Opioid Inhibition of Oxytocin Release, but not Autoinhibition of Dopamine Release May Involve Activation of Potassium (K+) Channels

1991

ABSTRACT Release of oxytocin (Ox) or dopamine (DA) from the isolated neural lobes or neurointermediate lobes, respectively, was evoked by high K + (30 or 45 mM). Naloxone (1-10 μmol/l) which largely enhances the impulse-induced release of Ox had no effect on Ox release evoked by 30 or 45 mM K + . In the presence of 10 mM tetraethylammonium (TEA), Ox release evoked by 30 or 45 mM K + was increased 2-3fold; nevertheless, naloxone caused a further 2-3fold increase. Barium (500 μM) and quinidine (300 μM) antagonized the effect of naloxone observed in the presence of TEA. (-)-Sulpiride (10 μM) enhanced the release of DA evoked by 30 and 45 mM K + by 94 % and 19 %, respectively. TEA enhanced the …

Histamine inhibits 5-hydroxytryptamine release from the porcine small intestine: Involvement of H3 receptors

1992

Abstract Strips of the porcine small intestine were incubated in vitro, and the release of 5-hydroxytryptamine (5-HT) was determined by high-pressure liquid chromatography with electrochemical detection. Removal of the mucosa resulted in a large reduction (95%) of tissue 5-HT, suggesting that enterochromaffin cells are the main source of 5-HT. The release of 5-HT was reduced by 70% after omission of calcium. Tetrodotoxin and hexamethonium reduced the release of 5-HT by 30%–40% in a nonadditive manner, indicating a spontaneous neuronal (nicotinic) excitatory input to the enterochromaffin cells. Histamine inhibited the release of 5-HT by about 50%. This effect was not affected by mepyramine o…

Prostanoid receptors of the EP3 subtype mediate inhibition of evoked [3 H]acetylcholine release from isolated human bronchi

1998

1 The release of neuronal [3H]acetylcholine (ACh) from isolated human bronchi after labelling with [3H]choline was measured to investigate the effects of prostanoids. 2 A first period of electrical field stimulation (S1) caused a [3H]ACh release of 320±70 and 200±40 Becquerel (Bq) g−1 in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (Sn, n=2, 3, and 4) released less [3H]ACh, i.e. decreasing Sn/S1 values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively). 3 Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E2, nocloprost, and sulprostone (EP1 and EP3 selective) inhibited ev…

Release of endogenous 3,4-dihydroxyphenylethylamine and its metabolites from the isolated neurointermediate lobe of the rat pituitary gland. Effects …

1986

: Isolated rat neurointermediate lobes were incubated in vitro. The release of 3,4-dihydroxyphenylethylamine (dopamine, DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and methoxyphenylethanol (MOPET) was determined by HPLC with electrochemical detection. Under resting conditions, the outflow of metabolites was 35–50 times that of DA. HVA accounted for 50%, DOPAC for 45%, and MOPET for 5% of the metabolites. Although an equivalent of 40–50% of the tissue DA content was released per hour as metabolites, the tissue DA content was not reduced after 110 min of incubation. The spontaneous outflow of DA and its metabolites was not affected by the DA uptake inhibitor GBR 12921 (1…

Presynaptic regulation of the electrically evoked release of endogenous dopamine from the isolated neurointermediate lobe or isolated neural lobe of …

1988

Isolated neurointermediate lobes (NILs) or isolated neural lobes (NLs) of the rat pituitary gland were incubated in Krebs-HEPES solution which contained pargyline and the dopamine uptake inhibitor GBR 12921. The release of endogenous dopamine was determined by HPLC with electrochemical detection. Electrical stimulation of the pituitary stalk induced a frequency-dependent release of dopamine. The release of dopamine from the combined NIL evoked by stimulation at 15 Hz was increased by 130% in the presence of the dopamine D2 receptor antagonist, (-)-sulpiride; the (+)-enantiomer of sulpiride had virtually no effect. When the stimulation frequency was 3 Hz (-)-sulpiride caused an increase in d…

Frequency-dependent effects of activation and inhibition of protein kinase C on neurohypophysial release of oxytocin and vasopressin

1989

Isolated rat neurohypophyses were superfused in vitro and the release of vasopressin and oxytocin into the medium was determined by specific radioimmunoassays. Hormone secretion was increased by electrical stimulation of the pituitary stalk at different frequencies. The effects of several phorbol esters, known to activate (phorbol 12,13-dibutyrate, PDB) or not to affect (4a-phorbol 12,13-dideconate and phorbol 12-monoacetate) protein kinase C, and of the direct protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) were tested. Electrical stimulation with 450 pulses caused the release of about 45 μU vasopressin and 55 μU oxytocin, when a frequency of 3 Hz was applied…

Acetylcholine and nicotine stimulate the release of granulocyte-macrophage colony stimulating factor from cultured human bronchial epithelial cells.

1998

Primary cultures of human bronchial epithelial cells (HBE-cells) were established to measure granulocyte-macrophage colony stimulating factor (GM-CSF) release. HBE-cells showed a basal GM-CSF release (82+/-20 ng/well/24 h; 30 donors), which was increased by interleukin-1 beta(IL-1beta, 1 ng/ml) by 270%. This effect was blocked by 1 microM dactinomycin or 10 microM cycloheximide, i.e. the stimulatory effect of IL-1beta depended on de-novo synthesis. Histamine (100 microM) and acetylcholine ( 100 nM) stimulated GM-CSF release more than two-fold above the baseline. Nicotine (1 microM) increased GM-CSF release to a similar extent, and this effect was prevented by 30 microM (+)-tubocurarine. The…

Muscarinic inhibition of endogenous noradrenaline release from rabbit isolated trachea: receptor subtype and receptor reserve.

1994

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 trachea 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 mumol/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 m…

Adrenergic modulation of the release of 5-hydroxytryptamine from the vascularly perfused ileum of the guinea-pig

1988

1. Isolated segments of the guinea-pig ileum were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent was determined by h.p.l.c. with electrochemical detection. Test substances were applied via the arterial perfusion medium. 2. Isoprenaline (0.1 microM) increased the outflow of 5-HT and 5-HIAA maximally by about 75% and this was antagonized by propranolol (0.1 microM). Forskolin (1-10 microM) increased the outflow of 5-HT by approximately 105% and that of 5-HIAA by approximately 55%. The phosphodiesterase inhibitor AH 21-132 (0.1-1 microM) increased the outflow of 5-HT and 5-HIAA by about 70%. Isoprenaline…

Release of [3H]Acetylcholine in Human Isolated Bronchi: Effect of Indomethacin on Muscarinic Autoinhibition

1995

Receptor-mediated regulation of acetylcholine release in the airways, particularly in humans, remains unclear. In the present study, the tissue content of acetylcholine and release of [3H]acetylcholine were measured in freshly dissected human bronchi obtained at thoracotomy. Large (main and lobar bronchi) and small (segmental and subsegmental bronchi) airways contained considerable amounts of endogenous acetylcholine (300 +/- 50 pmol/100 mg wet weight), whereas significantly less was found in lung parenchyma (60 +/- 30 pmol/100 mg). Isolated small bronchi incubated in an organ bath with the precursor [3H]choline synthesized significant amounts of [3H]acetylcholine (26,000 +/- 4,000 dpm/100 …

Epithelium-derived inhibition of [3H]acetylcholine release from the isolated guinea-pig trachea

1990

To investigate presynaptic, regulatory mechanisms on parasympathetic nerve fibres innervating the airways, the release of newly-synthesized [3H]acetylcholine from the isolated trachea was studied. Reverse phase HPLC followed by liquid scintillation spectrometry was used to separate and quantify the radioactive compounds choline, phosphorylcholine and acetylcholine in the incubation medium and the tissue. During the incubation of the tracheae with [3H]choline a significant synthesis of [3H]acetylcholine (35,000 dpm/preparation) and [3H]phosphorylcholine (500,000 dpm/preparation) occurred. In epithelium-deficient tracheae the formation of [3H]phosphorylcholine was enhanced, whereas the conten…

Adrenoceptor- and cholinoceptor-mediated mechanisms in the regulation of 5-hydroxytryptamine release from isolated tracheae of newborn rabbits

1996

Abstract 1. Isolated tracheae of newborn rabbits were incubated in vitro and the outflow of 5-hydroxytryptamine (5-HT) was determined by h.p.l.c. with electrochemical detection. Evidence has previously been provided that this 5-HT outflow derives from neuroendocrine epithelial (NEE) cells of the airway mucosa. 2. Phenylephrine (1, 10 and 30 microM) enhanced the outflow of 5-HT by 80, 290 and 205%, respectively. 5-HT outflow evoked by 10 microM phenylephrine was not affected by the presence of the neurotoxin tetrodotoxin (1 microM). 3. Rauwolscine, ARC 239 (an alpha(2B)-adrenoceptor preferring antagonist), yohimbine and prazosin antagonized the effect of 10 microM phenylephrine in a concentr…

Inability of rat alveolar macrophages to recycle l-citrulline to l-arginine despite induction of argininosuccinate synthetase mRNA and protein, and i…

1998

In the present study it was tested whether rat alveolar macrophages (AMphi) convert L-citrulline to L-arginine to maintain nitric oxide (NO) synthesis under conditions of limited availability of L-arginine. Rat AMphi (0.5 x 10(6) cells/well, cultured for 20 h in the absence or presence of 1 microg/ml lipopolysaccharides, LPS), were incubated for 6 h in amino acid-free Krebs solution and nitrite accumulation was determined as a measure of NO synthesis. After culture in the absence of LPS, nitrite in the incubation media was at the detection limit, independent of the addition of L-arginine or L-citrulline. AMphi, cultured in the presence of LPS, produced about 4 nmol per 10(6) cells and 6 h n…

Release of [3H]acetylcholine from the isolated rat or guinea-pig trachea evoked by preganglionic nerve stimulation; a comparison with transmural stim…

1991

Basal and stimulated outflow of radioactive acetylcholine, phosphorylcholine and choline from rat and guinea-pig isolated tracheae were measured by reverse phase HPLC followed by liquid-scintillation-spectrometry. Tracheae were stimulated either by an electrical field (transmural stimulation) or by a local stimulation of the innervating parasympathetic nerves (preganglionic stimulation). Epithelium was removed in most experiments, as the epithelium inhibits acetylcholine release. The basal tritium efflux (1,600 dpm/3min) from rat isolated tracheae incubated with [3H]choline consisted of 56% [3H]phosphorylcholine and 38% [3H]choline. Preganglionic stimulation (15 Hz, 1,200 pulses) caused a 2…

Uptake of [3H]dopamine into dopaminergic and noradrenergic neurones of the isolated neurointermediate lobe of the rat hypophysis. Effects of desipram…

1983

The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [3H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [3H]Dopamine represented 70% and [3H]noradrenaline 30% of the [3H]catecholamines. Desipramine (1 microM) prevented the formation of [3H]noradrenaline without affecting the storage of [3H]dopamine. Nomifensine (10 microM) blocked the storage of [3H]dopamine and [3H]noradrenaline. Thus, in the NIL, [3H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [3H]dopamine is converted to [3H]noradrenaline, indicating a significant dopa…

Characterization of the muscarine receptors involved in the modulation of serotonin release from the vascularly perfused small intestine of guinea pi…

1989

Isolated small intestinal segments of the guinea pig were arterially perfused and the release of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent measured by HPLC with electrochemical detection. Test substances were applied via the arterial perfusion medium. McN-A-343, pilocarpine and oxotremorine inhibited concentration-dependently the outflow of 5-HT and 5-HIAA. Pirenzepine (0.03-0.1 mumol/l) which can discriminate between M1 and M2-receptor subtypes antagonized completely this inhibitory effect. In the presence of 1 mumol/l tetrodotoxin (TTx), all three muscarine receptor agonists increased the outflow of 5-HT and 5-HIAA. O…

Alternative mechanisms for tiotropium

2009

Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summ…

Differential effects of potassium channel blockers on neurohypophysial release of oxytocin and vasopressin. Evidence for frequency-dependent interact…

1988

Isolated rat neurohypophyses were fixed by their stalks to a platinum wire electrode and superfused with Krebs-HEPES solution. Vasopressin and oxytocin released into the medium were determined by specific radioimmunoassays. Hormone secretion was increased by electrical stimulation of the pituitary stalk at different frequencies. The effects of several potassium channel blockers, tetraethylammonium (TEA) ions, 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) were tested. The release of vasopressin and oxytocin evoked by electrical stimulation with 900 pulses at 15 Hz (about 900 and 1,000 μU, respectively) was about 10 times higher than that evoked by 900 pulses at 3 Hz. Both 10 and 3…

Effects of tetraethylammonium ions on frequency-dependent vasopressin release from the rat neurohypophysis.

1988

1. Isolated rat neurohypophyses were fixed by their stalks to a platinum wire electrode and superfused with oxygenated Krebs-HEPES solution. Vasopressin release into the medium was determined by radioimmunoassay. Vasopressin secretion was increased by electrical stimulation at different frequencies (3-30 Hz) and different train lengths (75-900 pulses). The effects of tetraethylammonium (TEA) ions and of enhanced calcium were tested. 2. Electrical stimulation at 7.5 or 15 Hz evoked a markedly larger release of vasopressin than stimulation at 3 Hz. During continuous stimulation at 7.5 and 15 Hz the evoked vasopressin release per pulse declined rapidly, but with similar time constants for both…

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

1995

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). …

Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig: Involvement of…

1991

Isolated segments of the guinea-pig small intestine were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Release of acetylcholine from isolated superfused intestinal segments was determined as outflow of [3H]radioactivity from preparations preincubated with [3H]choline. Cisplatin (3 microM) increased the outflow of 5-HT and 5-HIAA by about 90%. At 30 and 100 microM cisplatin decreased the outflow of 5-HT and its metabolite by 40%-50%. The stimulatory effect of cisplatin was consistently observed only when the bicarbonate-…

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

1993

Rat 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 mumol/l) mediated a concentration-dependent inhibition. The inhibitory effect of 1 mumol/l cromakalim was prevented by 0.1 mumol/l glibenclamide. Li…

Inhibition of arginase in rat and rabbit alveolar macrophages by Nω-hydroxy-D,L-indospicine, effects onL-arginine utilization by nitric oxide synthase

1997

1. Alveolar macrophages (AM phi) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate. L-arginine. The present study tested whether two recently described potent inhibitors of liver arginase (N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-ar…

Isoprenaline and forskolin increase evoked vasopressin release from rat pituitary

1982

Isolated neurointermediate lobes of rat pituitaries were incubated in Krebs solution. The vasopressin release evoked by electrical stimulation (0.2 ms, 80 V, 15 Hz, 10 s trains at 10 s intervals for a total of 10 min) was completely inhibited by tetrodotoxin. Isoprenaline increased the evoked vasopressin release to a maximum of 60% (EC50 10 nM) and this effect was antagonized surmountably by propranolol. Forskolin increased the vasopressin release by 98%. These results suggest the presence within the neurohypophysis of a beta-adrenoceptor-linked adenylate cyclase facilitating vasopressin secretion.

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

1992

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 microM) 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.

Regulation of serotonin release from the intestinal mucosa.

1991

Summary In the mammalian intestine serotonin (5-hydroxytryptamine, 5-HT) is present in high concentrations in the enterochromaffin cells. The release of 5-HT from the intestinal mucosa is regulated by a complex pattern of neuronal and humoral inputs to the enterochromaffin cells. The enterochromaffin cells appear to be endowed with different inhibitory ( α 2 -adrenoceptors, GABA A - and GABA B -receptors, histamine H 3 -receptors, receptors for vasoactive intestinal polypeptide and somatostatin) as well as stimulatory receptors ( β -adrenoceptors, muscarine and nicotine receptors). The physiological significance of this complex system of receptors is suggested by experiments which demonstra…

Lack of autoreceptor mediated regulation of the spontaneous dopamine turnover in the isolated neurointermediate lobe of the rat pituitary gland in vi…

1990

Isolated neurointermediate lobes of the rat pituitary gland were incubated in Krebs-HEPES solution and the spontaneous outflow of endogenous dopamine and its metabolites (DOPAC, HVA and MOPET) was determined by HPLC with electrochemical detection. The spontaneous outflow of dopamine metabolites (about 1500 fmol/10 min) largely exceeded that of dopamine (about 60 fmol/10 min). Apomorphine concentration-dependently (IC50, 205 nmol/l) reduced the spontaneous outflow of the dopamine metabolites. The effect of apomorphine developed slowly and was progressive over an observation period of 70 min. After 1 h of exposure to a maximall effective concentration of apomorphine (10 mumol/l), the outflow …

Characterization of endogenous noradrenaline release from intact and epithelium-denuded rat isolated trachea.

1991

1. Overflow of endogenous noradrenaline (NA) from the in vitro incubated rat trachea evoked by two periods of electrical field stimulation (S1, S2 at 3 or 15 Hz) or by high potassium (60 mM) was determined by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. In the presence of the neuronal uptake inhibitor desipramine, the alpha 2-adrenoceptor antagonist, yohimbine, enhanced the overflow of NA evoked by stimulation at 3 Hz by about 100% suggesting the presence of presynaptic inhibitory autoreceptors on the sympathetic nerves innervating the trachea. 3. When desipramine and yohimbine were present throughout the experiments, the overflow of NA evoked by the …

A New Culture Technique: An Approach to the in-Vitro Reconstitution of Endocrine Organs

1983

A new cell culture technique called three-dimensional-rotation culture (3dR-culture) has been developed. Its main characteristics are: (i) cultivation under constant rotation, and (ii) the use of a three-dimensional material that allows the cells to adhere and to grow in a three-dimensional arrangement. Two glassfibre materials and one gelatine-sponge proved suitable for the use in cell culture. The new technique has the advantage to yield in large numbers cells bound on a stable matrix that can easily be handled, e.g. for implantation.

Effect of vasoactive intestinal polypeptide on the release of serotonin from the in vitro vascularly perfused small intestine of guinea pig.

1989

Isolated segments of the guinea pig small intestine were vascularly perfused and the release of endogenous serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) into the portal vein was measured. All test substances were intraarterially perfused. Vasoactive intestinal polypeptide (VIP, 1 pmol/l-100 nmol/l) inhibited the spontaneous release of 5-HT and 5-HIAA. The maximal inhibitory effect (about 60%) was seen at 100 pmol/l. The effect of VIP on the spontaneous release of 5-HT and 5-HIAA was not changed in the presence of 1 mumol/l tetrodotoxin (TTX). Raising intraluminal pressure by 500 Pa for 5 min increased the release of 5-HT and 5-HIAA by about 25%. Raising the intralu…

Beta-adrenoceptor-mediated facilitation of endogenous noradrenaline release from rat isolated trachea.

1994

Overflow of endogenous noradrenaline from rat isolated trachea was evoked by electrical field stimulation (3 Hz, 540 pulses) in the presence of yohimbine, desipramine and tyrosine. Isoprenaline 100 nmol/l increased the evoked overflow of noradrenaline by about 65%. This effect was antagonized by propranolol (100 nmol/l) and the beta 2-selective adrenoceptor antagonist ICI 118,551 (100 nmol/l), but not by the beta 1-selective adrenoceptor antagonist CGP 20712 A (100 nmol/l). The beta 2-selective adrenoceptor agonist formoterol (1-100 nmol/l) also facilitated the evoked overflow of noradrenaline, but maximally by only about 25% at 10 nmol/l, i.e. formoterol behaved as a partial agonist at the…

Activation of L-arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages

1998

1 The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. 2 L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4β-Phorbol 12-myristate 13-acetate (PMA), but not 4α-phorbol 12-myristate 13-acetate (α-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. 3 L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. 4 Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This…

Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in humans.

1998

Acetylcholine acts as a neurotransmitter in the central and peripheral nervous systems in humans. However, recent experiments demonstrate a widespread expression of the cholinergic system in non-neuronal cells in humans. The synthesizing enzyme choline acetyltransferase, the signalling molecule acetylcholine, and the respective receptors (nicotinic or muscarinic) are expressed in epithelial cells (human airways, alimentary tract, epidermis). Acetylcholine is also found in mesothelial, endothelial, glial, and circulating blood cells (platelets, mononuclear cells), as well as in alveolar macrophages. The existence of non-neuronal acetylcholine explains the widespread expression of muscarinic …

Characterization of 5-hydroxytryptamine release from isolated rabbit and rat trachea: the role of neuroendocrine epithelial cells and mast cells

1995

Rabbit or rat isolated tracheae were incubated in vitro, and the release of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) was determined by HPLC with electrochemical detection. Release of 5-HT from rabbit tracheae could be evoked by the calcium ionophore A 23187 and, in a calcium-dependent manner, by depolarizing concentrations of potassium (45 mmol/l), but not by the mast cell degranulating drug compound 48/80. High potassium- and A 23187-evoked release of 5-HT was markedly higher from tracheae of newborn compared to adult rabbits. In rabbit tracheae, mechanical removal of the mucosa resulted in 80-90% reduction in tissue 5-HT and in a similar reduction …

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

1993

The 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 μm…

Cationic Proteins Inhibit l-Arginine Uptake in Rat Alveolar Macrophages and Tracheal Epithelial Cells

1999

Eosinophil-derived cationic proteins play an essential role in the pathogenesis of bronchial asthma. We tested whether cationic proteins interfere with the cationic amino-acid transport in alveolar macrophages (AMPhi) and tracheal epithelial cells, and whether L-arginine-dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [(3)H]-L-arginine, nitrite accumulation, and the turnover of [(3)H]-L-arginine by nitric oxide (NO) synthase and arginase (formation of [(3)H]-L-citrulline and [(3)H]-L-ornithine, respectively) were studied. Poly-L-arginine reduced [(3)H]-L-arginine uptake in rat AMPhi and tracheal epithelial cells in a concentration-dependent manner…

Nitric oxide, via activation of guanylyl cyclase, suppresses alpha2-adrenoceptor-mediated 5-hydroxytryptamine release from neuroendocrine epithelial …

1998

Isolated tracheae of newborn rabbits were incubated in vitro and the outflow of 5-hydroxytryptamine (5-HT) was determined by HPLC with electrochemical detection. Evidence has previously been provided that this 5-HT outflow derives from neuroendocrine epithelial (NEE) cells of the airway mucosa. Phenylephrine, at a maximally effective concentration of 10 microM, caused a transient increase in 5-HT outflow by about 250%, an effect mediated by alpha2B-adrenoceptors, as previously shown. The phenylephrine-induced 5-HT release remained unchanged in calcium-free medium, but was reduced by 75% when the tracheae were incubated in calcium-free medium which contained 0.5 mM EDTA, a treatment known to…

Phosphodiesterase inhibitors suppress alpha2-adrenoceptor-mediated 5-hydroxytryptamine release from tracheae of newborn rabbits.

1998

The outflow of 5-hydroxytryptamine (5-HT) from isolated tracheae of newborn rabbits was determined by high pressure liquid chromatography with electrochemical detection. This 5-HT outflow reflects release from neuroendocrine epithelial cells of the airway mucosa, as previously shown. Phenylephrine, via alpha2B-adrenoceptors, caused a transient increase in 5-HT outflow, maximally by about 250%, an effect mediated by liberation of intracellular Ca2+, as previously shown. The non-selective phosphodiesterase inhibitor 2-isobutyl-1-methylxanthine (IBMX) concentration-dependently inhibited phenylephrine-induced 5-HT release (completely at 100 microM, IC50: 1.3 microM). Likewise, benzafentrine (in…

Effects of the benzodiazepine receptor agonist midazolam and antagonist flumazenil on 5-hydroxytryptamine release from guinea-pig intestine in vitro

1990

Isolated segments of the guinea-pig small intestine and the guinea-pig stomach were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Test substances were applied intraarterially. The benzodiazepine receptor agonist, midazolam, concentration-dependently increased (by 58%, at 1 nmol/l) and decreased (by 32%, at 100 nmol/l) the release of 5-HT from small intestine preparations. Both effects were blocked by the benzodiazepine receptor antagonist flumazenil (10 nmol/l) The stimulatory effect of midazolam was also abolished in the presen…

Effects of bacterial lipopolysaccharides (LPS) and tumour necrosis factor-alpha (TNF alpha) on rat tracheal epithelial cells in culture: morphology, …

1996

Rat tracheal epithelial cells were cultured and the effects of LPS and TNF alpha on cell morphology, rate of proliferation and NO synthase activity were studied. NO synthase activity was determined by measuring the accumulation of 3H-L-citrulline during incubation of confluent monolayer with 3H-L-arginine. In untreated cells no significant 3H-L-citrulline formation was detected, and bradykinin and the calcium ionophore A 23187 failed to stimulate 3H-L-citrulline formation excluding a constitutively expressed, calcium-dependent NO synthase activity. After culturing the cells for 18 h in the presence of LPS (10 micrograms/ml) and TNF alpha (500 U/ml) a marked formation of 3H-L-citrulline coul…

Acetylcholine via Muscarinic Receptors Inhibits Histamine Release from Human Isolated Bronchi

1997

Human bronchi were incubated in organ baths to measure histamine release. The calcium ionophore A23187 (10 mumol/L; 1 min) stimulated histamine release by 148 +/- 28% (n = 11) above the prestimulation level but was ineffective in epithelium-denuded bronchi. Neither bradykinin (0.1 mumol/L) nor compound 48/80 (10 micrograms/ml) triggered the release of histamine from epithelium-intact bronchi. Acetylcholine did not affect spontaneous histamine release (about 2 nmol/g x 5 min) but inhibited A23187-evoked histamine release in an atropine-sensitive manner. Already a concentration as low as 0.1 nmol/L acetylcholine was effective, the maximal inhibition (by 89%) occurred at 100 nmol/L, whereas a …

Endogenous nitric oxide inhibits leukotriene B4 release from rat alveolar macrophages

1997

Effects of endogenous nitric oxide (NO) on the release of mediators of the lipoxygenase and cyclo-oxygenase pathway from rat alveolar macrophages were studied. Alveolar macrophages, freshly isolated or after 18-h culture, were incubated in (amino acid-free) Krebs medium and labelled with [3H]arachidonic acid. The release of [3H]leukotriene B4 and [3H]prostanoids (separated by high performance liquid chromatography) was determined. A 23187 was used as stimulus, as rising intracellular Ca2+ activates directly the phospholipase A2 and lipoxygenase pathway. A 23187 (10 microM) enhanced [3H]leukotriene B4 release from freshly prepared alveolar macrophages about 65-fold, but only 5- to 6-fold fro…

Temperature-dependent effects of increased intraluminal pressure on serotonin release from the vascularly perfused guinea pig ileum

1987

Isolated segments of the guinea pig ileum were vascularly perfused and the release of endogenous serotonin into the portal effluent was measured. Peristalsis was induced by raising the intraluminal hydrostatic pressure by 500 Pa for 5 min. Serotonin release increased during peristalsis induced by fluid of 37 degrees C, but decreased when the temperature of the intraluminal fluid was between 13 degrees C and 22 degrees C. In the presence of naloxone (0.3 mumol/l) raising the intraluminal pressure with fluid of 37 degrees C caused an inhibition of the serotonin release which was blocked by scopolamine (0.1 mumol/l). Naloxone did not affect the inhibition of serotonin release during peristalsi…

GABA receptors are involved in the modulation of the release of 5-hydroxytryptamine from the vascularly perfused small intestine of the guinea-pig

1989

Isolated small intestinal segments of the guinea-pig were perfused arterially and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent was determined by HPLC with electrochemical detection. Test substances were applied intraarterially. Muscimol (1 microM) time dependently first increased then decreased the release of 5-HT and 5-HIAA. The stimulatory effect was prevented by tetrodotoxin (TTx) or scopolamine, indicating that it was mediated by the release of acetylcholine. Bicuculline concentration dependently decreased (1 microM) or increased (10, 50 microM) the release of 5-HT and 5-HIAA, indicating that endogenous GABA also activ…

Mammalian glial cells in culture synthesize acetylcholine.

1997

In the present study we demonstrate that acetylcholine is synthesized by cultured mammalian glial cells identified by cell-type specific markers. Primary cultures of rat brain astrocytes or microglia contained 2.0 and 1.6 pmol acetylcholine/10(6) cells on average respectively. Astrocyte cultures established from neonatal mouse brain contained even more acetylcholine (about 80 pmol acetylcholine/10(6) cells). Primary cultures of rat brain astrocytes showed choline acetyltransferase (ChAT) enzyme activity of 3 nmol/mg protein/h; ChAT activity was blocked by 10 microM bromoacetylcholine. In conclusion, these data demonstrate the synthesis of the "neurotransmitter" acetylcholine in cultured gli…

The cholinergic 'pitfall': acetylcholine, a universal cell molecule in biological systems, including humans.

1999

1. Acetylcholine (ACh) represents one of the most exemplary neurotransmitters. In addition to its presence in neuronal tissue, there is increasing experimental evidence that ACh is widely expressed in pro- and eukaryotic non-neuronal cells. Thus, ACh has been detected in bacteria, algae, protozoa, tubellariae and primitive plants, suggesting an extremely early appearance of ACh in the evolutionary process. 2. In humans, ACh and/or the synthesizing enzyme, choline acetyltransferase, has been demonstrated in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium) and endothelial and muscle cells. In addition, immune cells express the non-ne…

Spontaneous release of endogenous 5-hydroxytryptamine and 5-hydroxyindoleacetic acid from the isolated vascularly perfused ileum of the guinea-pig

1987

The spontaneous release of 5-hydroxytryptamine and its metabolite 5-hydroxyindoleacetic acid from the enterochromaffin cells of the small intestine into the portal circulation was investigated in vitro using the vascularly perfused ileum of the guinea-pig. The release of 5-hydroxytryptamine decreased by 70% in a calcium-free medium and by 35% in the presence of tetrodotoxin. Inhibition of monoamine oxidase activity by pargyline (100 microM) had no effect on the spontaneous release of 5-hydroxytryptamine although it caused a 75% reduction in the outflow of 5-hydroxyindoleacetic acid. Imipramine (1 microM), an inhibitor of neuronal uptake of 5-hydroxytryptamine, reduced the 5-hydroxyindoleace…

Beta-adrenoceptors mediate inhibition of [3H]-acetylcholine release from the isolated rat and guinea-pig trachea: role of the airway mucosa and prost…

1994

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 microM) inhibited evoked [3H]-acetylcholine release, whereas beta 2-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 microM) or of the beta 1-selective antagonist CGP 20712 A (0.1 microM); (i…

The role of cytoplasmic (newly synthesized) dopamine for the spontaneous and electrically evoked release of dopamine and its metabolites from the iso…

1987

Isolated rat NILs were incubated in Krebs-HEPES solution. The release of dopamine and its metabolites (DOPAC, HVA and MOPET) was determined by HPLC with electrochemical detection. The spontaneous release of the sum of metabolites was about 40 times that of dopamine. The spontaneous outflow of dopamine metabolites was unaffected after inhibition of dopamine uptake (by GBR 12921) or after pretreatment with reserpine (5 mg/kg, 12 h before the experiments), but it was reduced by 50% after preincubation with the irreversible DOPA decarboxylase inhibitor, (MFMD, 10 microM, for 10 min). The combination of pretreatment with reserpine and preincubation with MFMD resulted in an 80% inhibition of the …

Airway epithelium: more than just a barrier!

1999

Abstract Airway epithelium: source of non-neuronal acetylcholine and modulator of neurotransmission

Inhibition of nitric oxide synthase abrogates lipopolysaccharides-induced up-regulation of L-arginine uptake in rat alveolar macrophages

2001

It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides-(LPS)-induced up-regulation of L-arginine transport in rat alveolar macrophages (AM). AM were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]-L-arginine uptake or to isolate RNA for RT - PCR. Culture in presence of LPS (1 microg ml(-1), 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]-L-arginine uptake. The inducible NO synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) present alone during culture had only marginal effects on [3H]-L-arginin…

Acetylcholine in isolated airways of rat, guinea pig, and human: species differences in role of airway mucosa

1996

Stored endogenous acetylcholine (ACh) and in vitro synthesis of [3H]ACh were measured in isolated, mucosa-intact and mucosa-denuded airways of rat, guinea pig, and humans. In addition, choline acetyltransferase (ChAT) activity and ACh content were measured in freshly isolated airway mucosa as well as in cultured epithelial cells of rat tracheas. Rat tracheas stored 25 nmol/g ACh, whereas guinea pig tracheas and human bronchi contained only 2-3 nmol/g ACh. When incubated with [3H]choline, the isolated airways of rat, guinea pig, and human synthesized significant amounts of [3H]ACh. In guinea pig and human airways, removal of the mucosa affected neither stored ACh nor in vitro synthesis of […

Cholinergic modulation of the release of 5-hydroxytryptamine from the guinea pig ileum.

1987

Isolated segments of the guinea pig ileum were vascularly perfused and the release of 5-HT and its metabolite 5-HIAA into the portal venous effluent determined by HPLC with electrochemical detection. Test substances were applied via the arterial perfusion medium. Oxotremorine inhibited concentration-dependently the release of 5-HT and 5-HIAA (by 47% at 1 mumol/l). Scopolamine (0.1 mumol/l) did not affect the release of 5-HT and 5-HIAA, but antagonized the effect of oxotremorine. In the presence of TTX (1 mumol/l), oxotremorine (1 mumol/l) increased the release of 5-HT by 150% and that of 5-HIAA by 220%. This increase was completely blocked by scopolamine. Hexamethonium (100 mumol/l) and TTX…

In vitro synthesis of dopamine and noradrenaline in the isolated rat pineal gland: day-night variations and effects of electrical stimulation.

1989

: Isolated rat pineal glands were incubated in vitro in a medium containing [I4C]dopamine or [14C]tyrosine, and the tissue contents of l4C-labelled and total dopamine and noradrenaline were determined by HPLC followed by electrochemical detection and scintillation spectrometry. During incubation with [l4C]dopamine, the labelled amine accumulated in pineal glands and was partially converted into [l4C]noradrenaline. Nomifensine, a neuronal amine uptake blocker, largely inhibited the accumulation of [l4C]dopamine and the formation of [14C]noradrenaline. These experiments demonstrated dopamine β-hydroxylase activity in the sympathetic nerves of the pineal gland. During incubation with [14C]tyro…

Mucosa-dependent muscarinic liberation of prostaglandins from rat isolated trachea.

1995

1. The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid-derived mediators from rat isolate tracheae. 2. Tracheae were preincubated with [3H]-arachidonic acid and the outflow of 3H-compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor-preferring antagonist rho-fluoro-hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine. 3. High performance liquid chromatography analysis (methanol gradient) of the released 3H-compounds showed that one peak, co-eluting with […

Calcium dependence of the evoked arginine vasopressin release by electrical stimulation and by L-glutamate.

1989

Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man.

1997

Acetylcholine acts as a prominent transmitter in the central and peripheral nervous system. The aim of the present study was to investigate whether mammalian non-neuronal cells can synthesize and store acetylcholine. A cotton tipped applicator (Q-tip) was used to collect surface cells from airways and alimentary tract. Histological inspection indicated that rubbing of the luminal surface of human bronchi did not penetrate the basal membrane. Acetylcholine was measured by an HPLC-method using substrate-specific enzyme reactor-columns. Non-neuronal acetylcholine was found in cells covering inner and outer surfaces of rat and man. For example, acetylcholine was detected in the surface epitheli…

Evidence that neuronally released vasoactive intestinal polypeptide inhibits the release of serotonin from enterochromaffin cells of the guinea pig s…

1991

Abstract. Isolated small intestinal segments of the guinea pig were arterially perfused and the release of serotonin (5-hydroxytryptamine) and 5-hydroxyindoleacetic acid into the portal venous effluent was determined by HPLC with electrochemical detection. Test substances were intra-arterially applied. The muscarine receptor agonist oxotremorine (1 μmol/l inhibited the release of 5-hydroxytryptamine by about 50%. In the presence of the neurotoxin tetrodotoxin, oxotremorine enhanced the release of 5-hydroxytryptamine by 145%, indicating that the inhibitory effect of oxotremorine was mediated by the release of a neurotransmitter. Exogenous vasoactive intestinal polypeptide ( 1-100 pmol/l inhi…

Glucocorticoids mediate reduction of epithelial acetylcholine content in the airways of rats and humans

1998

The cholinergic system in rat and human airways and the effects of glucocorticoids were investigated by assay of choline acetyltransferase activity, by high-pressure liquid chromatography measurement of acetylcholine, and by anti-choline acetyltransferase immunocyto-/histochemistry. Human bronchi were obtained at surgery from patients with lung cancer. Group 1 patients did not suffer from additional lung diseases and had not been treated with glucocorticoids. Group 2 patients, who suffered in addition to lung cancer from chronic obstructive bronchitis, had been treated for at least 6 weeks before surgery with four puffs of flusinolid daily. Isolated bronchial epithelial cells as well as int…

Effects of gadolinium and cadmium on the electrically evoked release of 45calcium from the isolated rat neurohypophysis.

1988

Isolated neural lobes of the rat pituitary gland were fixed by their stalks to a platinum wire electrode. They were loaded with 45calcium and then superfused with radioactivity-free Krebs-solution. The efflux of 45calcium into the superfusion medium was determined. After 54–60 min of superfusion the spontaneous outflow of 45calcium was 2.03%/min of the tissue 45calcium. It was not affected by cadmium (Cd2+, 0.03-3 mmol/1), but reduced by 40% in the presence of 1 mmol/1 gadolinium (Gd3). Electrical stimulation with pulses of 15 Hz (3 times for 1 min with intervals of 1 min) evoked a 45calcium release of 14.4% of the tissue radioactivity. The evoked release of 45calcium was reduced by 80% in …