No abstract

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)

Sjö blom, Markus, Bengt Sä fsten, and Gunnar Flemströ m. Melatonin-induced calcium signaling in clusters of human and rat duodenal enterocytes. Am J Physiol Gastrointest Liver Physiol 284: G1034-G1044, 2003. First published February 12, 2003 10.1152/ajpgi.00500.2002The amount of melatonin present in enterochromaffin cells in the alimentary tract is much higher than that in the central nervous system, and melatonin acting at MT2 receptors mediates neural stimulation of mucosal HCO 3 Ϫ secretion in duodenum in vivo. We have examined effects of melatonin and receptor ligands on intracellular free calcium concentration ([Ca 2ϩ ]i) signaling in human and rat duodenal enterocytes. Clusters of interconnecting enterocytes (10-50 cells) were isolated by mild digestion (collagenase/dispase) of human duodenal biopsies or rat duodenal mucosa loaded with fura-2 AM and attached to the bottom of a temperature-controlled perfusion chamber. Clusters provided viable preparations and respond to stimuli as a syncytium. Melatonin and melatonin receptor agonists 2-iodo-N-butanoyl-5-methoxytryptamine and 2-iodomelatonin (1.0-100 nM) increased enterocyte [Ca 2ϩ ]i, EC50 of melatonin being 17.0 Ϯ 2.6 nM. The MT2 receptor antagonists luzindole and N-pentanoyl-2-benzyltryptamine abolished the [Ca 2ϩ ]i responses. The muscarinic antagonist atropine (1.0 M) was without effect on basal [Ca 2ϩ ]i and did not affect the response to melatonin. In the main type of response, [Ca 2ϩ ]i spiked rapidly and returned to basal values within 4-6 min. In another type, the initial rise in [Ca 2ϩ ]i was followed by rhythmic oscillations of high amplitude. Melatonin-induced enterocyte [Ca 2ϩ ]i signaling as well as mucosal cell-to-cell communication may be involved in stimulation of duodenal mucosal HCO 3 Ϫ secretion. enterochromaffin cells; enterocyte clusters; intracellular calcium; mucosal protection; syncytium DUODENAL MUCOSAL HCO 3 Ϫ SECRETION has a key role in duodenal protection against the pulses of hydrochloric acid (and pepsin) intermittently discharged from the stomach. The duodenal enterocytes transport HCO 3 Ϫ into the continuous layer of viscoelastic mucus gel on top of the epithelial surface, maintaining pH in its cell-facing portion at neutrality despite high acidities (pH Յ 2.0) in the duodenal luminal bulk solution. The major physiological stimulant of the HCO 3 Ϫ secretion is gastric acid expelled into the duodenal lumen, and the acid-induced HCO 3 Ϫ response is mediated by enteric nervous pathways, involving release of vasoactive intestinal polypeptide and acetylcholine (11, 12), as well as by E-type prostaglandins released from mucosal cells (36). In addition to the local intestinal control, the alkaline secretion is under central nervous influence. Up to fourfold increases in secretion occur after intracerebroventricular infusion of the ␣ 1 -adrenoceptor agonist phenylephrine (20,34), and stimulation has also been observed after intracerebroventricular administration of some neuropeptides (13, 21) and benzodiazepines (29)....

Vagal stimuli increase duodenal mucosal[Formula: see text] 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 ∼3 μM. In a first type of signaling pattern, [Ca2+]ireturned 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 μM 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.

Stimulation of the duodenal protective secretion by 5-HT thus involves receptors of the 5-HT4 subtype as well as nicotinic transmission, the myenteric plexus being a likely location. In addition, serotonin acts on enterocyte membrane receptors, inducing intracellular calcium signalling.