoked by distension of the rat duodenum in vivo: both NK₁ and NK₂ receptors are activated by endogenous ligands to produce NANC contractions of rat duodenum in vivo. However, the contractile response to i.v. administered NK₁ receptor agonists, [Sar⁹] substance P sulfone and septide, may involve the release of mediators producing smooth muscle contraction via NK₂ receptors.     

Further evidence for the existence of two receptor sites for bradykinin responsible for the diphasic effect in the rat isolated duodenum.

Low doses of bradykinin (below 10 nM), as well as of K+ (below 10 mM) induced relaxation, whereas higher doses caused contraction of the rat duodenum. The relaxant responses induced by bradykinin and K+ were not affected by ouabain (1 microM), but pre-incubation with 5.9 mM K+ abolished the responses to that ion but not those to bradykinin. The contractile and relaxant components of the response to bradykinin (but not those to K+) increased with the time elapsed after mounting of the preparation, and this was due to stretching by the load of the recording system. Specific and reversible desensitization (tachyphylaxis) was observed with the contractile response (but not the relaxation) induced by bradykinin. Des-Arg9-bradykinin, an analogue specific for B1-receptors, was much less active than bradykinin, and elicited only a contractile response. Among four bradykinin potentiating peptides that were tested, potentiator C enhanced the relaxation only, whereas BPP5a and captopril potentiated only the contraction and BPP9a potentiated both types of response to bradykinin. Our results support the hypothesis that the relaxant and contractile components of the rat duodenum's response to bradykinin are due to actions at different receptor sites, which can be distinguished by their properties (desensitization) and their different apparent affinities for agonists and for potentiating peptides.     

Pharmacological evidence for the existence of two distinct serotonin receptors in rat brain

The effect of a series of indoleamines on the potassium-evoked release of previously accumulated [³H]serotonin from slices of rat raphe nuclei has been studied. Indoleamine agonists produced a dose-related inhibition of potassium-evoked tritium release which was reversed by methiothepin, metergoline minaserin and methysergide but not cyproheptadine or cinanserin. The relative order of antagonist potency for this effect was different from that obtained for the antagonism of indoleamine-induced inhibition of potassium-evoked tritium release from rat striatal slices previously loaded with [³H]-dopamine. The results show that the serotonin-autoreceptor located on cell bodies and dendrites in the raphe nucleus is different from the postsynaptic serotonin receptor located on dopamine nerve terminals in the striatum.     

Aluminum absorption by rat duodenum: further evidence of energy-dependent uptake

Duodenums of intact anaesthetised rats have been perfused with saline-bicarbonate buffer (pH 8.5), containing AlCl3, across a range of concentrations 0-300 microM. A linear relation between rate of uptake and perfusate Al concentration was found. The clearance of Al from the lumen was also studied with the mean perfusate concentration of 60 microM, in the presence of certain inhibitors. The clearance was significantly reduced by NaCN(1 mM) or dinitrophenol (0.1 mM) in the perfusate, and by prior perfusion with vanadate (10 microM) in saline. No effect was observed after colchicine (25 micrograms) was administered i.v. 3 h before perfusion, but vincristine (10 micrograms) given similarly reduced clearance significantly. Verapamil (25 micrograms/ml) in the perfusate caused a small, just significant, reduction in clearance. These data further characterise duodenal absorption of Al under physiological conditions and suggest that energy-dependent transport plays an important role in its uptake. Calcium channels may provide an additional entry site.     

Functional evidence for the existence of a capsaicin-sensitive innervation in the rat urinary bladder

Capsaicin (0.03-3 microM) induces contractions of the rat isolated bladder which are unaffected by either atropine (3 microM) or tetrodotoxin (0.5 microM). In the presence of capsaicin (0.1 microM) an enhancement of field stimulation-induced contractions was observed. Capsaicin-desensitization did not modify the height of these. The neurogenic nature of the capsaicin-induced contractions was proved by the observation that 'chronic' (48 h) denervation prevented, while 'acute' (2 h) denervation did not modify the effect of capsaicin. Denervated bladders maintained their responsiveness to acetylcholine but not to field stimulation. Isolated bladders from rat pups (1-2 days old) did not respond to capsaicin while strong contractile responses to acetylcholine or field stimulation were obtained in these preparations. In bladders from two week old animals, capsaicin produced similar contractions to those observed in preparations from adult animals. The bladders from rats receiving a high dose of capsaicin (50 mg kg-1 s.c.) at birth were heavier than those of their age-matched, vehicle-treated controls. Isolated bladders from 2 month old animals pretreated with capsaicin at birth were unresponsive to capsaicin while responsiveness to acetylcholine, substance P or field stimulation was unaffected compared with that of vehicle-treated controls. These experiments provide evidence that a capsaicin-sensitive innervation exists in the rat urinary bladder which undergoes a postnatal development at end organ level.     

Neurotensin: dual effect on the motor activity of rat duodenum.

The effects of neurotensin on mechanical activity of rat duodenum were investigated using an isometric-isovolumic preparation. Neurotensin (1 pM to 10 nM) induced a concentration-dependent, tetrodotoxin (TTX)-insensitive fall in both endoluminal pressure and isometric tension. At higher concentrations of neurotensin (1 nM to 1 microM) the relaxation was followed by a concentration-dependent TTX-insensitive contraction, detected only by an increase in endoluminal pressure. Different concentrations of neurotensin were required to desensitize the relaxant and the contractile actions of the neuropeptide. The relaxation was antagonized by apamin, while the contractile response was blocked by nifedipine. Neurotensin, when tested separately on longitudinal and circular muscular strips, caused relaxation of the longitudinal strips. Circular strips showed contractions in response to neurotensin, following an inhibitory phase, if the strips were spontaneously or pharmacologically activated. The results suggest the presence of two sets of neurotensin receptors with a differential localization between the two muscular layers in rat duodenum.     

Further evidence for the existence of a homogenous beta-endorphin-sensitive receptor population in the rat tail artery

Isolated rat tail arteries were perfused and vasoconstriction was evoked by electrical field stimulation (2 pulses at 1 Hz every 2 min). The vasoconstriction was depressed by DAGO (IC50 = 611 nM) and beta-endorphin (IC50 = 37 nM). Structuraly analogues and shorter fragments of beta-endorphin were also tested. beta-Endorphin and beta-endorphin-(1-26) were about equipotent whereas the beta-endorphin fragments 1-17, 1-16 and 6-31 were inactive. The potencies of beta-endorphin, beta-endorphin-(1-26), -(1-17) and -(1-16) were not changed in the presence of peptidase inhibitors. Structural analogues such as [D-Ala2]beta-endorphin or [Leu5]beta-endorphin had a somewhat lower potency than beta-endorphin itself. Naloxone 30 nM antagonized the effects of DAGO and beta-endorphin to a similar extent with dissociation constants 3.8 and 3.7 nM, respectively for the antagonist against the agonists. The results support the existence in the rat tail artery of a homogenous population of beta-endorphin-sensitive receptors which may belong to the epsilon-type.     

Further evidence for involvement of adenosine-5'-triphosphate in non-adrenergic non-cholinergic relaxation of the isolated rat duodenum

The nature of the inhbitory non-adrenergic non-cholinergic (NANC) neurotransmitter responsible for neurogenic relaxation of rat duodenum was studied with in vitro techniques. Adenosine 5'-triphosphate (ATP)(1 mM), gamma-aminobutyric acid (GABA, 1 mM), dimethylphenylpiperazinium (DMPP, 0.1 mM) and field stimulation (60 V, 2 ms, 0.1 Hz) produced transient relaxation followed by rebound contraction. In contrast vasoactive intestinal polypeptide (VIP) (0.3 microM) and noradrenaline (1 microM) induced relaxation which set in more slowly and lasted longer. Tetrodotoxin (0.85 microM) abolished field stimulation-induced relaxation but not ATP-, VIP- or noradrenaline-induced relaxation. Nucleotide pyrophosphatase (0.25 U/ml), but not the proteolytic enzyme alpha-chymotrypsin (2 U/ml), selectively antagonized NANC relaxation. The rank order of potency of various adenine derivatives for inducing relaxation was adenosine-5'-triphosphate greater than adenosine-5'-diphosphate much greater than adenosine greater than adenosine-5'-monophosphate. ATP-induced relaxation was selectively antagonized by the putative P2 purinoceptor antagonist reactive blue 2, but unaffected by the selective P1 purinoceptor antagonist 8-phenyltheophylline. The duration of ATP- as well as beta-gamma-methylene adenosine-5'-triphosphate (a stable analogue of ATP)-induced relaxation was similar and was unaffected by indomethacin 10 microM (which abolished the rebound contraction). In those preparations whose contractile tone was increased by using a high-K+ medium the ability of ATP to elicit relaxation was markedly reduced, while GABA- and DMPP-induced relaxation was abolished. On the other hand, ATP-, GABA- and DMPP-induced relaxation of the tonic component of 5-hydroxytryptamine (5-HT)(0.1 mM)-induced contraction was similar to that observed in control conditions. These findings add further weight to the proposal that endogenous ATP is involved in determining NANC relaxation of rat duodenum.     

Cholinergic activity in the rat hippocampus, cortex and striatum correlates with locomotor activity: an in vivo microdialysis study

The possible relationship between behavioral arousal and acetylcholine release in the striatum, hippocampus and frontal cortex was investigated in rats. In vivo microdialysate concentrations of acetylcholine and choline from these brain structures, and photocell beam interruptions (as a measure of behavioral arousal), were measured simultaneously under three conditions: after injections of 1) vehicle or 2) scopolamine (0.4 mg/kg), and 3) before and after the beginning of the rats' night cycle. Dialysate concentrations of ACh in all 3 brain structures and locomotor activity were increased after scopolamine and the onset of the lights out condition. Vehicle injections transiently increased ACh in the hippocampus and cortex and caused short-lasting increases in locomotor activity. Under all conditions, the release of ACh from each of the 3 brain structures correlated with the level of locomotor activity.     

In vivo inhibition of polymyxin B-induced hypotension: evidence of beta-adrenergic inhibitory activity on rat mast cells

In vitro, rat mast cells show a dose-inhibition profile when stimulated with compound 48/80 in the presence of isoprenaline. Isoprenaline-induced inhibition is stereoselective at low concentrations, being greater on pleural mast cells. In vitro, polymyxin B (0.02-0.05 mg/kg) induces a marked hypotension and slow heart rate, but not tachyphylaxis. Both effects were not suppressed in the presence of isoprenaline (with short half-life in vivo), but orciprenaline does induce a clear inhibition of Polymyxin B-induced hypotension, which correlates well with heart rate.     

Methoxytyrosine formation as an indicator of catechol-O-methyltransferase activity in rat liver in vivo

Summary The O-methyl derivative methoxytyrosine accumulated rapidly in rat liver after an intraperitoneal injection of l-dopa (50 mg/kg) in combination with 3-hydroxybenzylhydrazine, an inhibitor of the aromatic amino acid decarboxylase. Methoxytyrosine levels reached a plateau 40–60 min after an i.p. injection of l-dopa, of which the tissue concentration declined monoexponentially. Injection of various doses of l-dopa revealed that methoxytyrosine formation was saturable and followed enzyme kinetics in rat liver. The catechol-O-methyltransferase inhibitors pyrogallol, tropolone and -propyldopacetamide as well as the inhibitor of the aromatic amino acid decarboxylase benserazide inhibited the formation of methoxytyrosine dose-dependently and concomitantly increased the tissue concentration of dopa.The accumulation of methoxytyrosine from exogeneously applied l-dopa appears to be a reliable indicator of the in vivo activity of catechol-O-methyltransferase.Part of this paper has been presented at the Spring Meeting 1975 of the German Pharmacological Society at Mainz (Kehr et al., 1975)     

Abdominal migraine: evidence for existence and treatment options

There is evidence to suggest that, in children, episodic abdominal pain occurring in the absence of headache may be a migrainous phenomenon. There are four separate strands of evidence for this: the common co-existence of abdominal pain and migraine headaches; the similarity between children with episodic abdominal pain and children with migraine headaches, with respect to social and demographic factors, precipitating and relieving factors, and accompanying gastrointestinal, neurological and vasomotor features; the effectiveness of nonanalgesic migraine therapy (such as pizotifen, propanolol, cyproheptadine and the triptans) in abdominal migraine; and the finding of similar neurophysiological features in both migraine headache and abdominal migraine. Abdominal migraine is rare, but not unknown, in adults. Many families are content with a diagnosis and reassurance that the episodes, though distressing, are not the result of serious pathology. Some patients respond to simple dietary and other prophylactic measures. There is scant evidence on which to base recommendations for the drug management of abdominal migraine. What little literature exists suggests that the antimigraine drugs pizotifen, propanolol and cyproheptadine are effective prophylactics. Nasal sumatriptan (although not licensed for pediatric use) may be effective in relieving abdominal migraine attacks.     

Effect of an aqueous distillate of Origanum onites L. on isolated rat fundus, duodenum and ileum: evidence for the role of oxygenated monoterpenes

The aqueous distillate of Origanum onites L. (Labiatae) was reported to have various ethnomedical uses including effects on the gastrointestinal tract. The effects of three different doses (0.1, 0.2 and 0.4 mL) of the aqueous distillate of Origanum onites on isolated rat fundus, duodenum and ileum against acetylcholine induced contractions were investigated in this study. The aqueous distillate inhibited contractions in a dose dependent manner. Inhibitions were lowest on fundus. Carvacrol, being the major compound of the test substance, did not inhibit acetylcholine induced contractions of the isolated rat fundus, indicating the presence of other active principles including menthane diols. As being one of the first pharmacological studies on aqueous distillates, a possible pharmacological activity of cis-p-menth-4-ene-1,2-diol and 3,7-dimethyl-1-octen-3,7-diol is proposed in this study.     

Autoradiographic evidence for the occlusion of rat brain dopamine D3 receptors in vivo.

[125I]Iodosulpride binding was studied in frontal rat brain sections by quantitative autoradiography. Using preincubated (= washed) sections, selective labelling and identification of dopamine D3 receptors was obtained using 0.2 nM [125I]iodosulpride in the presence of 100 nM domperidone for the occlusion of the D2 receptors. A high density of D3 receptors was noticed in the islands of Calleja. When preincubation of the sections was omitted, no D3 receptor labelling could be achieved, indicating tight binding to the receptor of an endogenous inhibitor. Such a tight receptor occupancy was not observed for the D2 receptor and various other neurotransmitter receptors. The occlusion of the D3 receptor could be prevented by tetrabenazine-induced monoamine depletion of the rats. It can be concluded, therefore, that D3 receptors are massively occupied by a monoamine, likely to be dopamine. This observation prompts the question to what extent dopamine D3 receptors can become occupied in vivo by systematically applied exogenous compounds.     

Kinin-induced relaxations of the rat duodenum

Summary Both bradykinin (BK) and des-Arg⁹-BK induced relaxations of the isolated longitudinal smooth muscles of the rat duodenum. No contractile effects were observed with both peptides at concentrations up to 1 mol/l. Des-Arg⁹-BK was about 1000 times less potent than BK. The novel B₂ antagonist HOE 140 (d-Arg-[Hyp³, Thi⁵, D-Phe⁷, Oic⁸]-BK) potently inhibited the BK-induced relaxations, but did not affect the relaxations induced by des-Arg⁹-BK. Conversely, the B₁ receptor antagonist des-Arg⁹-[Leu⁸]-BK only inhibited des-Arg⁹-BK, but did not affect BK-induced relaxations.The relaxations induced by BK and by des-Arg⁹-BK were inhibited by apamin (1 mol/l) demonstrating that apamin-sensitive K⁺ channels are involved. In contrast, tetraethylammonium (1 mmol/l) did not inhibit the relaxations. BK-induced relaxations were reduced by about 25% in the presence of indomethacin (10 mol/l) although the concentration-response curve to BK was not shifted to the left. Prostaglandin E₁ caused relaxations with a pD₂ value of 9.2.It is concluded that both BK and des-Arg⁹-BK can elicit relaxations of the rat duodenum via pharmacologically distinct kinin receptor subtypes, but via similar effector mechanisms.     

Functional evidence for the existence of adenosine receptors in the human heart

Adenosine added to isolated electrically driven preparations of human ventricular heart muscle antagonized the positive inotropic effect of isoprenaline (mean EC50 19 mumol 1(-1), n = 9). Similar effects were observed with the adenosine receptor agonist (-)-N6-phenylisopropyladenosine (mean EC50 0.5 mumol 1(-1), n = 7). These data provide functional evidence for the existence of adenosine receptors in the human myocardium which may modulate the force of contraction during beta-adrenergic stimulation and thus could be involved in the autoregulation of myocardial contractility.