Eosinophil granulocytes are activated during the remission phase of ulcerative colitis.

AIM: The aim of this study was to establish a method of investigating intestinal eosinophil and neutrophil granulocytes by flow cytometry, and to compare the distribution and activity of these cells in different stages of ulcerative colitis (UC). METHODS: Biopsy samples were taken from six locations of the entire colon and from the terminal ileum in 10 patients with active total UC, 10 patients with inactive total UC, eight patients with active distal UC, and 11 control subjects. Cell suspensions from biopsies and from peripheral blood were incubated with fluorophore conjugated monoclonal antibodies. The use of scatter plot-gating and specific antibodies was established in a flow cytometry assay. RESULTS: Eosinophils were more numerous and more active in patients with active UC than in controls. Interestingly, during inactive UC, the number of activated eosinophils was even larger. Eosinophil activity was high in the rectum of patients with distal colitis but was also slightly elevated in the proximal colon. Neutrophils were increased in number and activity during active but not inactive UC. In patients with distal colitis, activated neutrophils were only found in the sigmoid colon and rectum. CONCLUSION: With this method, we confirm that neutrophils participate in the inflammatory process during active UC, and that they express a resting phenotype during remission. The finding of activated eosinophils in inflamed intestine strengthens the view of these cells as proinflammatory and tissue damaging. Nevertheless, our new finding of high eosinophil activation during inactive UC suggests that eosinophils play a role in repair of injured epithelium.

Effects of gamma-aminobutyric acid and peripheral benzodiazepine ligands on duodenal bicarbonate secretion.

BACKGROUND: The effects of gamma-aminobutyric (GABA(A))-receptor ligands and peripheral-type benzodiazepine (BZ) ligands on duodenal mucosal bicarbonate secretion (DBS) were studied in thiobarbiturate-anesthetized rats. METHODS: A segment of proximal duodenum was perfused, and bicarbonate secretion was continuously titrated by pH-stat. In some experiments the vagus nerves were dissected free and cut at the neck level. RESULTS: Luminal GABA (10(-8) to 10(-6) M) increased (P < 0.01) DBS dose-dependently, and the GABA(A) antagonist (+)-bicuculline (0.1-2.5 mg/kg intravenously) caused a similar increase (P < 0.01) in DBS. Vagotomy abolished the latter response, suggesting a centrally elicited nervous action of bicuculline mediated by the vagal nerves. This was supported by the absence of an effect of bicuculline administered intra-arterially close to the duodenum. The GABA(A) agonist muscimol had no effect on DBS when administered intravenously (0.01-0.25 mg/kg) or into a lateral brain ventricle (2 microg/kg/h) but counteracted any stimulation by subsequent intravenous bicuculline. The 'peripheral-type' BZ agonist 4'-chlorodiazepam increased DBS when infused close intra-arterially but had no effect when administered intravenously. CONCLUSIONS: Inhibition of GABA(A) receptors related to cholinergic excitatory pathways in the central nervous system stimulates duodenal mucosal bicarbonate secretion and may increase the local mucosal defense. The stimulation by luminal GABA may reflect modulation of the local mucosal control of duodenal bicarbonate secretion.

Calcium signaling in cultured human and rat duodenal enterocytes.

Vagal stimuli increase duodenal mucosal HCO-3 secretion and may provide anticipatory protection against acid injury, but duodenal enterocyte (duodenocyte) responses and cholinoceptor selectivity have not been defined. We therefore developed a stable primary culture model of duodenocytes from rats and humans. Brief digestion of scraped rat duodenal mucosa or human biopsies with collagenase/dispase yielded cells that attached to the extracellular matrix Matrigel within a few hours of plating. Columnar cells with villus enterocyte morphology that exhibited spontaneous active movement were evident between 1 and 3 days of culture. Rat duodenocytes loaded with fura 2 responded to carbachol with a transient increase in intracellular calcium concentration ([Ca2+]i), with an apparent EC50 of approximately 3 microM. In a first type of signaling pattern, [Ca2+]i returned to basal or near basal values within 3-5 min. In a second type, observed in cells with enlarged vacuoles characteristic of crypt cell morphology, the initial transient increase was followed by rhythmic oscillations. Human duodenocytes responded with a more sustained increase in [Ca2+]i, and oscillations were not observed. Rat as well as human duodenocytes also responded to CCK-octapeptide but not to vasoactive intestinal polypeptide. Equimolar concentrations (100 nM) of the subtype-independent muscarinic antagonist atropine and the M3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the response to 10 microM carbachol, whereas the M1 antagonist pirenzepine and the M2 antagonists methoctramine and AF-DX 116BS had no effect at similar concentrations. Responses in rat and human duodenocytes were similar. A new agonist-sensitive primary culture model for rat and human duodenocytes has thus been established and the presence of enterocyte CCK and muscarinic M3 receptors demonstrated.

Role of dopamine and other stimuli of mucosal bicarbonate secretion in duodenal protection.

Duodenal mucosal secretion of bicarbonate is one main mechanism in the protection of this epithelium against luminal acid. The duodenal secretagogue vasoactive intestinal polypeptide, at doses not affecting mucosal blood flow, protects against acid-induced morphological changes. Some sigma receptor ligands, which increase the duodenal alkaline secretion, prevent duodenal but not gastric mucosal ulceration. Dopamine D-1 receptor agonists and the peripherally acting catechol-O-methyl-transferase (COMT) inhibitor nitecapone stimulate the bicarbonate secretion in the rat and a similar increase in secretion has been observed in human volunteers. COMT inhibitors decrease tissue degradation of catecholamines, including dopamine. The D-2 agonist bromocriptine, in contrast, decreases the secretion. These results, indicating that the bicarbonate secretion is stimulated via peripheral dopamine D-1 receptors, are supported by the finding that dopamine D-1 (but not D-2) agonists increase the production of cyclic AMP in isolated duodenal enterocytes. The increase in mucosal alkaline secretion may contribute to the previously observed ulceroprotective actions of dopaminergic compounds.

Cholinergic influence on duodenal mucosal bicarbonate secretion in the anesthetized rat.

Bicarbonate secretion by duodenum distal to the Brunner's glands area was titrated in situ in rats anesthetized with thiobarbiturate. The unselective muscarinic antagonist atropine (0.4 mg/kg) inhibited secretion stimulated by bethanechol (15 micrograms.kg-1.h-1) but not that stimulated by carbachol (15 micrograms.kg-1.h-1). The nicotinic antagonist hexamethonium (10 mg.kg-1.h-1), however, abolished the latter response. The muscarinic M1-selective antagonists pirenzepine and telenzepine (0.025, 0.25 and 2.5 mg/kg) did not decrease but caused a dose-dependent rise in duodenal mucosal HCO3- secretion, an effect abolished by cervical vagotomy or infusion of the alpha-adrenoceptor antagonist phentolamine (0.1 mg.kg-1.h-1). Phentolamine alone caused a sustained increase in secretion. McN-A-343 (0.025, 0.25, and 2.5 mg/kg), an M1-selective agonist and ganglionic stimulator, increased the HCO3- secretion; this effect was not prevented by vagotomy but was attenuated by pirenzepine. Intracerebroventricular infusion of pirenzepine and telenzepine did not cause any changes in secretion. These findings suggest that peripheral muscarinic M1 and nicotinic receptors mediate cholinergic stimulation of duodenal mucosal HCO3- secretion. Pirenzepine and telenzepine may act stimulatory by antagonizing muscarinic M1-transmission in peripheral sympathetic ganglia, thus decreasing postsynaptic adrenergic inhibition.

Proximal duodenal enterocyte transport: evidence for Na(+)-H+ and Cl(-)-HCO3- exchange and NaHCO3 cotransport.

The duodenum, in contrast to the jejunum, actively secretes HCO3- at a high rate, a process that protects the mucosa from acid/peptic injury. Our purpose was to define the mechanisms involved in HCO3- transport by studying the acid-base transport processes in isolated duodenal enterocytes. Individual rat duodenocytes, isolated by a combination of Ca2+ chelation and collagenase, attached to a collagen matrix were loaded with the pH-sensitive fluoroprobe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), and intracellular pH was monitored by microfluorospectrophotometry. To identify Na(+)-H+ transport, cells in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 1) were pulsed with NH4Cl (40 mM) in the absence and presence of amiloride and 2) were removed of Na+. To examine Cl(-)-HCO3- exchange, Cl- was removed from Ringer-HCO3- superfusate in the presence and absence of dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS). The NaHCO3 cotransporter was studied by addition and subtraction of Na+ to amiloride-treated and Cl(-)-depleted enterocytes perfused with Na(+)- and Cl(-)-free Ringer-HCO3- buffer with and without H2DIDS. Mammalian duodenocytes contain at least three acid-base transporters: an amiloride-sensitive Na(+)-H+ exchanger that extrudes acid, a DIDS-sensitive Cl(-)-HCO3- exchanger that extrudes base, and a NaHCO3 cotransporter, also DIDS sensitive, that functions as a base loader. These acid-base transporters likely play a key role in duodenal mucosal HCO3- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine and vasoactive intestinal peptide stimulate cyclic adenosine-3',5'-monophosphate formation in isolated rat villus and crypt duodenocytes.

Secretion of bicarbonate increases the pH at the duodenal mucosal surface, a process which contributes to the protection against acid/pepsin injury. Previously, we have shown that dopaminergic compounds stimulate the duodenal bicarbonate secretion in situ, in the anaesthetized rat, through an action on peripheral dopamine D1 receptors. In order to study the possible involvement of cyclic adenosine-3',5'-monophosphate (cAMP) as an intracellular mediator in enterocytes isolated from rat duodenum, cells were collected by a combination of enzyme treatment and calcium chelation. Two major cell fractions, one mainly from villi and the other mainly of crypt origin, were studied. In the villus cell fraction, the activity of alkaline phosphatase was 1.6 +/- 0.2 mumol mg protein-1 min-1 and that of sucrase 98.8 +/- 16.4 nmol mg protein-1 min-1. In the crypt fraction, activities were 0.7 +/- 0.1 and 28 +/- 10.5, respectively. Effects of dopamine, two selective dopamine receptor agonists and vasoactive intestinal peptide (VIP) on intracellular accumulation of cAMP were examined by radio-immunoassay (RIA). In the crypt cell fraction, VIP (10(-7) M) caused an increase in cAMP which was maximal after 5 min (78 +/- 28% above control, P < 0.01). In the villus cell fraction, maximal responses to VIP (60 +/- 24% above control, P < 0.05), did not occur until after 60 min of incubation. In contrast, there were no significant differences between villi and crypt enterocytes in respect to effects of dopamine, the dopamine D1-receptor agonist SKF-38393 and the D2-receptor agonist quinpirole.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of mucosal alkaline secretion in rat duodenum by dopamine and dopaminergic compounds.

BACKGROUND: The catechol-O-methyl-transferase (COMT) inhibitor nitecapone, which prevents mucosal degradation of dopamine, and some dopamine receptor agonists ameliorate gastroduodenal mucosal damage. Therefore, their effects on mucosal bicarbonate secretion were studied. METHODS: Duodenum just distal to the Brunner's glands area was cannulated in situ in anesthetized rats. Bicarbonate secretion into the luminal perfusate and transmucosal electrical potential difference (PD) were recorded. RESULTS: Intravenous dopamine (50 micrograms.kg-1 x h-1) increased bicarbonate secretion twofold, and a higher rate of infusion (250 micrograms.kg-1 x h-1) resulted in a further increase. Neither dose affected the PD. The dopamine D1 agonist SKF-38393 (10-50 micrograms/kg) and the COMT inhibitor nitecapone (50-500 micrograms/kg) caused dose-dependent increases in secretion, similar to that observed with dopamine. Domperidone, a peripherally acting dopamine antagonist, inhibited the stimulatory effects of SKF-38393 and nitecapone. Hexamethonium or the alpha-adrenoceptor antagonist phentolamine, in contrast, did not affect the response to nitecapone. Intracerebroventricular administration of nitecapone was without effect. CONCLUSIONS: A probable electroneutral component of duodenal mucosal bicarbonate secretion is stimulated via peripheral dopamine D1 receptors. This may contribute to the previously observed ulceroprotective actions of dopaminergic compounds.

Duodenal bicarbonate secretion and mucosal protection. Neurohumoral influence and transport mechanisms.

Duodenal mucosal bicarbonate secretion (DMBS) plays an important role in the defence against acid discharged from the stomach. The secretion by duodenum immediately distal to the Brunner's glands area and devoid of pancreatic and biliary secretions, was investigated in vivo in anaesthetized Sprague-Dawley rats and in vitro in mucosae isolated from the American bullfrog. Transport mechanisms were studied in isolated rat duodenal enterocytes and identified by use of digitized microfluorometry and the fluoroprobe BCECF. Cyclic AMP production in enterocytes of villus vs. crypt origin was measured with radioimmunoassay. The benzodiazepines diazepam and Ro 15-1788 stimulated DMBS in the rat when administered intravenously or intracerebroventricularly; however, their stimulatory effect was abolished by bilateral proximal vagotomy, and they had no effect on the secretion by isolated bullfrog mucosa. It is concluded that these benzodiazepines stimulate secretion by acting upon the central nervous system and that their effects are vagally mediated. Dopamine, the catechol-O-methyl-transferase-inhibitor nitecapone, and the dopamine D1 agonist SKF-38393 all stimulated DMBS. The peripherally acting antagonist domperidone while having no influence on basal DMBS did prevent the influences of SKF-38393 and nitecapone. The alpha 1-antagonist prazosin had no such effects and the combined results suggest that DMBS is stimulated via peripheral dopamine D1 receptors. Intravenous, but not central nervous, administration of the muscarinic M1 receptor antagonists pirenzepine and telenzepine effectively stimulated DMBS; however their effectiveness was dependent on intact vagal nerves. Phentolamine, an unselective alpha-adrenergic antagonist, prevented the stimulation by pirenzepine and telenzepine and stimulation by carbachol was abolished by hexamethonium. It is concluded that peripheral nicotinergic and muscarinergic M1 receptors mediate stimulation of DMBS, in part by acting upon peripheral sympathetic ganglia. Whereas dopamine and SKF-38393 caused a time-dependent increase in the accumulation of cyclic AMP in duodenal enterocytes of crypt and villous origin, the D2 agonist quinpirole had an inhibitive influence. Crypt and villus cells differed in their respective time-courses in response to vasoactive intestinal polypeptide. Finally, Cl-/HCO3- exchange, Na+/H+ exchange and NaHCO3 cotransport were identified as membrane acid/base transport mechanisms in isolated duodenal enterocytes.

Effects of diazepam and Ro 15-1788 on duodenal bicarbonate secretion in the rat.

Bicarbonate secretion by duodenal mucosa just distal to the Brunner's glands area and devoid of pancreatic secretions was titrated in situ in anesthetized rats. Intravenous injection of diazepam (0.1 and 0.5 mg/kg) significantly increased the secretion; this stimulation was abolished by proximal bilateral vagotomy. Ro 15-1788, a benzodiazepine antagonist that also has well-known intrinsic activity, caused similar stimulation of the secretion when administered IV (0.01 and 0.1 mg/kg). Intracerebroventricular infusion of Ro 15-1788 (10 micrograms/h) resulted in a greater increase in secretion; again, this stimulation was prevented by vagotomy. Adrenoceptor blockade by phentolamine increased basal alkaline secretion but did not affect the stimulation by diazepam. The tricyclic antidepressant trimipramine (2.5 mg/kg IV) did not affect the duodenal bicarbonate secretion. For comparison, effects of diazepam and Ro 15-1788 (10(-6)-10(-4) mol/L) were also tested in isolated bullfrog duodenal mucosa. Neither drug effected the alkaline secretion in vitro. The combined results strongly suggest that benzodiazepines, as previously shown for certain brain peptides, influence the central nervous control of duodenal mucosal alkaline secretion and that their stimulation of secretion is vagally mediated. This action benzodiazepines might be used in modulating mucosal protection against acid.