Kurt Racké

Regulation of 5-HT release from enterochromaffin cells

Large amounts of 5-HT are present in the mammalian intestine where the amine is concentrated in the enterochromaffin cells (ECs) of the mucosa. ECs have the enzymes to synthesize 5-HT, are endowed with a specific, imipramine-sensitive 5-HT uptake mechanism and can store 5-HT in specific secretory vesicles. ECs can secrete 5-HT in a calcium-dependent manner. In particular, calcium influx through voltage-regulated channels and receptor-mediated liberation of intracellular calcium can evoke 5-HT release. 5-HT secretion from ECs occurs predominantly at the interstitial side and is controlled by a complex pattern of receptor-mediated mechanisms. Stimulatory receptors (beta-adrenoceptors, muscarine, nicotine and 5-HT3 receptors) and inhibitory receptors (alpha 2-adrenoceptors, histamine H3, GABAA- and GABAB-, A2 and P2y alpha purine and 5-HT4 receptors as well as receptors for vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase stimulating peptide (PACAP) and somatostatin) have been shown to be involved in the control of 5-HT release from the ECs.

Inhibition of arginase by NG-hydroxy-l-arginine in alveolar macrophages: implications for the utilization of l-arginine for nitric oxide synthesis

The hypothesis was investigated that the nitric oxide (NO) synthase intermediate, (HOArg), is an arginase inhibitor in rabbit or rat alveolar macrophages. Exogenously applied HOArg strongly inhibited the arginase activity present in these cells (IC50 ≥ 15 μM), and attenuated l‐[3H]arginine transport (IC50 ≥ 500 μM) in rabbit alveolar macrophages. Moreover, up to 37 μM HOArg were detected in the conditioned medium, but not in the lysate, of rat alveolar macrophages exposed to bacterial lipopolysaccharide for 18 h. HOArg may thus be a potent endogenous arginase inhibitor in these cells which increases the availability of l‐arginine for NO biosynthesis.

Nitric oxide synthase activity is inducible in rat, but not rabbit alveolar macrophages, with a concomitant reduction in arginase activity

Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 × 106 cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of 3H-citrulline (NO synthase activity) and 3H-ornithine (arginase activity) were determined.During incubation of rat alveolar macrophages with 3H-arginine clear amounts of 3H-citrulline and 3H-ornithine (3.8 and 4.6% of the added 3H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra-/intracellular of 17 and 70 for 3H-citrulline and 3H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of 3H-citrulline was increased about 30-fold and this was accompanied by a reduction in 3H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 μmol/1). Inhibition of NO synthase by NG-monomethyl-l,-arginine (l-NMMA, 100 μmol/1) in LPS treated alveolar macrophages reduced the formation 3H-citrulline by more than 90% and restored the 3H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of 3H-citrulline and 3H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive. During incubation of rabbit alveolar macrophages a marked formation of 3H-ornithine (about 5.3% of the added 3H-arginine), but no significant formation of 3H-citrulline could be detected. Pretreatment with LPS tended to enhance the formation of 3H-ornithine (by 50%) without effects on 3H-citrulline. Rabbit-interferon and/or tumor necrosis factor-α present together with LPS during the culture period did not result in a significant 3H-citrulline formation. Under all conditions tested, culture media of rabbit alveolar macrophages did not contain significant amounts of nitrite (less than 0.5 nmol) whereas in culture media of untreated rat alveolar macrophages 22 nmol nitrite (per 18 h) were detected, and LPS induced a 3-fold nitrite accumulation, an effect prevented by dexamethasone.In conclusion, in rabbit alveolar macrophages NO synthase activity was not detectable and could also not be induced by LPS and different cytokines, whereas in rat alveolar macrophages NO synthase was readily inducible. Alveolar macrophages of both species showed marked arginase activity. After induction of marked NO synthase activity, ornithine formation was largely reduced possibly by concomitant inhibition of arginase and/or “withdrawn” of arginine from arginase.

Characterization of histamine H3 receptors inhibiting 5-HT release from porcine enterochromaffin cells: Further evidence for H3 receptor heterogeneity

The nature of the histamine receptor mediating inhibition of 5-HT release was investigated in strips of the porcine small intestine by investigating the effects of histamine ligands on the overflow of endogenous 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). The overflow was measured by HPLC, combined with electrochemical detection and represents calcium-sensitive 5-HT release from enterochromaffin cells, as reported previously. The histamine H3 receptor selective agonists (R)-α-methyl-histamine and imetit inhibited the overflow of 5-HT maximally by 50–60%, with EC50 values of 48 and 3.2 nmol/l, respectively. Effects on 5-HT overflow were always accompanied by similar effects on the overflow of 5-HIAA. Thioperamide (100 nmol/l) shifted the concentration response curve of (R)-α-methyl-histamine to the right (pKB value 8.38). The inhibitory effect of 1 μmol/l (R)-α-methyl-histamine was antagonized in a concentration-dependent manner by thioperamide (IC50: 65 nmol/l) and dimaprit (IC50: 8.6 μmol/l); however, the effect of (R)-α-methyl-histamine was weakly antagonized by burimamide (by 38% at 100 μmol/l) and not significantly affected by other H3 receptor antagonists, such as impromidine, betahistine and phenylbutanoyl-histamine (each up to 100 μmol/l). In conclusion, H3 receptors mediating inhibition of 5-HT release from porcine enterochromaffin cells have a particular pharmacological profile indicating that heterogeneity of H3 receptors may exist. The data suggest that histamine H3 receptors modulating 5-HT release in pig small intestine do not belong to either H3A or H3B receptors as defined in rat tissue.

Characterization of the role of calcium and sodium channels in the stimulus secretion coupling of 5-hydroxytryptamine release from porcine enterochromaffin cells

SummaryStrips of the porcine small intestine were incubated in vitro and the outflow of 5-hydroxytryptamine (5-HT) was determined by HPLC with electrochemical detection.Spontaneous outflow of 5-HT from the porcine small intestine was reduced by about 70% after removal of the extracellular calcium or by addition of 1 mM gadolinium. Tetrodotoxin reduced the outflow of 5-HT by 30%, an effect which has previously been shown to be caused by inhibition of an excitatory cholinergic input. The sodium channel opener veratridine (up to 100 μM) did not affect the outflow of 5-HT. ω-Conotoxin GVIA (500 nM) or nifedipine (10 μM) reduced the outflow of 5-HT only by about 50%, and their effects were not additive. The inhibitory effects of ω-conotoxin GVIA occurred also in the presence of tetrodotoxin. Elevation of extracellular potassium to 40 mM caused a marked and sustained increase in 5-HT outflow. High potassium evoked release of 5-HT was blocked by ω-conotoxin GVIA, nifedipine and gadolinium. When ω-conotoxin GVIA and nifedipine were present in combination, their inhibitory effects on the high potassium evoked 5-HT release vanished. BAY K 8644 (1–10 μM) did not facilitate 5-HT release, but markedly reduced the spontaneous and high potassium evoked release of 5-HT.In conclusion, the enterochromaffm cells are endowed with multiple calcium channels, but voltage-sensitive calcium channels of a neuronal L-type which are sensitive to dihydropyridines and ω-conotoxin GVIA appear to play a major role.

β-Adrenoceptors mediate inhibition of [3H]-acetylcholine release from the isolated rat and guinea-pig trachea: role of the airway mucosa and prostaglandins

1 Rat or guinea pig isolated tracheae were labelled with [3H]‐choline to measure evoked tritium outflow, which reflects neuronal release of [3H]‐acetylcholine. Tritium outflow was evoked either by electrical stimulation of the extrinsic vagal nerve (rat tracheae) or by 27 mm potassium (guinea pig tracheae). 2 In rat tracheae isoprenaline (0.01, 0.1 μm) inhibited evoked [3H]‐acetylcholine release, whereas β‐adrenoceptor‐selective agonists (fenoterol, formoterol, salbutamol) were ineffective. 3 The inhibitory effect of isoprenaline was abolished under the following conditions: (i) presence of propranolol (1 μm) or of the β‐selective antagonist CGP 20712 A (0.1 μm); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 μm indomethacin. 4 In rat isolated tracheae prelabelled with [3H]‐arachidonic acid, isoprenaline (0.1 μm) but not formoterol (0.01 μm) enhanced the outflow of [3H]‐prostaglandins (PGD2, PGE2). This effect was blocked by 0.1 μm CGP 20712 A. 5 In guinea pig tracheae electrical stimulation of the extrinsic vagal nerve did not cause a constant release of [3H]‐acetylcholine, but 27 mm potassium elicited a reproducible release of [3H]‐acetylcholine. In this species both isoprenaline (0.1 μm) and formoterol (0.01 μm) inhibited evoked [3H]‐acetylcholine release. Inhibition was abolished under the following conditions: (i) presence of propranolol (1 μm) or of the β2‐selective antagonist ICI 118551 (0.3 μm); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 μm indomethacin. 6 In conclusion, the present experiments have demonstrated that activation of β‐adrenoceptors localized in the mucosa mediates inhibition of [3H]‐acetylcholine release from the neuroeffector junctions of the pulmonary, parasympathetic nerves most probably by the liberation of inhibitory prostaglandins from the airway mucosa. The adrenoceptor subtype involved differs in rat (β1 subtype) and guinea pig (β 2subtype) airways.

Endogenous noradrenaline release from guinea-pig isolated trachea is inhibited by activation of M2 receptors

Overflow of endogenous noradrenaline (NA) from guinea‐pig isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses). The muscarinic receptor agonist oxotremorine inhibited the evoked overflow of NA in a concentration‐dependent manner (EC50 84 nm). Methoctramine, pirenzepine and p‐fluoro‐hexahydrosiladiphenidol (each 1 μm) shifted the concentration‐response curves of oxotremorine to the right with apparent pA2 values of 7.60, 6.74 and 6.18, respectively. It is concluded that sympathetic nerve terminals in the guinea‐pig trachea are endowed with inhibitory muscarinic M2 receptors.

Effects of indomethacin on muscarinic inhibition of endogenous noradrenaline release from rat isolated trachea

SummaryThe release of endogenous noradrenaline from rat isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) in the presence of yohimbine, desipramine and tyrosine. The muscarine receptor agonist oxotremorine concentration-dependently inhibited the evoked release of noradrenaline by 95% at 1 μmol/l, EC50 values in two series of experiments 41 and 57 nmol/l, respectively. The effect of oxotremorine was antagonized by the non-selective muscarine receptor antagonist scopolamine (10–1000 nmol/l) in a manner suggesting a simple competitive interaction (slope of Schild plot −0.94; pA2 value 8.88). However, the M2 selective muscarine receptor antagonist methoctramine (0.1–10 μmol/l) affected the action of oxotremorine in a manner suggesting a complex interaction (slope of Schild plot −0.47). Addition of indomethacin (3 μmol/l) caused an increase of the evoked release of noradrenaline by 45% and low concentrations of oxotremorine (0.01 and 0.1 μmol/l, but not 1 μmol/l) became less effective resulting in a slight shift to the right of the concentration response curve (EC50 169 nmol/l). Moreover, in the presence of indomethacin methoctramine (0.1–10 μmol/l) antagonized the effects of oxotremorine in a manner suggesting a simple competitive interaction (slope of Schild plot −0.93, pA2 value 7.61). In the presence of indomethacin, the concentration response curve of oxotremorine was only slightly shifted to the right in the presence of the M1 receptor selective antagonist pirenzepine (1 μmol/l, −log KB 6.1) and not significantly affected by the M3 receptor selective antagonist pfluoro-hexahydrosiladifenidol (1 μmol/l).In conclusion, the release of noradrenaline in the rat trachea is inhibited via presynaptic muscarine heteroreceptors of the M2 subtype. In addition, activation of muscarine receptors affects the release of noradrenaline also indirectly, probably via the release of inhibitory prostanoids, and this involves muscarine receptors which are pharmacologically different from those at the sympathetic nerve terminals.

Cromakalim inhibits electrically-evoked [3H] acetylcholine release from a tube-preparation of the rat isolated trachea by an epithelium-dependent mechanism

SummaryRat isolated tracheae were labelled by incubation with [3H]choline to measure the tritium efflux elicited by electrical stimulation of the extrinsic parasympathetic nerves in vitro. Stimulated tritium efflux reflects the neuronal release of newly synthesized acetylcholine; the effects of potassium channel openers on the stimulated tritium efflux were investigated. In tracheae opened longitudinally neither cromakalim nor its 3S,4R-enantiomer, BRL 38227, reduced the stimulated tritium efflux, whereas in intact tube-preparations cromakalim (0.01–1 μmol/l) mediated a concentration-dependent inhibition. The inhibitory effect of 1 μmol/l cromakalim was prevented by 0.1 μmol/l glibenclamide. Likewise, BRL 38227 (0.01 and 0.1 μmol/l) inhibited the stimulated tritium efflux, but the inhibitory effect vanished at high concentrations (1 and 10 μmol/l). The 3R,4S-enantiomer of cromakalim, BRL 38226 (0.1, 1 and 10 μmol/l), on its own did not significantly inhibit the stimulated tritium efflux, but a combination of both enantiomers (0.5 or 1 μmol/l of each) produced an inhibition similar to that caused by 1 μmol/l cromakalim. In epithelium-denuded tube-preparations neither cromakalim nor BRL 38227 reduced the stimulated tritium efflux. The mucosal/submucosal microenvironment is better preserved in intact tube-preparations than in longitudinally-opened tracheae which are cut along their whole length so that the luminal surface is exposed directly to the surrounding medium. The present experiments show an neuronal inhibitory effect of cromakalim which is mediated by an epithelium-dependent mechanism.

MUSCARINIC INHIBITION OF ENDOGENOUS NORADRENALINE RELEASE FROM RABBIT ISOLATED TRACHEA : RECEPTOR SUBTYPE AND RECEPTOR RESERVE

The aim of the present study was to characterize putative muscarine receptors on sympathetic nerve terminals in the rabbit trachea. Release of endogenous noradrenaline from in vitro incubated rabbit tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) and quantified by high performance liquid chromatography with electrochemical detection.The muscarine receptor agonist oxotremorine inhibited the evoked release of noradrenaline completely at 1 μol/l (EC50: 64 nmol/l). The concentration response curve was very steep (Hill coefficient of 2.3). Scopolamine shifted the concentration response curve of oxotremorine to the right (−log KB 8.48) demonstrating specific, inhibitory muscarine receptors. Several subtype-preferring muscarine receptor antagonists also shifted the concentration response curve of oxotremorine to the right. The rank order of potency was (−log KB or pA*2): scopolamine (8.48) > AF DX 384 (7.88*; slope of Schild plot 1.1) > (R)-trihexyphenidyl (7.87) > 4-DAMP (7.85) > AQ-RA 741 (7.77) ≫ methoctramine 6.18 > pirenzepine (6.0) >p-fluoro-hexahydrosiladifenidol (p-FHHSiD, 5.68). When these affinity constants were plotted against reported −log Ki values determined in binding studies on human cloned muscarine receptor subtypes (m1-m5), the best correlation was obtained for m2. Indomethacin (3 μmol/l), which on its own increased the evoked noradrenaline release by about 45%, affected neither the inhibitory effect of oxotremorine nor the antagonistic potency of methoctramine or p-FHHSiD. After preincubation for 48 min with 300 μmol/l phenoxybenzamine, which has been shown to inactivate muscarine receptors irreversibly, the concentration response curve of oxotremorine was shifted 5.2 fold to the right and the maximal inhibition was reduced by 50%, whereas the slope remained steep (Hill coefficient 2.6). These experiments indicated that a fraction of about 22% of the muscarine receptors has to be occupied by oxotremorine to produce half-maximum inhibition of noradrenaline release; the dissociation constant of oxotremorine at the prejunctional muscarine receptors was 0.33 μmol/l.In conclusion, the sympathetic nerve terminals in the rabbit trachea are endowed with inhibitory M2-like muscarine receptors for which methoctramine displayed a low affinity. Since a large receptor reserve could be excluded, the steep concentration response curve of oxotremorine suggests that activation of muscarine receptors has to reach a threshold level before the onset of an inhibitory effect.