K. Racké

REGULATION OF SEROTONIN RELEASE FROM THE INTESTINAL MUCOSA

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 (alpha 2-adrenoceptors, GABAA- and GABAB-receptors, histamine H3-receptors, receptors for vasoactive intestinal polypeptide and somatostatin) as well as stimulatory receptors (beta-adrenoceptors, muscarine and nicotine receptors). The physiological significance of this complex system of receptors is suggested by experiments which demonstrate that the respective intrinsic neurotransmitters (catecholamines, acetylcholine, GABA and vasoactive intestinal polypeptide) released within the gut are involved in the regulation of the release of 5-HT from the enterochromaffin cells.

Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig : involvement of 5-HT3 receptors

SummaryIsolated 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 μM) increased the outflow of 5-HT and 5-HIAA by about 90%. At 30 and 100 μM 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-phosphate buffer of the Tyrodes solution was replaced by HEPES-buffer. The stimulatory effect of cisplatin was abolished in the absence of extracellular calcium or presence of tetrodotoxin (1 μM). The stimulatory effect of cisplatin was also prevented by hexamethonium (100 μM) or scopolamine (100 nM). The 5-HT3 receptor antagonists ondansetron and ICS 205-930 in concentrations as low as 1 pM also abolished the stimulatory effect of cisplatin. The 5-HT3 receptor antagonist MDL 72222 prevented the stimulatory effect of cisplatin only at a concentration of 1 μM. None of the 5-HT3 receptor antagonists alone significantly altered the outflow of 5-HT and 5-HIAA.Cisplatin (3 μM) enhanced the outflow of [3H]radioactivity from intestinal segments and caused longitudinal muscle contractions that were abolished by 100 nM scopolamine.In conclusion, cisplatin, at concentrations which occur during anti-cancer therapy in humans and induce emesis, increases the release of 5-HT from the enterochromaffin cells of the small intestine of the guinea-pig. This effect of cisplatin is mediated by a cascade of events which involves release of acetylcholine and stimulation of 5-HT3 receptors.

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

SummaryIsolated segements 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 μmol/l). Scopolamine (0.1 μmol/1) did not affect the release of 5-HT and 5-HIAA, but antagonized the effect of oxotremorine. In the presence of TTX (1 μmol/1) oxotremorine (1 pmol/1) increased the release of 5-HT by 150% and that of 5-HIAA by 220%. This increase was completely blocked by scopolamine. Hexamethonium (100 pmol/1) and TTX (1 pmol/1) reduced the release of 5-HT by 32 and 40%, respectively. DMPP (10 pmol/1) increased the release of 5-HT by 57%, and this effect was prevented by hexamethonium. Neither DMPP nor hexamethonium significantly affected the release of 5-HIAA. The enhancing effect of DMPP on 5-HT release was increased and prolonged in the presence of TTX or scopolamine. Nicotine (1, 10 or 30 μmol/l) alone did not cause a consistent increase in the release of 5-HT. However, in the presence of scopolamine nicotine increased the release of 5-HT by 57%. In conclusion, the release of intestinal 5-HT is facilitated via muscarine and nicotine receptors located on the enterochromaffin cells. Indirect evidence suggests that the release of 5-HT is additionally modulated by an as yet unknown inhibitory neurotransmitter released by muscarine receptor activation.

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

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 μm) increased the outflow of 5‐HT and 5‐HIAA maximally by about 75% and this was antagonized by propranolol (0.1 μm). Forskolin (1–10 μm) 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 μm) increased the outflow of 5‐HT and 5‐HIAA by about 70%. Isoprenaline (1 nm) and AH 21–132 (10 nm), which alone had no effect, increased the outflow of 5‐HT and 5‐HIAA by 75%, when applied in combination. 3 Clonidine (1 μm) reduced the outflow of 5‐HT by 45%, an effect blocked by tolazoline (1 μm), but not by prazosin (0.1μm). 4 The effects of isoprenaline, forskolin and clonidine were also observed in the presence of tetrodotoxin (1 μm) demonstrating a direct modulation of 5‐HT release from the enterochromaffin cells. 5 In conclusion, the release of 5‐HT from enterochromaffin cells is facilitated by activation of β‐adrenoceptors and inhibited via α2‐adrenoceptors. Enhancing intracellular cyclic AMP, by direct stimulation of adenylate cyclase with forskolin or by inhibition of phosphodiesterase, also facilitates the release of 5‐HT. The β‐adrenoceptor‐mediated effect on 5‐HT release appears to involve an increase in cyclic AMP, as the effect of isoprenaline was potentiated after inhibition of phosphodiesterase.

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

SummaryTo 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 content of [3H]acetylcholine remained unchanged. The spontaneous outflow of tritium consisted mainly of [3H]phosphorylcholine (900 dpm/3 min) and [3H]choline (800 dpm/3 min); [3H]acetylcholine was only a minor fraction (50 dpm/3 min). Electrical stimulation of tracheae with intact epithelium caused only a small release of [3H]acetylcholine (460 dpm in the sample obtained during stimulation), but a considerable outflow of [3H]phosphorylcholine (1,900 dpm) without affecting the outflow of [3H]choline. Electrical stimulation of epithelium-deficient tracheae, however, induced a substantial release of [3H]acetylcholine (2,400 dpm), but only a small outflow of [3H]phosphorylcholine. Chemical stimulation (30 μmol/1 veratridine) also caused a large release of [3H]acetylcholine (1,700 dpm) without affecting the outflow of [3H]phosphorylcholine or [3H]choline. Indomethacin (3 μmol/1) enhanced the electrically-evoked release of [3H]acetylcholine from tracheae with intact epithelium by 89%.The present experiments demonstrate a strong inhibition by the epithelium of the electrically-evoked release of [3H]acetylcholine from the isolated guinea-pig trachea. Cyclooxygenase products of arachidonic acid do not appear as the main mediators of the epithelium-derived inhibition of acetylcholine release.

Release of Endogenous 3,4‐Dihydroxyphenylethylamine and Its Metabolites from the Isolated Neurointermediate Lobe of the Rat Pituitary Gland. Effects of Electrical Stimulation and of Inhibition of Monoamine Oxidase and Reuptake

Abstract: 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 (100 μM). Pargyline (10 μM) caused a time‐dependent decrease of all metabolites (up to 90%). In the presence of GBR 12921 and pargyline, the spontaneous outflow of DA increased sevenfold. Removal of the intermediate lobe caused a 78% reduction in tissue DA content and a corresponding reduction of the outflow of metabolites. Electrical stimulation of the pituitary stalk (0.2 ms, 10 V, 15 Hz, three times for 1 min at intervals of 1 min) induced an increase in outflow of DA and all metabolites. DA accounted for 15%, HVA for 41%, DOPAC for 32%, and MOPET for 12% of the evoked release. The electrically evoked release of DA increased fourfold in the presence of GBR 12921 or pargyline and the effects of both drugs were additive. The evoked release of metabolites was not significantly affected by GBR 12921 but completely abolished by pargyline. In conclusion, oxidative deamination and O‐methylation are important pathways for the catabolism of DA in the neurointermediate lobe. Most of the DA metabolites released spontaneously originate from DA that had been degraded intraneuronally without undergoing prior release. Reuptake and extraneuronal metabolism contribute to the inactivation of DA released during electrical stimulation.

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

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 activates stimulatory and inhibitory GABAA receptors. Bicuculline antagonized the stimulatory and inhibitory effect of muscimol. (-)-Baclofen, but not its (+) enantiomer, inhibited the release of 5-HT in the absence and presence of TTx. It was concluded that the release of 5-HT from enterochromaffin cells is directly inhibited by GABAA and GABAB receptors. In addition, acetylcholine released after activation of GABAA receptors stimulates 5-HT release.

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

SummaryBasal 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-fold increase in tritium outflow that was abolished by the removal of extracellular calcium or by the addition of tetrodotoxin. The stimulated outflow of tritium induced by preganglionic nerve stimulation was caused by an exclusive release of [3H]acetylcholine, whereas the efflux of [3H]phosphorylcholine and [3H]choline remained unaffected by this stimulation mode. Transmural stimulation of the rat or guinea-pig trachea, however, caused, in addition to the release of [3H]acetylcholine, the outflow of [3H]phosphorylcholine. Hexamethonium (300 μmol/l) or tubocurarine (100 μmol/l) inhibited (80%) the increase in tritium outflow evoked by preganglionic stimulation, but did not affect tritium outflow evoked by transmural stimulation. Oxotremorine reduced [3H]acetylcholine release evoked by both stimulation modes, but oxotremorine was less potent with transmural stimulation. Scopolamine (0.3 μmol/l) enhanced (120%) the release of [3H]acetylcholine evoked by preganglionic nerve stimulation indicating the blockade of an endogenous negative muscarinic feedback mechanism. Epithelium-dependent inhibition of [3H]acetylcholine release was evident with both preganglionic and transmural stimulation.The present experiments demonstrate the release of [3H]acetylcholine evoked from the isolated trachea by stimulation of the preganglionic trunk of the parasympathetic cholinergic nerves. Qualitative and quantitative differences were observed in comparison to transmural stimulation. Preganglionic nerve stimulation allows a selective excitation of pulmonary, parasympathetic nerve fibres, mimics the physiological excitation of intramural neurones and is not followed by the liberation of phosphorylcholine from non-neuronal cells.

Dopamine release and synthesis in the neurointermediate lobe of the rat hypophysis in vitro after electrical stimulation of the pituitary stalk

The isolated neurointermediate lobe of the rat hypophysis was incubated in Krebs solution and the stalk was electrically stimulated. The endogenous dopamine (DA) released into the medium was estimated by HPLC with electrochemical detection. Stimulation with biphasic pulses (1 ms, 10 Hz) in the presence of pargyline elicited a mean DA output of 200 fg X pulse-1. This release was calcium-dependent but was only partially inhibited by tetrodotoxin (TTX) (1 microM), effects typical for direct electrical depolarization of the nerve endings. Reducing the duration of the electric pulses to 0.2 ms (15 Hz) caused a reduction in DA output to about 40 fg X pulse-1 which was completely blocked by TTX indicating that it was evoked by propagated action potentials. DA overflow was enhanced when the action potential was prolonged with tetraethylammonium (TEA) or when DA uptake was inhibited with GBR 12921. Evidence for a calcium-dependent increase in DA synthesis in electrically stimulated NILs has been obtained when monoamine oxidase was inhibited and TEA or GBR 12921 was present in the Krebs solution. The present results complete the requirements for the DA in the NIL to be classified as a neurotransmitter substance. The NIL-pituitary stalk preparation is a useful model for studying regulatory mechanisms in dopaminergic nerve terminals.

Isoprenaline and forskolin increase evoked vasopressin release from rat pituitary

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.